Dan Rudolf Littman, M.D., Ph.D.

RUNX Transcription Factor-Mediated Association of Cd4 and Cd8 Enables Coordinate Gene Regulation
Collins, Amelie; Hewitt, Susannah L; Chaumeil, Julie; Sellars, Maclean; Micsinai, Mariann; Allinne, Jeanne; Parisi, Fabio; Nora, Elphege P; Bolland, Dan J; Corcoran, Anne E; Kluger, Yuval; Bosselut, Remy; Ellmeier, Wilfried; Chong, Mark M W; Littman, Dan R; Skok, Jane A
2011 Mar 25;34(3):303-314, Immunity
T cell fate is associated with mutually exclusive expression of CD4 or CD8 in helper and cytotoxic T cells, respectively. How expression of one locus is temporally coordinated with repression of the other has been a long-standing enigma, though we know RUNX transcription factors activate the Cd8 locus, silence the Cd4 locus, and repress the Zbtb7b locus (encoding the transcription factor ThPOK), which is required for CD4 expression. Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart. Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8. Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells
— id: 129323, year: 2011, vol: 34, page: 303, stat: Journal Article,

CXCR7 influences leukocyte entry into the CNS parenchyma by controlling abluminal CXCL12 abundance during autoimmunity
Cruz-Orengo, Lillian; Holman, David W; Dorsey, Denise; Zhou, Liang; Zhang, Penglie; Wright, Melissa; McCandless, Erin E; Patel, Jigisha R; Luker, Gary D; Littman, Dan R; Russell, John H; Klein, Robyn S
2011 Feb 14;208(2):327-339, Journal of experimental medicine
Loss of CXCL12, a leukocyte localizing cue, from abluminal surfaces of the blood-brain barrier occurs in multiple sclerosis (MS) lesions. However, the mechanisms and consequences of reduced abluminal CXCL12 abundance remain unclear. Here, we show that activation of CXCR7, which scavenges CXCL12, is essential for leukocyte entry via endothelial barriers into the central nervous system (CNS) parenchyma during experimental autoimmune encephalomyelitis (EAE), a model for MS. CXCR7 expression on endothelial barriers increased during EAE at sites of inflammatory infiltration. Treatment with a CXCR7 antagonist ameliorated EAE, reduced leukocyte infiltration into the CNS parenchyma and parenchymal VCAM-1 expression, and increased abluminal levels of CXCL12. Interleukin 17 and interleukin 1beta increased, whereas interferon-gamma decreased, CXCR7 expression on and CXCL12 internalization in primary brain endothelial cells in vitro. These findings identify molecular requirements for the transvascular entry of leukocytes into the CNS and suggest that CXCR7 blockade may have therapeutic utility for the treatment of MS
— id: 134177, year: 2011, vol: 208, page: 327, stat: Journal Article,

Transcription factor AP4 modulates reversible and epigenetic silencing of the Cd4 gene
Egawa, Takeshi; Littman, Dan R
2011 Sep 6;108(36):14873-14878, Proceedings of the National Academy of Sciences of the United States of America
CD4 coreceptor expression is negatively regulated through activity of the Cd4 silencer in CD4(-)CD8(-) double-negative (DN) thymocytes and CD8(+) cytotoxic lineage T cells. Whereas Cd4 silencing is reversed during transition from DN to CD4(+)CD8(+) double-positive stages, it is maintained through heritable epigenetic processes following its establishment in mature CD8(+) T cells. We previously demonstrated that the Runx family of transcription factors is required for Cd4 silencing both in DN thymocytes and CD8(+) T cells. However, additional factors that cooperate with Runx proteins in the process of Cd4 silencing remain unknown. To identify collaborating factors, we used microarray and RNAi-based approaches and found the basic helix-loop-helix ZIP transcription factor AP4 to have an important role in Cd4 regulation. AP4 interacts with Runx1 in cells in which Cd4 is silenced, and is required for Cd4 silencing in immature DN thymocytes through binding to the proximal enhancer. Furthermore, although AP4-deficient CD8(+) T cells appeared to normally down-regulate CD4 expression, AP4 deficiency significantly increased the frequency of CD4-expressing effector/memory CD8(+) T cells in mice harboring point mutations in the Cd4 silencer. Our results suggest that AP4 contributes to Cd4 silencing both in DN and CD8(+) T cells by enforcing checkpoints for appropriate timing of CD4 expression and its epigenetic silencing
— id: 137074, year: 2011, vol: 108, page: 14873, stat: Journal Article,

Characterization of interleukin-17-producing regulatory T cells in inflamed intestinal mucosa from patients with inflammatory bowel diseases
Hovhannisyan, Zaruhi; Treatman, Jacquelyn; Littman, Dan R; Mayer, Lloyd
2011 Mar;140(3):957-965, Gastroenterology
BACKGROUND & AIMS: Interleukin (IL)-17-producing CD4(+) helper T cells (Th17) mediate mucosal immunity and are involved in the pathogenesis of inflammatory bowel disease (IBD). They are believed to arise from the same precursor population as regulatory T (Treg) cells, but little is known about how these T-cell subsets interact under chronic inflammatory conditions. We studied Th17 and Treg cells isolated from intestinal lamina propria of patients with IBD to investigate their role in pathogenesis. METHODS: FoxP3 expression (a marker of Treg cells) and IL-17 production were assessed in CD4(+) lamina propria lymphocytes isolated from IBD patients and healthy subjects. IL-17(+)FoxP3(+) and IL-17(+) CD4(+) T-cell clones were generated by limiting dilution. An in vitro suppression assay was performed to assess the functional capacity of derived T-cell clones. RESULTS: IL-17(+)FoxP3(+) T cells were identified in inflamed intestinal mucosa of patients with Crohn disease (CD), but not in patients with ulcerative colitis (UC) or healthy controls. These cells shared phenotypic characteristics of Th17 and Treg cells, and showed potent suppressor activity in vitro. Transforming growth factor-beta was necessary and sufficient to induce development of an IL-17(+) FoxP3(+) cell population in CD4(+) lamina propria lymphocytes derived from patients with UC. CONCLUSIONS: The inflammatory environment in the intestinal mucosa of patients with CD contributes to the generation of a distinct population of Treg cells that are FoxP3(+) and produce IL-17. These cells are likely to arise during differentiation of Th17 and Treg cells. Specific microenvironmental cues from tissues are likely to determine their commitment to either lineage and affect the balance between regulation and inflammation in the intestine
— id: 134124, year: 2011, vol: 140, page: 957, stat: Journal Article,

Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORgammat activity
Huh, Jun R; Leung, Monica W L; Huang, Pengxiang; Ryan, Daniel A; Krout, Michael R; Malapaka, Raghu R V; Chow, Jonathan; Manel, Nicolas; Ciofani, Maria; Kim, Sangwon V; Cuesta, Adolfo; Santori, Fabio R; Lafaille, Juan J; Xu, H Eric; Gin, David Y; Rastinejad, Fraydoon; Littman, Dan R
2011 Apr 28;472(7344):486-490, Nature
CD4(+) T helper lymphocytes that express interleukin-17 (T(H)17 cells) have critical roles in mouse models of autoimmunity, and there is mounting evidence that they also influence inflammatory processes in humans. Genome-wide association studies in humans have linked genes involved in T(H)17 cell differentiation and function with susceptibility to Crohn's disease, rheumatoid arthritis and psoriasis. Thus, the pathway towards differentiation of T(H)17 cells and, perhaps, of related innate lymphoid cells with similar effector functions, is an attractive target for therapeutic applications. Mouse and human T(H)17 cells are distinguished by expression of the retinoic acid receptor-related orphan nuclear receptor RORgammat, which is required for induction of IL-17 transcription and for the manifestation of T(H)17-dependent autoimmune disease in mice. By performing a chemical screen with an insect cell-based reporter system, we identified the cardiac glycoside digoxin as a specific inhibitor of RORgammat transcriptional activity. Digoxin inhibited murine T(H)17 cell differentiation without affecting differentiation of other T cell lineages and was effective in delaying the onset and reducing the severity of autoimmune disease in mice. At high concentrations, digoxin is toxic for human cells, but non-toxic synthetic derivatives 20,22-dihydrodigoxin-21,23-diol and digoxin-21-salicylidene specifically inhibited induction of IL-17 in human CD4(+) T cells. Using these small-molecule compounds, we demonstrate that RORgammat is important for the maintenance of IL-17 expression in mouse and human effector T cells. These data indicate that derivatives of digoxin can be used as chemical templates for the development of RORgammat-targeted therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease
— id: 131813, year: 2011, vol: 472, page: 486, stat: Journal Article,

Modulation of immune homeostasis by commensal bacteria
Ivanov, Ivaylo I; Littman, Dan R
2011 Feb;14(1):106-114, Current opinion in microbiology
Intestinal bacteria form a resident community that has co-evolved with the mammalian host. In addition to playing important roles in digestion and harvesting energy, commensal bacteria are crucial for the proper functioning of mucosal immune defenses. Most of these functions have been attributed to the presence of large numbers of 'innocuous' resident bacteria that dilute or occupy niches for intestinal pathogens or induce innate immune responses that sequester bacteria in the lumen, thus quenching excessive activation of the mucosal immune system. However it has recently become obvious that commensal bacteria are not simply beneficial bystanders, but are important modulators of intestinal immune homeostasis and that the composition of the microbiota is a major factor in pre-determining the type and robustness of mucosal immune responses. Here we review specific examples of individual members of the microbiota that modify innate and adaptive immune responses, and we focus on potential mechanisms by which such species-specific signals are generated and transmitted to the host immune system
— id: 134125, year: 2011, vol: 14, page: 106, stat: Journal Article,

DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration
Kaneko, Hiroki; Dridi, Sami; Tarallo, Valeria; Gelfand, Bradley D; Fowler, Benjamin J; Cho, Won Gil; Kleinman, Mark E; Ponicsan, Steven L; Hauswirth, William W; Chiodo, Vince A; Kariko, Katalin; Yoo, Jae Wook; Lee, Dong-ki; Hadziahmetovic, Majda; Song, Ying; Misra, Smita; Chaudhuri, Gautam; Buaas, Frank W; Braun, Robert E; Hinton, David R; Zhang, Qing; Grossniklaus, Hans E; Provis, Jan M; Madigan, Michele C; Milam, Ann H; Justice, Nikki L; Albuquerque, Romulo J C; Blandford, Alexander D; Bogdanovich, Sasha; Hirano, Yoshio; Witta, Jassir; Fuchs, Elaine; Littman, Dan R; Ambati, Balamurali K; Rudin, Charles M; Chong, Mark M W; Provost, Patrick; Kugel, Jennifer F; Goodrich, James A; Dunaief, Joshua L; Baffi, Judit Z; Ambati, Jayakrishna
2011 Mar 17;471(7338):325-330, Nature
Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness
— id: 134204, year: 2011, vol: 471, page: 325, stat: Journal Article,

Role of the commensal microbiota in normal and pathogenic host immune responses
Littman, Dan R; Pamer, Eric G
2011 Oct 4;10(4):311-323, Cell Host & Microbe
The commensal microbiota that inhabit different parts of the gastrointestinal (GI) tract have been shaped by coevolution with the host species. The symbiotic relationship of the hundreds of microbial species with the host requires a tuned response that prevents host damage, e.g., inflammation, while tolerating the presence of the potentially beneficial microbes. Recent studies have begun to shed light on immunological processes that participate in maintenance of homeostasis with the microbiota and on how disturbance of host immunity or the microbial ecosystem can result in disease-provoking dysbiosis. Our growing appreciation of this delicate host-microbe relationship promises to influence our understanding of inflammatory diseases and infection by microbial pathogens and to provide new therapeutic opportunities
— id: 139746, year: 2011, vol: 10, page: 311, stat: Journal Article,

Hiding in Plain Sight: How HIV Evades Innate Immune Responses
Manel, Nicolas; Littman, Dan R.
2011 OCT 14 ;147(2):271-274, Cell
Two groups have identified SAMHD1, a protein encoded by an Aicardi-Goutie` res Syndrome susceptibility gene, as the factor that restricts infection of macrophages and dendritic cells with HIV-1. Here we discuss implications of this discovery for induction of antiviral protective immunity
— id: 145691, year: 2011, vol: 147, page: 271, stat: Journal Article,

LYMPHOTOXIN-beta RECEPTOR SIGNALING SUPPORTS A UNIQUE STROMAL CELL NICHE THAT SUPPORTS IGA CLASS SWITCH RECOMBINATION IN THE INTESTINAL LAMINA PROPRIA
McCarthy, Douglas D.; Ivanov, Ivaylo I.; Tumanov, Alexei V.; Shi, Hang; Koroleva, Ekaterina P.; Summers-deLuca, Leslie; Ward, Lesley A.; Casellas, Rafael; Nedospovav, Sergei A.; Fu, Yang-Xin; Littman, Dan R.; Gommerman, Jennifer L.
2011 JUN ;691(3):776-776, Advances in experimental medicine & biology
— id: 134680, year: 2011, vol: 691, page: 776, stat: Journal Article,

Immunomodulatory functions of segmented filamentous bacteria
Sczesnak A.; Segata N.; Qin X.; Petrosino J.F.; Huttenhower C.; Littman D.R.; Ivanov I.
2011 ;135:13-13, Immunology
Commensal intestinal bacteria are indispensible for the proper functioning of the mammalian immune system. Both mucosal and systemic immune mechanisms are profoundly affected by the gut microbiota. Commensals provide not only essential immune protection against life-threatening infections, but shape pre-existing immune responses and thus modulate the ability of the host to respond to environmental challenges. These immunomodulatory functions are dependent on the composition of the commensal community and seem to be a property of individual members of this community. Investigation of the underlying mechanisms requires the identification of specific examples of such interactions. We have identified the murine commensal segmented filamentous bacteria (SFB) as capable of specifically shifting the homeostasis of effector T cell subsets in the small intestinal lamina propria. SFB induce preferentially Th17 cells and augment Th17 celldependent immune responses. As a first step toward the identification of immunomodulatory SFB products, we have sequenced the SFB genome and have performed initial analysis based on its annotation. This information will hopefully help the investigation of the mechanisms by which SFB affect intestinal immune responses and the development of new genetic tools for the study of the biology of this unique microorganism
— id: 147764, year: 2011, vol: 135, page: 13, stat: Journal Article,

The genome of th17 cell-inducing segmented filamentous bacteria reveals extensive auxotrophy and adaptations to the intestinal environment
Sczesnak, Andrew; Segata, Nicola; Qin, Xiang; Gevers, Dirk; Petrosino, Joseph F; Huttenhower, Curtis; Littman, Dan R; Ivanov, Ivaylo I
2011 Sep 15;10(3):260-272, Cell Host & Microbe
Perturbations of the composition of the symbiotic intestinal microbiota can have profound consequences for host metabolism and immunity. In mice, segmented filamentous bacteria (SFB) direct the accumulation of potentially proinflammatory Th17 cells in the intestinal lamina propria. We present the genome sequence of SFB isolated from monocolonized mice, which classifies SFB phylogenetically as a unique member of Clostridiales with a highly reduced genome. Annotation analysis demonstrates that SFB depend on their environment for amino acids and essential nutrients and may utilize host and dietary glycans for carbon, nitrogen, and energy. Comparative analyses reveal that SFB are functionally related to members of the genus Clostridium and several pathogenic or commensal 'minimal' genera, including Finegoldia, Mycoplasma, Borrelia, and Phytoplasma. However, SFB are functionally distinct from all 1200 examined genomes, indicating a gene complement representing biology relatively unique to their role as a gut commensal closely tied to host metabolism and immunity
— id: 137848, year: 2011, vol: 10, page: 260, stat: Journal Article,

Regulatory T cells suppress development of colitis, blocking differentiation of T-helper 17 into alternative T-helper 1 cells
Sujino, Tomohisa; Kanai, Takanori; Ono, Yuichi; Mikami, Yohei; Hayashi, Atsushi; Doi, Tomomitsu; Matsuoka, Katsuyoshi; Hisamatsu, Tadakazu; Takaishi, Hiromasa; Ogata, Haruhiko; Yoshimura, Akihiko; Littman, Dan R; Hibi, Toshifumi
2011 Sep;141(3):1014-1023, Gastroenterology
BACKGROUND & AIMS: Although T-helper (Th) 17 and Th1 cells are involved in pathogenesis of intestinal inflammation, their developmental pathways and sufficiency to promote disease are not known; nor are the roles of CD4(+)CD25(+) regulatory T (T(R)) cells in their development. METHODS: We performed adoptive transfer experiments to investigate the induction and suppression of colitis using naive CD4(+)CD45RB(high) T cells and/or CD4(+)CD25(+) T(R) cells that were obtained from retinoid-related orphan receptor gamma t (RORgammat) gfp/(+) or Ly5.1/Ly5.2 congenic mice. RESULTS: We observed 3 types of colitogenic CD4(+) Th1 cells (interleukin [IL]-17A(-)interferon [IFN]-gamma(+)): RORgammat(-) classical Th1 cells that differentiated directly from naive T cells; RORgammat(+) Th1-like cells; and RORgammat(-) alternative Th1 cells that were terminally differentiated from RORgammat(+) cells via Th17 (IL-17A(+)IFN-gamma(-)), Th17/Th1 (IL-17A(+)IFN-gamma(+)), or Th1-like (IL-17A(-)IFN-gamma(+)) cells. In this pathway, CD4(+)CD25(+) T(R) cells suppress the development of not only classical Th1 cells, but also alternative Th1 cells at the transition of Th17/Th1 into alternative Th1 cells, resulting in accumulation of Th17 and Th17/Th1 cells in mice in which the development of colitis was suppressed. Furthermore, T(R) cells regulated the established balance of Th17 and Th1 cells under colitic conditions to yield a high ratio of Th17 and Th17/Th1 cells to Th1 cells in noncolitic conditions. CONCLUSIONS: Th17 and Th17/Th1 cells become colitogenic alternative Th1 cells via Th17, Th17/Th1, and Th1-like cells, independently of classical Th1 cells. T(R) cells suppress this pathway, resulting in accumulation of Th17 and Th17/Th1 cells
— id: 137118, year: 2011, vol: 141, page: 1014, stat: Journal Article,

The inducible deletion of Drosha and microRNAs in mature podocytes results in a collapsing glomerulopathy
Zhdanova, Olga; Srivastava, Shekhar; Di, Lie; Li, Zhai; Tchelebi, Leila; Dworkin, Sara; Johnstone, Duncan B; Zavadil, Jiri; Chong, Mark M; Littman, Dan R; Holzman, Lawrence B; Barisoni, Laura; Skolnik, Edward Y
2011 Oct;80(7):719-730, Kidney international
Micro-RNAs (miRNAs) are short (average 22 nucleotides) noncoding regulatory RNAs that inhibit gene expression by targeting complementary 3'-untranslated regions of protein-encoding mRNAs for translational repression or degradation. miRNAs play key roles in both the function and differentiation of many cell types. Drosha and Dicer, two RNAase III enzymes, function in a stepwise manner to generate a mature miRNA. Previous studies have shown that podocyte-specific deletion of Dicer during development results in proteinuric renal disease and collapsing glomerulopathy (CG); however, Dicer has functions other than the generation of miRNAs. Here we found that the podocyte-specific deletion of Drosha results in a similar phenotype to Dicer mutants, confirming that the Dicer mutant phenotype is due to the loss of miRNAs. Moreover, the inducible deletion of Drosha in 2- to 3-month-old mice (Tet-On system) resulted in CG. Thus, continuous generation of miRNAs are required for the normal function of mature podocytes and their loss leads to CG. Identifying these miRNAs may provide new insight into disease pathogenesis and novel therapeutic targets in various podocytopathies
— id: 137467, year: 2011, vol: 80, page: 719, stat: Journal Article,

Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology
Buonocore, Sofia; Ahern, Philip P; Uhlig, Holm H; Ivanov, Ivaylo I; Littman, Dan R; Maloy, Kevin J; Powrie, Fiona
2010 Apr 29;464(7293):1371-1375, Nature
The key role of interleukin (IL)-23 in the pathogenesis of autoimmune and chronic inflammatory disorders is supported by the identification of IL-23 receptor (IL-23R) susceptibility alleles associated with inflammatory bowel disease, psoriasis and ankylosing spondylitis. IL-23-driven inflammation has primarily been linked to the actions of T-helper type 17 (TH17) cells. Somewhat overlooked, IL-23 also has inflammatory effects on innate immune cells and can drive T-cell-independent colitis. However, the downstream cellular and molecular pathways involved in this innate intestinal inflammatory response are poorly characterized. Here we show that bacteria-driven innate colitis is associated with an increased production of IL-17 and interferon-gamma in the colon. Stimulation of colonic leukocytes with IL-23 induced the production of IL-17 and interferon-gamma exclusively by innate lymphoid cells expressing Thy1, stem cell antigen 1 (SCA-1), retinoic-acid-related orphan receptor (ROR)-gammat and IL-23R, and these cells markedly accumulated in the inflamed colon. IL-23-responsive innate intestinal cells are also a feature of T-cell-dependent models of colitis. The transcription factor ROR-gammat, which controls IL-23R expression, has a functional role, because Rag-/-Rorc-/- mice failed to develop innate colitis. Last, depletion of Thy1+ innate lymphoid cells completely abrogated acute and chronic innate colitis. These results identify a previously unrecognized IL-23-responsive innate lymphoid population that mediates intestinal immune pathology and may therefore represent a target in inflammatory bowel disease
— id: 137120, year: 2010, vol: 464, page: 1371, stat: Journal Article,

Epigenetic propagation of CD4 expression is established by the Cd4 proximal enhancer in helper T cells
Chong, Mark M W; Simpson, Natalie; Ciofani, Maria; Chen, Grace; Collins, Amelie; Littman, Dan R
2010 Apr 1;24(7):659-669, Genes & development
The stability of a lineage program (cellular memory) is dependent on mechanisms that epigenetically maintain active or repressed states of gene expression (transcriptional memory). Although epigenetic silencing of genes has been clearly demonstrated from yeast to mammals, heritable maintenance of active transcription has been less clearly defined. To investigate the potential role of active transcriptional memory during lineage diversification, we employed targeted mutation of a positive-acting cis element in the Cd4 locus to determine the impact on CD4 expression and the differentiation of CD4(+) helper T cells in mice. We show that the proximal enhancer (E4(P)) of Cd4 is essential for CD4 expression in immature CD4(+)8(+) thymocytes. Furthermore, its loss resulted in reduced and unstable expression of CD4 in mature T cells. However, if the enhancer was deleted after cells had already committed to the helper T-cell lineage, CD4 expression remained high and was stable upon cell division. 'Active' histone modifications, once initiated by E4(P), were also propagated independently of the enhancer. Thus, E4(P) is responsible for establishing an epigenetically inherited active Cd4 locus in the helper T-cell lineage. To our knowledge, this is the first genetic demonstration of active transcriptional memory in mammalian cells
— id: 109058, year: 2010, vol: 24, page: 659, stat: Journal Article,

Canonical and alternate functions of the microRNA biogenesis machinery
Chong, Mark M W; Zhang, Guoan; Cheloufi, Sihem; Neubert, Thomas A; Hannon, Gregory J; Littman, Dan R
2010 Sep 1;24(17):1951-1960, Genes & development
The canonical microRNA (miRNA) biogenesis pathway requires two RNaseIII enzymes: Drosha and Dicer. To understand their functions in mammals in vivo, we engineered mice with germline or tissue-specific inactivation of the genes encoding these two proteins. Changes in proteomic and transcriptional profiles that were shared in Dicer- and Drosha-deficient mice confirmed the requirement for both enzymes in canonical miRNA biogenesis. However, deficiency in Drosha or Dicer did not always result in identical phenotypes, suggesting additional functions. We found that, in early-stage thymocytes, Drosha recognizes and directly cleaves many protein-coding messenger RNAs (mRNAs) with secondary stem-loop structures. In addition, we identified a subset of miRNAs generated by a Dicer-dependent but Drosha-independent mechanism. These were distinct from previously described mirtrons. Thus, in mammalian cells, Dicer is required for the biogenesis of multiple classes of miRNAs. Together, these findings extend the range of function of RNaseIII enzymes beyond canonical miRNA biogenesis, and help explain the nonoverlapping phenotypes caused by Drosha and Dicer deficiency
— id: 112041, year: 2010, vol: 24, page: 1951, stat: Journal Article,

Susceptibility of Human Th17 Cells to Human Immunodeficiency Virus and Their Perturbation during Infection
El Hed, Aimee; Khaitan, Alka; Kozhaya, Lina; Manel, Nicolas; Daskalakis, Demetre; Borkowsky, William; Valentine, Fred; Littman, Dan R; Unutmaz, Derya
2010 Mar 15;201(6):843-854, Journal of infectious diseases
Background. Identification of the Th17 T cell subset as important mediators of host defense and pathology prompted us to determine their susceptibility to human immunodeficiency virus (HIV) infection. Methods and results. We found that a sizeable portion of Th17 cells express HIV coreceptor CCR5 and produce very low levels of CCR5 ligands macrophage inflammatory protein (MIP)-1alpha and MIP-1beta. Accordingly, CCR5(+) Th17 cells were efficiently infected with CCR5-tropic HIV and were depleted during viral replication in vitro. Remarkably, HIV-infected individuals receiving treatment had significantly reduced Th17 cell counts, compared with HIV-uninfected subjects, regardless of viral load or CD4 cell count, whereas treatment-naive subjects had normal levels. However, there was a preferential reduction in CCR5(+) T cells that were also CCR6 positive, which is expressed on all Th17 cells, compared with CCR6(-)CCR5(+) cells, in both treated and untreated HIV-infected subjects. This observation suggests preferential targeting of CCR6(+)CCR5(+) Th17 cells by CCR5-tropic viruses in vivo. Th17 cell levels also inversely correlated with activated CD4(+) T cells in HIV-infected individuals who are receiving treatment. Conclusions. Our findings suggest a complex perturbation of Th17 subsets during the course of HIV disease potentially through both direct viral infection and virus indirect mechanisms, such as immune activation
— id: 107380, year: 2010, vol: 201, page: 843, stat: Journal Article,

Attenuated Acute Graft Versus Host Disease Following Allogeneic Stem Cell Transplantation In the Absence of ROR gamma t
Fulton, LeShara M.; Carlson, Michael J.; Coghill, James; West, Michelle L.; Mortari, Angela Panoskaltisis; Blazar, Bruce R.; Littman, Dan; Serody, Jonathan
2010 NOV 19 ;116(21):1535-1535, Blood
— id: 130871, year: 2010, vol: 116, page: 1535, stat: Journal Article,

Segmented filamentous bacteria take the stage
Ivanov, I I; Littman, D R
2010 May;3(3):209-212, Mucosal immunology
Commensal bacteria are crucial for maturation and function of the mucosal immune system. However, the mechanisms of these interactions are poorly understood. In addition, the role of the composition of the microbiota and the importance of individual species in this community in stimulating different types of immunity are major unanswered questions. We recently showed that the balance between two major effector T cell populations in the intestine, IL-17(+) Th17 cells and Foxp3(+) Tregs, requires signals from commensal bacteria and is dependent on the composition of the intestinal microbiota. Comparison of microbiota from Th17 cell-deficient and Th17 cell-sufficient mice identified segmented filamentous bacteria (SFB) as capable of specifically inducing Th17 cells in the gut. SFB represent the first example of a commensal species that can skew the mucosal effector T cell balance and thus affect the immune fitness of the individual
— id: 109204, year: 2010, vol: 3, page: 209, stat: Journal Article,

Stem cell exhaustion due to Runx1 deficiency is prevented by Evi5 activation in leukemogenesis
Jacob, Bindya; Osato, Motomi; Yamashita, Namiko; Wang, Chelsia Qiuxia; Taniuchi, Ichiro; Littman, Dan R; Asou, Norio; Ito, Yoshiaki
2010 Feb 25;115(8):1610-1620, Blood
The RUNX1/AML1 gene is the most frequently mutated gene in human leukemia. Conditional deletion of Runx1 in adult mice results in an increase of hematopoietic stem cells (HSCs), which serve as target cells for leukemia; however, Runx1(-/-) mice do not develop spontaneous leukemia. Here we show that maintenance of Runx1(-/-) HSCs is compromised, progressively resulting in HSC exhaustion. In leukemia development, the stem cell exhaustion was rescued by additional genetic changes. Retroviral insertional mutagenesis revealed Evi5 activation as a cooperating genetic alteration and EVI5 overexpression indeed prevented Runx1(-/-) HSC exhaustion in mice. Moreover, EVI5 was frequently overexpressed in human RUNX1-related leukemias. These results provide insights into the mechanism for maintenance of pre-leukemic stem cells and may provide a novel direction for therapeutic applications
— id: 137121, year: 2010, vol: 115, page: 1610, stat: Journal Article,

Flexible use of nuclear import pathways by HIV-1
Lee, KyeongEun; Ambrose, Zandrea; Martin, Thomas D; Oztop, Ilker; Mulky, Alok; Julias, John G; Vandegraaff, Nick; Baumann, Joerg G; Wang, Rui; Yuen, Wendy; Takemura, Taichiro; Shelton, Kenneth; Taniuchi, Ichiro; Li, Yuan; Sodroski, Joseph; Littman, Dan R; Coffin, John M; Hughes, Stephen H; Unutmaz, Derya; Engelman, Alan; KewalRamani, Vineet N
2010 Mar 18;7(3):221-233, Cell Host & Microbe
HIV-1 replication requires transport of nascent viral DNA and associated virion proteins, the retroviral preintegration complex (PIC), into the nucleus. Too large for passive diffusion through nuclear pore complexes (NPCs), PICs use cellular nuclear transport mechanisms and nucleoporins (NUPs), the NPC components that permit selective nuclear-cytoplasmic exchange, but the details remain unclear. Here we identify a fragment of the cleavage and polyadenylation factor 6, CPSF6, as a potent inhibitor of HIV-1 infection. When enriched in the cytoplasm, CPSF6 prevents HIV-1 nuclear entry by targeting the viral capsid (CA). HIV-1 harboring the N74D mutation in CA fails to interact with CPSF6 and evades the nuclear import restriction. Interestingly, whereas wild-type HIV-1 requires NUP153, N74D HIV-1 mimics feline immunodeficiency virus nuclear import requirements and is more sensitive to NUP155 depletion. These findings reveal a remarkable flexibility in HIV-1 nuclear transport and highlight a single residue in CA as essential in regulating interactions with NUPs
— id: 119325, year: 2010, vol: 7, page: 221, stat: Journal Article,

Th17 and regulatory T cells in mediating and restraining inflammation
Littman, Dan R; Rudensky, Alexander Y
2010 Mar 19;140(6):845-858, Cell
The vertebrate immune system is poised in a state of equilibrium that permits accurate and rapid protective responses against pathogens but curtails potential for causing harm to the host through targeting of 'self' and provoking overexuberant inflammatory processes. In this Review we discuss this balance achieved in large part by interactions of different classes of T lymphocytes that have potent pro- or anti-inflammatory activity in the context of genetic and environmental factors, particularly the commensal microbiota
— id: 108798, year: 2010, vol: 140, page: 845, stat: Journal Article,

A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells
Manel, Nicolas; Hogstad, Brandon; Wang, Yaming; Levy, David E; Unutmaz, Derya; Littman, Dan R
2010 Sep 9;467(7312):214-217, Nature
Dendritic cells serve a key function in host defence, linking innate detection of microbes to activation of pathogen-specific adaptive immune responses. Whether there is cell-intrinsic recognition of human immunodeficiency virus (HIV) by host innate pattern-recognition receptors and subsequent coupling to antiviral T-cell responses is not yet known. Dendritic cells are largely resistant to infection with HIV-1, but facilitate infection of co-cultured T-helper cells through a process of trans-enhancement. Here we show that, when dendritic cell resistance to infection is circumvented, HIV-1 induces dendritic cell maturation, an antiviral type I interferon response and activation of T cells. This innate response is dependent on the interaction of newly synthesized HIV-1 capsid with cellular cyclophilin A (CYPA) and the subsequent activation of the transcription factor IRF3. Because the peptidylprolyl isomerase CYPA also interacts with HIV-1 capsid to promote infectivity, our results indicate that capsid conformation has evolved under opposing selective pressures for infectivity versus furtiveness. Thus, a cell-intrinsic sensor for HIV-1 exists in dendritic cells and mediates an antiviral immune response, but it is not typically engaged owing to the absence of dendritic cell infection. The virulence of HIV-1 may be related to evasion of this response, the manipulation of which may be necessary to generate an effective HIV-1 vaccine
— id: 112431, year: 2010, vol: 467, page: 214, stat: Journal Article,

The aryl hydrocarbon receptor modulates the Th17 and Treg balance and gut immunity
Qiu, Ju; Dieh, Gretchen; Fish, Kamonwan; Gong, Xing; Littman, Dan; Zhou, Liang
2010 OCT-NOV ;52(1-2):21-21, Cytokine
— id: 113932, year: 2010, vol: 52, page: 21, stat: Journal Article,

CXCR4 acts as a costimulator during thymic beta-selection
Trampont, Paul C; Tosello-Trampont, Annie-Carole; Shen, Yuelei; Duley, Amanda K; Sutherland, Ann E; Bender, Timothy P; Littman, Dan R; Ravichandran, Kodi S
2010 Feb;11(2):162-170, Nature immunology
Passage through the beta-selection developmental checkpoint requires productive rearrangement of segments of the T cell antigen receptor-beta gene (Tcrb) and formation of a pre-TCR on the surface of CD4(-)CD8(-) thymocytes. How other receptors influence betabeta-selection is less well understood. Here we define a new role for the chemokine receptor CXCR4 during T cell development. CXCR4 functionally associated with the pre-TCR and influenced beta-selection by regulating the steady-state localization of immature thymocytes in thymic subregions, by facilitating optimal pre-TCR-induced survival signals, and by promoting thymocyte proliferation. We also characterize functionally relevant signaling molecules downstream of CXCR4 and the pre-TCR in thymocytes. Our data designate CXCR4 as a costimulator of the pre-TCR during beta-selection
— id: 134426, year: 2010, vol: 11, page: 162, stat: Journal Article,

Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells
Wu, Hsin-Jung; Ivanov, Ivaylo I; Darce, Jaime; Hattori, Kimie; Shima, Tatsuichiro; Umesaki, Yoshinori; Littman, Dan R; Benoist, Christophe; Mathis, Diane
2010 Jun 25;32(6):815-827, Immunity
Commensal microbes can have a substantial impact on autoimmune disorders, but the underlying molecular and cellular mechanisms remain largely unexplored. We report that autoimmune arthritis was strongly attenuated in the K/BxN mouse model under germ-free (GF) conditions, accompanied by reductions in serum autoantibody titers, splenic autoantibody-secreting cells, germinal centers, and the splenic T helper 17 (Th17) cell population. Neutralization of interleukin-17 prevented arthritis development in specific-pathogen-free K/BxN mice resulting from a direct effect of this cytokine on B cells to inhibit germinal center formation. The systemic deficiencies of the GF animals reflected a loss of Th17 cells from the small intestinal lamina propria. Introduction of a single gut-residing species, segmented filamentous bacteria, into GF animals reinstated the lamina propria Th17 cell compartment and production of autoantibodies, and arthritis rapidly ensued. Thus, a single commensal microbe, via its ability to promote a specific Th cell subset, can drive an autoimmune disease
— id: 137119, year: 2010, vol: 32, page: 815, stat: Journal Article,

Myd88 is required for an antibody response to retroviral infection
Browne, Edward P; Littman, Dan R
2009 Feb;5(2):e1000298-e1000298, PLoS pathogens
Although retroviruses have been extensively studied for many years, basic questions about how retroviral infections are detected by the immune system and which innate pathways are required for the generation of immune responses remain unanswered. Defining these pathways and how they contribute to the anti-retroviral immune responses would assist in the development of more effective vaccines for retroviral pathogens such as HIV. We have investigated the roles played by CD11c(+) dendritic cells (DCs) and by Toll-like receptor (TLR) signaling pathways in the generation of an anti-retroviral immune response against a mouse retroviral pathogen, Friend murine leukemia virus (F-MLV). Specific deletion of DCs during F-MLV infection caused a significant increase in viral titers at 14 days post-infection, indicating the importance of DCs in immune control of the infection. Similarly, Myd88 knockout mice failed to control F-MLV, and sustained high viral titers (10(7) foci/spleen) for several months after infection. Strikingly, both DC-depleted mice and Myd88 knockout mice exhibited only a partial reduction of CD8(+) T cell responses, while the IgG antibody response to F-MLV was completely lost. Furthermore, passive transfer of immune serum from wild-type mice to Myd88 knockout mice rescued control of F-MLV. These results identify TLR signaling and CD11c(+) DCs as playing critical roles in the humoral response to retroviruses
— id: 95887, year: 2009, vol: 5, page: e1000298, stat: Journal Article,

RUNX proteins in transcription factor networks that regulate T-cell lineage choice
Collins, Amelie; Littman, Dan R; Taniuchi, Ichiro
2009 Feb;9(2):106-115, Nature reviews. Immunology
Recent research has uncovered complex transcription factor networks that control the processes of T-cell development and differentiation. RUNX (runt-related transcription factor) proteins are among the many factors that have crucial roles in these networks. In this Review, we examine the mechanisms by which RUNX complexes act together with other transcription factors, such as Th-POK (T-helper-inducing POZ/Kruppel-like factor) and GATA-binding protein 3 (GATA3) in determining the CD4/CD8 lineage choice of developing thymocytes. In addition, we discuss evidence indicating that RUNX complexes are also involved in the differentiation of effector T-cell subsets and that the molecular mechanisms by which RUNX proteins regulate T-cell fate decisions are conserved between the thymus and periphery
— id: 95888, year: 2009, vol: 9, page: 106, stat: Journal Article,

How punctual ablation of regulatory T cells unleashes an autoimmune lesion within the pancreatic islets
Feuerer, Markus; Shen, Yuelei; Littman, Dan R; Benoist, Christophe; Mathis, Diane
2009 Oct 16;31(4):654-664, Immunity
CD4(+)Foxp3(+) regulatory T cells (Treg cells) are known to control the progression of autoimmune diabetes, but when, where, and how they exert their influence in this context are questions still under vigorous debate. Exploiting a transgene encoding the human diphtheria toxin receptor, we punctually and specifically ablated Foxp3(+) cells in the BCD2.5/NOD mouse model of autoimmune diabetes. Strikingly, overt disease developed within 3 days. The earliest detectable event was the activation of natural killer (NK) cells directly within the insulitic lesion, particularly the induction of Ifng gene expression within 7 hours of Treg cell ablation. Interferon-gamma had a strong impact on the gene-expression program of the local CD4(+) T effector cell population, unleashing it to aggressively attack the islets, which was required for the development of diabetes. Thus, Treg cells regulate pancreatic autoimmunity in situ through control of a central innate immune system player, NK cells
— id: 133735, year: 2009, vol: 31, page: 654, stat: Journal Article,

Role of microbiota and transcription factors in control of Th17 cell differentiation
Ivanov, I; Zhou, L; Huh, J; Santori, F; Manel, N; Chong, M; Umesaki, Y; Brodie, E; Honda, K; Littman, DR
2009 OCT-NOV ;48(1-2):18-18, Cytokine
— id: 105954, year: 2009, vol: 48, page: 18, stat: Journal Article,

Induction of intestinal Th17 cells by segmented filamentous bacteria
Ivanov, Ivaylo I; Atarashi, Koji; Manel, Nicolas; Brodie, Eoin L; Shima, Tatsuichiro; Karaoz, Ulas; Wei, Dongguang; Goldfarb, Katherine C; Santee, Clark A; Lynch, Susan V; Tanoue, Takeshi; Imaoka, Akemi; Itoh, Kikuji; Takeda, Kiyoshi; Umesaki, Yoshinori; Honda, Kenya; Littman, Dan R
2009 Oct 30;139(3):485-498, Cell
The gastrointestinal tract of mammals is inhabited by hundreds of distinct species of commensal microorganisms that exist in a mutualistic relationship with the host. How commensal microbiota influence the host immune system is poorly understood. We show here that colonization of the small intestine of mice with a single commensal microbe, segmented filamentous bacterium (SFB), is sufficient to induce the appearance of CD4(+) T helper cells that produce IL-17 and IL-22 (Th17 cells) in the lamina propria. SFB adhere tightly to the surface of epithelial cells in the terminal ileum of mice with Th17 cells but are absent from mice that have few Th17 cells. Colonization with SFB was correlated with increased expression of genes associated with inflammation and antimicrobial defenses and resulted in enhanced resistance to the intestinal pathogen Citrobacter rodentium. Thus, manipulation of this commensal-regulated pathway may provide new opportunities for enhancing mucosal immunity and treating autoimmune disease
— id: 105170, year: 2009, vol: 139, page: 485, stat: Journal Article,

Impact of the TCR signal on regulatory T cell homeostasis, function, and trafficking
Kim, Joong Kyu; Klinger, Mark; Benjamin, Jonathan; Xiao, Yuanyuan; Erle, David J; Littman, Dan R; Killeen, Nigel
2009 ;4(8):e6580-e6580, PLoS ONE
Signaling through the T cell antigen receptor (TCR) is important for the homeostasis of naive and memory CD4(+) T cells. The significance of TCR signaling in regulatory T (Treg) cells has not been systematically addressed. Using an Ox40-cre allele that is prominently expressed in Treg cells, and a conditional null allele of the gene encoding p56(Lck), we have examined the importance of TCR signaling in Treg cells. Inactivation of p56(Lck) resulted in abnormal Treg homeostasis characterized by impaired turnover, preferential redistribution to the lymph nodes, loss of suppressive function, and striking changes in gene expression. Abnormal Treg cell homeostasis and function did not reflect the involvement of p56(Lck) in CD4 function because these effects were not observed when CD4 expression was inactivated by Ox40-cre.The results make clear multiple aspects of Treg cell homeostasis and phenotype that are dependent on a sustained capacity to signal through the TCR
— id: 102577, year: 2009, vol: 4, page: e6580, stat: Journal Article,

Transcription factors RUNX1 and RUNX3 in the induction and suppressive function of Foxp3+ inducible regulatory T cells
Klunker, Sven; Chong, Mark M W; Mantel, Pierre-Yves; Palomares, Oscar; Bassin, Claudio; Ziegler, Mario; Ruckert, Beate; Meiler, Flurina; Akdis, Mubeccel; Littman, Dan R; Akdis, Cezmi A
2009 Nov 23;206(12):2701-2715, Journal of experimental medicine
Forkhead box P3 (FOXP3)(+)CD4(+)CD25(+) inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis. In this study, we show that the transforming growth factor-beta (TGF-beta) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4(+) T cells. This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter. Inactivation of the gene encoding RUNX cofactor core-binding factor-beta (CBFbeta) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3. The in vivo conversion of naive CD4(+) T cells into Foxp3(+) iT reg cells was significantly decreased in adoptively transferred Cbfb(F/F) CD4-cre naive T cells into Rag2(-/-) mice. Both RUNX1 and RUNX3 siRNA silenced human T reg cells and Cbfb(F/F) CD4-cre mouse T reg cells showed diminished suppressive function in vitro. Circulating human CD4(+) CD25(high) CD127(-) T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-beta mRNA compared with CD4(+)CD25(-) cells. Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells. These data demonstrate Runx transcription factors as a molecular link in TGF-beta-induced Foxp3 expression in iT reg cell differentiation and function
— id: 137122, year: 2009, vol: 206, page: 2701, stat: Journal Article,

RORgamma-expressing Th17 cells induce murine chronic intestinal inflammation via redundant effects of IL-17A and IL-17F
Leppkes, Moritz; Becker, Christoph; Ivanov, Ivaylo I; Hirth, Sebastian; Wirtz, Stefan; Neufert, Clemens; Pouly, Sandrine; Murphy, Andrew J; Valenzuela, David M; Yancopoulos, George D; Becher, Burkhard; Littman, Dan R; Neurath, Markus F
2009 Jan;136(1):257-267, Gastroenterology
BACKGROUND AND AIMS: IL-17-producing CD4(+) T-helper cells (Th17) contribute to chronic autoimmune inflammation in the brain, and levels of Th17-derived cytokines increase in patients with colitis, suggesting a role in pathogenesis. We analyzed the roles of Th17 cells and the transcription factor retinoic acid receptor-related organ receptor (ROR)gamma, which regulates Th17 differentiation, in chronic intestinal inflammation. METHODS: Using an adoptive transfer model of colitis, we compared the colitogenic potential of wild-type, interleukin-17A (IL-17A)-, IL-17F-, IL-22-, and RORgamma-deficient CD4(+)CD25(-) T cells in RAG1-null mice. RESULTS: Adoptive transfer of IL-17A-, IL-17F-, or IL-22-deficient T lymphocytes into RAG1-null mice caused severe colitis that was indistinguishable from that caused by wild-type cells. In contrast, transfer of RORgamma-null T cells failed to increase mucosal IL-17 cytokine levels and did not induce colitis. Treatment with IL-17A was able to restore colitis after transfer of RORgamma-null T cells, indicating a crucial role for Th17 cells in pathogenesis. Treatment of RAG1 mice that received IL-17F-null (but not wild-type) T cells with a neutralizing anti-IL-17A antibody significantly suppressed disease, indicating redundant biological effects of IL-17A and IL-17F. CONCLUSIONS: We have identified a crucial role of RORgamma-expressing Th17 cells in chronic intestinal inflammation. RORgamma controls IL-17A and IL-17F production, and these cytokines have a redundant but highly pathogenic role in gut inflammation. Reagents that target RORgamma or a combination of anti-IL-17A and anti-IL-17F might be developed as therapeutics for chronic colitis
— id: 95893, year: 2009, vol: 136, page: 257, stat: Journal Article,

Influence of the transcription factor RORgammat on the development of NKp46+ cell populations in gut and skin
Luci, Carmelo; Reynders, Ana; Ivanov, Ivaylo I; Cognet, Celine; Chiche, Laurent; Chasson, Lionel; Hardwigsen, Jean; Anguiano, Esperanza; Banchereau, Jacques; Chaussabel, Damien; Dalod, Marc; Littman, Dan R; Vivier, Eric; Tomasello, Elena
2009 Jan;10(1):75-82, Nature immunology
NKp46+CD3- natural killer lymphocytes isolated from blood, lymphoid organs, lung, liver and uterus can produce granule-dependent cytotoxicity and interferon-gamma. Here we identify in dermis, gut lamina propria and cryptopatches distinct populations of NKp46+CD3- cells with a diminished capacity to degranulate and produce interferon-gamma. In the gut, expression of the transcription factor RORgammat, which is involved in the development of lymphoid tissue-inducer cells, defined a previously unknown subset of NKp46+CD3- lymphocytes. Unlike RORgammat- lamina propria and dermis natural killer cells, gut RORgammat+NKp46+ cells produced interleukin 22. Our data show that lymphoid tissue-inducer cells and natural killer cells shared unanticipated similarities and emphasize the heterogeneity of NKp46+CD3- cells in innate immunity, lymphoid organization and local tissue repair
— id: 95892, year: 2009, vol: 10, page: 75, stat: Journal Article,

Human cyclin T1 expression ameliorates a T-cell-specific transcriptional limitation for HIV in transgenic rats, but is not sufficient for a spreading infection of prototypic R5 HIV-1 strains ex vivo
Michel, Nico; Goffinet, Christine; Ganter, Kerstin; Allespach, Ina; Kewalramani, Vineet N; Saifuddin, Mohammed; Littman, Dan R; Greene, Warner C; Goldsmith, Mark A; Keppler, Oliver T
2009 ;6:2-2, Retrovirology
BACKGROUND: Cells derived from native rodents have limits at distinct steps of HIV replication. Rat primary CD4 T-cells, but not macrophages, display a profound transcriptional deficit that is ameliorated by transient trans-complementation with the human Tat-interacting protein Cyclin T1 (hCycT1). RESULTS: Here, we generated transgenic rats that selectively express hCycT1 in CD4 T-cells and macrophages. hCycT1 expression in rat T-cells boosted early HIV gene expression to levels approaching those in infected primary human T-cells. hCycT1 expression was necessary, but not sufficient, to enhance HIV transcription in T-cells from individual transgenic animals, indicating that endogenous cellular factors are critical co-regulators of HIV gene expression in rats. T-cells from hCD4/hCCR5/hCycT1-transgenic rats did not support productive infection of prototypic wild-type R5 HIV-1 strains ex vivo, suggesting one or more significant limitation in the late phase of the replication cycle in this primary rodent cell type. Remarkably, we identify a replication-competent HIV-1 GFP reporter strain (R7/3 YU-2 Env) that displays characteristics of a spreading, primarily cell-to-cell-mediated infection in primary T-cells from hCD4/hCCR5-transgenic rats. Moreover, the replication of this recombinant HIV-1 strain was significantly enhanced by hCycT1 transgenesis. The viral determinants of this so far unique replicative ability are currently unknown. CONCLUSION: Thus, hCycT1 expression is beneficial to de novo HIV infection in a transgenic rat model, but additional genetic manipulations of the host or virus are required to achieve full permissivity
— id: 95889, year: 2009, vol: 6, page: 2, stat: Journal Article,

Runx-CBFbeta complexes control expression of the transcription factor Foxp3 in regulatory T cells
Rudra, Dipayan; Egawa, Takeshi; Chong, Mark M W; Treuting, Piper; Littman, Dan R; Rudensky, Alexander Y
2009 Nov;10(11):1170-1177, Nature immunology
The transcription factor Foxp3 has an indispensable role in establishing stable transcriptional and functional programs of regulatory T cells (T(reg) cells). Loss of Foxp3 expression in mature T(reg) cells results in a failure of suppressor function, yet the molecular mechanisms that ensure steady, heritable Foxp3 expression in the T(reg) cell lineage remain unknown. Using T(reg) cell-specific gene targeting, we found that complexes of the transcription factors Runx and CBFbeta were required for maintenance of Foxp3 mRNA and protein expression in T(reg) cells. Consequently, mice lacking CBFbetab exclusively in the T(reg) cell lineage had a moderate lymphoproliferative syndrome. Thus, Runx-CBFbeta complexes maintain stable high expression of Foxp3 and serve as an essential determinant of T(reg) cell lineage stability
— id: 137123, year: 2009, vol: 10, page: 1170, stat: Journal Article,

Lymphoid tissue inducer-like cells are an innate source of IL-17 and IL-22
Takatori, Hiroaki; Kanno, Yuka; Watford, Wendy T; Tato, Cristina M; Weiss, Greta; Ivanov, Ivaylo I; Littman, Dan R; O'Shea, John J
2009 Jan 16;206(1):35-41, Journal of experimental medicine
The interleukin (IL) 17 family of cytokines has emerged to be critical for host defense as well as the pathogenesis of autoimmune and autoinflammatory disorders, and serves to link adaptive and innate responses. Recent studies have identified a new subset of T cells that selectively produce IL-17 (Th17 cells; Bettelli, E., T. Korn, and V.K. Kuchroo. 2007. Curr. Opin. Immunol. 19:652-657; Kolls, J.K., and A. Linden. 2004. Immunity. 21:467-476), but the regulation of IL-17 production by innate immune cells is less well understood. We report that in vitro stimulation with IL-23 induced IL-17 production by recombination activating gene (Rag) 2(-/-) splenocytes but not Rag2(-/-) common gamma chain(-/-) splenocytes. We found that a major source of IL-17 was CD4(+)CD3(-)NK1.1(-)CD11b(-)Gr1(-)CD11c(-)B220(-) cells, a phenotype that corresponds to lymphoid tissue inducer-like cells (LTi-like cells), which constitutively expressed the IL-23 receptor, aryl hydrocarbon receptor, and CCR6. In vivo challenge with the yeast cell wall product zymosan rapidly induced IL-17 production in these cells. Genetic deletion of signal transducer and activator of transcription 3 reduced but did not abrogate IL-17 production in LTi-like cells. Thus, it appears that splenic LTi-like cells are a rapid source of IL-17 and IL-22, which might contribute to dynamic organization of secondary lymphoid organ structure or host defense
— id: 95890, year: 2009, vol: 206, page: 35, stat: Journal Article,

Identification of IL-17-producing FOXP3+ regulatory T cells in humans
Voo, Kui Shin; Wang, Yui-Hsi; Santori, Fabio R; Boggiano, Cesar; Wang, Yi-Hong; Arima, Kazuhiko; Bover, Laura; Hanabuchi, Shino; Khalili, Jahan; Marinova, Ekaterina; Zheng, Biao; Littman, Dan R; Liu, Yong-Jun
2009 Mar 24;106(12):4793-4798, Proceedings of the National Academy of Sciences of the United States of America
IL-17-producing CD4(+) T helper (Th17) cells have recently been defined as a unique subset of proinflammatory helper cells whose development depends on signaling initiated by IL-6 and TGF-beta, autocrine activity of IL-21, activation of STAT3, and induction of the orphan nuclear receptor RORgammat. The maintenance, expansion, and further differentiation of the committed Th17 cells depend on IL-1beta and IL-23. IL-17 was originally found produced by circulating human CD45RO(+) memory T cells. A recent study found that human Th17 memory cells selectively express high levels of CCR6. In this study, we report that human peripheral blood and lymphoid tissue contain a significant number of CD4(+)FOXP3(+) T cells that express CCR6 and have the capacity to produce IL-17 upon activation. These cells coexpress FOXP3 and RORgammat transcription factors. The CD4(+)FOXP3(+)CCR6(+) IL-17-producing cells strongly inhibit the proliferation of CD4(+) responder T cells. CD4(+)CD25(high)-derived T-cell clones express FOXP3, RORgammat, and IL-17 and maintain their suppressive function via a cell-cell contact mechanism. We further show that human CD4(+)FOXP3(+)CCR6(-) regulatory T (Treg) cells differentiate into IL-17 producer cells upon T-cell receptor stimulation in the presence of IL-1beta, IL-2, IL-21, IL-23, and human serum. This, together with the finding that human thymus does not contain IL-17-producing Treg cells, suggests that the IL-17(+)FOXP3(+) Treg cells are generated in the periphery. IL-17-producing Treg cells may play critical roles in antimicrobial defense, while controlling autoimmunity and inflammation
— id: 95886, year: 2009, vol: 106, page: 4793, stat: Journal Article,

Plasticity of CD4+ T cell lineage differentiation
Zhou, Liang; Chong, Mark M W; Littman, Dan R
2009 May;30(5):646-655, Immunity
The differentiation of naive CD4(+) T cells into lineages with distinct effector functions has been considered to be an irreversible event. T helper type 1 (Th1) cells stably express IFN-gamma, whereas Th2 cells express IL-4. The discovery and investigation of two other CD4(+) T cell subsets, induced regulatory T (iTreg) cells and Th17 cells, has led to a rethinking of the notion that helper T cell subsets represent irreversibly differentiated endpoints. Accumulating evidence suggests that CD4(+) T cells, particularly iTreg and Th17 cells, are more plastic than previously appreciated. It appears that expression of Foxp3 by iTreg cells or IL-17 by Th17 cells may not be stable and that there is a great degree of flexibility in their differentiation options. Here, we will discuss recent findings that demonstrate the plasticity of CD4(+) T cell differentiation and the biological implications of this flexibility
— id: 99221, year: 2009, vol: 30, page: 646, stat: Journal Article,

Transcriptional regulatory networks in Th17 cell differentiation
Zhou, Liang; Littman, Dan R
2009 Apr;21(2):146-152, Current opinion in immunology
Upon encountering antigen in the context of antigen presenting cells, naive CD4(+) T cells undergo differentiation into effector T helper (Th) cells, which can secrete high levels of cytokines and other immunomodulators to mediate host defense and tissue inflammation. During the past three years, the immunology field has witnessed an explosion of research advances in the biology of Th17 cells, the most recently described subset of T helper cells, which play crucial roles in host immunity and inflammation. Here we review emerging data on transcriptional regulatory networks that govern the differentiation program of Th17 cells, and focus on how the orphan nuclear receptor RORgammat coordinates this process in concert with diverse cytokine-induced transcription factors
— id: 100594, year: 2009, vol: 21, page: 146, stat: Journal Article,

Species-specific restriction of apobec3-mediated hypermutation
Browne, Edward P; Littman, Dan R
2008 Feb;82(3):1305-1313, Journal of virology
Apobec proteins are a family of cellular cytidine deaminases, among which several members have been shown to have potent antiviral properties. This antiviral activity is associated with the ability to cause hypermutation of retroviral cDNA. However, recent research has indicated that Apobec proteins are also able to inhibit retroviruses by other mechanisms that are independent of their deaminase activity. We have compared the antiviral activities of human and murine Apobec3 (A3) proteins, and we have found that, consistent with previous reports, human immunodeficiency virus (HIV) is able to resist human A3G but is sensitive to murine A3, whereas murine leukemia virus (MLV) is relatively resistant to murine A3 (mA3) but sensitive to human A3G. In contrast to previous studies, we observed that mA3 is packaged efficiently into MLV particles. The C-terminal cytidine deaminase domain (CDD2) is required for packaging of mA3 into MLV particles, and packaging did not depend on the MLV viral RNA. However, mA3 packed into MLV particles failed to cause hypermutation of viral DNA, indicating that its deaminase activity is blocked or inhibited. hA3G also caused significantly less hypermutation of MLV than of HIV DNA. Both mA3 and the splice variant mA3Delta5 exhibited some residual antiviral activity against MLV and caused a reduction in the ability of MLV particles to generate reverse transcription products. These results suggest that MLV has evolved specific mechanisms to block the ability of Apobec proteins to mediate deaminase-dependent hypermutation
— id: 75770, year: 2008, vol: 82, page: 1305, stat: Journal Article,

The RNAseIII enzyme Drosha is critical in T cells for preventing lethal inflammatory disease
Chong, Mark M W; Rasmussen, Jeffrey P; Rudensky, Alexander Y; Littman, Dan R
2008 Sep 1;205(9):2005-2017, Journal of experimental medicine
MicroRNAs (miRNAs) are implicated in the differentiation and function of many cell types. We provide genetic and in vivo evidence that the two RNaseIII enzymes, Drosha and Dicer, do indeed function in the same pathway. These have previously been shown to mediate the stepwise maturation of miRNAs (Lee, Y., C. Ahn, J. Han, H. Choi, J. Kim, J. Yim, J. Lee, P. Provost, O. Radmark, S. Kim, and V.N. Kim. 2003. Nature. 425:415-419), and genetic ablation of either within the T cell compartment, or specifically within Foxp3(+) regulatory T (T reg) cells, results in identical phenotypes. We found that miRNA biogenesis is indispensable for the function of T reg cells. Specific deletion of either Drosha or Dicer phenocopies mice lacking a functional Foxp3 gene or Foxp3(+) cells, whereas deletion throughout the T cell compartment also results in spontaneous inflammatory disease, but later in life. Thus, miRNA-dependent regulation is critical for preventing spontaneous inflammation and autoimmunity
— id: 86558, year: 2008, vol: 205, page: 2005, stat: Journal Article,

Lineage diversion of T cell receptor transgenic thymocytes revealed by lineage fate mapping
Egawa, Takeshi; Kreslavsky, Taras; Littman, Dan R; von Boehmer, Harald
2008 ;3(1):e1512-e1512, PLoS ONE
BACKGROUND: The binding of the T cell receptor (TCR) to major histocompatibility complex (MHC) molecules in the thymus determines fates of TCRalphabeta lymphocytes that subsequently home to secondary lymphoid tissue. TCR transgenic models have been used to study thymic selection and lineage commitment. Most TCR transgenic mice express the rearranged TCRalphabeta prematurely at the double negative stage and abnormal TCRalphabeta populations of T cells that are not easily detected in non-transgenic mice have been found in secondary lymphoid tissue of TCR transgenic mice. METHODOLOGY AND PRINCIPAL FINDINGS: To determine developmental pathways of TCR-transgenic thymocytes, we used Cre-LoxP-mediated fate mapping and show here that premature expression of a transgenic TCRalphabeta diverts some developing thymocytes to a developmental pathway which resembles that of gamma delta cells. We found that most peripheral T cells with the HY-TCR in male mice have bypassed the RORgammat-positive CD4(+)8(+) (double positive, DP) stage to accumulate either as CD4(-)8(-) (double negative, DN) or as CD8alpha(+) T cells in lymph nodes or gut epithelium. Likewise, DN TCRalphabeta cells in lymphoid tissue of female mice were not derived from DP thymocytes. CONCLUSION: The results further support the hypothesis that the premature expression of the TCRalphabeta can divert DN thymocytes into gamma delta lineage cells
— id: 78849, year: 2008, vol: 3, page: e1512, stat: Journal Article,

ThPOK acts late in specification of the helper T cell lineage and suppresses Runx-mediated commitment to the cytotoxic T cell lineage
Egawa, Takeshi; Littman, Dan R
2008 Oct;9(10):1131-1139, Nature immunology
The transcription factor ThPOK is required and sufficient for the generation of CD4(+)CD8(-) thymocytes, yet the mechanism by which ThPOK orchestrates differentiation into the CD4(+) helper T cell lineage remains unclear. Here we used reporter mice to track the expression of transcription factors in developing thymocytes. Distal promoter-driven expression of the gene encoding the transcription factor Runx3 was restricted to major histocompatibility complex (MHC) class I-selected thymocytes. In ThPOK-deficient mice, such expression was derepressed in MHC class II-selected thymocytes, which contributed to their redirection to the CD8(+) T cell lineage. In the absence of both ThPOK and Runx, redirection was prevented and cells potentially belonging to the CD4(+) lineage, presumably specified independently of ThPOK, were generated. Our results suggest that MHC class II-selected thymocytes are directed toward the CD4(+) lineage independently of ThPOK but require ThPOK to prevent Runx-dependent differentiation toward the CD8(+) lineage
— id: 93366, year: 2008, vol: 9, page: 1131, stat: Journal Article,

Multiple ITAM-coupled NK cell receptors engage the Bcl10/Malt1 complex via Carma1 for NF-{kappa}B and MAPK activation to selectively control cytokine production
Gross, Olaf; Grupp, Christina; Steinberg, Christian; Zimmermann, Stephanie; Strasser, Dominikus; Hannesschlager, Nicole; Reindl, Wolfgang; Jonsson, Helena; Huo, Hairong; Littman, Dan R; Peschel, Christian; Yokoyama, Wayne M; Krug, Anne; Ruland, Jurgen
2008 Sep 15;112(6):2421-2428, Blood
Natural Killer (NK) cells are innate immune cells that mediate resistance against viruses and tumors. They express multiple activating receptors that couple to immunoreceptor tyrosine-based activation motif (ITAM) containing signaling chains for downstream cell activation. Ligation of activating NK cell receptors induces NK cell cytotoxicity and cytokine release. How these distinct events are selectively controlled is not well defined. Here we report the identification of a specific signaling pathway that operates downstream of the ITAM coupled NK cell receptors NK1.1, Ly49D, Ly49H and NKG2D. Using primary NK cells from Bcl10(-/-), Malt1(-/-), Carma1(-/-), and Card9(-/-) mice we demonstrate a key role for Bcl10 signalosomes in the activation of canonical NF-kappaB signaling as well as JNK and p38 MAPK upon NK cell triggering. Bcl10 directly cooperates with Malt1 and depends on Carma1 (Card11) but not on Card9 for NK cell activation. These Bcl10-dependent cascades selectively control cytokine and chemokine production but do not affect NK cell differentiation or killing. Thus, we identify a molecular basis for the segregation of NK cell receptor induced signals for cytokine release and target cell killing and extend the previously recognized roles for CARD-protein/Bcl10/Malt1 complexes in ITAM receptor signaling in innate and adaptive immune cells
— id: 78850, year: 2008, vol: 112, page: 2421, stat: Journal Article,

Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine
Ivanov, Ivaylo I; Frutos, Rosa de Llanos; Manel, Nicolas; Yoshinaga, Keiji; Rifkin, Daniel B; Sartor, R Balfour; Finlay, B Brett; Littman, Dan R
2008 Oct 16;4(4):337-349, Cell Host & Microbe
The requirements for in vivo steady state differentiation of IL-17-producing T-helper (Th17) cells, which are potent inflammation effectors, remain obscure. We report that Th17 cell differentiation in the lamina propria (LP) of the small intestine requires specific commensal microbiota and is inhibited by treating mice with selective antibiotics. Mice from different sources had marked differences in their Th17 cell numbers and animals lacking Th17 cells acquired them after introduction of bacteria from Th17 cell-sufficient mice. Differentiation of Th17 cells correlated with the presence of cytophaga-flavobacter-bacteroidetes (CFB) bacteria in the intestine and was independent of toll-like receptor, IL-21 or IL-23 signaling, but required appropriate TGF-beta activation. Absence of Th17 cell-inducing bacteria was accompanied by increase in Foxp3+ regulatory T cells (Treg) in the LP. Our results suggest that composition of intestinal microbiota regulates the Th17:Treg balance in the LP and may thus influence intestinal immunity, tolerance, and susceptibility to inflammatory bowel diseases
— id: 93379, year: 2008, vol: 4, page: 337, stat: Journal Article,

Regulated movement of CD4 in and out of the immunological synapse
Kao, Henry; Lin, Joseph; Littman, Dan R; Shaw, Andrey S; Allen, Paul M
2008 Dec 15;181(12):8248-8257, Journal of immunology
The mechanism underlying the transient accumulation of CD4 at the immunological synapse (IS) and its significance for T cell activation are not understood. To investigate these issues, we mutated a serine phosphorylation site (S408) in the cytoplasmic tail of murine CD4. Preventing phosphorylation of S408 did not block CD4 recruitment to the IS; rather, it blocked the ability of CD4 to leave the IS. Surprisingly, enhanced and prolonged CD4 accumulation at the supramolecular activation cluster in the contact area had no functional consequence for T cell activation, cytokine production, or proliferation. Protein kinase C theta (PKCtheta)-deficient T cells also displayed enhanced and prolonged accumulation of wild-type CD4 at the IS, indicating that theta is the critical PKC isoform involved in CD4 movement. These findings suggest a model wherein recruitment of CD4 to the IS allows its phosphorylation by PKCtheta and subsequent removal from the IS. Thus, an important role for PKCtheta in T cell activation involves its recruitment to the IS, where it phosphorylates specific substrates that help to maintain the dynamism of protein turnover at the IS
— id: 95891, year: 2008, vol: 181, page: 8248, stat: Journal Article,

IL-17 IS REQUIRED FOR CD4-MEDIATED GVHD
Kappel, LW; Goldberg, GL; Ivanov, II; Na, IK; King, C; Suh, D; Smith, OM; Ligh, C; Littman, DR; van den Brink, MRM
2008 FEB ;14(2):12-13, Biology of blood & marrow transplantation
— id: 91478, year: 2008, vol: 14, page: 12, stat: Journal Article,

The differentiation of human T(H)-17 cells requires transforming growth factor-beta and induction of the nuclear receptor RORgammat
Manel, Nicolas; Unutmaz, Derya; Littman, Dan R
2008 Jun;9(6):641-649, Nature immunology
T(H)-17 cells are interleukin 17 (IL-17)-secreting CD4+ T helper cells involved in autoimmune disease and mucosal immunity. In naive CD4+ T cells from mice, IL-17 is expressed in response to a combination of IL-6 or IL-21 and transforming growth factor-beta (TGF-beta) and requires induction of the nuclear receptor RORgammat. It has been suggested that the differentiation of human T(H)-17 cells is independent of TGF-beta and thus differs fundamentally from that in mice. We show here that TGF-beta, IL-1beta and IL-6, IL-21 or IL-23 in serum-free conditions were necessary and sufficient to induce IL-17 expression in naive human CD4+ T cells from cord blood. TGF-beta upregulated RORgammat expression but simultaneously inhibited its ability to induce IL-17 expression. Inflammatory cytokines relieved this inhibition and increased RORgammat-directed IL-17 expression. Other gene products detected in T(H)-17 cells after RORgammat induction included the chemokine receptor CCR6, the IL-23 receptor, IL-17F and IL-26. Our studies identify RORgammat as having a central function in the differentiation of human T(H)-17 cells from naive CD4+ T cells and suggest that similar cytokine pathways are involved in this process in mice and humans
— id: 78844, year: 2008, vol: 9, page: 641, stat: Journal Article,

HIV immunology needs a new direction
Medzhitov, Ruslan; Littman, Dan
2008 Oct 2;455(7213):591-591, Nature
— id: 137124, year: 2008, vol: 455, page: 591, stat: Journal Article,

Limited tumor infiltration by activated T effector cells restricts the therapeutic activity of regulatory T cell depletion against established melanoma
Quezada, Sergio A; Peggs, Karl S; Simpson, Tyler R; Shen, Yuelei; Littman, Dan R; Allison, James P
2008 Sep 1;205(9):2125-2138, Journal of experimental medicine
Interference with inhibitory immunological checkpoints controlling T cell activation provides new opportunities to augment cancer immunotherapies. Whereas cytotoxic T lymphocyte-associated antigen-4 blockade has shown promising preclinical and clinical results, therapeutic CD4(+)CD25(+) T reg cell depletion has failed to consistently enhance immune-based therapies. Using B16/BL6, a transplantable murine melanoma model, we show a dichotomy between the effects of T reg cell depletion on tumor rejection dependent on whether depletion occurs before (prophylactic) or after (therapeutic) tumor engraftment. Failure to promote rejection with therapeutic depletion is not related to lack of T reg cell depletion, to elimination of CD25(+) effector T cells, or to a failure to enhance systemic antitumor T cell responses, but correlates with failure of effector cells to infiltrate the tumor and increase the intratumor ratio of effector T cell/T reg cell. Finally, systemic antitumor responses generated upon therapeutic T reg cell depletion are significantly stronger than those generated in the presence of T reg cells, and are capable of eliciting rejection of established tumors after transfer into immunoablated recipients receiving combination immunotherapy. The data demonstrate a dissociation between measurable systemic responses and tumor rejection during CD25-directed T reg cell depletion, and suggest an alternative, clinically applicable strategy for the treatment of established tumors
— id: 95895, year: 2008, vol: 205, page: 2125, stat: Journal Article,

Restoration of lymphoid organ integrity through the interaction of lymphoid tissue-inducer cells with stroma of the T cell zone
Scandella, Elke; Bolinger, Beatrice; Lattmann, Evelyn; Miller, Simone; Favre, Stephanie; Littman, Dan R; Finke, Daniela; Luther, Sanjiv A; Junt, Tobias; Ludewig, Burkhard
2008 Jun;9(6):667-675, Nature immunology
The generation of lymphoid microenvironments in early life depends on the interaction of lymphoid tissue-inducer cells with stromal lymphoid tissue-organizer cells. Whether this cellular interface stays operational in adult secondary lymphoid organs has remained elusive. We show here that during acute infection with lymphocytic choriomeningitis virus, antiviral cytotoxic T cells destroyed infected T cell zone stromal cells, which led to profound disruption of secondary lymphoid organ integrity. Furthermore, the ability of the host to respond to secondary antigens was lost. Restoration of the lymphoid microanatomy was dependent on the proliferative accumulation of lymphoid tissue-inducer cells in secondary lymphoid organs during the acute phase of infection and lymphotoxin alpha(1)beta(2) signaling. Thus, crosstalk between lymphoid tissue-inducer cells and stromal cells is reactivated in adults to maintain secondary lymphoid organ integrity and thereby contributes to the preservation of immunocompetence
— id: 78846, year: 2008, vol: 9, page: 667, stat: Journal Article,

NK cell-activating receptors require PKC-theta for sustained signaling, transcriptional activation, and IFN-gamma secretion
Tassi, Ilaria; Cella, Marina; Presti, Rachel; Colucci, Angela; Gilfillan, Susan; Littman, Dan R; Colonna, Marco
2008 Nov 15;112(10):4109-4116, Blood
Natural killer (NK) cell sense virally infected cells and tumor cells through multiple cell surface receptors. Many NK cell-activating receptors signal through immunoreceptor tyrosine-based activation motif (ITAM)-containing adapters, which trigger both cytotoxicy and secretion of interferon-gamma (IFN-gamma). Within the ITAM pathway, distinct signaling intermediates are variably involved in cytotoxicity and/or IFN-gamma secretion. In this study, we have evaluated the role of protein kinase C- (PKC-) in NK-cell secretion of lytic mediators and IFN-gamma. We found that engagement of NK-cell receptors that signal through ITAMs results in prompt activation of PKC-. Analyses of NK cells from PKC--deficient mice indicated that PKC- is absolutely required for ITAM-mediated IFN-gamma secretion, whereas it has no marked influence on the release of cytolytic mediators. Moreover, we found that PKC- deficiency preferentially impairs sustained extracellular-regulated kinase signaling as well as activation of c-Jun N-terminal kinase and the transcription factors AP-1 and NFAT but does not affect activation of NF-kappaB. These results indicate that NK cell-activating receptors require PKC- to generate sustained intracellular signals that reach the nucleus and promote transcriptional activation, ultimately inducing IFN-gamma production
— id: 95894, year: 2008, vol: 112, page: 4109, stat: Journal Article,

Requirement for lymphoid tissue-inducer cells in isolated follicle formation and T cell-independent immunoglobulin A generation in the gut
Tsuji, Masayuki; Suzuki, Keiichiro; Kitamura, Hiroshi; Maruya, Mikako; Kinoshita, Kazuo; Ivanov, Ivaylo I; Itoh, Kikuji; Littman, Dan R; Fagarasan, Sidonia
2008 Aug;29(2):261-271, Immunity
Immunoglobulin A (IgA) is generated in the gut by both T cell-dependent and T cell-independent processes. The sites and the mechanisms for T cell-independent IgA synthesis remain elusive. Here we show that isolated lymphoid follicles (ILFs) were sites where induction of activation-induced cytidine deaminase (AID) and IgA class switching of B cells took place in the absence of T cells. We also show that formation of ILFs was regulated by interactions between lymphoid tissue-inducer cells expressing the nuclear receptor ROR gamma t (ROR gamma t(+)LTi cells) and stromal cells (SCs). Activation of SCs by ROR gamma t(+)LTi cells through lymphotoxin (LT)-beta receptor (LT beta R) and simultaneously by bacteria through TLRs induced recruitment of dendritic cells (DCs) and B cells and formation of ILFs. These findings provide insight into the crosstalk between bacteria, ROR gamma t(+)LTi cells, SCs, DCs, and B cells required for ILF formation and establish a critical role of ILFs in T cell-independent IgA synthesis in gut
— id: 95896, year: 2008, vol: 29, page: 261, stat: Journal Article,

Nramp1 expression by dendritic cells modulates inflammatory responses during Salmonella Typhimurium infection
Valdez, Yanet; Diehl, Gretchen E; Vallance, Bruce A; Grassl, Guntram A; Guttman, Julian A; Brown, Nat F; Rosenberger, Carrie M; Littman, Dan R; Gros, Philippe; Finlay, B Brett
2008 Aug;10(8):1646-1661, Cellular microbiology
Host resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) is mediated by natural resistance-associated macrophage protein 1 (Nramp1/Slc11a1). Nramp1 is critical to host defence, as mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S. Typhimurium. Despite this crucial role, the mechanisms underlying Nramp1's protective effects are unclear. Dendritic cells (DCs) that sample the intestinal lumen are among the first cells encountered by S. Typhimurium following oral infection and act as a conduit for S. Typhimurium to cross the intestinal epithelial barrier. We report that DCs, including intestinal, splenic and bone marrow-derived DCs (BMDCs), express Nramp1 protein. In the small intestine, Nramp1 expression is greater in a subset of DCs (CD11c(+)CD103(-)) characterized by the elevated expression of pro-inflammatory cytokines in response to bacterial products. While Nramp1 expression did not affect S. Typhimurium replication in BMDCs, infected Nramp1+/+ BMDCs and intestinal CD11c(+)CD103(-) DCs secreted more inflammatory cytokines (IL-6, IL-12 and TNF-alpha) than Nramp1-/-, suggesting that Nramp1 expression may promote a more rapid inflammatory response following infection. Collectively, these findings reveal a new role for DCs and Nramp1 in modulating the host inflammatory response to S. Typhimurium
— id: 78847, year: 2008, vol: 10, page: 1646, stat: Journal Article,

Relief of preintegration inhibition and characterization of additional blocks for HIV replication in primary mouse T cells
Zhang, Jing-xin; Diehl, Gretchen E; Littman, Dan R
2008 ;3(4):e2035-e2035, PLoS ONE
Development of a small animal model to study HIV replication and pathogenesis has been hampered by the failure of the virus to replicate in non-primate cells. Most studies aimed at achieving replication in murine cells have been limited to fibroblast cell lines, but generating an appropriate model requires overcoming blocks to viral replication in primary T cells. We have studied HIV-1 replication in CD4(+) T cells from human CD4/CCR5/Cyclin T1 transgenic mice. Expression of hCD4 and hCCR5 in mouse CD4(+) T cells enabled efficient entry of R5 strain HIV-1. In mouse T cells, HIV-1 underwent reverse transcription and nuclear import as efficiently as in human T cells. In contrast, chromosomal integration of HIV-1 proviral DNA was inefficient in activated mouse T cells. This process was greatly enhanced by providing a secondary T cell receptor (TCR) signal after HIV-1 infection, especially between 12 to 24 h post infection. This effect was specific for primary mouse T cells. The pathways involved in HIV replication appear to be PKCtheta-, CARMA1-, and WASp-independent. Treatment with Cyclosporin A (CsA) further relieved the pre-integration block. However, transcription of HIV-1 RNA was still reduced in mouse CD4(+) T cells despite expression of the hCyclin T1 transgene. Additional post-transcriptional defects were observed at the levels of Gag expression, Gag processing, Gag release and virus infectivity. Together, these post-integration defects resulted in a dramatically reduced yield of infectious virus (300-500 fold) after a single cycle of HIV-1 replication. This study implies the existence of host factors, in addition to those already identified, that are critical for HIV-1 replication in mouse cells. This study also highlights the differences between primary T cells and cell lines regarding pre-integration steps in the HIV-1 replication cycle
— id: 78845, year: 2008, vol: 3, page: e2035, stat: Journal Article,

TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function
Zhou, Liang; Lopes, Jared E; Chong, Mark M W; Ivanov, Ivaylo I; Min, Roy; Victora, Gabriel D; Shen, Yuelei; Du, Jianguang; Rubtsov, Yuri P; Rudensky, Alexander Y; Ziegler, Steven F; Littman, Dan R
2008 May 8;453(7192):236-240, Nature
T helper cells that produce IL-17 (T(H)17 cells) promote autoimmunity in mice and have been implicated in the pathogenesis of human inflammatory diseases. At mucosal surfaces, T(H)17 cells are thought to protect the host from infection, whereas regulatory T (T(reg)) cells control immune responses and inflammation triggered by the resident microflora. Differentiation of both cell types requires transforming growth factor-beta (TGF-beta), but depends on distinct transcription factors: RORgammat (encoded by Rorc(gammat)) for T(H)17 cells and Foxp3 for T(reg) cells. How TGF-beta regulates the differentiation of T cells with opposing activities has been perplexing. Here we demonstrate that, together with pro-inflammatory cytokines, TGF-beta orchestrates T(H)17 cell differentiation in a concentration-dependent manner. At low concentrations, TGF-beta synergizes with interleukin (IL)-6 and IL-21 (refs 9-11) to promote IL-23 receptor (Il23r) expression, favouring T(H)17 cell differentiation. High concentrations of TGF-beta repress IL23r expression and favour Foxp3+ T(reg) cells. RORgammat and Foxp3 are co-expressed in naive CD4+ T cells exposed to TGF-beta and in a subset of T cells in the small intestinal lamina propria of the mouse. In vitro, TGF-beta-induced Foxp3 inhibits RORgammat function, at least in part through their interaction. Accordingly, lamina propria T cells that co-express both transcription factors produce less IL-17 (also known as IL-17a) than those that express RORgammat alone. IL-6, IL-21 and IL-23 relieve Foxp3-mediated inhibition of RORgammat, thereby promoting T(H)17 cell differentiation. Therefore, the decision of antigen-stimulated cells to differentiate into either T(H)17 or T(reg) cells depends on the cytokine-regulated balance of RORgammat and Foxp3
— id: 78848, year: 2008, vol: 453, page: 236, stat: Journal Article,

HIV's vagina travelogue
Boggiano, Cesar; Littman, Dan R
2007 Feb;26(2):145-147, Immunity
Details of how HIV-1 is transmitted across mucosal barriers remain sparse. In this issue of Immunity, Hladik et al. (2007) describe an organ culture system for imaging HIV-1 interaction with vaginal epithelial T cells and Langerhans cells early after infection
— id: 71929, year: 2007, vol: 26, page: 145, stat: Journal Article,

Dendritic cell-mediated trans-enhancement of HIV-1 infectivity is independent of DC-SIGN
Boggiano, Cesar; Manel, Nicolas; Littman, Dan R
2007 Mar;81(5):2519-2523, Journal of virology
Dendritic cells (DCs) enhance HIV-1 infection of CD4(+) T lymphocytes in trans. The C-type lectin DC-SIGN, expressed on DCs, binds to the HIV-1 envelope glycoprotein gp120 and confers upon some cell lines the capacity to enhance trans-infection. Using an shRNA approach, we demonstrate that DC-SIGN is not required for efficient trans-enhancement by DCs. In addition, the DC-SIGN ligand mannan and an anti-DC-SIGN antibody did not inhibit DC-mediated enhancement. HIV-1 particles were internalized and were protected from protease treatment following binding to DCs, but not to DC-SIGN-expressing Raji cells. Thus DC-SIGN is not required for DC-mediated trans-enhancement of HIV infectivity
— id: 69501, year: 2007, vol: 81, page: 2519, stat: Journal Article,

The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells
Egawa, Takeshi; Tillman, Robert E; Naoe, Yoshinori; Taniuchi, Ichiro; Littman, Dan R
2007 Aug 6;204(8):1945-1957, Journal of experimental medicine
Members of the Runx family of transcriptional regulators are required for the appropriate expression of CD4 and CD8 at discrete stages of T cell development. The roles of these factors in other aspects of T cell development are unknown. We used a strategy to conditionally inactivate the genes encoding Runx1 or Runx3 at different stages of thymocyte development, demonstrating that Runx1 regulates the transitions of developing thymocytes from the CD4(-)CD8(-) double-negative stage to the CD4(+)CD8(+) double-positive (DP) stage and from the DP stage to the mature single-positive stage. Runx1 and Runx3 deficiencies caused marked reductions in mature thymocytes and T cells of the CD4(+) helper and CD8(+) cytotoxic T cell lineages, respectively. Runx1-deficient CD4(+) T cells had markedly reduced expression of the interleukin 7 receptor and exhibited shorter survival. In addition, inactivation of both Runx1 and Runx3 at the DP stages resulted in a severe block in development of CD8(+) mature thymocytes. These results indicate that Runx proteins have important roles at multiple stages of T cell development and in the homeostasis of mature T cells
— id: 73912, year: 2007, vol: 204, page: 1945, stat: Journal Article,

Transcriptional regulation of Th17 cell differentiation
Ivanov, Ivaylo I; Zhou, Liang; Littman, Dan R
2007 Dec;19(6):409-417, Seminars in immunology
The paradigm of effector T helper cell differentiation into either Th1 or Th2 lineages has been profoundly shaken by the discovery of T cells that secrete IL-17 and other inflammatory cytokines. This subset, referred to as Th17, is centrally involved in autoimmune disease and is important in host defense at mucosal surfaces. In mouse, a series of cytokines, including IL-6, IL-21, IL-23, and TGF-beta, function sequentially or synergistically to induce the Th17 lineage. Other cytokines, including IL-2, IL-4, IFNgamma, and IL-27, inhibit differentiation of this lineage. Here we review how the nuclear orphan receptor RORgammat functions to coordinate the diverse cytokine-induced signals and thus controls Th17 cell differentiation
— id: 78851, year: 2007, vol: 19, page: 409, stat: Journal Article,

Immunology. Asymmetry and immune memory
Littman, Dan R; Singh, Harinder
2007 Mar 23;315(5819):1673-1674, Science
— id: 71351, year: 2007, vol: 315, page: 1673, stat: Journal Article,

Caspase-8 and c-FLIPL associate in lipid rafts with NF-kappaB adaptors during T cell activation
Misra, Ravi S; Russell, Jennifer Q; Koenig, Andreas; Hinshaw-Makepeace, Jennifer A; Wen, Renren; Wang, Demin; Huo, Hairong; Littman, Dan R; Ferch, Uta; Ruland, Jurgen; Thome, Margot; Budd, Ralph C
2007 Jul 6;282(27):19365-19374, Journal of biological chemistry
Humans and mice lacking functional caspase-8 in T cells manifest a profound immunodeficiency syndrome due to defective T cell antigen receptor (TCR)-induced NF-kappaB signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that is necessary to initiate T cell cycling. We observe that following TCR ligation, a small portion of total cellular caspase-8 and c-FLIP(L) rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIP(L) at a known caspase-8 cleavage site. The active caspase.c-FLIP complex forms in the absence of Fas (CD95/APO1) and associates with the NF-kappaB signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NF-kappaB regulators PKC theta, CARMA1, Bcl-10, and MALT1, which connect to the TCR. The lack of caspase-8 results in the absence of MALT1 and Bcl-10 in the active caspase complex. Consistent with this observation, inhibition of caspase activity attenuates NF-kappaB activation. The current findings define a link among TCR, caspases, and the NF-kappaB pathway that occurs in a sequestered lipid raft environment in T cells
— id: 78853, year: 2007, vol: 282, page: 19365, stat: Journal Article,

Runx1 protects hematopoietic stem/progenitor cells from oncogenic insult
Motoda, Lena; Osato, Motomi; Yamashita, Namiko; Jacob, Bindya; Chen, Lynnette Q; Yanagida, Masatoshi; Ida, Hiroshi; Wee, Hee-Jun; Sun, Alfred X; Taniuchi, Ichiro; Littman, Dan; Ito, Yoshiaki
2007 Dec;25(12):2976-2986, Stem cells
The RUNX1/AML1 gene encodes a transcription factor essential for the generation of hematopoietic stem cells and is frequently targeted in human leukemia. In human RUNX1-related leukemias, the RAS pathway is often concurrently mutated, but the mechanism of the synergism remains elusive. Here, we found that inactivation of Runx1 in mouse bone marrow cells results in an increase in the stem/progenitor cell fraction due to suppression of apoptosis and elevated expression of the polycomb gene Bmi-1, which is important for stem cell self-renewal. Introduction of oncogenic N-RAS into wild-type cells, in contrast, reduced the stem/progenitor cell fraction because of senescence, apoptosis, and differentiation. Such detrimental events presumably occurred because of the cellular fail-safe program, although hyperproliferation was initially induced by an oncogenic stimulus. Runx1 insufficiency appears to impair such a fail-safe mechanism, particularly in the stem/progenitor cells, thereby supporting the clonal maintenance of leukemia-initiating cells expressing an activated oncogene. Disclosure of potential conflicts of interest is found at the end of this article
— id: 78852, year: 2007, vol: 25, page: 2976, stat: Journal Article,

Repression of interleukin-4 in T helper type 1 cells by Runx/Cbf beta binding to the Il4 silencer
Naoe, Yoshinori; Setoguchi, Ruka; Akiyama, Kaori; Muroi, Sawako; Kuroda, Masahiko; Hatam, Farah; Littman, Dan R; Taniuchi, Ichiro
2007 Aug 6;204(8):1749-1755, Journal of experimental medicine
Interferon gamma (IFN gamma) is the hallmark cytokine produced by T helper type 1 (Th1) cells, whereas interleukin (IL)-4 is the hallmark cytokine produced by Th2 cells. Although previous studies have revealed the roles of cytokine signaling and of transcription factors during differentiation of Th1 or Th2 cells, it is unclear how the exclusive expression pattern of each hallmark cytokine is established. The DNaseI hypersensitivity site IV within the mouse Il4 locus plays an important role in the repression of Il4 expression in Th1 cells, and it has been named the Il4 silencer. Using Cbf beta- or Runx3-deficient T cells, we show that loss of Runx complex function results in derepression of IL-4 in Th1 cells. Binding of Runx complexes to the Il4 silencer was detected in naive CD4(+) T cells and Th1 cells, but not in Th2 cells. Furthermore, enforced expression of GATA-3 in Th1 cells inhibited binding of Runx complexes to the Il4 silencer. Interestingly, T cell-specific inactivation of the Cbf beta gene in mice led to elevated serum immunoglobulin E and airway infiltration. These results demonstrate critical roles of Runx complexes in regulating immune responses, at least in part, through the repression of the Il4 gene
— id: 73914, year: 2007, vol: 204, page: 1749, stat: Journal Article,

Opposing effects of PKCtheta and WASp on symmetry breaking and relocation of the immunological synapse
Sims, Tasha N; Soos, Timothy J; Xenias, Harry S; Dubin-Thaler, Benjamin; Hofman, Jake M; Waite, Janelle C; Cameron, Thomas O; Thomas, V Kaye; Varma, Rajat; Wiggins, Chris H; Sheetz, Michael P; Littman, Dan R; Dustin, Michael L
2007 May 18;129(4):773-785, Cell
The immunological synapse (IS) is a junction between the T cell and antigen-presenting cell and is composed of supramolecular activation clusters (SMACs). No studies have been published on naive T cell IS dynamics. Here, we find that IS formation during antigen recognition comprises cycles of stable IS formation and autonomous naive T cell migration. The migration phase is driven by PKCtheta, which is localized to the F-actin-dependent peripheral (p)SMAC. PKCtheta(-/-) T cells formed hyperstable IS in vitro and in vivo and, like WT cells, displayed fast oscillations in the distal SMAC, but they showed reduced slow oscillations in pSMAC integrity. IS reformation is driven by the Wiscott Aldrich Syndrome protein (WASp). WASp(-/-) T cells displayed normal IS formation but were unable to reform IS after migration unless PKCtheta was inhibited. Thus, opposing effects of PKCtheta and WASp control IS stability through pSMAC symmetry breaking and reformation
— id: 73235, year: 2007, vol: 129, page: 773, stat: Journal Article,

IL-6 programs TH-17 cell differentiation by promoting the sequential engagement of the IL-21 and IL-23 pathways
Zhou, L; Ivanov, II; Spolski, R; Min, R; Shenderov, K; Egawa, T; Levy, DE; Leonard, WJ; Littman, DR
2007 JUL ;39(1):49-49, Cytokine
— id: 87205, year: 2007, vol: 39, page: 49, stat: Journal Article,

IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways
Zhou, Liang; Ivanov, Ivaylo I; Spolski, Rosanne; Min, Roy; Shenderov, Kevin; Egawa, Takeshi; Levy, David E; Leonard, Warren J; Littman, Dan R
2007 Sep;8(9):967-974, Nature immunology
T helper cells that produce interleukin 17 (IL-17; 'T(H)-17 cells') are a distinct subset of proinflammatory cells whose in vivo function requires IL-23 but whose in vitro differentiation requires only IL-6 and transforming growth factor-beta (TGF-beta). We demonstrate here that IL-6 induced expression of IL-21 that amplified an autocrine loop to induce more IL-21 and IL-23 receptor in naive CD4(+) T cells. Both IL-21 and IL-23, along with TGF-beta, induced IL-17 expression independently of IL-6. The effects of IL-6 and IL-21 depended on STAT3, a transcription factor required for the differentiation of T(H)-17 cells in vivo. IL-21 and IL-23 induced the orphan nuclear receptor RORgammat, which in synergy with STAT3 promoted IL-17 expression. IL-6 therefore orchestrates a series of 'downstream' cytokine-dependent signaling pathways that, in concert with TGF-beta, amplify RORgammat-dependent differentiation of T(H)-17 cells
— id: 74681, year: 2007, vol: 8, page: 967, stat: Journal Article,

A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development
Burns, Jennifer M; Summers, Bretton C; Wang, Yu; Melikian, Anita; Berahovich, Rob; Miao, Zhenhua; Penfold, Mark E T; Sunshine, Mary Jean; Littman, Dan R; Kuo, Calvin J; Wei, Kevin; McMaster, Brian E; Wright, Kim; Howard, Maureen C; Schall, Thomas J
2006 Sep 4;203(9):2201-2213, Journal of experimental medicine
The chemokine stromal cell-derived factor (SDF-1; also known as chemokine ligand 12 [CXCL12]) regulates many essential biological processes, including cardiac and neuronal development, stem cell motility, neovascularization, angiogenesis, apoptosis, and tumorigenesis. It is generally believed that SDF-1 mediates these many disparate processes via a single cell surface receptor known as chemokine receptor 4 (CXCR4). This paper characterizes an alternate receptor, CXCR7, which binds with high affinity to SDF-1 and to a second chemokine, interferon-inducible T cell alpha chemoattractant (I-TAC; also known as CXCL11). Membrane-associated CXCR7 is expressed on many tumor cell lines, on activated endothelial cells, and on fetal liver cells, but on few other cell types. Unlike many other chemokine receptors, ligand activation of CXCR7 does not cause Ca2+ mobilization or cell migration. However, expression of CXCR7 provides cells with a growth and survival advantage and increased adhesion properties. Consistent with a role for CXCR7 in cell survival and adhesion, a specific, high affinity small molecule antagonist to CXCR7 impedes in vivo tumor growth in animal models, validating this new receptor as a target for development of novel cancer therapeutics
— id: 69502, year: 2006, vol: 203, page: 2201, stat: Journal Article,

Control of microglial neurotoxicity by the fractalkine receptor
Cardona, Astrid E; Pioro, Erik P; Sasse, Margaret E; Kostenko, Volodymyr; Cardona, Sandra M; Dijkstra, Ineke M; Huang, Deren; Kidd, Grahame; Dombrowski, Stephen; Dutta, RanJan; Lee, Jar-Chi; Cook, Donald N; Jung, Steffen; Lira, Sergio A; Littman, Dan R; Ransohoff, Richard M
2006 Jul;9(7):917-924, Nature neuroscience
Microglia, the resident inflammatory cells of the CNS, are the only CNS cells that express the fractalkine receptor (CX3CR1). Using three different in vivo models, we show that CX3CR1 deficiency dysregulates microglial responses, resulting in neurotoxicity. Following peripheral lipopolysaccharide injections, Cx3cr1-/- mice showed cell-autonomous microglial neurotoxicity. In a toxic model of Parkinson disease and a transgenic model of amyotrophic lateral sclerosis, Cx3cr1-/- mice showed more extensive neuronal cell loss than Cx3cr1+ littermate controls. Augmenting CX3CR1 signaling may protect against microglial neurotoxicity, whereas CNS penetration by pharmaceutical CX3CR1 antagonists could increase neuronal vulnerability
— id: 69503, year: 2006, vol: 9, page: 917, stat: Journal Article,

A clonogenic bone marrow progenitor specific for macrophages and dendritic cells
Fogg, Darin K; Sibon, Claire; Miled, Chaouki; Jung, Steffen; Aucouturier, Pierre; Littman, Dan R; Cumano, Ana; Geissmann, Frederic
2006 Jan 6;311(5757):83-87, Science
Macrophages and dendritic cells (DCs) are crucial for immune and inflammatory responses and belong to a network of cells that has been termed the mononuclear phagocyte system (MPS). However, the origin and lineage of these cells remain poorly understood. Here, we describe the isolation and clonal analysis of a mouse bone marrow progenitor that is specific for monocytes, several macrophage subsets, and resident spleen DCs in vivo. It was also possible to recapitulate this differentiation in vitro by using treatment with the cytokines macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Thus, macrophages and DCs appear to renew from a common progenitor, providing a cellular and molecular basis for the concept of the MPS
— id: 69506, year: 2006, vol: 311, page: 83, stat: Journal Article,

A clonogenic bone marrow progenitor specific for macrophages and dendritic cells (vol 311, pg 1908, 2005)
Fogg, DK; Sibon, C; Miled, C; Jung, S; Aucouturier, P; Littman, DR; Cumano, A; Geissmann, F
2006 MAR 3 ;311(5765):1242-1242, Science
— id: 62766, year: 2006, vol: 311, page: 1242, stat: Journal Article,

The neuronal chemokine CX3CL1/fractalkine selectively recruits NK cells that modify experimental autoimmune encephalomyelitis within the central nervous system
Huang, DeRen; Shi, Fu-Dong; Jung, Steffen; Pien, Gary C; Wang, Jintang; Salazar-Mather, Thais P; He, Toby T; Weaver, Jennifer T; Ljunggren, Hans-Gustaf; Biron, Christine A; Littman, Dan R; Ransohoff, Richard M
2006 May;20(7):896-905, FASEB journal
Leukocyte trafficking to the central nervous system (CNS), regulated in part by chemokines, determines severity of the demyelinating diseases multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). To examine chemokine receptor CX3CR1 in EAE, we studied CX3CR1(GFP/GFP) mice, in which CX3CR1 targeting by insertion of Green Fluorescent Protein (GFP) allowed tracking of CX3CR1+ cells in CX3CR1(+/GFP) animals and cells destined to express CX3CR1 in CX3CR1(GFP/GFP) knockouts. NK cells were markedly reduced in the inflamed CNS of CX3CR1-deficient mice with EAE, whereas recruitment of T cells, NKT cells and monocyte/macrophages to the CNS during EAE did not require CX3CR1. Impaired recruitment of NK cells in CX3CR1(GFP/GFP) mice was associated with increased EAE-related mortality, nonremitting spastic paraplegia and hemorrhagic inflammatory lesions. The absence of CD1d did not affect the severity of EAE in CX3CR1(GFP/GFP) mice, arguing against a role for NKT cells. Accumulation of NK cells in livers of wild-type (WT) and CX3CR1(GFP/GFP) mice with cytomegalovirus hepatitis was equivalent, indicating that CX3CL1 mediated chemoattraction of NK cells was relatively specific for the CNS. These results are the first to define a chemokine that governs NK cell migration to the CNS, and the findings suggest novel therapeutic manipulation of CX3CR1+ NK cells
— id: 69504, year: 2006, vol: 20, page: 896, stat: Journal Article,

Embryonic stage-specific inactivation of glucocorticoid receptor in thymic development results in differential postnatal immune responses
Ismaili, N; Pineda-Torra, I; Shen, YL; Littman, DR; Lee, MJ; Garabedian, MJ
2006 FEB ;13(2):203A-203A, Journal of the Society for Gynecologic Investigation
— id: 62829, year: 2006, vol: 13, page: 203A, stat: Journal Article,

Lymphoid tissue inducer cells in intestinal immunity
Ivanov, I I; Diehl, G E; Littman, D R
2006 ;308:59-82, Current topics in microbiology & immunology
During fetal development, lymphoid tissue inducer cells (LTis) seed the developing lymph node and Peyer's patch anlagen and initiate the formation of both types of lymphoid organs. In the adult, a similar population of cells, termed lymphoid tissue inducer-like cells (LTi-like cells), supports the formation of organized gut-associated lymphoid tissue (GALT) in the intestine, including both isolated lymphoid follicles (ILFs) and cryptopatches (CPs). Both LTi and LTi-like cells require expression of the transcription factor RORgammat for their differentiation and function, and mice lacking RORgammat lack lymph nodes, Peyer's patches, and other organized GALT. In ILFs and cryptopatches, LTi-like cells are in close contact with different populations of intestinal dendritic cells (DCs), including a subpopulation recently shown to extend dendrites and sample luminal microflora. This interaction may allow for communication between the intestinal lumen and the immune cells in the lamina propria, which is necessary for maintaining homeostasis between the commensal microflora and the intestinal immune system. The potential functional implications of the organization of LTi-like cells, DCs, and lymphocytes in the lamina propria are discussed in the context of maintenance of homeostasis and of infectious diseases, particularly HIV infection
— id: 68779, year: 2006, vol: 308, page: 59, stat: Journal Article,

The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells
Ivanov, Ivaylo I; McKenzie, Brent S; Zhou, Liang; Tadokoro, Carlos E; Lepelley, Alice; Lafaille, Juan J; Cua, Daniel J; Littman, Dan R
2006 Sep 22;126(6):1121-1133, Cell
IL-17-producing T lymphocytes have been recently shown to comprise a distinct lineage of proinflammatory T helper cells, termed Th17 cells, that are major contributors to autoimmune disease. We show here that the orphan nuclear receptor RORgammat is the key transcription factor that orchestrates the differentiation of this effector cell lineage. RORgammat induces transcription of the genes encoding IL-17 and the related cytokine IL-17F in naive CD4(+) T helper cells and is required for their expression in response to IL-6 and TGF-beta, the cytokines known to induce IL-17. Th17 cells are constitutively present throughout the intestinal lamina propria, express RORgammat, and are absent in mice deficient for RORgammat or IL-6. Mice with RORgammat-deficient T cells have attenuated autoimmune disease and lack tissue-infiltrating Th17 cells. Together, these studies suggest that RORgammat is a key regulator of immune homeostasis and highlight its potential as a therapeutic target in inflammatory diseases
— id: 69079, year: 2006, vol: 126, page: 1121, stat: Journal Article,

CX3CR1+ interstitial dendritic cells form a contiguous network throughout the entire kidney
Soos, T J; Sims, T N; Barisoni, L; Lin, K; Littman, D R; Dustin, M L; Nelson, P J
2006 Aug;70(3):591-596, Kidney international
Dendritic cells (DCs) interface innate and adaptive immunity in nonlymphoid organs; however, the exact distribution and types of DC within the kidney are not known. We utilized CX3CR1GFP/+ mice to characterize the anatomy and phenotype of tissue-resident CX3CR1+ DCs within normal kidney. Laser-scanning confocal microscopy revealed an extensive, contiguous network of stellate-shaped CX3CR1+ DCs throughout the interstitial and mesangial spaces of the entire kidney. Intravital microscopy of the superficial cortex showed stationary interstitial CX3CR1+ DCs that continually probe the surrounding tissue environment through dendrite extensions. Flow cytometry of renal CX3CR1+ DCs showed significant coexpression of CD11c and F4/80, high major histocompatibility complex class II and FcR expression, and immature costimulatory but competent phagocytic ability indicative of tissue-resident, immature DCs ready to respond to environment cues. Thus, within the renal parenchyma, there exists little immunological privilege from the surveillance provided by renal CX3CR1+ DCs, a major constituent of the heterogeneous mononuclear phagocyte system populating normal kidney
— id: 66675, year: 2006, vol: 70, page: 591, stat: Journal Article,

CD4-specific transgenic expression of human cyclin T1 markedly increases human immunodeficiency virus type 1 (HIV-1) production by CD4+ T lymphocytes and myeloid cells in mice transgenic for a provirus encoding a monocyte-tropic HIV-1 isolate
Sun, Jinglin; Soos, Timothy; Kewalramani, Vineet N; Osiecki, Kristin; Zheng, Jian Hua; Falkin, Laurie; Santambrogio, Laura; Littman, Dan R; Goldstein, Harris
2006 Feb;80(4):1850-1862, Journal of virology
Human immunodeficiency virus type 1 (HIV-1)-encoded Tat provides transcriptional activation critical for efficient HIV-1 replication by interacting with cyclin T1 and recruiting P-TEFb to efficiently elongate the nascent HIV transcript. Tat-mediated transcriptional activation in mice is precluded by species-specific structural differences that prevent Tat interaction with mouse cyclin T1 and severely compromise HIV-1 replication in mouse cells. We investigated whether transgenic mice expressing human cyclin T1 under the control of a murine CD4 promoter/enhancer cassette that directs gene expression to CD4(+) T lymphocytes and monocytes/macrophages (hu-cycT1 mice) would display Tat responsiveness in their CD4-expressing mouse cells and selectively increase HIV-1 production in this cellular population, which is infected primarily in HIV-1-positive individuals. To this end, we crossed hu-cycT1 mice with JR-CSF transgenic mice carrying the full-length HIV-1(JR-CSF) provirus under the control of the endogenous HIV-1 long terminal repeat and demonstrated that human cyclin T1 expression is sufficient to support Tat-mediated transactivation in primary mouse CD4 T lymphocytes and monocytes/macrophages and increases in vitro and in vivo HIV-1 production by these stimulated cells. Increased HIV-1 production by CD4(+) T lymphocytes was paralleled with their specific depletion in the peripheral blood of the JR-CSF/hu-cycT1 mice, which increased over time. In addition, increased HIV-1 transgene expression due to human cyclin T1 expression was associated with increased lipopolysaccharide-stimulated monocyte chemoattractant protein 1 production by JR-CSF mouse monocytes/macrophages in vitro. Therefore, the JR-CSF/hu-cycT1 mice should provide an improved mouse system for investigating the pathogenesis of various aspects of HIV-1-mediated disease and the efficacies of therapeutic interventions
— id: 69505, year: 2006, vol: 80, page: 1850, stat: Journal Article,

Virus-host interactions: new insights from the small RNA world
Browne, Edward P; Li, Junjie; Chong, Mark; Littman, Dan R
2005 ;6(11):238-238, Genome biology
RNA silencing has a known role in the antiviral responses of plants and insects. Recent evidence, including the finding that the Tat protein of human immunodeficiency virus (HIV) can suppress the host's RNA-silencing pathway and may thus counteract host antiviral RNAs, suggests that RNA-silencing pathways could also have key roles in mammalian virus-host interactions
— id: 64454, year: 2005, vol: 6, page: 238, stat: Journal Article,

Regulation of microglia neurotoxicity by CX3CR1 and CX3CL1
Cardona, AE; Huang, DR; Sasse, ME; Kidd, G; Lira, S; Cook, D; Jung, S; Littman, D; Ransohoff, R
2005 MAR 4 ;19(4):A940-A940, FASEB journal
— id: 55697, year: 2005, vol: 19, page: A940, stat: Journal Article,

Selection and lineage specification in the thymus: commitment 4-stalled
Collins, Amelie; Littman, Dan R
2005 Jul;23(1):4-5, Immunity
How CD4(+)CD8(+) thymocytes commit to CD4 helper versus CD8 cytotoxic lineages is a central unresolved question in developmental immunology. In this issue, show that engineering CD4 for shutoff immediately after positive selection misdirects cells to the cytotoxic lineage. The result highlights the distinction between positive selection and lineage commitment and provides new impetus for reexamining lineage models
— id: 57847, year: 2005, vol: 23, page: 4, stat: Journal Article,

ATP mediates rapid microglial response to local brain injury in vivo
Davalos, Dimitrios; Grutzendler, Jaime; Yang, Guang; Kim, Jiyun V; Zuo, Yi; Jung, Steffen; Littman, Dan R; Dustin, Michael L; Gan, Wen-Biao
2005 Jun;8(6):752-758, Nature neuroscience
Parenchymal microglia are the principal immune cells of the brain. Time-lapse two-photon imaging of GFP-labeled microglia demonstrates that the fine termini of microglial processes are highly dynamic in the intact mouse cortex. Upon traumatic brain injury, microglial processes rapidly and autonomously converge on the site of injury without cell body movement, establishing a potential barrier between the healthy and injured tissue. This rapid chemotactic response can be mimicked by local injection of ATP and can be inhibited by the ATP-hydrolyzing enzyme apyrase or by blockers of G protein-coupled purinergic receptors and connexin channels, which are highly expressed in astrocytes. The baseline motility of microglial processes is also reduced significantly in the presence of apyrase and connexin channel inhibitors. Thus, extracellular ATP regulates microglial branch dynamics in the intact brain, and its release from the damaged tissue and surrounding astrocytes mediates a rapid microglial response towards injury
— id: 56024, year: 2005, vol: 8, page: 752, stat: Journal Article,

Response to comment on "Thymic origin of intestinal up T cells revealed by fate mapping of ROR gamma t(+) cells"
Eberl, G; Littman, DR
2005 JUN 10 ;308(5728):45-54, Science
— id: 55929, year: 2005, vol: 308, page: 45, stat: Journal Article,

Genetic evidence supporting selection of the Valpha14i NKT cell lineage from double-positive thymocyte precursors
Egawa, Takeshi; Eberl, Gerard; Taniuchi, Ichiro; Benlagha, Kamel; Geissmann, Frederic; Hennighausen, Lothar; Bendelac, Albert; Littman, Dan R
2005 Jun;22(6):705-716, Immunity
Invariant Valpha14i NKT (iNKT) cells are a specialized subset of T lymphocytes with regulatory functions. They coexpress TCRalphabeta and natural killer cell markers. They differentiate through interaction of their Valpha14-Jalpha18 invariant TCRalpha chains with CD1d expressed on double-positive (DP) thymocytes. Although their development has been shown to be thymus dependent, their developmental pathway has not been definitively established. By using genetic analyses, we show here that all iNKT cells are selected from a pool of DP thymocytes. Their development is absolutely dependent on Runx1 and ROR(gamma)t, transcription factors that influence, but are not required for, development of conventional T cells. Our results indicate that even though CD1d binding DP thymocytes have yet to be observed, Valpha14-Jalpha18 rearrangement in these cells is required for development of iNKT cells
— id: 69511, year: 2005, vol: 22, page: 705, stat: Journal Article,

Functional and molecular analysis of the double-positive stage-specific CD8 enhancer E8III during thymocyte development
Feik, Nicholas; Bilic, Ivan; Tinhofer, Johanna; Unger, Bernd; Littman, Dan R; Ellmeier, Wilfried
2005 Feb 1;174(3):1513-1524, Journal of immunology
Several developmental stage-, subset-, and lineage-specific Cd8 cis-regulatory regions have been identified. These include the E8(III) enhancer, which directs expression in double-positive (DP) thymocytes, and E8(II), which is active in DP cells and CD8(+) T cells. Using a transgenic reporter expression assay, we identified a 285-bp core fragment of the E8(III) enhancer that retains activity in DP thymocytes. In vitro characterization of the core enhancer revealed five regulatory elements that are required for full enhancer activity, suggesting that multiple factors contribute to the developmental stage-specific activity. Furthermore, deletion of E8(III) in the mouse germline showed that this enhancer is required for nonvariegated expression of CD8 in DP thymocytes when E8(II) is also deleted. These results indicate that E8(III) is one of the cis-elements that contribute to the activation of the Cd8a and Cd8b gene complex during T cell development
— id: 69512, year: 2005, vol: 174, page: 1513, stat: Journal Article,

Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids
Geissmann, Frederic; Cameron, Thomas O; Sidobre, Stephane; Manlongat, Natasha; Kronenberg, Mitchell; Briskin, Michael J; Dustin, Michael L; Littman, Dan R
2005 Apr;3(4):e113-e113, PLoS biology
We examined the in vivo behavior of liver natural killer T cells (NKT cells) by intravital fluorescence microscopic imaging of mice in which a green fluorescent protein cDNA was used to replace the gene encoding the chemokine receptor CXCR6. NKT cells, which account for most CXCR6(+) cells in liver, were found to crawl within hepatic sinusoids at 10-20 microm/min and to stop upon T cell antigen receptor activation. CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. CXCL16, the cell surface ligand for CXCR6, is expressed on sinusoidal endothelial cells, and CXCR6 deficiency resulted in reduced survival, but not in altered speed or pattern of patrolling of NKT cells. Thus, NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance
— id: 56025, year: 2005, vol: 3, page: e113, stat: Journal Article,

Runx3 regulates integrin alpha E/CD103 and CD4 expression during development of CD4-/CD8+ T cells
Grueter, Baerbel; Petter, Michaela; Egawa, Takeshi; Laule-Kilian, Kirsten; Aldrian, Christine J; Wuerch, Andreas; Ludwig, Yvonne; Fukuyama, Hidehiro; Wardemann, Hedda; Waldschuetz, Ralph; Moroy, Tarik; Taniuchi, Ichiro; Steimle, Viktor; Littman, Dan R; Ehlers, Marc
2005 Aug 1;175(3):1694-1705, Journal of immunology
During thymic T cell development, immature CD4+CD8+ double-positive (DP) thymocytes develop either into CD4+CD8- Th cells or CD4-CD8+ CTLs. Differentially expressed primary factors inducing the fate of these cell types are still poorly described. The transcription factor Runx3/AML-2 Runx, runt [corrected] dominant factor; AML, acute myeloid leukemia is expressed specifically during the development of CD8 single-positive (SP) thymocytes, where it silences CD4 expression. Deletion of murine Runx3 results in a reduction of CD8 SP T cells and concomitant accumulation of CD4+CD8+ T cells, which cannot down-regulate CD4 expression in the thymus and periphery. In this study we have investigated the role of Runx3 during thymocyte development and CD4 silencing and have identified integrin alpha(E)/CD103 on CD8 SP T cells as a new potential target gene of Runx3. We demonstrate that Runx3 is necessary not only to repress CD4, but also to induce CD103 expression during development of CD8 SP T cells. In addition, transgenic overexpression of Runx3 reduced CD4 expression during development of DP thymocytes, leading to a reduced number of CD4 SP thymocytes and an increased number of CD8 SP thymocytes. This reversal is not caused by redirection of specific MHC class II-restricted cells to the CD8 lineage. Overexpression of Runx3 also up-regulated CD103 expression on a subpopulation of CD4 SP T cells with characteristics of regulatory T cells. Thus, Runx3 is a main regulator of CD4 silencing and CD103 induction and thus contributes to the phenotype of CD8 SP T cells during thymocyte development
— id: 69510, year: 2005, vol: 175, page: 1694, stat: Journal Article,

CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance
Niess, Jan Hendrik; Brand, Stephan; Gu, Xiubin; Landsman, Limor; Jung, Steffen; McCormick, Beth A; Vyas, Jatin M; Boes, Marianne; Ploegh, Hidde L; Fox, James G; Littman, Dan R; Reinecker, Hans-Christian
2005 Jan 14;307(5707):254-258, Science
Dendritic cells (DCs) and macrophages are critical to innate and adaptive immunity to the intestinal bacterial microbiota. Here, we identify a myeloid-derived mucosal DC in mice, which populates the entire lamina propria of the small intestine. Lamina propria DCs were found to depend on the chemokine receptor CX3CR1 to form transepithelial dendrites, which enable the cells to directly sample luminal antigens. CX3CR1 was also found to control the clearance of entero-invasive pathogens by DCs. Thus, CX3CR1-dependent processes, which control host interactions of specialized DCs with commensal and pathogenic bacteria, may regulate immunological tolerance and inflammation
— id: 69513, year: 2005, vol: 307, page: 254, stat: Journal Article,

Mice deficient in the chemokine receptor CXCR4 exhibit impaired limb innervation and myogenesis
Odemis, Veysel; Lamp, Elke; Pezeshki, Gita; Moepps, Barbara; Schilling, Karl; Gierschik, Peter; Littman, Dan R; Engele, Jurgen
2005 Dec;30(4):494-505, Molecular & cellular neurosciences
The chemokine CXCL12/SDF-1 and its receptor CXCR4 regulate the development and the function of the hematopoietic system and control morphogenesis of distinct brain areas. Here, we demonstrate that inactivation of CXCR4 results in a massive loss of spinal cord motoneurons and dorsal root ganglion neurons and, subsequently, in a reduced innervation of the developing mouse fore- and hindlimbs. However, only the death of sensory neurons seems to be a direct consequence of receptor inactivation as suggested by the observations that DRG neurons, but not motoneurons, of wild-type animals express CXCR4 and respond to CXCL12 with an increase in cell survival. In contrast, the increased death of motoneurons in CXCR4-deficient animals seems to result from impaired limb myogenesis and a subsequent loss of muscle-derived neurotrophic support. In summary, our findings unravel a previously unrecognized complex role of CXCL12/CXCR4 in the control of limb neuromuscular development
— id: 69509, year: 2005, vol: 30, page: 494, stat: Journal Article,

Role for CXCR6 in recruitment of activated CD8+ lymphocytes to inflamed liver
Sato, Tohru; Thorlacius, Henrik; Johnston, Brent; Staton, Tracy L; Xiang, Wenkai; Littman, Dan R; Butcher, Eugene C
2005 Jan 1;174(1):277-283, Journal of immunology
Hepatic infiltration of activated CD8 lymphocytes is a major feature of graft-vs-host disease (GvHD). Chemoattractant cytokines and their receptors are key regulators of lymphocyte trafficking, but the involvement of chemoattractant receptors in the physiologic recruitment of cells into the inflamed liver has not been defined. The present study examines the role of the chemokine receptor CXCR6, which is highly expressed by liver-infiltrating CD8 T cells. Hepatic accumulation of donor CD8, but not donor CD4, lymphocytes was significantly reduced in GvHD induced by transfer of CXCR6(-/-), H-2D(b) lymphocytes into BDF(1), H-2D(bxd) recipients. To determine whether altered recruitment contributes to the reduced accumulation, CXCR6(-/-) or wild-type splenic lymphocytes participating in an active GvHD response were isolated and transferred i.v. into secondary recipients with active GvHD, and the short term (6-h) recruitment of transferred cells to the inflamed liver was assessed. CXCR6(-/-) CD8 (but not CD4) cells displayed a significant (33%) reduction in liver localization, whereas frequencies in blood of CXCR6(-/-) and wild-type CD8 cells were similar. Proliferation and apoptosis of liver-infiltrating donor CD8 cells were unaffected. We conclude that CXCR6 helps mediate the recruitment of activated CD8 lymphocytes in GvHD-induced hepatitis and may be a useful target to treat pathological inflammation in the liver
— id: 69514, year: 2005, vol: 174, page: 277, stat: Journal Article,

The SDF-1/CXCR4 pathway and the development of the cerebellar system
Vilz, Tim O; Moepps, Barbara; Engele, Jurgen; Molly, Sabine; Littman, Dan R; Schilling, Karl
2005 Oct;22(8):1831-1839, European journal of neuroscience
Mice deficient for the chemokine receptor CXCR4 show premature translocation of granule cell neuroblasts from their germinal zone into the nascent cerebellum [Y.-R. Zuo et al. (1998) Nature, 393, 595-599]. Here, we used CXCR4-null mice to analyse the early development of cerebellar cortical inhibitory interneurons and pontine neurons which, in the adult, are synaptically integrated with granule cells. Cortical inhibitory interneuronal precursors normally invade the cerebellar anlage of CXCR4-deficient mice, but their dispersal is impeded by dislocated foci of proliferating granule cells, from which they are excluded. This is reminiscent of the strict exclusion of inhibitory interneuronal precursors from the superficial external granule cell layer. As inhibitory interneuronal precursors readily mingle with post-mitotic granule cells both in wild-type and CXCR4-null mice, these findings indicate that the developmentally regulated interactions between granule and inhibitory interneuronal precursors are independent of SDF-1/CXCR4 signalling. In contrast, the transit of pontine neurons from the rhombic lip through the anterior extramural stream to the basilar pons is disrupted in CXCR4-deficient animals. Migrating pontine neurons express CXCR4, and in CXCR4-null animals these cells are found displaced deep into the brainstem. Consequently, nascent pontine nuclei in CXCR4-deficient animals are hypoplastic. Moreover, they fail to express plexin D1, suggesting that SDF-1/CXCR4 signalling may also impinge on axon guidance critical to the orderly formation of granule cell mossy fibre afferents
— id: 69508, year: 2005, vol: 22, page: 1831, stat: Journal Article,

Runx1 prevents wasting, myofibrillar disorganization, and autophagy of skeletal muscle
Wang, Xiaoxia; Blagden, Chris; Fan, Jihua; Nowak, Scott J; Taniuchi, Ichiro; Littman, Dan R; Burden, Steven J
2005 Jul 15;19(14):1715-1722, Genes & development
Disruptions in the use of skeletal muscle lead to muscle atrophy. After short periods of disuse, muscle atrophy is reversible, and even after prolonged periods of inactivity, myofiber degeneration is uncommon. The pathways that regulate atrophy, initiated either by peripheral nerve damage, immobilization, aging, catabolic steroids, or cancer cachexia, however, are poorly understood. Previously, we found that Runx1 (AML1), a DNA-binding protein that is homologous to Drosophila Runt and has critical roles in hematopoiesis and leukemogenesis, is poorly expressed in innervated muscle, but strongly induced in muscle shortly after denervation. To determine the function of Runx1 in skeletal muscle, we generated mice in which Runx1 was selectively inactivated in muscle. Here, we show that Runx1 is required to sustain muscle by preventing denervated myofibers from undergoing myofibrillar disorganization and autophagy, structural defects found in a variety of congenital myopathies. We find that only 29 genes, encoding ion channels, signaling molecules, and muscle structural proteins, depend upon Runx1 expression, suggesting that their misregulation causes the dramatic muscle wasting. These findings demonstrate an unexpected role for electrical activity in regulating muscle wasting, and indicate that muscle disuse induces compensatory mechanisms that limit myofiber atrophy. Moreover, these results suggest that reduced muscle activity could cause or contribute to congenital myopathies if Runx1 or its target genes were compromised
— id: 57720, year: 2005, vol: 19, page: 1715, stat: Journal Article,

CD11chigh dendritic cell ablation impairs lymphopenia-driven proliferation of naive and memory CD8+ T cells
Zaft, Tami; Sapoznikov, Anita; Krauthgamer, Rita; Littman, Dan R; Jung, Steffen
2005 Nov 15;175(10):6428-6435, Journal of immunology
The peripheral lymphocyte pool size is governed by homeostatic mechanisms. Thus, grafted T cells expand and replenish T cell compartments in lymphopenic hosts. Lymphopenia-driven proliferation of naive CD8+ T cells depends on self-peptide/MHC class I complexes and the cytokine IL-7. Lymphopenia-driven proliferation and maintenance of memory CD8+ T cells are MHC independent, but are believed to require IL-7 and contact with a bone marrow-derived cell that presents the cytokine IL-15 by virtue of its high affinity receptor (IL-15Ralpha). In this study we show that optimal spontaneous proliferation of grafted naive and memory CD8+ T cells in mice rendered lymphopenic through gene ablation or irradiation requires the presence of CD11chigh dendritic cells. Our results suggest a dual role of CD11chigh dendritic cells as unique APC and cytokine-presenting cells
— id: 69507, year: 2005, vol: 175, page: 6428, stat: Journal Article,

Murine T cells potently restrict human immunodeficiency virus infection
Baumann, Jorg G; Unutmaz, Derya; Miller, Michael D; Breun, Sabine K J; Grill, Stacy M; Mirro, Jane; Littman, Dan R; Rein, Alan; KewalRamani, Vineet N
2004 Nov;78(22):12537-12547, Journal of virology
Development of a mouse model for human immunodeficiency virus type 1 (HIV-1) infection has advanced through the progressive identification of host cell factors required for HIV-1 replication. Murine cells lack HIV-1 receptor molecules, do not support efficient viral gene expression, and lack factors necessary for the assembly and release of virions. Many of these blocks have been described using mouse fibroblast cell lines. Here we identify a postentry block to HIV-1 infection in mouse T-cell lines that has not been detected in mouse fibroblasts. While murine fibroblastic lines are comparable to human T-cell lines in permissivity to HIV-1 transduction, infection of murine T cells is 100-fold less efficient. Virus entry occurs efficiently in murine T cells. However, reduced efficiency of the completion of reverse transcription and nuclear transfer of the viral preintegration complex are observed. Although this block has similarities to the restriction of murine retroviruses by Fv1, there is no correlation of HIV-1 susceptibility with cellular Fv1 genotypes. In addition, the block to HIV-1 infection in murine T-cell lines cannot be saturated by a high virus dose. Further studies of this newly identified block may lend insight into the early events of retroviral replication and reveal new targets for antiretroviral interventions
— id: 69516, year: 2004, vol: 78, page: 12537, stat: Journal Article,

PKC{theta} Signals Activation versus Tolerance In Vivo
Berg-Brown, Nancy N; Gronski, Matthew A; Jones, Russell G; Elford, Alisha R; Deenick, Elissa K; Odermatt, Bernhard; Littman, Dan R; Ohashi, Pamela S
2004 Mar 15;199(6):743-752, Journal of experimental medicine
Understanding the pathways that signal T cell tolerance versus activation is key to regulating immunity. Previous studies have linked CD28 and protein kinase C-theta (PKCtheta) as a potential signaling pathway that influences T cell activation. Therefore, we have compared the responses of T cells deficient for CD28 and PKCtheta in vivo and in vitro. Here, we demonstrate that the absence of PKCtheta leads to the induction of T cell anergy, with a phenotype that is comparable to the absence of CD28. Further experiments examined whether PKCtheta triggered other CD28-dependent responses. Our data show that CD4 T cell-B cell cooperation is dependent on CD28 but not PKCtheta, whereas CD28 costimulatory signals that augment proliferation can be uncoupled from signals that regulate anergy. Therefore, PKCtheta relays a defined subset of CD28 signals during T cell activation and is critical for the induction of activation versus tolerance in vivo
— id: 42239, year: 2004, vol: 199, page: 743, stat: Journal Article,

Role of fractalkine and fractalkine receptor in microglia neurotoxicity
Cardona, AE; Huang, D; Kidd, G; Sasse, ME; Lira, S; Cook, D; Jung, S; Littman, DR; Ransohoff, RM
2004 MAY ;63(5):525-525, Journal of neuropathology & experimental neurology
— id: 46514, year: 2004, vol: 63, page: 525, stat: Journal Article,

Neural and immune synaptic relations
Dustin, ML; Jiyun, K; Lieberthal, J; Littman, DR; Davalos, D; Wenbiao, G
2004 SEP ;154(1-2):5-5, Journal of neuroimmunology
— id: 48924, year: 2004, vol: 154, page: 5, stat: Journal Article,

Thymic origin of intestinal alphabeta T Cells Revealed by Fate Mapping of RORgammat+ Cells
Eberl, Gerard; Littman, Dan R
2004 Aug 9;305(5681):248-251, Science
Intestinal intraepithelial T lymphocytes (IELs) are likely to play a key role in host mucosal immunity and, unlike other T cells, have been proposed to differentiate from local precursors rather than from thymocytes. We show here that IELs expressing the alphabeta T cell receptor are derived from precursors that express RORgammat, an orphan nuclear hormone receptor detected only in immature CD4+CD8+ thymocytes, fetal lymphoid tissue-inducer (LTi) cells, and LTi-like cells in cryptopatches within the adult intestinal lamina propria. Using cell fate mapping, we found that all intestinal alphabeta T cells are progeny of CD4+CD8+ thymocytes, indicating that the adult intestine is not a significant site for alphabeta T cell development. Our results suggest that intestinal RORgammat+ cells are local organizers of mucosal lymphoid tissue
— id: 46159, year: 2004, vol: 305, page: 248, stat: Journal Article,

An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells
Eberl, Gerard; Marmon, Shana; Sunshine, Mary-Jean; Rennert, Paul D; Choi, Yongwon; Littman, Dan R
2004 Jan;5(1):64-73, Nature immunology
Lymphoid tissue inducer (LTi) cells are associated with early development of lymph nodes and Peyer's patches. We show here that during fetal life the nuclear hormone receptor RORgamma(t) is expressed exclusively in and is required for the generation of LTi cells. RORgamma(t+) LTi cells provide essential factors, among which lymphotoxin-alpha1beta2 is necessary but not sufficient for activation of the mesenchyma in lymph node and Peyer's patch anlagen. This early LTi cell-mediated activation of lymph node and Peyer's patch mesenchyma forms the necessary platform for the subsequent development of mature lymphoid tissues
— id: 42235, year: 2004, vol: 5, page: 64, stat: Journal Article,

Human immunodeficiency virus type 1 activates plasmacytoid dendritic cells and concomitantly induces the bystander maturation of myeloid dendritic cells
Fonteneau, Jean-Francois; Larsson, Marie; Beignon, Anne-Sophie; McKenna, Kelli; Dasilva, Ida; Amara, Ali; Liu, Yong-Jun; Lifson, Jeffrey D; Littman, Dan R; Bhardwaj, Nina
2004 May;78(10):5223-5232, Journal of virology
In this study, we analyzed the phenotypic and physiological consequences of the interaction of plasmacytoid dendritic cells (pDCs) with human immunodeficiency virus type 1 (HIV-1). pDCs are one cellular target of HIV-1 and respond to the virus by producing alpha/beta interferon (IFN-alpha/beta) and chemokines. The outcome of this interaction, notably on the function of bystander myeloid DC (CD11c+ DCs), remains unclear. We therefore evaluated the effects of HIV-1 exposure on these two DC subsets under various conditions. Blood-purified pDCs and CD11c+ DCs were exposed in vitro to HIV-1, after which maturation markers, cytokine production, migratory capacity, and CD4 T-cell stimulatory capacity were analyzed. pDCs exposed to different strains of infectious or even chemically inactivated, nonreplicating HIV-1 strongly upregulated the expression of maturation markers, such as CD83 and functional CCR7, analogous to exposure to R-848, a synthetic agonist of toll-like receptor-7 and -8. In addition, HIV-1-activated pDCs produced cytokines (IFN-alpha and tumor necrosis factor alpha), migrated in response to CCL19 and, in coculture, matured CD11c+ DCs, which are not directly activated by HIV. pDCs also acquired the ability to stimulate naive CD4+ T cells, albeit less efficiently than CD11c+ DCs. This HIV-1-induced maturation of both DC subsets may explain their disappearance from the blood of patients with high viral loads and may have important consequences on HIV-1 cellular transmission and HIV-1-specific T-cell responses
— id: 44440, year: 2004, vol: 78, page: 5223, stat: Journal Article,

Protein kinase C betaII regulates Akt phosphorylation on Ser-473 in a cell type- and stimulus-specific fashion
Kawakami, Yuko; Nishimoto, Hajime; Kitaura, Jiro; Maeda-Yamamoto, Mari; Kato, Roberta M; Littman, Dan R; Leitges, Michael; Rawlings, David J; Kawakami, Toshiaki
2004 Nov 12;279(46):47720-47725, Journal of biological chemistry
Akt (= protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKCbetaII, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKCbeta is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKCbetaII appears to work as a cell type- and stimulus-specific PDK2
— id: 69519, year: 2004, vol: 279, page: 47720, stat: Journal Article,

PKC-theta knockout mice are protected from fat-induced insulin resistance
Kim, Jason K; Fillmore, Jonathan J; Sunshine, Mary Jean; Albrecht, Bjoern; Higashimori, Takamasa; Kim, Dong-Wook; Liu, Zhen-Xiang; Soos, Timothy J; Cline, Gary W; O'Brien, William R; Littman, Dan R; Shulman, Gerald I
2004 Sep;114(6):823-827, Journal of clinical investigation
Insulin resistance plays a primary role in the development of type 2 diabetes and may be related to alterations in fat metabolism. Recent studies have suggested that local accumulation of fat metabolites inside skeletal muscle may activate a serine kinase cascade involving protein kinase C-theta (PKC-theta), leading to defects in insulin signaling and glucose transport in skeletal muscle. To test this hypothesis, we examined whether mice with inactivation of PKC-theta are protected from fat-induced insulin resistance in skeletal muscle. Skeletal muscle and hepatic insulin action as assessed during hyperinsulinemic-euglycemic clamps did not differ between WT and PKC-theta KO mice following saline infusion. A 5-hour lipid infusion decreased insulin-stimulated skeletal muscle glucose uptake in the WT mice that was associated with 40-50% decreases in insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-1-associated PI3K activity. In contrast, PKC-theta inactivation prevented fat-induced defects in insulin signaling and glucose transport in skeletal muscle. In conclusion, our findings demonstrate that PKC-theta is a crucial component mediating fat-induced insulin resistance in skeletal muscle and suggest that PKC-theta is a potential therapeutic target for the treatment of type 2 diabetes
— id: 69517, year: 2004, vol: 114, page: 823, stat: Journal Article,

Protein kinase C Theta inhibits insulin signaling by phosphorylating IRS1 at Ser(1101)
Li, Yu; Soos, Timothy J; Li, Xinghai; Wu, Jiong; Degennaro, Matthew; Sun, Xiaojian; Littman, Dan R; Birnbaum, Morris J; Polakiewicz, Roberto D
2004 Oct 29;279(44):45304-45307, Journal of biological chemistry
Obesity and stress inhibit insulin action by activating protein kinases that enhance serine phosphorylation of IRS1 and have been thus associated to insulin resistance and the development of type II diabetes. The protein kinase C (PKC) is activated by free-fatty acids, and its activity is higher in muscle from obese diabetic patients. However, a molecular link between PKC and insulin resistance has not been defined yet. Here we show that PKC phosphorylates IRS1 at serine 1101 blocking IRS1 tyrosine phosphorylation and downstream activation of the Akt pathway. Mutation of Ser(1101) to alanine makes IRS1 insensitive to the effect of PKC and restores insulin signaling in culture cells. These results provide a novel mechanism linking the activation of PKC to the inhibition of insulin signaling
— id: 69518, year: 2004, vol: 279, page: 45304, stat: Journal Article,

CD8alphaalpha-mediated survival and differentiation of CD8 memory T cell precursors
Madakamutil, Loui T; Christen, Urs; Lena, Christopher J; Wang-Zhu, Yiran; Attinger, Antoine; Sundarrajan, Monisha; Ellmeier, Wilfried; von Herrath, Matthias G; Jensen, Peter; Littman, Dan R; Cheroutre, Hilde
2004 Apr 23;304(5670):590-593, Science
Memory T cells are long-lived antigen-experienced T cells that are generally accepted to be direct descendants of proliferating primary effector cells. However, the factors that permit selective survival of these T cells are not well established. We show that homodimeric alpha chains of the CD8 molecule (CD8alphaalpha) are transiently induced on a selected subset of CD8alphabeta+ T cells upon antigenic stimulation. These CD8alphaalpha molecules promote the survival and differentiation of activated lymphocytes into memory CD8 T cells. Thus, memory precursors can be identified among primary effector cells and are selected for survival and differentiation by CD8alphaalpha
— id: 69523, year: 2004, vol: 304, page: 590, stat: Journal Article,

Protein kinase C theta is critical for the development of in vivo T helper (Th)2 cell but not Th1 cell responses
Marsland, Benjamin J; Soos, Timothy J; Spath, Gerald; Littman, Dan R; Kopf, Manfred
2004 Jul 19;200(2):181-189, Journal of experimental medicine
The serine/threonine-specific protein kinase C (PKC)-theta is predominantly expressed in T cells and localizes to the center of the immunological synapse upon T cell receptor (TCR) and CD28 signaling. T cells deficient in PKC-theta exhibit reduced interleukin (IL)-2 production and proliferative responses in vitro, however, its significance in vivo remains unclear. We found that pkc-theta(-/-) mice were protected from pulmonary allergic hypersensitivity responses such as airway hyperresponsiveness, eosinophilia, and immunoglobulin E production to inhaled allergen. Furthermore, T helper (Th)2 cell immune responses against Nippostrongylus brasiliensis were severely impaired in pkc-theta(-/-) mice. In striking contrast, pkc-theta(-/-) mice on both the C57BL/6 background and the normally susceptible BALB/c background mounted protective Th1 immune responses and were resistant against infection with Leishmania major. Using in vitro TCR transgenic T cell-dendritic cell coculture systems and antigen concentration-dependent Th polarization, PKC-theta-deficient T cells were found to differentiate into Th1 cells after activation with high concentrations of specific peptide, but to have compromised Th2 development at low antigen concentration. The addition of IL-2 partially reconstituted Th2 development in pkc-theta(-/-) T cells, consistent with an important role for this cytokine in Th2 polarization. Taken together, our results reveal a central role for PKC-theta signaling during Th2 responses
— id: 69520, year: 2004, vol: 200, page: 181, stat: Journal Article,

The role of CXCR4 in maintaining peripheral B cell compartments and humoral immunity
Nie, Yuchun; Waite, Janelle; Brewer, Faraha; Sunshine, Mary-Jean; Littman, Dan R; Zou, Yong-Rui
2004 Nov 1;200(9):1145-1156, Journal of experimental medicine
The chemokine receptor CXCR4 is expressed in B cells at multiple stages of their development. CXCR4 function in humoral immunity has not been fully investigated. We have generated gene-targeted mice in which CXCR4 can be selectively inactivated in B cells and have shown that it is required for retention of B cell precursors in the bone marrow. CXCR4-deficient B cell precursors that migrated prematurely became localized in splenic follicles despite their unresponsiveness to CXCL13. Concomitantly, mature B cell populations were reduced in the splenic marginal zone and primary follicles, and in the peritoneal cavity in the mutant animals, as were T-independent antibody responses. In addition, aberrant B cell follicles formed ectopically in intestinal lamina propria around Peyer's patches. These findings establish an important role for CXCR4 in regulating homeostasis of B cell compartmentalization and humoral immunity
— id: 69515, year: 2004, vol: 200, page: 1145, stat: Journal Article,

The CD4/CD8 lineage choice: new insights into epigenetic regulation during T cell development
Taniuchi, Ichiro; Ellmeier, Wilfried; Littman, Dan R
2004 ;83:55-89, Advances in immunology
— id: 69522, year: 2004, vol: 83, page: 55, stat: Journal Article,

Epigenetic gene silencing by Runx proteins
Taniuchi, Ichiro; Littman, Dan R
2004 May 24;23(24):4341-4345, Oncogene
Runx family proteins have the potential for either activating or suppressing gene expression in a context-dependent manner. There are several mechanisms by which transcriptional repression can occur. A wide range of locus inactivation, that is often called gene silencing, is thought to be achieved by chromatin modifications. Recently, Runx family proteins were found to have an essential role in either temporal transcriptional repression or irreversible epigenetic silencing at the CD4 locus through binding to a CD4 silencer at different stages of development. These findings link Runx function to epigenetic gene regulation, and shed new light on the mechanisms by which Runx represses target gene expression
— id: 69521, year: 2004, vol: 23, page: 4341, stat: Journal Article,

After Hrs with HIV
Amara, Ali; Littman, Dan R
2003 Aug 4;162(3):371-375, Journal of cell biology
To efficiently bud off from infected cells, HIV and other enveloped viruses hijack the host cellular machinery that is normally involved in vacuolar protein sorting and multivesicular body (MVB) biogenesis. The HIV Gag protein mimics hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a modular adaptor protein that links membrane cargo recognition to its degradation after delivery to MVBs. In contrast to T cells, where HIV budding occurs at the plasma membrane, virus buds into vacuoles of macrophages, a process that may facilitate its spread within the infected host
— id: 39117, year: 2003, vol: 162, page: 371, stat: Journal Article,

The chemokine stromal cell-derived factor-1 promotes the survival of embryonic retinal ganglion cells
Chalasani, Sreekanth H; Baribaud, Frederic; Coughlan, Christine M; Sunshine, Mary J; Lee, Virginia M Y; Doms, Robert W; Littman, Dan R; Raper, Jonathan A
2003 Jun 1;23(11):4601-4612, Journal of neuroscience
The chemokine receptor CXCR4 is expressed in the embryonic and mature CNS, yet its normal physiological function in neurons remains obscure. Here, we show that its cognate chemokine, stromal cell-derived factor-1 (SDF-1), promotes the survival of cultured embryonic retinal ganglion cell neurons even in the absence of other neurotrophic factors. This survival effect is mediated primarily through a cAMP-dependent pathway that acts through protein kinase A and MAP kinase. Addition of SDF-1 to a human neuronal cell line induces phosphorylation of p44/p42 MAP kinase and GSK3beta. Mouse embryos lacking the CXCR4 receptor have a reduced number of retinal ganglion cells. The ligand of CXCR4, SDF-1, may therefore provide generalized trophic support to neurons during their development and maturation
— id: 42236, year: 2003, vol: 23, page: 4601, stat: Journal Article,

A chemokine, SDF-1, reduces the effectiveness of multiple axonal repellents and is required for normal axon pathfinding
Chalasani, Sreekanth H; Sabelko, Kimberly A; Sunshine, Mary J; Littman, Dan R; Raper, Jonathan A
2003 Feb 15;23(4):1360-1371, Journal of neuroscience
Altering the concentrations of cyclic nucleotides within nerve cells can dramatically change their responses to axonal guidance cues, but the physiological signals that might induce such alterations are unknown. Here we show that the chemokine stromal cell-derived factor 1 (SDF-1) reduces the repellent activities of slit-2 on cultured retinal ganglion cell axons, of semaphorin 3A on dorsal root ganglion sensory axons, and of semaphorin 3C on sympathetic axons. This is a modulatory effect because SDF-1 has no detectable attractive or repellent effects on retinal or DRG axons by itself. This modulation is mediated through CXCR4, the receptor of SDF-1, and a pertussis toxin-sensitive G-protein-coupled signaling pathway that induces an elevation of cAMP. The spinal cords of CXCR4 mutant mice contain hyperfasciculated and aberrantly projecting axons. These results suggest that SDF-1 plays an essential role in modulating axonal responsiveness to various known guidance cues through a cyclic nucleotide-dependent signaling pathway
— id: 42237, year: 2003, vol: 23, page: 1360, stat: Journal Article,

The role of the nuclear hormone receptor RORgammat in the development of lymph nodes and Peyer's patches
Eberl, Gerard; Littman, Dan R
2003 Oct;195(19):81-90, Immunological reviews
The nuclear hormone receptor retinoic acid-related orphan receptor (ROR)gammat is required for the development of lymph nodes (LNs) and Peyer's patches (PPs), as these organs are absent in RORgammat-deficient mice. During fetal life, RORgammat is expressed exclusively in lymphoid tissue-inducer (LTi) cells, a cell type that localizes in developing LNs and PPs. LTi cells express surface lymphotoxin alpha1beta2 that activates specialized mesenchymal cells to produce chemokines, upregulate adhesion molecules and induce further maturation of lymphoid organs. RORgammat inhibits nuclear factor of activated T-cell (NFAT) function in cell lines and induces the expression of Bcl-xL and p27kip1 in the adult thymus, suggesting that RORgammat prevents cell activation, cell-cycle progression, and apoptosis. We propose that RORgammat, together with the inhibitor of basic helix-loop-helix transcription factor Id2, ensures generation and survival of fetal LTi cells necessary for the development of LNs and PPs
— id: 39077, year: 2003, vol: 195, page: 81, stat: Journal Article,

Requirement for CARMA1 in antigen receptor-induced NF-kappa B activation and lymphocyte proliferation
Egawa, Takeshi; Albrecht, Bjorn; Favier, Benoit; Sunshine, Mary-Jean; Mirchandani, Kanchan; O'Brien, William; Thome, Margot; Littman, Dan R
2003 Jul 15;13(14):1252-1258, Current biology. CB
Ligation of antigen receptors (TCR, BCR) on T and B lymphocytes leads to the activation of new transcriptional programs and cell cycle progression. Antigen receptor-mediated activation of NF-kappa B, required for proliferation of B and T cells, is disrupted in T cells lacking PKC theta and in B and T cells lacking Bcl10, a caspase recruitment domain (CARD)-containing adaptor protein. CARMA1 (also called CARD11 and Bimp3), the only lymphocyte-specific member in a family of membrane-associated guanylate kinase (MAGUK) scaffolding proteins that interact with Bcl10 by way of CARD-CARD interactions, is required for TCR-induced NF-kappa B activation in Jurkat T lymphoma cells. Here we show that T cells from mice lacking CARMA1 expression were defective in recruitment of Bcl10 to clustered TCR complexes and lipid rafts, in activation of NF-kappa B, and in induction of IL-2 production. Development of CD5(+) peritoneal B cells was disrupted in these mice, as was B cell proliferation in response to both BCR and CD40 ligation. Serum immunoglobulin levels were also markedly reduced in the mutant mice. Together, these results show that CARMA1 has a central role in antigen receptor signaling that results in activation and proliferation of both B and T lymphocytes
— id: 39138, year: 2003, vol: 13, page: 1252, stat: Journal Article,

Blood monocytes consist of two principal subsets with distinct migratory properties
Geissmann, Frederic; Jung, Steffen; Littman, Dan R
2003 Jul;19(1):71-82, Immunity
Peripheral blood monocytes are a heterogeneous population of circulating leukocytes. Using a murine adoptive transfer system to probe monocyte homing and differentiation in vivo, we identified two functional subsets among murine blood monocytes: a short-lived CX(3)CR1(lo)CCR2(+)Gr1(+) subset that is actively recruited to inflamed tissues and a CX(3)CR1(hi)CCR2(-)Gr1(-) subset characterized by CX(3)CR1-dependent recruitment to noninflamed tissues. Both subsets have the potential to differentiate into dendritic cells in vivo. The level of CX(3)CR1 expression also defines the two major human monocyte subsets, the CD14(+)CD16(-) and CD14(lo)CD16(+) monocytes, which share phenotype and homing potential with the mouse subsets. These findings raise the potential for novel therapeutic strategies in inflammatory diseases
— id: 39135, year: 2003, vol: 19, page: 71, stat: Journal Article,

Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E
Huo, Yuqing; Schober, Andreas; Forlow, S Bradley; Smith, David F; Hyman, Matthew Craig; Jung, Steffen; Littman, Dan R; Weber, Christian; Ley, Klaus
2003 Jan;9(1):61-67, Nature medicine
We studied whether circulating activated platelets and platelet-leukocyte aggregates cause the development of atherosclerotic lesions in apolipoprotein-E-deficient (Apoe(-/-)) mice. Circulating activated platelets bound to leukocytes, preferentially monocytes, to form platelet-monocyte/leukocyte aggregates. Activated platelets and platelet-leukocyte aggregates interacted with atherosclerotic lesions. The interactions of activated platelets with monocytes and atherosclerotic arteries led to delivery of the platelet-derived chemokines CCL5 (regulated on activation, normal T cell expressed and secreted, RANTES) and CXCL4 (platelet factor 4) to the monocyte surface and endothelium of atherosclerotic arteries. The presence of activated platelets promoted leukocyte binding of vascular cell adhesion molecule-1 (VCAM-1) and increased their adhesiveness to inflamed or atherosclerotic endothelium. Injection of activated wild-type, but not P-selectin-deficient, platelets increased monocyte arrest on the surface of atherosclerotic lesions and the size of atherosclerotic lesions in Apoe(-/-) mice. Our results indicate that circulating activated platelets and platelet-leukocyte/monocyte aggregates promote formation of atherosclerotic lesions. This role of activated platelets in atherosclerosis is attributed to platelet P-selectin-mediated delivery of platelet-derived proinflammatory factors to monocytes/leukocytes and the vessel wall
— id: 42238, year: 2003, vol: 9, page: 61, stat: Journal Article,

The chemokine SDF1/CXCL12 and its receptor CXCR4 regulate mouse germ cell migration and survival
Molyneaux, Kathleen A; Zinszner, Helene; Kunwar, Prabhat S; Schaible, Kyle; Stebler, Jurg; Sunshine, Mary Jean; O'Brien, William; Raz, Erez; Littman, Dan; Wylie, Chris; Lehmann, Ruth
2003 Sep;130(18):4279-4286, Development
In mouse embryos, germ cells arise during gastrulation and migrate to the early gonad. First, they emerge from the primitive streak into the region of the endoderm that forms the hindgut. Later in development, a second phase of migration takes place in which they migrate out of the gut to the genital ridges. There, they co-assemble with somatic cells to form the gonad. In vitro studies in the mouse, and genetic studies in other organisms, suggest that at least part of this process is in response to secreted signals from other tissues. Recent genetic evidence in zebrafish has shown that the interaction between stromal cell-derived factor 1 (SDF1) and its G-protein-coupled receptor CXCR4, already known to control many types of normal and pathological cell migrations, is also required for the normal migration of primordial germ cells. We show that in the mouse, germ cell migration and survival requires the SDF1/CXCR4 interaction. First, migrating germ cells express CXCR4, whilst the body wall mesenchyme and genital ridges express the ligand SDF1. Second, the addition of exogenous SDF1 to living embryo cultures causes aberrant germ cell migration from the gut. Third, germ cells in embryos carrying targeted mutations in CXCR4 do not colonize the gonad normally. However, at earlier stages in the hindgut, germ cells are unaffected in CXCR4(-/-) embryos. Germ cell counts at different stages suggest that SDF1/CXCR4 interaction also mediates germ cell survival. These results show that the SDF1/CXCR4 interaction is specifically required for the colonization of the gonads by primordial germ cells, but not for earlier stages in germ cell migration. This demonstrates a high degree of evolutionary conservation of part of the mechanism, but also an area of evolutionary divergence
— id: 52649, year: 2003, vol: 130, page: 4279, stat: Journal Article,

Intestinal antigen sampling dendritic cells are characterized by the expression of the fractalkine receptor CX3CR1
Niess, JH; Brand, S; Gu, XB; Jung, S; Littman, DR; Reinecker, HC
2003 APR 14 ;17(7):C231-C231, FASEB journal
— id: 37134, year: 2003, vol: 17, page: C231, stat: Journal Article,

A critical role for CXCR4/CCL12 signaling in progenitor localization and differentiation in the post-natal thymus
Petrie, HT; Plotkin, J; Prockop, SE; Lepique, AP; Zou, YR; Littman, DR
2003 APR 14 ;17(7):C65-C65, FASEB journal
— id: 37132, year: 2003, vol: 17, page: C65, stat: Journal Article,

Protein kinase C-theta;: signaling from the center of the T-cell synapse
Arendt, Christopher W; Albrecht, Bjorn; Soos, Timothy J; Littman, Dan R
2002 Jun;14(3):323-330, Current opinion in immunology
The hypothesis that protein kinase C (PKC)-theta; plays an important role in T-lymphocyte activation, as indicated by numerous studies in cell lines, was recently confirmed in mice deficient in the expression of this enzyme. In response to TCR stimulation, peripheral T cells lacking PKC-theta; failed to activate NF-kappaB and AP-1, and to express IL-2. This revealed a critical function for this PKC family member in linking membrane-proximal activation cascades to transcriptional responses governing T-cell activation. Although the molecular interactions in which PKC-theta; engages have not been fully delineated, insights from a variety of recent studies have permitted new models to be formulated regarding the mechanisms through which it achieves its unique effector functions
— id: 32454, year: 2002, vol: 14, page: 323, stat: Journal Article,

The chemokine SDF1 regulates migration of dentate granule cells
Bagri, Anil; Gurney, Theresa; He, Xiaoping; Zou, Yong-Rui; Littman, Dan R; Tessier-Lavigne, Marc; Pleasure, Samuel J
2002 Sep;129(18):4249-4260, Development
The dentate gyrus is the primary afferent pathway into the hippocampus, but there is little information concerning the molecular influences that govern its formation. In particular, the control of migration and cell positioning of dentate granule cells is not clear. We have characterized more fully the timing and route of granule cell migration during embryogenesis using in utero retroviral injections. Using this information, we developed an in vitro assay that faithfully recapitulates important events in dentate gyrus morphogenesis. In searching for candidate ligands that may regulate dentate granule cell migration, we found that SDF1, a chemokine that regulates cerebellar and leukocyte migration, and its receptor CXCR4 are expressed in patterns that suggest a role in dentate granule cell migration. Furthermore, CXCR4 mutant mice have a defect in granule cell position. Ectopic expression of SDF1 in our explant assay showed that it directly regulates dentate granule cell migration. Our study shows that a chemokine is necessary for the normal development of the dentate gyrus, a forebrain structure crucial for learning and memory
— id: 69525, year: 2002, vol: 129, page: 4249, stat: Journal Article,

Reciprocal regulation of CD4/CD8 expression by SWI/SNF-like BAF complexes
Chi, Tian H; Wan, Mimi; Zhao, Keji; Taniuchi, Ichiro; Chen, Lei; Littman, Dan R; Crabtree, Gerald R
2002 Jul 11;418(6894):195-199, Nature
Thymic development produces two sub-lineages of T cells expressing either CD4 or CD8 co-receptors that assist antibody production and mediate cell killing, respectively. The mechanisms for mutually exclusive co-receptor expression remain poorly defined. We find that mutations in the high mobility group (HMG) domain of BAF57--a DNA-binding subunit of the mammalian SWI/SNF-like chromatin-remodelling BAF complexes--or in the BAF complex ATPase subunit Brg, impair both CD4 silencing and CD8 activation. Brg is haploinsufficient for CD8 activation, but not for CD4 silencing, whereas BAF57 mutations preferentially impair CD4 silencing, pointing to target- and subunit-specific mechanisms of chromatin remodelling. BAF complexes directly bind the CD4 silencer, but the BAF57 HMG domain is dispensable for tethering BAF complexes to the CD4 silencer or other chromatin loci in vivo, or for remodelling reconstituted templates in vitro, suggesting that chromatin remodelling in vivo requires HMG-dependent DNA bending. These results indicate that BAF complexes contribute to lineage bifurcation by reciprocally regulating lineage-specific genes, reminiscent of the role of the yeast SWI/SNF complex in mediating mating-type switching
— id: 69526, year: 2002, vol: 418, page: 195, stat: Journal Article,

Combined deletion of CD8 locus cis-regulatory elements affects initiation but not maintenance of CD8 expression
Ellmeier, Wilfried; Sunshine, Mary Jean; Maschek, Romana; Littman, Dan R
2002 May;16(5):623-634, Immunity
Developmental stage-, subset-, and lineage-specific CD8 enhancers have been identified recently by transgenic reporter analyses. Enhancer E8(II) (CIV-4,5) is active in both immature double-positive thymocytes (DP) and mature CD8 single-positive (SP) thymocytes and T cells, whereas E8(I) (CIII-1,2) directs expression only in mature cells. In mice lacking either E8(I) (CIII-1,2) or E8(II) (CIV-4,5), there was no effect on CD8 expression in DP thymocytes. However, deletion of both enhancers resulted in variegated expression of CD8, with appearance of CD4(+)CD8(-) SP thymocytes expressing surface markers characteristic of DP thymocytes. Consequently, fewer mature CD8(+) T cells developed from the reduced pool of DP cells. These results suggest that the initiation of CD8 expression is mediated by cis-regulatory elements that are distinct from any that may be involved in maintenance of expression
— id: 69528, year: 2002, vol: 16, page: 623, stat: Journal Article,

Regulation of the TCRalpha repertoire by the survival window of CD4(+)CD8(+) thymocytes
Guo, Jian; Hawwari, Abbas; Li, Hong; Sun, Zuoming; Mahanta, Sanjeev K; Littman, Dan R; Krangel, Michael S; He, You-Wen
2002 May;3(5):469-476, Nature immunology
T cell receptor (TCR) alpha alleles undergo primary and secondary rearrangement in double-positive (DP) thymocytes. By analyzing TCRalpha rearrangement in orphan nuclear receptor RORgamma-deficient mice, in which the DP lifespan is shorter, and in Bcl-x(L)-transgenic mice, in which the DP lifespan is extended, we show that the progression of secondary V(alpha) to J(alpha) rearrangements is controlled by DP thymocyte survival. In addition, because Bcl-x(L) induces a bias towards 3' J(alpha) usage in peripheral T cells, we conclude that the programmed cell death of DP thymocytes is not simply a consequence of failed positive selection. Rather, it limits the progression of rearrangement along the J(alpha) locus and the opportunities for positive selection, thereby regulating the TCRalpha repertoire
— id: 69530, year: 2002, vol: 3, page: 469, stat: Journal Article,

Cutting edge: organogenesis of nasal-associated lymphoid tissue (NALT) occurs independently of lymphotoxin-alpha (LT alpha) and retinoic acid receptor-related orphan receptor-gamma, but the organization of NALT is LT alpha dependent
Harmsen, Allen; Kusser, Kimberley; Hartson, Louise; Tighe, Michael; Sunshine, Mary Jean; Sedgwick, Jonathon D; Choi, Yongwon; Littman, Dan R; Randall, Troy D
2002 Feb 1;168(3):986-990, Journal of immunology
Peyer's patch and nasal-associated lymphoid tissue (NALT) are mucosal lymphoid tissues that appear similar in structure and function. Surprisingly, we found that NALT, unlike Peyer's patch, was formed independently of lymphotoxin (LT)alpha. Furthermore, using mice deficient in the retinoic acid receptor-related orphan receptor-gamma, we found that NALT was formed in the absence of CD4+CD3- cells, which are thought to be the embryonic source of LTalpha. However, we also found that NALT of LTalpha-/- animals was disorganized and lymphopenic, suggesting that the organization and recruitment of lymphocytes within NALT remained dependent on LTalpha. Finally, we demonstrated that both the structure and function of NALT were restored in LTalpha-/- animals upon reconstitution with normal bone marrow. These results demonstrate that the organogenesis of NALT occurs through unique mechanisms
— id: 69532, year: 2002, vol: 168, page: 986, stat: Journal Article,

In Vivo Depletion of CD11c(+) Dendritic Cells Abrogates Priming of CD8(+) T Cells by Exogenous Cell-Associated Antigens
Jung, Steffen; Unutmaz, Derya; Wong, Phillip; Sano, Gen-Ichiro; De los Santos, Kenia; Sparwasser, Tim; Wu, Shengji; Vuthoori, Sri; Ko, Kyung; Zavala, Fidel; Pamer, Eric G; Littman, Dan R; Lang, Richard A
2002 Aug;17(2):211-211, Immunity
Cytotoxic T lymphocytes (CTL) respond to antigenic peptides presented on MHC class I molecules. On most cells, these peptides are exclusively of endogenous, cytosolic origin. Bone marrow-derived antigen-presenting cells, however, harbor a unique pathway for MHC I presentation of exogenous antigens. This mechanism permits cross-presentation of pathogen-infected cells and the priming of CTL responses against intracellular microbial infections. Here, we report a novel diphtheria toxin-based system that allows the inducible, short-term ablation of dendritic cells (DC) in vivo. We show that in vivo DC are required to cross-prime CTL precursors. Our results thus define a unique in vivo role of DC, i.e., the sensitization of the immune system for cell-associated antigens. DC-depleted mice fail to mount CTL responses to infection with the intracellular bacterium Listeria monocytogenes and the rodent malaria parasite Plasmodium yoelii
— id: 32272, year: 2002, vol: 17, page: 211, stat: Journal Article,

Progress toward a human CD4/CCR5 transgenic rat model for de novo infection by human immunodeficiency virus type 1
Keppler, Oliver T; Welte, Frank J; Ngo, Tuan A; Chin, Peggy S; Patton, Kathryn S; Tsou, Chia-Lin; Abbey, Nancy W; Sharkey, Mark E; Grant, Robert M; You, Yun; Scarborough, John D; Ellmeier, Wilfried; Littman, Dan R; Stevenson, Mario; Charo, Israel F; Herndier, Brian G; Speck, Roberto F; Goldsmith, Mark A
2002 Mar 18;195(6):719-736, Journal of experimental medicine
The development of a permissive small animal model for the study of human immunodeficiency virus type (HIV)-1 pathogenesis and the testing of antiviral strategies has been hampered by the inability of HIV-1 to infect primary rodent cells productively. In this study, we explored transgenic rats expressing the HIV-1 receptor complex as a susceptible host. Rats transgenic for human CD4 (hCD4) and the human chemokine receptor CCR5 (hCCR5) were generated that express the transgenes in CD4(+) T lymphocytes, macrophages, and microglia. In ex vivo cultures, CD4(+) T lymphocytes, macrophages, and microglia from hCD4/hCCR5 transgenic rats were highly susceptible to infection by HIV-1 R5 viruses leading to expression of abundant levels of early HIV-1 gene products comparable to those found in human reference cultures. Primary rat macrophages and microglia, but not lymphocytes, from double-transgenic rats could be productively infected by various recombinant and primary R5 strains of HIV-1. Moreover, after systemic challenge with HIV-1, lymphatic organs from hCD4/hCCR5 transgenic rats contained episomal 2-long terminal repeat (LTR) circles, integrated provirus, and early viral gene products, demonstrating susceptibility to HIV-1 in vivo. Transgenic rats also displayed a low-level plasma viremia early in infection. Thus, transgenic rats expressing the appropriate human receptor complex are promising candidates for a small animal model of HIV-1 infection
— id: 69531, year: 2002, vol: 195, page: 719, stat: Journal Article,

DC-SIGN-mediated internalization of HIV is required for trans-enhancement of T cell infection
Kwon, Douglas S; Gregorio, Glenn; Bitton, Natacha; Hendrickson, Wayne A; Littman, Dan R
2002 Jan;16(1):135-144, Immunity
Fusion of the human immunodeficiency virus (HIV) to the plasma membrane of target cells is mediated by interaction of its envelope glycoprotein, gp120, with CD4 and appropriate chemokine receptors. gp120 additionally binds to DC-SIGN, a C-type lectin expressed on immature dendritic cells. This interaction does not result in viral fusion, but instead contributes to enhanced infection in trans of target cells that express CD4 and chemokine receptors. Here we show that DC-SIGN mediates rapid internalization of intact HIV into a low pH nonlysosomal compartment. Internalized virus retains competence to infect target cells. Removal of the DC-SIGN cytoplasmic tail reduced viral uptake and abrogated the trans-enhancement of T cell infection. We propose that HIV binds to DC-SIGN to gain access to an intracellular compartment that contributes to augmentation or retention of viral infectivity
— id: 39719, year: 2002, vol: 16, page: 135, stat: Journal Article,

Epigenetic Regulation in T Cell Development
Littman, Dan
[S.l.] : NIH, 2002,
The majority of mature T lymphocytes fall into one of two functional categories: helper cells, with T cell antigen receptors specific for peptides complexed to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells, and cytotoxic cells, which recognize peptides bound to MHC class I molecules on target cells
— id: 1424, year: 2002, vol: , page: , stat: ,

Generation and characterization of ecto-ADP-ribosyltransferase ART2.1/ART2.2-deficient mice
Ohlrogge, Wiebke; Haag, Friedrich; Lohler, Jurgen; Seman, Michel; Littman, Dan R; Killeen, Nigel; Koch-Nolte, Friedrich
2002 Nov;22(21):7535-7542, Molecular & cellular biology
This is the first study reporting the inactivation of a member of the mouse gene family of toxin-related ecto-ADP-ribosyltransferases (ARTs). Transfer of the ADP-ribose moiety from NAD onto extracellular arginine residues on T-cell membrane proteins is mediated by glycosylphosphatidylinositol-linked cell surface ARTs. Exposure of T cells to ecto-NAD blocks T-cell activation and induces T-cell apoptosis. To determine a possible role of ecto-ART2.1 and ART2.2 in these processes, we generated ART2.1/ART2.2 double-knockout mice. ART2-deficient mice were healthy and fertile and showed normal development of lymphoid organs. ART2-deficient T cells showed a dramatically reduced capacity to ADP-ribosylate cell surface proteins, indicating that most if not all ART activity on the T-cell surface can be attributed to the ART2s. Moreover, ART2-deficient T cells were completely resistant to NAD-induced apoptosis and partially resistant to NAD-mediated suppression of proliferation. These results demonstrate that the ART2 ectoenzymes are an essential component in the regulation of T-cell functions by extracellular NAD, e.g., following release of NAD upon lysis of cells in tissue injury and inflammation
— id: 69524, year: 2002, vol: 22, page: 7535, stat: Journal Article,

Chemokine requirements for B cell entry to lymph nodes and Peyer's patches
Okada, Takaharu; Ngo, Vu N; Ekland, Eric H; Forster, Reinhold; Lipp, Martin; Littman, Dan R; Cyster, Jason G
2002 Jul 1;196(1):65-75, Journal of experimental medicine
B cell entry to lymph nodes and Peyer's patches depends on chemokine receptor signaling, but the principal chemokine involved has not been defined. Here we show that the homing of CXCR4-/- B cells is suppressed in CCL19 (ELC)- and CCL21 (SLC)-deficient paucity of lymph node T cells mice, but not in wild-type mice. We also find that CXCR4 can contribute to T cell homing. Using intravital microscopy, we find that B cell adhesion to high endothelial venules (HEVs) is disrupted when CCR7 and CXCR4 are predesensitized. In Peyer's patches, B cell entry is dependent on CXCR5 in addition to CCR7/CXCR4. CXCL12 (SDF1) is displayed broadly on HEVs, whereas CXCL13 (BLC) is found selectively on Peyer's patch follicular HEVs. These findings establish the principal chemokine and chemokine receptor requirements for B cell entry to lymph nodes and Peyer's patches
— id: 69527, year: 2002, vol: 196, page: 65, stat: Journal Article,

Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development
Taniuchi, Ichiro; Osato, Motomi; Egawa, Takeshi; Sunshine, Mary Jean; Bae, Suk Chul; Komori, Toshihisa; Ito, Yoshiaki; Littman, Dan R
2002 Nov 27;111(5):621-633, Cell
T lymphocytes differentiate in discrete stages within the thymus. Immature thymocytes lacking CD4 and CD8 coreceptors differentiate into double-positive cells (CD4(+)CD8(+)), which are selected to become either CD4(+)CD8(-)helper cells or CD4(-)CD8(+) cytotoxic cells. A stage-specific transcriptional silencer regulates expression of CD4 in both immature and CD4(-)CD8(+) thymocytes. We show here that binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and that different Runx family members are required to fulfill unique functions at each stage. Runx1 is required for active repression in CD4(-)CD8(-) thymocytes whereas Runx3 is required for establishing epigenetic silencing in cytotoxic lineage thymocytes. Runx3-deficient cytotoxic T cells, but not helper cells, have defective responses to antigen, suggesting that Runx proteins have critical functions in lineage specification and homeostasis of CD8-lineage T lymphocytes
— id: 39359, year: 2002, vol: 111, page: 621, stat: Journal Article,

Evidence for distinct CD4 silencer functions at different stages of thymocyte differentiation
Taniuchi, Ichiro; Sunshine, Mary Jean; Festenstein, Richard; Littman, Dan R
2002 Nov;10(5):1083-1096, Molecular cell
An intronic silencer within the CD4 gene is the critical cis regulatory element for T cell subset-specific expression of CD4. We have combined transfection studies with gene targeting in mice to identify several key sequences within the silencer core that are required for gene silencing during thymocyte development. In mice, mutations in individual sites resulted in variegated, but heritable, derepression of CD4 in mature CD8(+) T lymphocytes, whereas compound mutations resulted in full derepression. These results indicate that there is partial redundancy in recruiting a chromatin remodeling machinery that results in epigenetic silencing. Mutations in single sites also resulted in partial derepression of CD4 in immature double-negative thymocytes, but there was no apparent variegation. These findings suggest two distinct modes of CD4 silencer function at different developmental stages: active repression in CD4(-)CD8(-) thymocytes, in which silencing must be reversible, and epigenetic gene silencing upon differentiation to the CD8(+) cytotoxic T cell lineage
— id: 39365, year: 2002, vol: 10, page: 1083, stat: Journal Article,

Nuclear hormone receptors in T lymphocytes
Winoto, Astar; Littman, Dan R
2002 Apr;109 Suppl:S57-S66, Cell
Among the numerous steroid and orphan nuclear receptors encoded within mammalian genomes, several are involved in regulating immune system functions. We review here recent studies on the glucocorticoid receptor and the orphan receptors Nur77 and RORgamma. These molecules play key roles in the development and the effector functions of T lymphocytes
— id: 69529, year: 2002, vol: 109 Suppl, page: S57, stat: Journal Article,

HIV: master of the host cell
Arendt CW; Littman DR
2001 ;2(11):1030-1030, Genome biology
The human immunodeficiency virus has evolved various mechanisms to exploit its host cells, including the interruption and augmentation of signal transduction pathways. Recently, two DNA microarray studies have illustrated a remarkably broad-based perturbation in host transcriptional responses, which is in part mediated by the HIV-encoded Nef protein. HIV therefore seems to function as a 'master regulator' of cellular gene expression
— id: 26504, year: 2001, vol: 2, page: 1030, stat: Journal Article,

Functional and antigenic characterization of human, rhesus macaque, pigtailed macaque, and murine DC-SIGN
Baribaud, FD; Pohlmann, S; Sparwasser, T; Kimata, MTY; Choi, YK; Haggarty, BS; Ahmad, N; MacFarlan, A; Edwards, TG; Leslie, GJ; Arnason, J; Reinhart, TA; Kimata, JT; Littman, DR; Hoxie, JA; Doms, RW
2001 NOV ;75(21):10281-10289, Journal of virology
DC-SIGN, a type II membrane protein with a C-type lectin binding domain that is highly expressed on mucosal dendritic cells (DCs) and certain macrophages in vivo, binds to ICAM-3, ICAM-2, and human and simian immunodeficiency viruses (HIV and SIV). Virus captured by DC-SIGN can be presented to T cells, resulting in efficient virus infection, perhaps representing a mechanism by which virus can be ferried via normal DC trafficking from mucosal tissues to lymphoid organs in vivo. To develop reagents needed to characterize the expression and in vivo functions of DC-SIGN, we cloned, expressed, and analyzed rhesus macaque, pigtailed macaque, and murine DC-SIGN and made a panel of monoclonal antibodies (MAbs) to human DC-SIGN. Rhesus and pigtailed macaque DC-SIGN proteins were highly similar to human DC-SIGN and bound and transmitted HIV type 1 (HIV-1), HIV-2, and SIV to receptor-positive cells. In contrast, while competent to bind virus, murine DC-SIGN did not transmit virus to receptor-positive cells under the conditions tested. Thus, mere binding of virus to a C-type lectin does not necessarily mean that transmission will occur. The murine and macaque DC-SIGN molecules all bound ICAM-3. We mapped the determinants recognized by a panel of 16 MAbs to the repeat region, the lectin binding domain, and the extreme C terminus of DC-SIGN. One MAb was specific for DC-SIGN, failing to cross-react with DC-SIGNR. Most MAbs crossreacted with rhesus and pigtailed macaque DC-SIGN, although none recognized murine DC-SIGN. Fifteen of the MAbs recognized DC-SIGN on DCs, with MAbs to the repeat region generally reacting most strongly. We conclude that rhesus and pigtailed macaque DC-SIGN proteins are structurally and functionally similar to human DC-SIGN and that the reagents that we have developed will make it possible to study the expression and function of this molecule in vivo
— id: 54882, year: 2001, vol: 75, page: 10281, stat: Journal Article,

Human GLI-2 is a Tat activation response element-independent Tat cofactor
Browning, CM; Smith, MJ; Clark, NM; Lane, BR; Parada, C; Montano, M; Kewalramani, VN; Littman, DR; Essex, M; Roeder, RG; Markovitz, DM
2001 MAR ;75(5):2314-2323, Journal of virology
Zinc finger-containing GLI proteins are involved in the development of Caenorhabditis elegans, Xenopus, Drosophila, zebrafish, mice, and humans. In this study, we show that an isoform of human GLI-2 strongly synergizes with the Tat transactivating proteins of human immunodeficiency virus types 1 and 2 (HIV-1 and -2) and markedly stimulates viral replication. GLI-2 also synergizes with the previously described Tat cofactor cyclin T1 to stimulate Tat function. Surprisingly, GLI-2/Tat synergy is not dependent on either a typical GLI DNA binding site or an intact Tat activation response element but does require an intact TATA box. Thus, GLI-2/Tat synergy results from a mechanism of action which is novel both for a GLI protein and for a Tat cofactor. These findings link the GLI family of transcriptional and developmental regulatory proteins to Tat function and HIV replication
— id: 55202, year: 2001, vol: 75, page: 2314, stat: Journal Article,

A coordinated change in chemokine responsiveness guides plasma cell movements
Hargreaves, DC; Hyman, PL; Lu, TT; Ngo, VN; Bidgol, A; Suzuki, G; Zou, YR; Littman, DR; Cyster, JG
2001 JUL 2 ;194(1):45-56, Journal of experimental medicine
Antibody-secreting plasma cells are nonrecirculatory and lodge in splenic red pulp, lymph node medullary cords, and bone marrow. The factors that regulate plasma cell localization are poorly defined. Here we demonstrate that, compared with their B cell precursors, plasma cells exhibit increased chemotactic sensitivity to the CXCR4 ligand CXCL12. At the same time, they downregulate CXCR5 and CCR7 and have reduced responsiveness to the B and T zone chemokines CXCL13, CCL19, and CCL21. We demonstrate that CXCL12 is expressed within splenic red pulp and lymph node medullary cords as well as in bone marrow. In chimeric mice reconstituted with CXCR4-deficient fetal liver cells, plasma cells are mislocalized in the spleen, found in elevated numbers in blood, and fail to accumulate normally in the bone marrow. Our findings indicate that as B cells differentiate into plasma cells they undergo a coordinated change in chemokine responsiveness that regulates their movements in secondary lymphoid organs and promotes lodgment within the bone marrow
— id: 55012, year: 2001, vol: 194, page: 45, stat: Journal Article,

Control of interneuron fate in the developing spinal cord by the progenitor homeodomain protein Dbx1
Pierani, A; Moran-Rivard, L; Sunshine, M J; Littman, D R; Goulding, M; Jessell, T M
2001 Feb;29(2):367-384, Neuron
Spinal interneurons help to coordinate motor behavior. During spinal cord development, distinct classes of interneurons are generated from progenitor cells located at different positions within the ventral neural tube. V0 and V1 interneurons derive from adjacent progenitor domains that are distinguished by expression of the homeodomain proteins Dbx1 and Dbx2. The spatially restricted expression of Dbx1 has a critical role in establishing the distinction in V0 and V1 neuronal fate. In Dbx1 mutant mice, neural progenitors fail to generate V0 neurons and instead give rise to interneurons that express many characteristics of V1 neurons-their transcription factor profile, neurotransmitter phenotype, migratory pattern, and aspects of their axonal trajectory. Thus, a single progenitor homeodomain transcription factor coordinates many of the differentiated properties of one class of interneurons generated in the ventral spinal cord
— id: 104486, year: 2001, vol: 29, page: 367, stat: Journal Article,

Inactivation of Notch I in immature thymocytes does not perturb CD4 or CD8T cell development
Wolfer, A; Bakker, T; Wilson, A; Nicolas, M; Ioannidis, V; Littman, DR; Wilson, CB; Held, W; MacDonald, HR; Radtke, F
2001 MAR ;2(3):235-241, Nature immunology
Notch proteins influence cell-fate decisions in many developing systems. Several gain-of-function studies have suggested a critical role for Notch1 signaling in CD4-CD8 lineage commitment, maturation and survival in the thymus, However, we show here that tissue-specific inactivation of the gene encoding Notch1 in immature (CD25(+)CD44(-))T cell precursors does not affect subsequent thymocyte development, Neither steady-state numbers nor the rate of production of CD4(+) and CD8(+) mature thymocytes is perturbed in the absence of Notch1, In addition, Notch1-deficient thymocytes are normally sensitive to spontaneous or glucocorticoid-induced apoptosis, In contrast to earlier reports, these data formally exclude an essential role for Notch1 in CD4-CD8 lineage commitment, maturation or survival
— id: 55143, year: 2001, vol: 2, page: 235, stat: Journal Article,

Epigenetic silencing of CD4 in T cells committed to the cytotoxic lineage
Zou YR; Sunshine MJ; Taniuchi I; Hatam F; Killeen N; Littman DR
2001 Nov;29(3):332-336, Nature genetics
The process of thymocyte development culminates in the maturation of helper (CD4+) and cytotoxic (CD8+) T cells from their common precursors, the CD4+CD8+ double-positive cells. A crucial step during lineage specification is the termination of expression of either the CD4 or the CD8 coreceptor. A silencer element within the first intron of the CD4 gene is sufficient for CD4 transcriptional repression in cells of the cytotoxic lineage, as well as in thymocytes at earlier stages of differentiation. Here we show that the function of the CD4 silencer is required only at distinct stages of development. Its deletion before the initiation of lineage specification resulted in CD4 derepression throughout thymocyte differentiation. By contrast, once cells committed to the cytotoxic CD8+ lineage, the CD4 locus remained silent through subsequent mitoses, even when the silencer element was excised. The epigenetic inheritance of the silenced CD4 locus was not affected by the inhibition of DNA methylation or histone deacetylation, and may thus involve other mechanisms that ensure a stable state of gene expression
— id: 26588, year: 2001, vol: 29, page: 332, stat: Journal Article,

Severe B cell deficiency in mice lacking the tec kinase family members tec and Btk [In Process Citation]
Ellmeier W; Jung S; Sunshine MJ; Hatam F; Xu Y; Baltimore D; Mano H; Littman DR
2000 Dec 4;192(11):1611-1624, Journal of experimental medicine
The cytoplasmic protein tyrosine kinase Tec has been proposed to have important functions in hematopoiesis and lymphocyte signal transduction. Here we show that Tec-deficient mice developed normally and had no major phenotypic alterations of the immune system. To reveal potential compensatory roles of other Tec kinases such as Bruton's tyrosine kinase (Btk), Tec/Btk double-deficient mice were generated. These mice exhibited a block at the B220(+)CD43(+) stage of B cell development and displayed a severe reduction of peripheral B cell numbers, particularly immunoglobulin (Ig)M(lo)IgD(hi) B cells. Although Tec/Btk(null) mice were able to form germinal centers, the response to T cell-dependent antigens was impaired. Thus, Tec and Btk together have an important role both during B cell development and in the generation and/or function of the peripheral B cell pool. The ability of Tec to compensate for Btk may also explain phenotypic differences in X-linked immunodeficiency (xid) mice compared with human X-linked agammaglobulinemia (XLA) patients
— id: 15113, year: 2000, vol: 192, page: 1611, stat: Journal Article,

Severe B cell-deficiency in mice lacking the Tec kinase family members Btk and Tec
Ellmeier, W; Sunshine, M J; Jung, S; Xu, Y; Baltimore, D; Mano, H; Littman, D R
2000 Sept 23-26;73(2-3):131-131, Immunology letters
— id: 15800, year: 2000, vol: 73, page: 131, stat: Journal Article,

DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells [see comments]
Geijtenbeek TB; Kwon DS; Torensma R; van Vliet SJ; van Duijnhoven GC; Middel J; Cornelissen IL; Nottet HS; KewalRamani VN; Littman DR; Figdor CG; van Kooyk Y
2000 Mar 3;100(5):587-597, Cell
Dendritic cells (DC) capture microorganisms that enter peripheral mucosal tissues and then migrate to secondary lymphoid organs, where they present these in antigenic form to resting T cells and thus initiate adaptive immune responses. Here, we describe the properties of a DC-specific C-type lectin, DC-SIGN, that is highly expressed on DC present in mucosal tissues and binds to the HIV-1 envelope glycoprotein gp120. DC-SIGN does not function as a receptor for viral entry into DC but instead promotes efficient infection in trans of cells that express CD4 and chemokine receptors. We propose that DC-SIGN efficiently captures HIV-1 in the periphery and facilitates its transport to secondary lymphoid organs rich in T cells, to enhance infection in trans of these target cells
— id: 8528, year: 2000, vol: 100, page: 587, stat: Journal Article,

Apoptotic signaling through the beta -adrenergic receptor. A new Gs effector pathway
Gu C; Ma YC; Benjamin J; Littman D; Chao MV; Huang XY
2000 Jul 7;275(27):20726-20733, Journal of biological chemistry
Stimulation of beta-adrenergic receptor normally results in signaling by the heterotrimeric G protein G(s), leading to the activation of adenylyl cyclase, production of cAMP, and activation of cAMP-dependent protein kinase (PKA). Here we report that cell death of thymocytes can be induced after stimulation of beta-adrenergic receptor, or by addition of exogenous cAMP. Apoptotic cell death in both cases was observed with the appearance of terminal deoxynucleotidyl transferase-mediated UTP end labeling reactivity and the activation of caspase-3 in S49 T cells. Using thymocytes deficient in either Galpha(s) or PKA, we find that engagement of beta-adrenergic receptors initiated a Galpha(s)-dependent, PKA-independent pathway leading to apoptosis. This alternative pathway involves Src family tyrosine kinase Lck. Furthermore, we show that Lck protein kinase activity can be directly stimulated by purified Galpha(s). Our data reveal a new signaling pathway for Galpha(s), distinct from the classical PKA pathway, that accounts for the apoptotic action of beta-adrenergic receptors
— id: 14641, year: 2000, vol: 275, page: 20726, stat: Journal Article,

Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion
Jung S; Aliberti J; Graemmel P; Sunshine MJ; Kreutzberg GW; Sher A; Littman DR
2000 Jun;20(11):4106-4114, Molecular & cellular biology
The seven-transmembrane receptor CX(3)CR1 is a specific receptor for the novel CX(3)C chemokine fractalkine (FKN) (neurotactin). In vitro data suggest that membrane anchoring of FKN, and the existence of a shed, soluble FKN isoform allow for both adhesive and chemoattractive properties. Expression on activated endothelium and neurons defines FKN as a potential target for therapeutic intervention in inflammatory conditions, particularly central nervous system diseases. To investigate the physiological function of CX(3)CR1-FKN interactions, we generated a mouse strain in which the CX(3)CR1 gene was replaced by a green fluorescent protein (GFP) reporter gene. In addition to the creation of a mutant CX(3)CR1 locus, this approach enabled us to assign murine CX(3)CR1 expression to monocytes, subsets of NK and dendritic cells, and the brain microglia. Analysis of CX(3)CR1-deficient mice indicates that CX(3)CR1 is the only murine FKN receptor. Yet, defying anticipated FKN functions, absence of CX(3)CR1 interferes neither with monocyte extravasation in a peritonitis model nor with DC migration and differentiation in response to microbial antigens or contact sensitizers. Furthermore, a prominent response of CX(3)CR1-deficient microglia to peripheral nerve injury indicates unimpaired neuronal-glial cross talk in the absence of CX(3)CR1
— id: 11707, year: 2000, vol: 20, page: 4106, stat: Journal Article,

Analysis of CX3C chemokine receptor function by GFP reporter gene insertion and targeted deletion
Jung, S; Sunshine, MJ; Littman, DR
2000 JAN ;114(1):219-219, Journal of investigative dermatology
— id: 54772, year: 2000, vol: 114, page: 219, stat: Journal Article,

PKC-theta is required for TCR-induced NF-kappaB activation in mature but not immature T lymphocytes
Sun Z; Arendt CW; Ellmeier W; Schaeffer EM; Sunshine MJ; Gandhi L; Annes J; Petrzilka D; Kupfer A; Schwartzberg PL; Littman DR
2000 Mar 23;404(6776):402-407, Nature
Productive interaction of a T lymphocyte with an antigen-presenting cell results in the clustering of the T-cell antigen receptor (TCR) and the recruitment of a large signalling complex to the site of cell-cell contact. Subsequent signal transduction resulting in cytokine gene expression requires the activation of one or more of the multiple isoenzymes of serine/threonine-specific protein kinase C (PKC). Among the several PKC isoenzymes expressed in T cells, PKC-theta is unique in being rapidly recruited to the site of TCR clustering. Here we show that PKC-theta is essential for TCR-mediated T-cell activation, but is dispensable during TCR-dependent thymocyte development. TCR-initiated NF-kappaB activation was absent from PKC-theta(-/-) mature T lymphocytes, but was intact in thymocytes. Activation of NF-kappaB by tumour-necrosis factor alpha and interleukin-1 was unaffected in the mutant mice. Although studies in T-cell lines had suggested that PKC-theta regulates activation of the JNK signalling pathway, induction of JNK was normal in T cells from mutant mice. These results indicate that PKC-theta functions in a unique pathway that links the TCR signalling complex to the activation of NF-kappaB in mature T lymphocytes
— id: 11778, year: 2000, vol: 404, page: 402, stat: Journal Article,

Requirement for RORgamma in thymocyte survival and lymphoid organ development
Sun Z; Unutmaz D; Zou YR; Sunshine MJ; Pierani A; Brenner-Morton S; Mebius RE; Littman DR
2000 Jun 30;288(5475):2369-2373, Science
Most developing thymocytes undergo apoptosis because they cannot interact productively with molecules encoded by the major histocompatibility complex. Here, we show that mice lacking the orphan nuclear hormone receptor RORgamma lose thymic expression of the anti-apoptotic factor Bcl-xL. RORgamma thus regulates the survival of CD4+8+ thymocytes and may control the temporal window during which thymocytes can undergo positive selection. RORgamma was also required for development of lymph nodes and Peyer's patches, but not splenic follicles. In its absence, there was loss of a population of CD3-CD4+CD45+ cells that normally express RORgamma and that are likely early progenitors of lymphoid organs. Hence, RORgamma has critical functions in T cell repertoire selection and lymphoid organogenesis
— id: 11628, year: 2000, vol: 288, page: 2369, stat: Journal Article,

Characterization of functional sites within the CD4 silencer
Taniuchi, I; Sunshine, MJ; Sawada, S; Littman, DR
2000 APR 20 ;14(6):A922-A922, FASEB journal
— id: 54629, year: 2000, vol: 14, page: A922, stat: Journal Article,

The primate lentiviral receptor Bonzo/STRL33 is coordinately regulated with CCR5 and its expression pattern Is conserved between human and mouse [In Process Citation]
Unutmaz D; Xiang W; Sunshine MJ; Campbell J; Butcher E; Littman DR
2000 Sep 15;165(6):3284-3292, Journal of immunology
Chemokines play necessary and important roles in regulating the trafficking of lymphocytes to intra- or interlymphoid tissues as well as to sites of inflammation. The complex migratory patterns of lymphoid lineage cells is governed by subset-specific expression of chemokine receptors and their access to specific ligands. Several chemokine receptors and chemokine receptor-like orphan receptors also serve, in conjunction with CD4, as coreceptors for infection by human and simian immunodeficiency viruses (HIV and SIV). Here we show that the expression pattern of Bonzo/STRL33, an orphan SIV/HIV coreceptor, is highly restricted to the memory subset of T cells and is up-regulated upon stimulation of these cells with IL-2 or IL-15. Both the pattern and the regulation of Bonzo expression closely paralleled that of CC family chemokine receptors CCR5 or CCR6 and inversely correlated with CXCR4 expression. However, in striking contrast to CCR5, Bonzo expression was not down-modulated by PMA or mitogen stimulation of T cells. Targeted replacement of the Bonzo gene with a gene encoding green fluorescent protein in mice revealed that the expression and cytokine regulation of mouse Bonzo are comparable to those of its human counterpart. The similar expression and regulation patterns of Bonzo and the HIV coreceptor CCR5 may have implications for understanding the role of HIV/SIV receptors in viral evolution and pathogenesis
— id: 11512, year: 2000, vol: 165, page: 3284, stat: Journal Article,

The regulation of CD4 and CD8 coreceptor gene expression during T cell development
Ellmeier W; Sawada S; Littman DR
1999 ;17:523-554, Annual review of immunology
The two major subsets of T lymphocytes in the peripheral immune system, the helper and cytotoxic T cells, are defined by their expression of either the CD4 or the CD8 glycoproteins, respectively. Expression of these molecules, which serve as coreceptors by interacting specifically with either MHC class II or class I molecules, also defines discrete stages of T cell development within the thymus. Thus, CD4+ and CD8+ single-positive (SP) thymocytes arise from common progenitor double positive (DP) cells that express both CD4 and CD8, during a process known as positive selection. The molecular mechanisms underlying the developmental choice toward the helper or cytotoxic lineage remain poorly understood. Because regulation of coreceptor gene expression appears to be coupled to the phenotypic choice of the differentiating T cell, it is likely that shared signaling pathways direct CD4 and CD8 transcription and the development of an uncommited DP thymocyte toward either the helper or cytotoxic lineage. Therefore, an understanding of how CD4 and CD8 expression is regulated will not only provide insights into transcriptional control mechanisms in T cells, but may also result in the identification of molecular factors that are involved in lineage choices during T cell development. In this review, we summarize recent progress that has been made toward an understanding of how CD4 and CD8 gene expression is regulated
— id: 6131, year: 1999, vol: 17, page: 523, stat: Journal Article,

Impaired NFATc translocation and failure of Th2 development in Itk-deficient CD4+ T cells
Fowell DJ; Shinkai K; Liao XC; Beebe AM; Coffman RL; Littman DR; Locksley RM
1999 Oct;11(4):399-409, Immunity
Naive Itk-deficient CD4+ T cells were unable to establish stable IL-4 production, even when primed in Th2-inducing conditions. In contrast, IFNgamma production was little affected. Failure to express IL-4 occurred even among cells that had gone through multiple cell divisions and was associated with a delay in the kinetics and magnitude of NFATc nuclear localization. IL-4 production was restored genetically by retroviral reconstitution of Itk or biochemically by augmenting the calcium flux with ionomycin. In vivo, Itk-deficient mice were unable to establish functional Th2 cells. Development of protective Th1 cells was unimpeded. These data define a nonredundant role for Itk in modulating signals from the TCR/CD28 pathways that are specific for the establishment of stable IL-4 but not IFNgamma expression
— id: 15114, year: 1999, vol: 11, page: 399, stat: Journal Article,

Chemokine receptors in lymphoid organ homeostasis
Jung S; Littman DR
1999 Jun;11(3):319-325, Current opinion in immunology
Leukocytes respond to complex patterns of chemoattractant cytokine (chemokine) gradients that guide them to their destinations in secondary lymphoid organs. This directed movement of multiple cell types requires the choreographed expression of specific G-protein-coupled chemokine receptors and both positive and negative regulation of the signal transduction pathways emanating from them
— id: 6143, year: 1999, vol: 11, page: 319, stat: Journal Article,

Coreceptor specificity of temporal variants of simian immunodeficiency virus Mne
Kimata JT; Gosink JJ; KewalRamani VN; Rudensey LM; Littman DR; Overbaugh J
1999 Feb;73(2):1655-1660, Journal of virology
The simian immunodeficiency virus (SIV) Mne envelope undergoes genetic changes that alter tropism, syncytium-inducing capacity, and antigenic properties of the emerging variant virus population during the course of an infection. Here we investigated whether the mutations in envelope of SIVMne also influence coreceptor usage. The data demonstrate that the infecting macrophage-tropic SIVMne clone as well as the envelope variants that are selected during the course of disease progression all recognize both CCR5 and Bob (GPR15) but not Bonzo (STRL33), CXCR4, or CCR3. Although it remains to be determined if there are other coreceptors specific for dualtropic or T-cell-tropic variants of SIVMne that emerge during late stages of infection, these data suggest that such SIV variants that evolve in pathogenic infections do not lose the ability to recognize CCR5 or Bob/GPR15
— id: 57042, year: 1999, vol: 73, page: 1655, stat: Journal Article,

Fusion-competent vaccines: broad neutralization of primary isolates of HIV
LaCasse RA; Follis KE; Trahey M; Scarborough JD; Littman DR; Nunberg JH
1999 Jan 15;283(5400):357-362, Science
Current recombinant human immunodeficiency virus (HIV) gp120 protein vaccine candidates are unable to elicit antibodies capable of neutralizing infectivity of primary isolates from patients. Here, 'fusion-competent' HIV vaccine immunogens were generated that capture the transient envelope-CD4-coreceptor structures that arise during HIV binding and fusion. In a transgenic mouse immunization model, these formaldehyde-fixed whole-cell vaccines elicited antibodies capable of neutralizing infectivity of 23 of 24 primary HIV isolates from diverse geographic locations and genetic clades A to E. Development of these fusion-dependent immunogens may lead to a broadly effective HIV vaccine
— id: 7380, year: 1999, vol: 283, page: 357, stat: Journal Article,

Role of the nuclear hormone receptor ROR gamma in transcriptional regulation, thymocyte survival, and lymphoid organogenesis
Littman DR; Sun Z; Unutmaz D; Sunshine MJ; Petrie HT; Zou YR
1999 ;64(3):373-381, Cold Spring Harbor symposia on quantitative biology
— id: 39445, year: 1999, vol: 64, page: 373, stat: Journal Article,

Primary human immunodeficiency virus type 2 (HIV-2) isolates, like HIV-1 isolates, frequently use CCR5 but show promiscuity in coreceptor usage
Morner A; Bjorndal A; Albert J; Kewalramani VN; Littman DR; Inoue R; Thorstensson R; Fenyo EM; Bjorling E
1999 Mar;73(3):2343-2349, Journal of virology
Coreceptor usage of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to biological phenotype. The chemokine receptors CCR5 and CXCR4 are the major coreceptors that, together with CD4, govern HIV-1 entry into cells. Since CXCR4 usage determines the biological phenotype for HIV-1 isolates and is more frequent in patients with immunodeficiency, it may serve as a marker for viral virulence. This possibility prompted us to study coreceptor usage by HIV-2, known to be less pathogenic than HIV-1. We tested 11 primary HIV-2 isolates for coreceptor usage in human cell lines: U87 glioma cells, stably expressing CD4 and the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, and GHOST(3) osteosarcoma cells, coexpressing CD4 and CCR5, CXCR4, or the orphan receptor Bonzo or BOB. The indicator cells were infected by cocultivation with virus-producing peripheral blood mononuclear cells and by cell-free virus. Our results show that 10 of 11 HIV-2 isolates were able to efficiently use CCR5. In contrast, only two isolates, both from patients with advanced disease, used CXCR4 efficiently. These two isolates also promptly induced syncytia in MT-2 cells, a pattern described for HIV-1 isolates that use CXCR4. Unlike HIV-1, many of the HIV-2 isolates were promiscuous in their coreceptor usage in that they were able to use, apart from CCR5, one or more of the CCR1, CCR2b, CCR3, and BOB coreceptors. Another difference between HIV-1 and HIV-2 was that the ability to replicate in MT-2 cells appeared to be a general property of HIV-2 isolates. Based on BOB mRNA expression in MT-2 cells and the ability of our panel of HIV-2 isolates to use BOB, we suggest that HIV-2 can use BOB when entering MT-2 cells. The results indicate no obvious link between viral virulence and the ability to use a multitude of coreceptors
— id: 57056, year: 1999, vol: 73, page: 2343, stat: Journal Article,

Requirement for Tec kinases Rlk and Itk in T cell receptor signaling and immunity
Schaeffer EM; Debnath J; Yap G; McVicar D; Liao XC; Littman DR; Sher A; Varmus HE; Lenardo MJ; Schwartzberg PL
1999 Apr 23;284(5414):638-641, Science
T cell receptor (TCR) signaling requires activation of Zap-70 and Src family tyrosine kinases, but requirements for other tyrosine kinases are less clear. Combined deletion in mice of two Tec kinases, Rlk and Itk, caused marked defects in TCR responses including proliferation, cytokine production, and apoptosis in vitro and adaptive immune responses to Toxoplasma gondii in vivo. Molecular events immediately downstream from the TCR were intact in rlk-/-itk-/- cells, but intermediate events including inositol trisphosphate production, calcium mobilization, and mitogen-activated protein kinase activation were impaired, establishing Tec kinases as critical regulators of TCR signaling required for phospholipase C-gamma activation
— id: 15116, year: 1999, vol: 284, page: 638, stat: Journal Article,

A Huntington's disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice
Shelbourne PF; Killeen N; Hevner RF; Johnston HM; Tecott L; Lewandoski M; Ennis M; Ramirez L; Li Z; Iannicola C; Littman DR; Myers RM
1999 May;8(5):763-774, Human molecular genetics
Huntington's disease (HD) is a dominant disorder characterized by premature and progressive neurodegeneration. In order to generate an accurate model of the disease, we introduced an HD-like mutation (an extended stretch of 72-80 CAG repeats) into the endogenous mouse Hdh gene. Analysis of the mutation in vivo reveals significant levels of germline instability, with expansions, contractions and sex-of-origin effects in evidence. Mice expressing full-length mutant protein display abnormal social behaviour in the absence of acute neurodegeneration. Given that psychiatric changes, including irritability and aggression, are common findings in HD patients, our data are consistent with the hypothesis that some clinical features of HD may be caused by pathological processes that precede gross neuronal cell death. This implies that effective treatment of HD may require an understanding and amelioration of these dysfunctional processes, rather than simply preventing the premature death of neurons in the brain. These mice should facilitate the investigation of the molecular mechanisms that underpin the pathway from genotype to phenotype in HD
— id: 15117, year: 1999, vol: 8, page: 763, stat: Journal Article,

Cytokine signals are sufficient for HIV-1 infection of resting human T lymphocytes
Unutmaz D; KewalRamani VN; Marmon S; Littman DR
1999 Jun 7;189(11):1735-1746, Journal of experimental medicine
Lentiviral vectors have been advocated to be effective vehicles for the delivery and stable expression of genes in nondividing primary cells. However, certain cell types, such as resting T lymphocytes, are resistant to infection with HIV-1. Establishing parameters for stable gene delivery into primary human lymphocytes and approaches to overcome the resistance of resting T cells to HIV infection may permit potential gene therapy applications, genetic studies of primary cells in vitro, and a better understanding of the stages of the lentiviral life cycle. Here we demonstrate that an HIV-1-derived vector can be used for stable delivery of genes into activated human T cells as well as natural killer and dendritic cells. Remarkably, a sizeable fraction of resting T cells was stably transduced with the HIV-1 vector when cultured with the cytokine interleukin (IL)-2, IL-4, IL-7, or IL-15, or, at a lower level, with IL-6, in the absence of any other stimuli. Resting T cells stimulated with these cytokines could also be infected with replication-competent HIV-1. To test the utility of this system for performing structure-function analysis in primary T cells, we introduced wild-type as well as a mutant form of murine CD28 into human T cells and showed a requirement for the CD28 cytoplasmic domain in costimulatory signaling. The ability to stably express genes of interest in primary T cells will be a valuable tool for genetic and structure-function studies that previously have been limited to transformed cell lines. In addition, the finding that cytokine signals are sufficient to permit transduction of resting T cells with HIV may be relevant for understanding mechanism of HIV-1 transmission and pathogenesis
— id: 6133, year: 1999, vol: 189, page: 1735, stat: Journal Article,

Differential requirements for CD4 in TCR-ligand interactions
Vidal K; Daniel C; Hill M; Littman DR; Allen PM
1999 Nov 1;163(9):4811-4818, Journal of immunology
The coreceptor molecule, CD4, plays an integral part in T cell activation; it is involved in both extracellular Ag recognition and intracellular signaling. We wanted to examine the functional role of CD4 in the recognition of agonist and altered peptide ligands (APLs). We generated two CD4-deficient T cell lines expressing well-characterized TCRs specific for Hb(64-76)/I-Ek. Although the responsiveness of the T cell lines to the agonist peptide was differently affected by the loss of CD4 expression, the recognition of APLs was in both cases dramatically reduced. Nearly full responsiveness to the agonist peptide was achieved by expression of a CD4 variant that did not associate with p56lck; however, the stimulation by APLs was only partially restored. Importantly, the expression of a CD4 variant in which domains interacting with MHC class II molecules have been mutated failed to restore the reactivity to all ligands. CD4-deficient T cells were able to be antagonized by APLs, indicating that CD4 was not required for antagonism. Overall, these findings support the concepts that CD4 is an integral part of the initial formation of the immunological synapse, and that the requirement for different CD4 functions in T cell activation varies depending upon the potency of the ligand
— id: 15115, year: 1999, vol: 163, page: 4811, stat: Journal Article,

A new classification for HIV-1
Berger EA; Doms RW; Fenyo EM; Korber BT; Littman DR; Moore JP; Sattentau QJ; Schuitemaker H; Sodroski J; Weiss RA
1998 Jan 15;391(6664):240-240, Nature
— id: 57407, year: 1998, vol: 391, page: 240, stat: Journal Article,

Neutralization profiles of primary human immunodeficiency virus type 1 isolates in the context of coreceptor usage
Cecilia D; KewalRamani VN; O'Leary J; Volsky B; Nyambi P; Burda S; Xu S; Littman DR; Zolla-Pazner S
1998 Sep;72(9):6988-6996, Journal of virology
Most strains of human immunodeficiency virus type 1 (HIV-1) which have only been carried in vitro in peripheral blood mononuclear cells (primary isolates) can be neutralized by antibodies, but their sensitivity to neutralization varies considerably. To study the parameters that contribute to the differential neutralization sensitivity of primary HIV-1 isolates, we developed a neutralization assay with a panel of genetically engineered cell lines (GHOST cells) that express CD4, one of eight chemokine receptors which function as HIV-1 coreceptors, and a Tat-dependent green fluorescent protein reporter cassette which permits the evaluation and quantitation of HIV-1 infection by flow cytometry. All 21 primary isolates from several clades could grow in the various GHOST cell lines, and their use of one or more coreceptors could easily be defined by flow cytometric analysis. Ten of these primary isolates, three that were CXCR4 (X4)-tropic, three that were CCR5 (R5)-tropic, and four that were dual- or polytropic were chosen for study of their sensitivity to neutralization by human monoclonal and polyclonal antibodies. Viruses from the X4-tropic category of viruses were first tested since they have generally been considered to be particularly neutralization sensitive. It was found that the X4-tropic virus group contained both neutralization-sensitive and neutralization-resistant viruses. Similar results were obtained with R5-tropic viruses and with dual- or polytropic viruses. Within each category of viruses, neutralization sensitivity and resistance could be observed. Therefore, sensitivity to neutralization appears to be the consequence of factors that influence the antibody-virus interaction and its sequelae rather than coreceptor usage. Neutralization of various viruses by the V3-specific monoclonal antibody, 447-52D, was shown to be dependent not only on the presence of the relevant epitope but also on its presentation. An epitope within the envelope of a particular virus is not sufficient to render a virus sensitive to neutralization by an antibody that recognizes that epitope. Moreover, conformation-dependent factors may overcome the need for absolute fidelity in the match between an antibody and its core epitope, permitting sufficient affinity between the viral envelope protein and the antibody to neutralize the virus. The studies indicate that the neutralization sensitivity of HIV-1 primary isolates is a consequence of the complex interaction between virus, antibody, and target cell
— id: 7527, year: 1998, vol: 72, page: 6988, stat: Journal Article,

Multiple developmental stage-specific enhancers regulate CD8 expression in developing thymocytes and in thymus-independent T cells
Ellmeier W; Sunshine MJ; Losos K; Littman DR
1998 Oct;9(4):485-496, Immunity
We and others have recently identified a CD8 locus enhancer (E8) that directs expression in mature CD8 single-positive thymocytes and peripheral CD8+ T cells and in extrathymically derived intestinal intraepithelial lymphocytes (IEL). In this study, we show that deletion of E8, by homologous recombination results in reduced CD8alphaalpha homodimer expression on IEL. Since CD8 expression on thymus-derived T cells was normal, other enhancers regulate CD8 expression in these cells. By exploiting a transgenic reporter expression assay, we identified three additional enhancers that directed expression in diverse thymocyte subsets and mature T cells but not in CD8alphaalpha+ IEL. The results suggest that CD8alpha expression is primarily regulated by E8, in IEL and by the novel enhancers in the thymus-dependent lineages
— id: 57144, year: 1998, vol: 9, page: 485, stat: Journal Article,

The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein
Garber ME; Wei P; KewalRamani VN; Mayall TP; Herrmann CH; Rice AP; Littman DR; Jones KA
1998 Nov 15;12(22):3512-3527, Genes & development
HIV-1 Tat activates transcription through binding to human cyclin T1, a regulatory subunit of the TAK/P-TEFb CTD kinase complex. Here we show that the cyclin domain of hCycT1 is necessary and sufficient to interact with Tat and promote cooperative binding to TAR RNA in vitro, as well as mediate Tat transactivation in vivo. A Tat:TAR recognition motif (TRM) was identified at the carboxy-terminal edge of the cyclin domain, and we show that hCycT1 can interact simultaneously with Tat and CDK9 on TAR RNA in vitro. Alanine-scanning mutagenesis of the hCycT1 TRM identified residues that are critical for the interaction with Tat and others that are required specifically for binding of the complex to TAR RNA. Interestingly, we find that the interaction between Tat and hCycT1 requires zinc as well as essential cysteine residues in both proteins. Cloning and characterization of the murine CycT1 protein revealed that it lacks a critical cysteine residue (C261) and forms a weak, zinc-independent complex with HIV-1 Tat that greatly reduces binding to TAR RNA. A point mutation in mCycT1 (Y261C) restores high-affinity, zinc-dependent binding to Tat and TAR in vitro, and rescues Tat transactivation in vivo. Although overexpression of hCycT1 in NIH3T3 cells strongly enhances transcription from an integrated proviral promoter, we find that this fails to overcome all blocks to productive HIV-1 infection in murine cells
— id: 7572, year: 1998, vol: 12, page: 3512, stat: Journal Article,

The amino terminus of human CCR5 is required for its function as a receptor for diverse human and simian immunodeficiency virus envelope glycoproteins
Hill CM; Kwon D; Jones M; Davis CB; Marmon S; Daugherty BL; DeMartino JA; Springer MS; Unutmaz D; Littman DR
1998 Sep 1;248(2):357-371, Virology
The chemokine receptor CCR5 plays a key role in the CD4-dependent entry of human and simian immunodeficiency viruses into target cells. We have mapped the interaction sites on CCR5 for a number of novel anti-CCR5 monoclonal antibodies and have used these to study the role of the CCR5 N-terminal ectodomain in viral entry and to demonstrate differential CCR5 epitope expression on different cell types. Deletions of the CCR5 amino terminal domain or substitution with equivalent regions from other chemokine receptors did not affect cell surface expression or reactivity with loop-specific antibodies, suggesting that the loop regions remained conformationally intact. Exchanges of the amino terminal segment of CCR5 with the equivalent domains of CCR1, CCR2, and CXCR4 did not significantly affect infection with virus pseudotyped with envelope glycoproteins (Envs) from HIV-2 and SIV, but substitution with the CXCR4 sequence abrogated entry mediated by Env from HIV-1. In contrast, deletion of the amino terminus abrogated CCR5 receptor activity for all viral Envs examined. These data indicate that the amino terminus of CCR5 has an essential role in entry mediated by diverse viral Envs but that the sequence requirements are more relaxed for the HIV-2 and SIV Envs compared to the HIV-1 Env examined. This suggests that different viral Envs make distinct and specific interactions with the amino terminus of CCR5. Viral Env utilization of CCR5 expressed on 293-T cells does not always correlate with the cellular tropism of the virus, and one possible explanation is that Env-accessible interaction sites on CCR5 differ on different cell types. We therefore analyzed binding of several anti-CCR5 monoclonal antibodies to cell lines and primary cells that express this chemokine receptor and found that whereas all antibodies bound to CCR5-transfected 293T cells, several did not bind to PBMC. The results suggest that CCR5 undergoes cell type specific structural modifications which may affect interaction with different HIV and SIV envelope glycoproteins.
— id: 7599, year: 1998, vol: 248, page: 357, stat: Journal Article,

The cytoplasmic domain of CD8 beta regulates Lck kinase activation and CD8 T cell development
Irie HY; Mong MS; Itano A; Crooks ME; Littman DR; Burakoff SJ; Robey E
1998 Jul 1;161(1):183-191, Journal of immunology
Previous studies have shown that CD8 beta plays a role in both enhancing CD8 alpha-associated Lck kinase activity and promoting the development of CD8-lineage T cells. To examine the role of this enhancement in the maturation of CD8-lineage cells, we assessed CD8 alpha-associated Lck kinase activity in both T cell hybridomas and thymocytes of mice expressing CD8 beta mutations known to impair CD8 T cell development. Lack of CD8 beta expression or expression of a cytoplasmic domain-deleted CD8 beta resulted in a severalfold reduction in CD8 alpha-associated Lck kinase activity compared with that observed with cells expressing wild-type CD8 beta chain. This analysis indicated a critical role for the cytoplasmic domain of CD8 beta in the regulation of CD8 alpha-associated Lck activity. Decreased CD8 alpha-associated Lck activity observed with the various CD8 beta mutations also correlated with diminished in vivo cellular tyrosine phosphorylation. In addition, analysis of CD8 beta mutant mice (CD8 beta-/- or cytoplasmic domain-deleted CD8 beta transgenic) indicated that the degree of reduction in CD8 alpha-associated Lck activity associated with each mutation correlated with the severity of developmental impairment. These results support the importance of CD8 beta-mediated enhancement of CD8 alpha-associated Lck kinase activity in the differentiation of CD8 single-positive thymocytes
— id: 7614, year: 1998, vol: 161, page: 183, stat: Journal Article,

Neutralizing antibodies in sera from macaques immunized with attenuated simian immunodeficiency virus
Langlois AJ; Desrosiers RC; Lewis MG; KewalRamani VN; Littman DR; Zhou JY; Manson K; Wyand MS; Bolognesi DP; Montefiori DC
1998 Aug;72(8):6950-6955, Journal of virology
Infection with attenuated simian immunodeficiency virus (SIV) in rhesus macaques has been shown to raise antibodies capable of neutralizing an animal challenge stock of primary SIVmac251 in CEMx174 cells that correlate with resistance to infection after experimental challenge with this virulent virus (M. S. Wyand, K. H. Manson, M. Garcia-Moll, D. C. Montefiori, and R. C. Desrosiers, J. Virol. 70:3724-3733, 1996). Here we show that these neutralizing antibodies are not detected in human and rhesus peripheral blood mononuclear cells (PBMC). In addition, neutralization of primary SIVmac251 in human and rhesus PBMC was rarely detected with plasma samples from a similar group of animals that had been infected either with SIVmac239Deltanef for 1.5 years or with SIVmac239Delta3 for 3.2 years, although low-level neutralization was detected in CEMx174 cells. Potent neutralization was detected in CEMx174 cells when the latter plasma samples were assessed with laboratory-adapted SIVmac251. In contrast to primary SIVmac251, laboratory-adapted SIVmac251 did not replicate in human and rhesus PBMC despite its ability to utilize CCR5, Bonzo/STRL33, and BOB/gpr15 as coreceptors for virus entry. These results illustrate the importance of virus passage history and the choice of indicator cells for making assessments of neutralizing antibodies to lentiviruses such as SIV. They also demonstrate that primary SIVmac251 is less sensitive to neutralization in human and rhesus PBMC than it is in established cell lines. Results obtained in PBMC did not support a role for neutralizing antibodies as a mechanism of protection in animals immunized with attenuated SIV and challenged with primary SIVmac251
— id: 57190, year: 1998, vol: 72, page: 6950, stat: Journal Article,

Chemokine receptors: keys to AIDS pathogenesis?
Littman DR
1998 May 29;93(5):677-680, Cell
— id: 12106, year: 1998, vol: 93, page: 677, stat: Journal Article,

Exclusive and persistent use of the entry coreceptor CXCR4 by human immunodeficiency virus type 1 from a subject homozygous for CCR5 delta32
Michael NL; Nelson JA; KewalRamani VN; Chang G; O'Brien SJ; Mascola JR; Volsky B; Louder M; White GC 2nd; Littman DR; Swanstrom R; O'Brien TR
1998 Jul;72(7):6040-6047, Journal of virology
Individuals who are homozygous for the 32-bp deletion in the gene coding for the chemokine receptor and major human immunodeficiency virus type 1 (HIV-1) coreceptor CCR5 (CCR5 -/-) lack functional cell surface CCR5 molecules and are relatively resistant to HIV-1 infection. HIV-1 infection in CCR5 -/- individuals, although rare, has been increasingly documented. We now report that the viral quasispecies from one such individual throughout disease is homogenous, T cell line tropic, and phenotypically syncytium inducing (SI); exclusively uses CXCR4; and replicates well in CCR5 -/- primary T cells. The recently discovered coreceptors BOB and Bonzo are not used. Although early and persistent SI variants have been described in longitudinal studies, this is the first demonstration of exclusive and persistent CXCR4 usage. With the caveat that the earliest viruses available from this subject were from approximately 4 years following primary infection, these data suggest that HIV-1 infection can be mediated and persistently maintained by viruses which exclusively utilize CXCR4. The lack of evolution toward the available minor coreceptors in this subject underscores the dominant biological roles of the major coreceptors CCR5 and CXCR4. This and two similar subjects (R. Biti, R. Ffrench, J. Young, B. Bennetts, G. Stewart, and T. Liang, Nat. Med. 3:252-253, 1997; I. Theodoreu, L. Meyer, M. Magierowska, C. Katlama, and C. Rouzioux, Lancet 349:1219-1220, 1997) showed relatively rapid CD4+ T-cell declines despite average or low initial viral RNA load. Since viruses which use CXCR4 exclusively cannot infect macrophages, these data have implications for the relative infection of the T-cell compartment versus the macrophage compartment in vivo and for the development of CCR5-based therapeutics
— id: 57343, year: 1998, vol: 72, page: 6040, stat: Journal Article,

Regulation of IL-4 expression by activation of individual alleles
Riviere I; Sunshine MJ; Littman DR
1998 Aug;9(2):217-228, Immunity
To study the in vivo role of IL-4-expressing cells, we developed a strategy to tag these cells, by generating mice in which one IL-4 allele was replaced with a cDNA encoding the human CD2 (huCD2) cell-surface molecule. Expression of the huCD2 reporter was, like IL-4, restricted to the appropriately polarized T helper 2 cells. However, most of the cells expressed only the IL-4 or the targeted allele. Analysis of the frequency of monoallelic versus biallelic expression suggests that the activation of each individual allele is regulated by a stochastic process whose probability can be augmented by increasing the strength of signal delivered through the TCR. Allele-specific activation may be a general feature of cytokine regulation that contributes to the functional diversity within T helper cell subpopulations
— id: 57232, year: 1998, vol: 9, page: 217, stat: Journal Article,

Neutralization sensitivity of human immunodeficiency virus type 1 primary isolates to antibodies and CD4-based reagents is independent of coreceptor usage
Trkola A; Ketas T; Kewalramani VN; Endorf F; Binley JM; Katinger H; Robinson J; Littman DR; Moore JP
1998 Mar;72(3):1876-1885, Journal of virology
We have investigated whether the identity of the coreceptor (CCR5, CXCR4, or both) used by primary human immunodeficiency virus type 1 (HIV-1) isolates to enter CD4+ cells influences the sensitivity of these isolates to neutralization by monoclonal antibodies and CD4-based agents. Coreceptor usage was not an important determinant of neutralization titer for primary isolates in peripheral blood mononuclear cells. We also studied whether dualtropic primary isolates (able to use both CCR5 and CXCR4) were differentially sensitive to neutralization by the same antibodies when entering U87MG-CD4 cells stably expressing either CCR5 or CXCR4. Again, we found that the coreceptor used by a virus did not greatly affect its neutralization sensitivity. Similar results were obtained for CCR5- or CXCR4-expressing HOS cell lines engineered to express green fluorescent protein as a reporter of HIV-1 entry. Neutralizing antibodies are therefore unlikely to be the major selection pressure which drives the phenotypic evolution (change in coreceptor usage) of HIV-1 that can occur in vivo. In addition, the increase in neutralization sensitivity found when primary isolates adapt to growth in transformed cell lines in vitro has little to do with alterations in coreceptor usage
— id: 15118, year: 1998, vol: 72, page: 1876, stat: Journal Article,

Differences in chemokine coreceptor usage between genetic subtypes of HIV-1
Tscherning C; Alaeus A; Fredriksson R; Bjorndal A; Deng H; Littman DR; Fenyo EM; Albert J
1998 Feb 15;241(2):181-188, Virology
HIV-1 uses chemokine coreceptors for cell entry. CXCR4 is the major coreceptor for T-cell-line-adapted isolates and CCR5 for non-T-cell-line-adapted isolates. This study investigated if coreceptor usage differs between genetic subtypes of HIV-1. Eighty-one primary isolates representing nine different genetic subtypes (A-J, except I) were tested on U87.CD4 glioma cells stably expressing chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4. Coreceptor usage was compared to biological phenotype of the isolates (rapid/high, syncytium-inducing or slow/low, non-syncytium-inducing) and to clinical and immunological status of the study subjects. CXCR4 usage was perfectly correlated to the biological phenotype for all subtypes; all of 26 isolates with rapid/high phenotype and none of 55 isolates with slow/low phenotype could infect the CXCR4 expressing cell line. Importantly, the CXCR4-positive, rapid/high phenotype was underrepresented among subtype C isolates. Furthermore, dual tropism for CXCR4 and CCR5 was not found among subtype D isolates. Uni- and multivariate analyses indicated that these subtype-specific differences in coreceptor usage were not due to differences in clinical status, CD4 counts, or treatment. This study shows that CXCR4 usage determines the biological phenotype for all subtypes, but that there appear to exist subtype-dependent differences in frequency of usage of certain coreceptors. This opens up the possibility that genetic subtypes may differ in important biological properties such as virulence, tissue tropism, and transmissibility
— id: 7978, year: 1998, vol: 241, page: 181, stat: Journal Article,

G protein-coupled receptors in HIV and SIV entry: new perspectives on lentivirus-host interactions and on the utility of animal models
Unutmaz D; KewalRamani VN; Littman DR
1998 Jun;10(3):225-236, Seminars in immunology
Entry of primate lentiviruses into target cells has recently been shown to depend upon the interaction of the viral envelope glycoprotein with CD4 and one or more members of the G protein-coupled receptor (GPCR) family of transmembrane proteins. In vivo, the transmission of HIV-1 infection generally requires viral strains that utilise chemokine recep- tor CCR5, and these strains prevail during the early course of infection. Strains isolated later, in the course of progression to immunodeficiency, are often CXCR4-tropic or are dual tropic for both chemokine receptors. SIV isolates also use CCR5 but are only rarely specific for CXCR4. Instead, SIVs use two orphan members of the GPCR family, named Bonzo/STRL33/TYMSTR and BOB/GPR15. Strains of HIV-2, which are closely related to the SIVs, also often utilise CXCR4, CCR5, BOB and/or Bonzo. Additional GPCR family members have also been shown to be utilised by various strains of HIV and SIV, albeit less efficiently and less frequently. Here we discuss the potential relationship between receptor specificity and viral pathogenesis as well as efforts to develop animal model systems to study the mechanism of disease progression.
— id: 7833, year: 1998, vol: 10, page: 225, stat: Journal Article,

Use of coreceptors other than CCR5 by non-syncytium-inducing adult and pediatric isolates of human immunodeficiency virus type 1 is rare in vitro
Zhang YJ; Dragic T; Cao Y; Kostrikis L; Kwon DS; Littman DR; KewalRamani VN; Moore JP
1998 Nov;72(11):9337-9344, Journal of virology
We have tested a panel of pediatric and adult human immunodeficiency virus type 1 (HIV-1) primary isolates for the ability to employ the following proteins as coreceptors during viral entry: CCR1, CCR2b, CCR3, CCR4, CCR5, CCR8, CXCR4, Bonzo, BOB, GPR1, V28, US28, and APJ. Most non-syncytium-inducing isolates could utilize only CCR5. All syncytium-inducing viruses used CXCR4, some also employed V28, and one (DH123) used CCR8 and APJ as well. A longitudinal series of HIV-1 subtype B isolates from an infected infant and its mother utilized Bonzo efficiently, as well as CCR5. The maternal isolates, which were syncytium inducing, also used CXCR4, CCR8, V28, and APJ
— id: 57289, year: 1998, vol: 72, page: 9337, stat: Journal Article,

Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development
Zou YR; Kottmann AH; Kuroda M; Taniuchi I; Littman DR
1998 Jun 11;393(6685):595-599, Nature
Chemokines and their receptors are important in cell migration during inflammation, in the establishment of functional lymphoid microenvironments, and in organogenesis. The chemokine receptor CXCR4 is broadly expressed in cells of both the immune and the central nervous systems and can mediate migration of resting leukocytes and haematopoietic progenitors in response to its ligand, SDF-1. CXCR4 is also a major receptor for strains of human immunodeficiency virus-1 (HIV-1) that arise during progression to immunodeficiency and AIDS dementia. Here we show that mice lacking CXCR4 exhibit haematopoietic and cardiac defects identical to those of SDF-1-deficient mice, indicating that CXCR4 may be the only receptor for SDF-1. Furthermore, fetal cerebellar development in mutant animals is markedly different from that in wild-type animals, with many proliferating granule cells invading the cerebellar anlage. This is, to our knowledge, the first demonstration of the involvement of a G-protein-coupled chemokine receptor in neuronal cell migration and patterning in the central nervous system. These results may be important for designing strategies to block HIV entry into cells and for understanding mechanisms of pathogenesis in AIDS dementia
— id: 57298, year: 1998, vol: 393, page: 595, stat: Journal Article,

Antiviral immune responses in Itk-deficient mice
Bachmann MF; Littman DR; Liao XC
1997 Oct;71(10):7253-7257, Journal of virology
Mice lacking Itk, a T-cell-specific protein tyrosine kinase, have reduced numbers of T cells and reduced responses to allogeneic major histocompatibility molecules. This study analyzed antiviral immune responses in mice deficient for Itk. Primary cytotoxic T-lymphocyte (CTL) responses were analyzed after infection with lymphocytic choriomeningitis virus (LCMV), vaccinia virus (VV), and vesicular stomatitis virus (VSV). Ex vivo CTL activity was consistently reduced by a factor of two to six for the different viruses. CTL responses after restimulation in vitro were similarly reduced unless exogenous cytokines were added. In the presence of interleukin-2 or concanavalin A supernatant, Itk-deficient and control mice responded similarly. Interestingly, while LCMV was completely eliminated by day 8 in both Itk-deficient and control mice, VV cleared from itk-/- mice with delayed kinetics. Antibody responses were evaluated after VSV infection. Both the T-cell-independent neutralizing immunoglobulin M (IgM) and the T-cell-dependent IgG responses were similar in Itk-deficient and control mice. Taken together, the results show that CTL responses are reduced in the absence of Itk whereas antiviral B-cell responses are not affected
— id: 15121, year: 1997, vol: 71, page: 7253, stat: Journal Article,

Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype
Bjorndal A; Deng H; Jansson M; Fiore JR; Colognesi C; Karlsson A; Albert J; Scarlatti G; Littman DR; Fenyo EM
1997 Oct;71(10):7478-7487, Journal of virology
The biological phenotype of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to the severity of the HIV infection. Here we show that the two previously described groups of rapid/high, syncytium-inducing (SI) and slow/low, non-syncytium-inducing (NSI) isolates are distinguished by their ability to utilize different chemokine receptors for entry into target cells. Recent studies have identified the C-X-C chemokine receptor CXCR4 (also named fusin or Lestr) and the C-C chemokine receptor CCR5 as the principal entry cofactors for T-cell-line-tropic and non-T-cell-line-tropic HIV-1, respectively. Using U87.CD4 glioma cell lines, stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, we have tested chemokine receptor specificity for a panel of genetically diverse envelope glycoprotein genes cloned from primary HIV-1 isolates and have found that receptor usage was closely associated with the biological phenotype of the virus isolate but not the genetic subtype. We have also analyzed a panel of 36 well-characterized primary HIV-1 isolates for syncytium induction and replication in the same series of cell lines. Infection by slow/low viruses was restricted to cells expressing CCR5, whereas rapid/high viruses could use a variety of chemokine receptors. In addition to the regular use of CXCR4, many rapid/high viruses used CCR5 and some also used CCR3 and CCR2b. Progressive HIV-1 infection is characterized by the emergence of viruses resistant to inhibition by beta-chemokines, which corresponded to changes in coreceptor usage. The broadening of the host range may even enable the use of uncharacterized coreceptors, in that two isolates from immunodeficient patients infected the parental U87.CD4 cell line lacking any engineered coreceptor. Two primary isolates with multiple coreceptor usage were shown to consist of mixed populations, one with a narrow host range using CCR5 only and the other with a broad host range using CCR3, CCR5, or CXCR4, similar to the original population. The results show that all 36 primary HIV-1 isolates induce syncytia, provided that target cells carry the particular coreceptor required by the virus
— id: 15120, year: 1997, vol: 71, page: 7478, stat: Journal Article,

Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5
Davis CB; Dikic I; Unutmaz D; Hill CM; Arthos J; Siani MA; Thompson DA; Schlessinger J; Littman DR
1997 Nov 17;186(10):1793-1798, Journal of experimental medicine
Infection with HIV-1 requires expression of CD4 and the chemokine receptors CXCR4 or CCR5 at the target cell surface. Engagement of these receptors by the HIV-1 envelope glycoprotein is essential for membrane fusion, but may additionally activate intracellular signaling pathways. In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2. The response requires CXCR4 and CCR5 to be accessible on the cell surface. The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors
— id: 12196, year: 1997, vol: 186, page: 1793, stat: Journal Article,

Expression cloning of new receptors used by simian and human immunodeficiency viruses
Deng HK; Unutmaz D; KewalRamani VN; Littman DR
1997 Jul 17;388(6639):296-300, Nature
Several members of the chemokine-receptor family serve, in conjunction with CD4, as receptors for the entry of human immunodeficiency virus type I (HIV-1) into cells. The principal receptor for entry of macrophage-tropic (M-tropic) HIV-1 strains is CCR5, whereas that for T-cell-line-tropic (T-tropic) strains is CXCR4. Unlike HIV-1, infection with either M-tropic or T-tropic strains of simian immunodeficiency virus (SIV) can be mediated by CCR5, but not CXCR4. SIV strains will also infect CD4+ cells that lack CCR5, which suggests that these strains use as yet unidentified receptors. Here we use an expression-cloning strategy to identify SIV receptors and have isolated genes encoding two members of the seven-transmembrane G-protein-coupled receptor family that are used not only by SIVs, but also by strains of HIV-2 and M-tropic HIV-1. Both receptors are closely related to the chemokine-receptor family and are expressed in lymphoid tissues. One of the receptors is also expressed in colon and may therefore be important in viral transmission. Usage of these new receptors following experimental infection of non-human primates with SIV strains may provide important insight into viral transmission and the mechanisms of SIV- and HIV-induced acquired immune-deficiency syndrome
— id: 56936, year: 1997, vol: 388, page: 296, stat: Journal Article,

An enhancer that directs lineage-specific expression of CD8 in positively selected thymocytes and mature T cells
Ellmeier W; Sunshine MJ; Losos K; Hatam F; Littman DR
1997 Oct;7(4):537-547, Immunity
Positive selection of CD4+CD8+ T cells to the CD4+CD8- helper and CD4- CD8+ cytotoxic lineages is a multistep process that involves complex regulation of coreceptor gene expression. By analyzing expression of a reporter gene in transgenic mice, we have identified a DNA segment, located between the murine CD8beta and CD8alpha genes, that has enhancer activity restricted to CD8 lineage cells. Remarkably, this enhancer functions in thymocytes undergoing positive selection to the CD4-CD8+ phenotype but not in immature double-positive thymocytes. The enhancer also functions in gut intraepithelial lymphocytes that express CD8alpha but not CD8beta, suggesting that it is specific for CD8alpha expression. The tight correlation between activation of this enhancer and the final step in positive selection has important implications for understanding the mechanism of lineage commitment in thymocytes
— id: 12237, year: 1997, vol: 7, page: 537, stat: Journal Article,

Envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus can use human CCR5 as a coreceptor for viral entry and make direct CD4-dependent interactions with this chemokine receptor
Hill CM; Deng H; Unutmaz D; Kewalramani VN; Bastiani L; Gorny MK; Zolla-Pazner S; Littman DR
1997 Sep;71(9):6296-6304, Journal of virology
Several members of the chemokine receptor family have recently been identified as coreceptors, with CD4, for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. In this report, we show that the envelope glycoproteins of several strains of HIV-2 and simian immunodeficiency virus (SIV) employ the same chemokine receptors for infection. Envelope glycoproteins from HIV-2 use CCR5 or CXCR4, while those from several strains of SIV use CCR5. Our data indicate also that some viral envelopes can use more than one coreceptor for entry and suggest that some of these coreceptors remain to be identified. To further understand how different envelope molecules use CCR5 as an entry cofactor, we show that soluble purified envelope glycoproteins (SU component) from CCR5-tropic HIV-1, HIV-2, and SIV can compete for binding of iodinated chemokine to CCR5. The competition is dependent on binding of the SU glycoprotein to cell surface CD4 and implies a direct interaction between envelope glycoproteins and CCR5. This interaction is specific since it is not observed with SU glycoprotein from a CXCR4-tropic virus or with a chemokine receptor that is not competent for viral entry (CCR1). For HIV-1, the interaction can be inhibited by antibodies specific for the V3 loop of SU. Soluble CD4 was found to potentiate binding of the HIV-2 ST and SIVmac239 envelope glycoproteins to CCR5, suggesting that a CD4-induced conformational change in SU is required for subsequent binding to CCR5. These data suggest a common fundamental mechanism by which structurally diverse HIV-1, HIV-2, and SIV envelope glycoproteins interact with CD4 and CCR5 to mediate viral entry
— id: 57413, year: 1997, vol: 71, page: 6296, stat: Journal Article,

Itk negatively regulates induction of T cell proliferation by CD28 costimulation
Liao XC; Fournier S; Killeen N; Weiss A; Allison JP; Littman DR
1997 Jul 21;186(2):221-228, Journal of experimental medicine
CD28 is a cell surface molecule that mediates a costimulatory signal crucial for T cell proliferation and lymphokine production. The signal transduction mechanisms of CD28 are not well understood. Itk, a nonreceptor protein tyrosine kinase specifically expressed in T cells and mast cells, has been implicated in the CD28 signaling pathway because of reports that it becomes phosphorylated on tyrosines and associates with CD28 upon cross-linking of the cell surface molecule. To determine whether Itk plays a functional role in CD28 signaling, we compared T cells from Itk-deficient mice and control mice for their responses to CD28 costimulation. T cells defective in Itk were found to be fully competent to respond to costimulation. Whereas the CD3-mediated proliferative response was severely compromised in the absence of Itk, the calcineurin-independent CD28-mediated response was significantly elevated when compared with cells from control animals. The augmented proliferation was not due to increased production of interleukin-2. The results suggest that Itk has distinct roles in the CD3 versus the CD28 signaling pathways. By negatively regulating the amplitude of signaling upon CD28 costimulation, Itk may provide a means for modulating the outcome of T cell activation during development and during antigen-driven immune responses
— id: 15123, year: 1997, vol: 186, page: 221, stat: Journal Article,

Itk and Fyn make independent contributions to T cell activation
Liao XC; Littman DR; Weiss A
1997 Dec 15;186(12):2069-2073, Journal of experimental medicine
Itk is a member of the Btk/Tec/Itk family of nonreceptor protein tyrosine kinases (PTKs), and has been implicated in T cell antigen receptor (TCR) signal transduction. Lck and Fyn are the Src-family nonreceptor PTKs that are involved in TCR signaling. To address the question of how these members of different families of PTKs functionally contribute to T cell development and to T cell activation, mice deficient for both Itk and either Lck or Fyn were generated. The Itk/Lck doubly deficient mice exhibited a phenotype similar to that of Lck-deficient mice. The phenotype of the Itk/Fyn doubly deficient mice was similar to that of Itk deficient mice. However the Itk/Fyn doubly deficient mice exhibited a more severe defect in TCR-induced proliferation of thymocytes and peripheral T cells than did mice deficient in either kinase alone. These data support the notion that Itk and Fyn both make independent contributions to TCR-induced T cell activation
— id: 7658, year: 1997, vol: 186, page: 2069, stat: Journal Article,

Chemokine receptors and animal models for HIV pathogenesis
Littman, DR; Davis, C; Deng, HK; Ellmeier, W; Hill, M; KewalRamani, V; Scarborough, J; Taniuchi, I; Unutmaz, D; Zou, YR
1997 NOV ;8(5):2026-2026, Molecular biology of the cell
— id: 53170, year: 1997, vol: 8, page: 2026, stat: Journal Article,

Cell and viral regulatory elements enhance the expression and function of a human immunodeficiency virus inhibitory gene
Ranga U; Woffendin C; Yang ZY; Xu L; Verma S; Littman DR; Nabel GJ
1997 Sep;71(9):7020-7029, Journal of virology
Regulated expression of recombinant genes in CD4+ cells is an important objective for gene therapy of AIDS, as these cells represent the principal target for viral replication of human immunodeficiency virus (HIV). We report here that specific combinations of CD4 cell-specific and viral regulatory elements can enhance expression of an antiviral gene product. Different viral regulatory elements were incorporated into a previously reported CD4 locus control region to increase the expression of reporter genes in T and monocytic cell lines. The CD4-specific regulatory elements were included to enhance expression in CD4 cells, and viral regulatory regions, including the cytomegalovirus immediate-early (CMV IE) upstream enhancer, which contains the kappa B and Ap1 regulatory elements and a Tat-responsive element of the HIV type 1 long terminal repeat, were used to increase gene expression and modulate its activity in response to viral infection. In transient transfection assays, this vector was 100- to 1,000-fold more active than the original CD4 regulatory elements alone. Expression of an inhibitory form of the Rev protein, Rev M10, was more effective than previously described vectors and protected against productive viral replication in CD4+ peripheral blood mononuclear cells. The combination of CD4 lineage-specific and viral regulatory elements will facilitate the development of more effective antiviral genetic strategies for AIDS
— id: 15122, year: 1997, vol: 71, page: 7020, stat: Journal Article,

In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression
Scarlatti G; Tresoldi E; Bjorndal A; Fredriksson R; Colognesi C; Deng HK; Malnati MS; Plebani A; Siccardi AG; Littman DR; Fenyo EM; Lusso P
1997 Nov;3(11):1259-1265, Nature medicine
Following the identification of the C-C chemokines RANTES, MIP-1alpha and MIP-1beta as major human immunodeficiency virus (HIV)-suppressive factors produced by CD8+ T cells, several chemokine receptors were found to serve as membrane co-receptors for primate immunodeficiency lentiretroviruses. The two most widely used co-receptors thus far recognized, CCR5 and CXCR4, are expressed by both activated T lymphocytes and mononuclear phagocytes. CCR5, a specific RANTES, MIP-1alpha and MIP-1 receptor, is used preferentially by non-MT2-tropic HIV-1 and HIV-2 strains and by simian immunodeficiency virus (SIV), whereas CXCR4, a receptor for the C-X-C chemokine SDF-1, is used by MT2-tropic HIV-1 and HIV-2, but not by SIV. Other receptors with a more restricted cellular distribution, such as CCR2b, CCR3 and STRL33, can also function as co-receptors for selected viral isolates. The third variable region (V3) of the gp120 envelope glycoprotein of HIV-1 has been fingered as a critical determinant of the co-receptor choice. Here, we document a consistent pattern of evolution of viral co-receptor usage and sensitivity to chemokine-mediated suppression in a longitudinal follow-up of children with progressive HIV-1 infection. Viral isolates obtained during the asymptomatic stages generally used only CCR5 as a co-receptor and were inhibited by RANTES, MIP-1alpha and MIP-1beta, but not by SDF-1. By contrast, the majority of the isolates derived after the progression of the disease were resistant to C-C chemokines, having acquired the ability to use CXCR4 and, in some cases, CCR3, while gradually losing CCR5 usage. Surprisingly, most of these isolates were also insensitive to SDF-1, even when used in combination with RANTES. An early acquisition of CXCR4 usage predicted a poor prognosis. In children who progressed to AIDS without a shift to CXCR4 usage, all the sequential isolates were CCR5-dependent but showed a reduced sensitivity to C-C chemokines. Discrete changes in the V3 domain of gp120 were associated with the loss of sensitivity to C-C chemokines and the shift in co-receptor usage. These results suggest an adaptive evolution of HIV-1 in vivo, leading to escape from the control of the antiviral C-C chemokines
— id: 15119, year: 1997, vol: 3, page: 1259, stat: Journal Article,

CD2 regulates the positive selection and function of antigen-specific CD4- CD8+ T cells
Teh SJ; Killeen N; Tarakhovsky A; Littman DR; Teh HS
1997 Feb 15;89(4):1308-1318, Blood
The CD2 glycoprotein has been implicated in both positive and negative regulation of T-cell mitogenesis. To study the involvement of CD2 in T-lymphocyte development and immune responses, we have analyzed two lines of CD2-null mice, each expressing a distinct class I major histocompatibility complex (MHC)-restricted T-cell receptor (TCR). In both situations, the absence of CD2 appeared to promote the positive selection of cells in a manner that is similar to that which occurs in the absence of CD5. Consistent with this, compound homozygotes that lacked both CD2 and CD5 showed evidence of enhanced positive selection even in the absence of a transgenic TCR. Despite the observed enhancement of positive selection, the lack of CD2 was associated with defects in proliferative responses and interferon-gamma production when transgenic thymocytes and mature T lymphocytes were stimulated with the appropriate antigens. These findings raise the possibility that impaired sensitivity to selecting ligands in the thymus may provide a selective advantage that improves the efficiency of positive selection for certain TCRs. Furthermore, the results highlight the potential for a differential role for CD2 in thymocyte selection and T-cell immune responses
— id: 15124, year: 1997, vol: 89, page: 1308, stat: Journal Article,

Expression pattern of HIV-1 coreceptors on T cells: implications for viral transmission and lymphocyte homing
Unutmaz D; Littman DR
1997 Mar 4;94(5):1615-1618, Proceedings of the National Academy of Sciences of the United States of America
— id: 12356, year: 1997, vol: 94, page: 1615, stat: Journal Article,

Identification of a major co-receptor for primary isolates of HIV-1
Deng H; Liu R; Ellmeier W; Choe S; Unutmaz D; Burkhart M; Di Marzio P; Marmon S; Sutton RE; Hill CM; Davis CB; Peiper SC; Schall TJ; Littman DR; Landau NR
1996 Jun 20;381(6584):661-666, Nature
Entry of HIV-1 into target cells requires cell-surface CD4 and additional host cell cofactors. A cofactor required for infection with virus adapted for growth in transformed T-cell lines was recently identified and named fusin. However, fusin does not promote entry of macrophage-tropic viruses, which are believed to be the key pathogenic strains in vivo. The principal cofactor for entry mediated by the envelope glycoproteins of primary macrophage-tropic strains of HIV-1 is CC-CKR-5, a receptor for the beta-chemokines RANTES, MIP-1alpha and MIP-1beta
— id: 57390, year: 1996, vol: 381, page: 661, stat: Journal Article,

Natural resistance to HIV?
Hill CM; Littman DR
1996 Aug 22;382(6593):668-669, Nature
— id: 57364, year: 1996, vol: 382, page: 668, stat: Journal Article,

The regulation and function of the CD4 coreceptor during T lymphocyte development
Killeen N; Littman DR
1996 ;205:89-106, Current topics in microbiology & immunology
The data reviewed in this chapter suggest that the primary developmental function of the CD4 and CD8 coreceptors is to improve the efficacy by which a thymocyte recognizes peptide/MHC. During positive selection, DP thymocytes down-regulate expression of either CD4 or CD8 in response to signals that originate from the TCR/coreceptor complex. Experiments with transgenic and MHC-null mice have shown that coreceptor down-regulation and lineage commitment can occur stochastically in a manner that is independent of TCR specificity for MHC. Nevertheless, the positive selection of a given thymocyte is contingent on sustained expression of the coreceptor that is appropriate for the MHC specificity of its TCR. In most cases, loss of the required coreceptor blocks developmental progression and results in thymocyte apoptosis. CD4 expression is controlled by both positive and negative regulatory sequences embedded in the CD4 gene and it is likely that similar sequences regulate the CD8 gene. The down-regulation of coreceptor expression is coupled to a functional commitment which ensures that mature CD4+ T cells have a helper phenotype and CD8+ T cells have a cytotoxic phenotype. The molecular basis for this coupling and the identity of the switching mechanism which governs coreceptor regulation remain to be determined
— id: 15128, year: 1996, vol: 205, page: 89, stat: Journal Article,

The CD4 molecule. Roles in T lymphocytes and in HIV disease. Introduction
Littman DR
1996 ;205:v-x, Current topics in microbiology & immunology
— id: 15129, year: 1996, vol: 205, page: v, stat: Journal Article,

Broad host range of human T-cell leukemia virus type 1 demonstrated with an improved pseudotyping system
Sutton RE; Littman DR
1996 Oct;70(10):7322-7326, Journal of virology
Studies of human T-cell leukemia virus type 1 (HTLV-1) have been hampered by the difficulty of achieving high cell-free and cell-associated infectious titers. Current retroviral pseudotyping systems using the HTLV-1 envelope generate titers of less than 200 infectious particles per ml. We describe here an improved system for pseudotyping using a defective human immunodeficiency virus (HIV) type 1 genome in combination with HTLV-1 env in 293T producer cells. Introduction of additional copies of rev and treatment of cells with sodium butyrate resulted in a cell-associated titer of 10(5)/ml and cell-free titers of greater than 10(4)/ml . By using this system, we found that the host range of HTLV-1 is even greater than previously suspected. Earlier studies which assigned a chromosomal location for the HTLV-1 receptor may therefore reflect cell-to-cell variation in receptor number rather than the absolute presence or absence of a receptor. The generation of higher-titer HIV(HTLV-1) may facilitate identification of the cellular receptor and investigations of the pathophysiology of HTLV-1 infection
— id: 15125, year: 1996, vol: 70, page: 7322, stat: Journal Article,

Studies of HIV-1 envelope glycoprotein-mediated fusion using a simple fluorescence assay
Weiss CD; Barnett SW; Cacalano N; Killeen N; Littman DR; White JM
1996 Mar;10(3):241-246, AIDS
OBJECTIVE: To study HIV envelope glycoprotein (Env)-mediated entry using a sensitive fusion assay. DESIGN AND METHODS: CD4+ lymphocytes or T-cell lines were labelled with fluorescent cytoplasm or membrane markers. Fusion with Env-expressing adherent cells was monitored by observing dye transfer from CD4+ cells to Env cells. RESULTS: Cell-cell fusion began 20-30 min after co-cultivation at 37 degrees C. Pre-binding at 4 degrees C was observed not to decrease the lag phase before fusion. Cells expressing envelope glycoproteins from non-syncytium-inducing (NSI) HIV strains showed dye transfer between two cells without progression to syncytia. A glycosylphosphatidylinositol anchored Env was found to be incapable of mediating membrane fusion, as measured either by lipid or cytoplasm contents mixing. Primary mouse cells expressing human CD4 and mouse 3T3 cells stably expressing both human CD4 and human CD26 did not support fusion with our Env-expressing cells. CONCLUSIONS: Env-mediated cell-cell fusion is a relatively slow process, probably reflecting a multi-step process occurring after CD4 binding and requiring the transmembrane domain of gp41. Env proteins are able to mediate cell-cell fusion at least under some experimental conditions, indicating that lack of a syncytia phenotype does not rule out the possibility of fusion occurring between only two or a few cells
— id: 15126, year: 1996, vol: 10, page: 241, stat: Journal Article,

Inhibition of thymocyte negative selection by T cell receptor antagonist peptides
Williams O; Tanaka Y; Bix M; Murdjeva M; Littman DR; Kioussis D
1996 Mar;26(3):532-538, European journal of immunology
The T cell receptor (TCR) recognizes antigenic peptide presented by major histocompatibility complex (MHC) molecules. Analogs of antigenic peptides have been shown to inhibit antigen-specific T cell responses, a phenomenon described as TCR antagonism. We have examined the effect of a natural variant of an antigenic peptide and a synthetic peptide analog, on the responses of mature T cells and immature thymocytes from an alpha-beta TCR-transgenic mouse (F5), the TCR of which recognizes a nonamer peptide from the nucleoprotein (NP) of influenza virus in the context of the H-2Db MHC molecule. Both peptides were shown to antagonize specifically the T cells cytolytic response without being able directly to stimulate mature T cells from these transgenic mice. Furthermore, a negative selection assay in vitro was used to demonstrate for the first time that antagonistic peptides are capable of antagonizing thymocyte deletion induced by antigenic peptides. These data suggest that the final selection of a T cell could be the result of a balance between the positive and negative influences of endogenous peptide ligands
— id: 15127, year: 1996, vol: 26, page: 532, stat: Journal Article,

The cytoplasmic tail of CD4 is required for inhibition of human immunodeficiency virus type 1 replication by antibodies that bind to the immunoglobulin CDR3-like region in domain 1 of CD4
Benkirane M; Schmid-Antomarchi H; Littman DR; Hirn M; Rossi B; Devaux C
1995 Nov;69(11):6904-6910, Journal of virology
Monoclonal antibodies (MAb) directed against the immunoglobulin complementary determining region 3 (CDR3)-like region of the CD4 molecule inhibit human immunodeficiency virus type 1 (HIV-1) transcription. We report here data showing that the cytoplasmic tail of CD4 is required for such inhibition to be achieved. To this aim, we studied the effect of MAb 13B8-2 treatment on (i) HIV-1 production in A2.01 cells, which express different forms of the CD4 gene, (ii) Tat-induced HIV-1 promoter activation, and (iii) mitogen-activated protein kinase (MAPK) activation, which is induced in CD4-positive cells by HIV-1 cross-linking of CD4. Inhibition of HIV production by 13B8-2 MAb treatment was consistently observed in cells expressing wild-type CD4 and cells expressing a hybrid CD4-CD8 molecule (amino acids 1 to 177 of CD4 fused to the hinge, transmembrane, and cytoplasmic domains of CD8). However, no delay in HIV-1 production was observed in cells expressing a truncated CD4 which lacks the cytoplasmic domain (CD4.401). Chloramphenicol acetyltransferase assays demonstrated that Tat-dependent activation of the HIV-1 long terminal repeat promoter was inhibited by MAb 13B8-2 in A2.01/CD4 and A2.01/CD4-CD8 but not in A2.01/CD4.401 cells. Finally, we found that MAb 13B8-2 treatment inhibited the activation of MAPK induced in A2.01/CD4 and A2.01/CD4-CD8 following cross-linking of CD4 by HIV-1
— id: 15132, year: 1995, vol: 69, page: 6904, stat: Journal Article,

CD28-mediated costimulation in the absence of phosphatidylinositol 3-kinase association and activation
Crooks ME; Littman DR; Carter RH; Fearon DT; Weiss A; Stein PH
1995 Dec;15(12):6820-6828, Molecular & cellular biology
T-cell activation involves two distinct signal transduction pathways. Antigen-specific signaling events are initiated by T-cell receptor recognition of cognate peptide presented by major histocompatibility complex molecules. Costimulatory signals, which are required for optimal T-cell activation and for overcoming the induction of anergy, can be provided by the homodimeric T-cell glycoprotein CD28 through its interaction with the counterreceptors B7-1 and B7-2 on antigen-presenting cells. Ligation of CD28 results in its phosphorylation on tyrosines and the subsequent recruitment and activation of phosphatidylinositol 3-kinase (PI 3-kinase). It has been suggested that the induced association of CD28 and PI 3-kinase is required for costimulation. We report here that ligation of CD19, a heterologous B-cell receptor that also associates with and activates PI 3-kinase upon ligation, failed to costimulate interleukin-2 production. Moreover, pharmacological inhibition of PI 3-kinase activity failed to block costimulation mediated by CD28. By mutational analysis, we demonstrate that disruption of PI 3-kinase association with CD28 also did not abrogate costimulation. These results argue that PI 3-kinase association with CD28 is neither necessary nor sufficient for costimulation of interleukin-2 production. Finally, we identify specific amino acid residues required for CD28-mediated costimulatory activity
— id: 15131, year: 1995, vol: 15, page: 6820, stat: Journal Article,

Disrupted development of thymocytes expressing a transgenic TCR upon CD4 overexpression
Davis CB; Littman DR
1995 Dec;7(12):1977-1986, International immunology
CD4 assists in T cell recognition of peptides bound to MHC class II molecules by binding to a non-polymorphic region on class II and stabilizing TCR recognition of the peptide-class II complex. Overexpression of CD4 in transgenic mice expressing a class II-restricted TCR resulted in a dramatic loss of thymocytes that became evident soon after the TCR and CD4 were co-expressed. Both the thymus and lymph nodes of the double-transgenic mice had reduced numbers of CD4 lineage T cells. A large proportion of the remaining CD4 lineage T cells lost either the transgenic TCR alpha or beta chains. The double-transgenic mice continued to generate thymocytes and T cells that expressed the transgenic TCR, but these cells did not express endogenous CD4. Overexpression of CD4 thus severely disrupts the normal developmental pathway of these thymocytes, supporting a model in which avidity of the TCR complex for self class II molecules determines the outcome of thymocyte development
— id: 12705, year: 1995, vol: 7, page: 1977, stat: Journal Article,

Phorbol ester-induced down modulation of tailless CD4 receptors requires prior binding of gp120 and suggests a role for accessory molecules
Golding H; Dimitrov DS; Manischewitz J; Broder CC; Robinson J; Fabian S; Littman DR; Lapham CK
1995 Oct;69(10):6140-6148, Journal of virology
The entry of human immunodeficiency virus type 1 into cells proceeds via a fusion mechanism that is initiated by binding of the viral glycoprotein gp120-gp41 to its cellular receptor CD4. Species- and tissue-specific restrictions to viral entry suggested the participation of additional membrane components in the postbinding fusion events. In a previous study (H. Golding, J. Manischewitz, L. Vujcic, R. Blumenthal, and D. Dimitrov, J. Virol. 68:1962-1968, 1994), it was found that phorbol myristate acetate (PMA) inhibits human immunodeficiency virus type 1 envelope-mediated cell fusion by inducing down modulation of an accessory component(s) in the CD4-expressing cells. The fusion inhibition was seen in a variety of cells, including T-cell transfectants expressing engineered CD4 receptors (CD4.401 and CD4.CD8) which are not susceptible to down modulation by PMA treatment. In the current study, it was found that preincubation of A2.01.CD4.401 cells with soluble monomeric gp120 for 1 h at 37 degrees C primed them for PMA-induced down modulation (up to 70%) of the tailless CD4 receptors. The gp120-priming effect was temperature dependent, and the down modulation may have occurred via clathrin-coated pits. Importantly, nonhuman cell lines expressing tailless CD4 molecules did not down modulate their CD4 receptors under the same conditions. The gp120-dependent PMA-induced down modulation of tailless CD4 receptors could be efficiently blocked by the human monoclonal antibodies 48D and 17B, which bind with increased avidity to gp120 that was previously bound to CD4 (M. Thali, J. P. Moore, C. Furman, M. Charles, D. D. Ho, J. Robinson, and J. Sodroski, J. Virol. 67:3978-3988, 1993). These findings suggest that gp120 binding to cellular CD4 receptors induces conformational changes leading to association of the gp120-CD4 complexes with accessory transmembrane molecules that are susceptible to PMA-induced down modulation and can target the virions to clathrin-coated pits
— id: 15133, year: 1995, vol: 69, page: 6140, stat: Journal Article,

The function of the CD4 coreceptor in the development of T cells
Killeen N; Littman DR
1995 ;13(1):15-27, International reviews of immunology
T cells with helper activity can be found in mice that lack expression of the CD4 glycoprotein. The CD4 promoter is active in these cells; they respond to antigens presented by MHC class II molecules; they do not express CD8 and they do not depend on MHC class I for their development. By such criteria, these CD8- T cells resemble normal CD4+ helper T cells. The development of the helper lineage in CD4-null mice can be potentiated by expression of transgenes that encode either wild type CD4, or a deletion mutant of CD4 that lacks the cytoplasmic tail and therefore cannot interact with the tyrosine kinase p56lck. These observations suggest that CD4 is not absolutely required for the specification of the helper cell lineage. The role of the CD4 molecule in the development of T cells and possible mechanisms by which it achieves its functions are discussed
— id: 15134, year: 1995, vol: 13, page: 15, stat: Journal Article,

Altered T cell receptor signaling and disrupted T cell development in mice lacking Itk
Liao XC; Littman DR
1995 Dec;3(6):757-769, Immunity
Itk is a T cell protein tyrosine kinase (PTK) that, along with Btk and Tec, belongs to a family of cytoplasmic PTKs with N-terminal pleckstrin homology domains. Btk plays a critical role in B lymphocyte development. To determine whether Itk has an analogous role in T lymphocytes, we used gene targeting to prepare mice lacking expression of Itk. Such animals had decreased numbers of mature thymocytes, an effect most clearly observed in mice expressing T cell receptor (TCR) transgenes. Mature T cells from Itk-deficient mice had reduced proliferative responses to allogeneic MHC stimulation and to anti-TCR cross-linking, but responded normally to stimulation with phorbol ester plus ionomycin or with IL-2. These results provide genetic evidence that Itk is involved in T cell development and also suggest that Itk has an important role in proximal events in TCR-mediated signaling pathways
— id: 15130, year: 1995, vol: 3, page: 757, stat: Journal Article,

The kinase-dependent function of Lck in T-cell activation requires an intact site for tyrosine autophosphorylation
Xu H; Littman DR
1995 Sep 7;766:99-116, Annals of the New York Academy of Sciences
The cytoplasmic protein tyrosine kinase p56lck (Lck) has important signaling roles in T-cell development and activation. We have mutated the two known regulatory tyrosine residues of CD4-associated Lck and examined the effects on its kinase-dependent function in an antigen-specific CD4-dependent T-cell hybridoma. Substitution of phenylalanine for the negative regulatory tyrosine-505 within a CD4/Lck chimera resulted in a slightly increased response to antigen, whereas mutation of the major in vitro autophosphorylation site (tyrosine-394) completely abolished the kinase-dependent function of Lck. Even though its kinase activity was only slightly affected, the F394 mutant behaved similarly to a catalytically inactive chimeric protein. Cross-linking of the F505 mutant, but not of wild-type Lck or F394 mutants, resulted in tyrosine phosphorylation of multiple cellular proteins. Although the pattern of tyrosine phosphorylation resembled that observed upon T-cell receptor cross-linking, there was no induction of interleukin-2 synthesis upon cross-linking of the chimeric protein. These results suggest that the activity of the Lck kinase domain in vivo is controlled by dephosphorylation at the negative regulatory site and phosphorylation at the positive regulatory (autophosphorylation) site. Additionally, our data show that the specific kinase activity of Lck towards an artificial substrate need not correlate with its ability to phosphorylate cellular proteins or its biological function
— id: 57371, year: 1995, vol: 766, page: 99, stat: Journal Article,

A subset of CD4+ thymocytes selected by MHC class I molecules
Bendelac A; Killeen N; Littman DR; Schwartz RH
1994 Mar 25;263(5154):1774-1778, Science
To complete their maturation, most immature thymocytes depend on the simultaneous engagement of their antigen receptor [alpha beta T cell receptor (TCR)] and their CD4 or CD8 coreceptors with major histocompatibility complex class II or I ligands, respectively. However, a normal subset of mature alpha beta TCR+ thymocytes did not follow these rules. These thymocytes expressed NK1.1 and a restricted set of alpha beta TCRs that are intrinsically class I-reactive because their positive selection was class I-dependent but CD8-independent. These cells were CD4+ and CD4-8- but never CD8+, because the presence of CD8 caused negative selection. Thus, neither CD4 nor CD8 contributes signals that direct their maturation into the CD4+ and CD4-8- lineages
— id: 15141, year: 1994, vol: 263, page: 1774, stat: Journal Article,

Requirement for kinase activity of CD4-associated p56lck in antibody-triggered T cell signal transduction
Chu K; Littman DR
1994 Sep 30;269(39):24095-24101, Journal of biological chemistry
The lymphoid-specific Src family protein tyrosine kinase p56lck (Lck) is non-covalently associated with the cytoplasmic tail of CD4 and has an essential role in T cell activation. Engagement of ligand by the T cell antigen receptor (TCR) is followed by rapid tyrosine phosphorylation of several cellular proteins, including phospholipase C gamma 1 (PLC) and the TCR-associated CD3 zeta polypeptides. Tyrosine phosphorylation of PLC gamma 1 results in activation of PLC and subsequent phosphatidylinositol turnover. We have studied the effects of the CD4-associated Lck molecule on TCR-mediated activation of the protein tyrosine kinase (PTK) pathway in a murine T cell hybridoma. Antibodies against CD3 elicited the expected PTK activation, which was enhanced upon co-cross-linking of CD4. In contrast, anti-TCR-alpha beta antibodies had no effect on the PTK pathway unless CD4 was co-cross-linked. Antibody cross-linking of CD4 alone failed to induce the same pattern of tyrosine phosphorylation. Similar results were obtained when a chimeric protein consisting of the extracellular and transmembrane domains of CD4 linked to the intracellular Lck molecule was used in place of CD4. The tyrosine kinase activity of Lck was essential for the activity of the chimeric protein. Cross-linking of the CD4/Lck chimera to a CD8/zeta chimeric molecule also facilitated induction of the PTK pathway with anti-CD8 antibodies. Moreover, the interaction of the two chimeric proteins, either in vitro or in vivo, resulted in tyrosine phosphorylation of CD8/zeta. The effects of CD4/Lck on tyrosine phosphorylation and activation of PLC correlated well with the effects on PTK activation. Our results suggest that the Lck molecule positively regulates the TCR-coupled PTK pathway by phosphorylating tyrosines on the TCR-associated CD3 zeta polypeptides
— id: 15135, year: 1994, vol: 269, page: 24095, stat: Journal Article,

Disruption of T lymphocyte positive and negative selection in mice lacking the CD8 beta chain
Crooks ME; Littman DR
1994 Jul;1(4):277-285, Immunity
The CD4 and CD8 coreceptors have been shown to play significant roles in the differentiation and activation of helper and cytotoxic T lymphocytes (CTLs), respectively. Coordinate binding of coreceptor and T cell receptor (TCR) to the same major histocompatibility complex (MHC) molecule and coreceptor interaction with the tyrosine kinase p56lck are required for effective signaling. Whereas CD4 is a monomer, CD8 consists of either alpha alpha homodimers or alpha beta heterodimers. Signaling properties of CD8 have been ascribed to the alpha chain, which binds to both the MHC class I and to p56lck, respectively. To study CD8 beta specifically, we have generated mice defective in its expression. We observe a significant reduction in the numbers of CD8+ T cells, but these cells have normal CTL activity. By breeding CD8 beta null mice with animals expressing a class I-specific TCR transgene, we show that CD8 beta plays a significant role in both positive and negative selection of developing thymocytes
— id: 15137, year: 1994, vol: 1, page: 277, stat: Journal Article,

Thymocyte lineage commitment: is it instructed or stochastic?
Davis CB; Littman DR
1994 Apr;6(2):266-272, Current opinion in immunology
Thymocytes co-expressing the CD4 and CD8 co-receptors differentiate into mature T cells that express either CD4 or CD8 and have helper or cytotoxic functions, respectively. Recent studies indicate that commitment to the CD4+ or CD8+ lineages occurs stochastically, but retention of the appropriate co-receptor is required to complete development
— id: 15139, year: 1994, vol: 6, page: 266, stat: Journal Article,

Lymphocyte development in genetically manipulated mice
Killeen N; Littman DR
1994 Apr;1(2):123-133, Therapeutic immunology
— id: 15140, year: 1994, vol: 1, page: 123, stat: Journal Article,

Immunodeficiency viruses. Not enough sans Nef
Littman DR
1994 Jul 1;4(7):618-620, Current biology. CB
The nef genes of HIV and SIV are dispensable in vitro, but are essential for viral spread and disease progression in vivo. Nef-induced down-regulation of CD4, the viral receptor, may be the key to this requirement
— id: 15136, year: 1994, vol: 4, page: 618, stat: Journal Article,

Signal transduction during T cell development
Littman DR; Davis CB; Killeen N; Xu H
1994 ;365:63-71, Advances in experimental medicine & biology
— id: 15143, year: 1994, vol: 365, page: 63, stat: Journal Article,

CD4+ T-lymphocytes are not required for murine resistance to the human filarial parasite, Brugia malayi
Rajan TV; Nelson FK; Killeen N; Shultz LD; Yates JA; Bailis JM; Littman DR; Greiner DL
1994 Jun;78(4):352-360, Experimental parasitology
Immunocompetent mice are nonpermissive for the development and maturation of the human filarial parasite, Brugia malayi. We and others have shown that the absence of T-lymphocytes, alone or in combination with B-lymphocytes, renders mice permissive to infection. In a previous study, we showed that mice lacking CD8+ T-lymphocytes are also completely nonpermissive for B. malayi, indicating that CD8+ T-lymphocytes are not an obligate requirement for resistance. In the present study, we have examined the role of CD4+ T-lymphocytes in resistance to filarial infection using two experimental systems. In the first, we used an anti-CD4 monoclonal antibody to deplete CD4+ T-cells in vivo in immunocompetent BALB/c mice. In the second system, we used mutant mice in which the gene encoding the CD4 antigen had been disrupted by homologous recombination, resulting in a lack of CD4+ T-cells. Challenge of either the anti-CD4 antibody depleted BALB/c mice or CD4 knockout mice with B. malayi infective-stage larvae demonstrated that mice lacking CD4+ T-lymphocytes were resistant to infection. These data indicate that CD4+ T-cells are not an obligate requirement for murine resistance to B. malayi
— id: 15138, year: 1994, vol: 78, page: 352, stat: Journal Article,

A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development
Sawada S; Scarborough JD; Killeen N; Littman DR
1994 Jun 17;77(6):917-929, Cell
During development of T lymphocytes, differential regulation of expression of the CD4 and CD8 glycoproteins is coupled to the choice of one of two pathways of differentiation. Thymocytes that express both of these coreceptors commit to either the helper lineage, shutting off CD8, or the cytotoxic lineage, shutting off CD4. We have used transgenic mice to identify an intronic regulatory region that controls CD4 gene expression during development. This region selectively extinguishes transgene expression in CD4-CD8+, but not CD4+CD8- nor CD4+CD8+ T cells. It also represses gene expression in CD4-CD8- immature thymocytes, indicating that the CD4 gene is derepressed during differentiation from the CD4-CD8- to the CD4+CD8+ stage. The negative element(s) is both orientation and position independent and acts also on heterologous regulatory sequences. Its properties are functionally similar to those of silencers described in yeast and in Drosophila, suggesting that we have identified a developmentally regulated vertebrate transcriptional silencer
— id: 57506, year: 1994, vol: 77, page: 917, stat: Journal Article,

Signal transduction by lymphocyte antigen receptors
Weiss A; Littman DR
1994 Jan 28;76(2):263-274, Cell
Despite the differences in the antigens that they recognize and in the effector functions they carry out, B and T lymphocytes utilize remarkably similar signal transduction components to initiate responses. They both use oligomeric receptors that contain distinct recognition and signal transduction subunits. Antigen receptors on both cells interact with at least two distinct families of PTKs via common sequence motifs, ARAMs, in the cytoplasmic tails of their invariant chains, which have likely evolved from a common evolutionary precursor. Coreceptors appear to serve to increase the sensitivity of both of these receptor systems through events that influence ligand binding and signal transduction. The critical role of tyrosine phosphorylation of downstream signaling components, such as phospholipase C, is the net result of changes in the balance of the action of antigen receptor-regulated PTKs and PTPases. The identification of downstream effectors, including calcineurin and Ras, that regulate cellular responses, such as lymphokine gene expression, promises the future possibility of connecting the complex pathway from the plasma membrane to the nucleus in lymphocytes. Insight gained from studies of the signaling pathways downstream of TCR and BCR stimulation is likely to contribute significantly to future understanding of mechanisms responsible for lymphocyte differentiation and for the discrimination of self from nonself in developing and mature cells
— id: 15142, year: 1994, vol: 76, page: 263, stat: Journal Article,

Evidence for a stochastic mechanism in the differentiation of mature subsets of T lymphocytes [see comments]
Davis CB; Killeen N; Crooks ME; Raulet D; Littman DR
1993 Apr 23;73(2):237-247, Cell
Thymocytes that coexpress the CD4 and CD8 glycoproteins differentiate into mature CD4+ helper or CD8+ cytotoxic cells depending on whether their antigen receptors are specific for MHC class II or class I molecules, respectively. The mechanism of this decision process was investigated in mice whose T cell development was biased toward the class II-specific lineage. We found that constitutive expression of CD4 allows a developmentally arrested population of thymocytes that have mismatched class II-specific TCRs and the CD8 coreceptor to be rescued and to acquire a cytotoxic phenotype. This result is consistent with a two-step process of thymocyte maturation, in which there is stochastic down-regulation of either CD4 or CD8 and subsequent selection based on the ability of the TCR and remaining coreceptor to engage the same MHC molecule
— id: 15151, year: 1993, vol: 73, page: 237, stat: Journal Article,

Cell fusion mediated by interaction of a hybrid CD4.CD8 molecule with the human immunodeficiency virus type 1 envelope glycoprotein does occur after a long lag time
Golding H; Blumenthal R; Manischewitz J; Littman DR; Dimitrov DS
1993 Nov;67(11):6469-6475, Journal of virology
Several domains of CD4 have been suggested to play a critical role in events that follow its binding to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41). It has been reported previously that cells expressing a chimeric molecule consisting of the first 177 residues of human CD4 attached to residues from the hinge, transmembrane, and cytoplasmic domains of human CD8 did not form syncytia with HIV-1-infected cells (L. Poulin, L.A. Evans, S. Tang, A. Barboza, H. Legg, D.R. Littman, and J.A. Levy, J. Virol. 65: 4893-4901, 1991). In contrast, we found that the hybrid CD4.CD8 molecule expressed in human cells did render them susceptible to fusion with cells expressing HIV-1IIIB or HIV-1RF envelope glycoproteins encoded by vaccinia virus recombinants, but only after long lag times. The lag time of membrane fusion mediated by the hybrid CD4.CD8 molecule was fivefold longer than that for the wild-type CD4 molecule. However, the rate of binding to and the affinity of soluble gp120 for membrane-associated CD4.CD8 were the same as for CD4. Both molecules were laterally mobile, as determined by patching experiments. Coexpression of the CD4.CD8 chimera with wild-type CD4 did not lead to interference in fusion but had an additive effect. Therefore, the proximal membrane domains of CD4 play an important role in determining the kinetics of postbinding events leading to membrane fusion. We hypothesize that the long lag time is due to the inability of the CD4.CD8-gp120-gp41 complex to undergo the rapid conformational changes which occur during the fusion mediated by wild-type CD4
— id: 15144, year: 1993, vol: 67, page: 6469, stat: Journal Article,

CD4 function in thymocyte differentiation and T cell activation
Killeen N; Davis CB; Chu K; Crooks ME; Sawada S; Scarborough JD; Boyd KA; Stuart SG; Xu H; Littman DR
1993 Oct 29;342(1299):25-34, Philosophical transactions of the Royal Society of London. Series B. Biological sciences
The ectodomains of the T cell surface glycoproteins CD4 and CD8 bind to membrane-proximal domains of MHC class II and class I molecules, respectively, while both cytoplasmic domains interact with the protein tyrosine kinase (PTK) p56lck (lck) through a shared cysteine-containing motif. Function of CD4 and CD8 requires their binding to the same MHC molecule as that recognized by the T cell antigen receptor (TCR). In vitro studies indicate that CD4-associated lck functions even in the absence of kinase activity. In vivo experiments show that, whereas helper T cell development is impaired in CD4-deficient mice, high level expression of a transgenic CD4 that cannot bind lck rescues development of this T cell subset. These studies suggest that CD4 is an adhesion molecule whose localization is regulated through protein-protein interactions of the associated PTK and whose function is to increase the stability of the TCR signalling complex by binding to the relevant MHC. The function of CD4 in development has been further studied in the context of how double positive (CD4+CD8+) thymocytes mature into either CD4+ T cells with helper function and TCR specificity for class II or into CD8+ T cells with cytotoxic function and specificity for class I. Studies using CD4-transgenic mice indicate that development of single positive T cells involves stochastic downregulation of either CD4 or CD8, coupled to activation of a cytotoxic or helper program, respectively, and subsequent selection based on the ability of the TCR and remaining co-receptor to engage the same MHC molecule
— id: 15145, year: 1993, vol: 342, page: 25, stat: Journal Article,

Helper T-cell development in the absence of CD4-p56lck association
Killeen N; Littman DR
1993 Aug 19;364(6439):729-732, Nature
The CD4 and CD8 glycoproteins are expressed on helper and cytoxic T lymphocytes, respectively, and have important functions in the differentiation and activation of these cells. These molecules are thought to participate in signal transduction by binding to the same class II or class I major histocompatibility complex molecules that are engaged by the T-cell antigen receptor. The cytoplasmic domains of both CD4 and CD8 interact with the protein tyrosine kinase p56lck (refs 14-17), an essential participant in thymocyte maturation and T-cell activation. This interaction is required for effective in vitro responses to antigen, suggesting that signalling through p56lck is a major function of CD4 and CD8. Here we investigate the role of the CD4-p56lck interaction during T-lymphocyte development by expressing wild-type and truncated products of CD4 transgenes in mice that lack endogenous CD4 and hence have defective helper-cell development. We find that transgenic CD4, which cannot associate with p56lck, can nevertheless rescue the helper-cell lineage when overexpressed. This result indicates that the contribution of CD4 to lineage development need not involve signalling through p56lck, and provides insight into the general function of CD4 and CD8
— id: 15149, year: 1993, vol: 364, page: 729, stat: Journal Article,

Regulated expression of human CD4 rescues helper T cell development in mice lacking expression of endogenous CD4
Killeen N; Sawada S; Littman DR
1993 Apr;12(4):1547-1553, EMBO journal
During T cell development, precursor thymocytes that co-express the CD4 and CD8 glycoproteins give rise to mature progeny expressing one of these molecules to the exclusion of the other. Continued expression of only CD4 is the hallmark of mature helper T cells, whereas cytotoxic T cells express CD8 and extinguish CD4. The differentiation program that generates the two T cell subsets is likely to be intimately tied to regulation of expression of these cell surface molecules. We now describe the use of a murine CD4 enhancer in the generation of transgenic mice expressing physiologic levels of human CD4. The transgene is appropriately regulated during T cell development and includes the necessary cis-acting sequences for extinguishing expression in the CD8 lineage. Furthermore, in mice whose endogenous CD4 gene is inactivated, the transgenic human CD4 mediates rescue of the CD4 lineage and restoration of normal helper cell functions. The generation of these mice exemplifies a general approach for developing reliable animal models for the human immune system
— id: 15152, year: 1993, vol: 12, page: 1547, stat: Journal Article,

Helper T cells without CD4: control of leishmaniasis in CD4-deficient mice
Locksley RM; Reiner SL; Hatam F; Littman DR; Killeen N
1993 Sep 10;261(5127):1448-1451, Science
Expression of either the CD4 or CD8 glycoproteins discriminates two functionally distinct lineages of T lymphocytes. A null mutation in the gene encoding CD4 impairs the development of the helper cell lineage that is normally defined by CD4 expression. Infection of CD4-null mice with Leishmania has revealed a population of functional helper T cells that develops despite the absence of CD4. These CD8- alpha beta T cell receptor+ T cells are major histocompatibility complex class II-restricted and produce interferon-gamma when challenged with parasite antigens. These results indicate that T lymphocyte lineage commitment and peripheral function need not depend on the function of CD4
— id: 15146, year: 1993, vol: 261, page: 1448, stat: Journal Article,

A heterodimer of HEB and an E12-related protein interacts with the CD4 enhancer and regulates its activity in T-cell lines
Sawada S; Littman DR
1993 Sep;13(9):5620-5628, Molecular & cellular biology
A T-lymphocyte-specific enhancer located 13 kb upstream of the murine CD4 gene was recently shown to be required for the developmentally regulated expression of CD4. We have previously identified three nuclear protein binding sites in this enhancer; one of these sites, CD4-3, is essential for expression and contains two E-box core motifs (CANNTG) adjacent to each other in the sequence TAACAGGTGTCAGCTGGT. In electrophoretic mobility shift assays using the CD4-3 oligonucleotide as a probe, three nuclear protein complexes, termed CD4-3A, -B, and -C, were detected with nuclear extracts from T-cell lines. CD4-3A, which involves nuclear protein binding to the 5' E-box, was detected only with nuclear extracts from lymphoid cells. Specific antisera were used to show that the CD4-3A complex contains a heterodimer or heterooligomer of basic helix-loop-helix transcriptional factors, E12 or a related factor and HEB, which is expressed predominantly in thymus. Consistent with this finding, in vitro-translated E12 and HEB proteins, as homodimers or heterodimers, bound preferentially to the 5' E-box. Point mutations in the 5' E-box, but not in the 3' E-box, abolished CD4 enhancer activity. Furthermore, overexpression of Id, a protein that forms inactive heterodimers with E12/E47, blocked CD4 enhancer activity in T cells. These results suggest that a heterodimer composed of HEB and E12 or a closely related protein plays a critical role in CD4 enhancer function by interacting with the 5' E-box motif of the CD4-3 site in vivo
— id: 15147, year: 1993, vol: 13, page: 5620, stat: Journal Article,

A kinase-independent function of Lck in potentiating antigen-specific T cell activation
Xu H; Littman DR
1993 Aug 27;74(4):633-643, Cell
The lymphocyte-specific cytoplasmic protein-tyrosine kinase p56lck (Lck) is essential for T cell development and activation. Its association with the co-receptor molecules, CD4 and CD8, is required for potentiation of antigen-specific signals through the T cell antigen receptor. To study the mechanism of action of Lck, hybrid molecules consisting of the extracellular and transmembrane domains of CD4 fused to Lck or other Src family kinases were analyzed in an antigen-specific, CD4-dependent T cell hybridoma. Surprisingly, a chimera with a deletion of the Lck kinase domain was more active than the full-length protein. In contrast, point mutations in residues required for SH2 or kinase function resulted in moderately decreased activity, while a combination of these mutations rendered the chimera largely inactive. Different domains of CD4-associated Lck therefore have distinct functions that can independently contribute to T cell activation
— id: 15148, year: 1993, vol: 74, page: 633, stat: Journal Article,

Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein increases env incorporation into particles and fusogenicity and infectivity
Zingler K; Littman DR
1993 May;67(5):2824-2831, Journal of virology
Growth of macaque simian immunodeficiency virus (SIVmac) in certain cloned human T-cell lines, such as HUT.78, selects for isolates containing a premature stop codon within the cytoplasmic domain of the transmembrane envelope glycoprotein. In contrast, propagation of virus in macaques or in their cultured T cells favors replication of virus containing the full-length envelope glycoprotein. To elucidate the causes of this phenomenon, we used a human immunodeficiency virus pseudotyping system to assess the effects on infectivity of the cytoplasmic domains of envelope glycoproteins obtained from SIVmac1A11 and SIVmac239. These envelopes contain truncated and full-length cytoplasmic domains, respectively. By analyzing human immunodeficiency virus particles containing selectable genes pseudotyped with each glycoprotein or with chimeric derivatives, we found that truncation of the cytoplasmic domain resulted in a significant advantage in viral entry into HUT.78 T cells and CD4+ U87.MG glial cells. Truncation of the cytoplasmic domain significantly enhanced both envelope density on particles and envelope-mediated cell-to-cell fusion. It is likely that one or both of these effects contribute to the observed differences in infectivity and to the selection of virions with short cytoplasmic tails in human T cells
— id: 15150, year: 1993, vol: 67, page: 2824, stat: Journal Article,

Requirement for CD8-major histocompatibility complex class I interaction in positive and negative selection of developing T cells
Killeen N; Moriarty A; Teh HS; Littman DR
1992 Jul 1;176(1):89-97, Journal of experimental medicine
The interaction of the T cell surface glycoprotein CD8 with major histocompatibility complex (MHC) class I molecules on target cells is required for effective T cell activation. Mutations in the alpha 3 domain of the MHC class I molecule can disrupt binding to CD8, yet leave antigen presentation unaffected. Here we show that such a mutation can interfere with positive and negative selection of T cells bearing T cell receptors (TCRs) that interact specifically with the mutant class I molecule. Autoreactive T cells in male mice expressing a transgenic TCR specific for the male antigen H-Y and H-2Db were not deleted in the context of a transgenic Db molecule bearing a mutation at residue 227. Similarly, CD8+ cells were not positively selected in female mice expressing both the TCR and mutant class I transgenes. Endogenous MHC class I molecules were competent to bind CD8, but were unable to rescue the defect, indicating a requirement for coordinate recognition of antigen/MHC by a complex of the TCR and CD8 coreceptor for both positive and negative selection of thymocytes
— id: 15155, year: 1992, vol: 176, page: 89, stat: Journal Article,

Development and function of T cells in mice with a disrupted CD2 gene
Killeen N; Stuart SG; Littman DR
1992 Dec;11(12):4329-4336, EMBO journal
CD2 is a T cell surface glycoprotein that mediates cellular adhesion and can participate in signal transduction. It is expressed early in thymocyte ontogeny and consequently has been proposed to participate in T cell development. To study the in vivo function of CD2, the murine gene was inactivated using the technique of homologous recombination in embryonic stem cells. Homozygous mutant mice are healthy and have an apparently normal complement of lymphocytes. They mount effective immune responses similar to those of wild type controls. In particular, the generation and function of cytotoxic T cells was found to be normal as was the production of antibodies following immunization. Selection of thymocytes expressing either MHC class I- or class II-restricted transgenic T cell receptors was also grossly normal in the absence of CD2. Thus, CD2 may be dispensable for the development and function of T cells. Within the context of other targetted mutations, these mice should be useful in defining the precise roles of various cell surface molecules involved in T cell responses
— id: 15153, year: 1992, vol: 11, page: 4329, stat: Journal Article,

Packaging system for rapid production of murine leukemia virus vectors with variable tropism
Landau NR; Littman DR
1992 Aug;66(8):5110-5113, Journal of virology
A method for rapidly producing helper-free murine leukemia virus (MLV) without using packaging cell lines is described. Viruses bearing ecotropic or amphotropic MLV or Rous sarcoma virus envelope glycoprotein and containing various retroviral vector genomes have been prepared with titers 30 to 40-fold higher than those produced by transient transfection of standard packaging cells. This system can be used to alter the cellular tropism of MLV by incorporating other envelope glycoproteins and to prepare retroviral vector stocks without establishing stable producer cell lines. This method will be particularly useful for preparing viruses that encode toxic proteins and for the rapid analysis of panels of mutant envelope glycoproteins
— id: 15154, year: 1992, vol: 66, page: 5110, stat: Journal Article,

Analysis of mutations in the V3 domain of gp160 that affect fusion and infectivity
Page KA; Stearns SM; Littman DR
1992 Jan;66(1):524-533, Journal of virology
The third hypervariable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been proposed to play an important role in mediating viral entry. Antibodies to the V3 domain block HIV-1 infection but not virus binding to CD4. At the center of the V3 domain is a relatively conserved sequence of amino acids, GPGRA. It has previously been shown that mutation of some of these amino acids reduced the ability of gp160 expressed on the surface of cells to induce fusion with CD4-bearing cells. In order to analyze the role of V3 domain sequences in mediating HIV entry, we introduced several amino acid substitution mutations in the GPGRA sequence of gp160 derived from HIV-1 strain HXB2 and in the analogous sequence of strain SF33, GPGKV. Virus was generated by cotransfecting the env constructs and a selectable env-negative HIV vector, HIV-gpt. When complemented with a retrovirus env gene, infectious virus capable of a single round of replication was produced. The viral particles produced were analyzed biochemically for core and envelope proteins and for infectious titer. The transfected envs were also analyzed for ability to bind to CD4 and mediate cell fusion. Several of the amino acid substitutions resulted in moderate to severe decreases in virus infectivity and fusion activity. Envelope glycoprotein assembly onto particles and CD4 binding were not affected. These results provide evidence that V3 sequences are involved in mediating the fusion step of HIV-1 entry
— id: 15158, year: 1992, vol: 66, page: 524, stat: Journal Article,

The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits
Pelchen-Matthews A; Boulet I; Littman DR; Fagard R; Marsh M
1992 Apr;117(2):279-290, Journal of cell biology
The lymphocyte glycoprotein CD4 is constitutively internalized and recycled in nonlymphoid cells, but is excluded from the endocytic pathway in lymphocytic cells (Pelchen-Matthews, A., J. E. Armes, G. Griffiths, and M. Marsh. 1991. J. Exp. Med. 173: 575-587). Inhibition of CD4 endocytosis is dependent on CD4 expressing an intact cytoplasmic domain and is only observed in cells where CD4 can interact with the protein tyrosine kinase p56lck, a member of the src gene family. We have expressed p56lck, p60c-src, or chimeras of the two proteins in CD4-transfected NIH-3T3 or HeLa cells. Immunoprecipitation of CD4 and in vitro kinase assays showed that p56lck and the lck/src chimera, which contains the NH2 terminus of p56lck, can associate with CD4. In contrast, p60c-src and the src/lck chimera, which has the NH2 terminus of p60c-src, do not associate with CD4. Endocytosis assays using radioiodinated anti-CD4 monoclonal antibodies demonstrated that coexpression of CD4 with p56lck, but not with p60c-src, inhibited CD4 endocytosis, and that the extent of the inhibition depended directly on the relative levels of CD4 and p56lck expressed. The uptake of mutant CD4 molecules which cannot interact with p56lck was not affected. Measurement of the fluid-phase endocytosis of HRP or the internalization of transferrin indicated that the effect of p56lck was specific for CD4, and did not extend to other receptor-mediated or fluid-phase endocytic processes. Immunogold labeling of CD4 at the cell surface and observation by electron microscopy demonstrated directly that p56lck inhibits CD4 endocytosis by preventing its entry into coated pits
— id: 15156, year: 1992, vol: 117, page: 279, stat: Journal Article,

Differential involvement of protein tyrosine kinases p56lck and p59fyn in T cell development
van Oers NS; Garvin AM; Cooke MP; Davis CB; Littman DR; Perlmutter RM; Teh HS
1992 ;323:89-99, Advances in experimental medicine & biology
— id: 15159, year: 1992, vol: 323, page: 89, stat: Journal Article,

Disruption of CD8-dependent negative and positive selection of thymocytes is correlated with a decreased association between CD8 and the protein tyrosine kinase, p56lck
Van Oers NS; Garvin AM; Davis CB; Forbush KA; Carlow DA; Littman DR; Perlmutter RM; Teh HS
1992 Mar;22(3):735-743, European journal of immunology
The CD4 and CD8 coreceptor molecules on immature thymocytes participate in T cell repertoire selection. To examine more definitively the role of CD4 and CD8 in the negative and positive selection of immature thymocytes, we generated transgenic mice with elevated surface CD4 expression and mated them with mice expressing a transgenic T cell receptor. Augmented CD4 expression was found to markedly alter CD8-dependent negative and positive selection of T cells specific for the male (H-Y) antigen presented by H-2Db major histocompatibility complex class I molecules. Moreover, the cytoplasmic tail of CD4 was essential for effecting these alterations, since the overexpression of tailless CD4 molecules failed to influence the outcome of CD8-dependent selection. The inhibition of positive and negative selection in double-transgenic mice expressing the full-length CD4 molecule was associated with a decreased interaction between the protein tyrosine kinase p56lck and CD8. These results strongly implicate p56lck in T cell repertoire selection
— id: 15157, year: 1992, vol: 22, page: 735, stat: Journal Article,

Requirement for association of p56lck with CD4 in antigen-specific signal transduction in T cells
Glaichenhaus N; Shastri N; Littman DR; Turner JM
1991 Feb 8;64(3):511-520, Cell
The T cell-specific transmembrane glycoprotein CD4 interacts with class II MHC molecules via its external domain and is associated with tyrosine kinase p56lck via a cysteine motif in its cytoplasmic domain. We have assessed the ability of CD4 to synergize with the antigen-specific T cell receptor (TCR) for induction of transmembrane signals that result in lymphokine production. Mutant CD4 molecules were introduced into T cells that lacked endogenous CD4 but expressed TCRs specific for lysozyme peptides or the superantigen SEA bound to Ab or Abm12 class II MHC molecules. With either ligand, T cell activation occurred only when CD4 was associated with p56lck. These results demonstrate that residues within the cytoplasmic domain of CD4 are required for its coreceptor function in TCR-mediated signal transduction and strongly support the notion that the association of CD4 with p56lck is critical in this process
— id: 15164, year: 1991, vol: 64, page: 511, stat: Journal Article,

Glycosylphosphatidylinositol-anchored CD4/Thy-1 chimeric molecules serve as human immunodeficiency virus receptors in human, but not mouse, cells and are modulated by gangliosides
Jasin M; Page KA; Littman DR
1991 Jan;65(1):440-444, Journal of virology
Human and mouse cell lines that expressed a CD4/Thy-1 fusion protein on the cell surface were constructed and tested for the capacity to be infected with human immunodeficiency virus. The human cell lines, in contrast to the mouse line, were infectable. The CD4/Thy-1 fusion, which is anchored to the membrane by a glycosylphosphatidylinositol tail rather than a peptide linkage, can therefore serve as a human immunodeficiency virus receptor. In addition, this molecule, like CD4, is down-modulated in its cell surface expression by exogenous gangliosides
— id: 15167, year: 1991, vol: 65, page: 440, stat: Journal Article,

Pseudotyping with human T-cell leukemia virus type I broadens the human immunodeficiency virus host range
Landau NR; Page KA; Littman DR
1991 Jan;65(1):162-169, Journal of virology
Several epidemiologic and clinical studies suggest that patients coinfected with human immunodeficiency virus (HIV), the primary etiologic agent in AIDS, and other viruses, such as cytomegalovirus or human T-cell leukemia virus (HTLV), have a more severe clinical course than those infected with HIV alone. Cells infected with two viruses can, in some cases, give rise to phenotypically mixed virions with altered or broadened cell tropism and could therefore account for some of these findings. Such pseudotypes could alter the course of disease by infecting more tissues than are normally infected by HIV. We show here that HIV type 1 (HIV-1) efficiently incorporates the HTLV type I (HTLV-I) envelope glycoprotein and that both HIV-1 and HTLV-II accept other widely divergent envelope glycoproteins to form infectious pseudotype viruses whose cellular tropisms and relative abilities to be transmitted by cell-free virions or by cell contact are determined by the heterologous envelope. We also show that the mechanism by which virions incorporate heterologous envelope glycoproteins is independent of the presence of the homologous glycoprotein or heterologous gag proteins. These results may have important implications for the mechanism of HIV pathogenesis
— id: 15166, year: 1991, vol: 65, page: 162, stat: Journal Article,

BCR first exon sequences specifically activate the BCR/ABL tyrosine kinase oncogene of Philadelphia chromosome-positive human leukemias
Muller AJ; Young JC; Pendergast AM; Pondel M; Landau NR; Littman DR; Witte ON
1991 Apr;11(4):1785-1792, Molecular & cellular biology
The c-abl proto-oncogene encodes a cytoplasmic tyrosine kinase which is homologous to the src gene product in its kinase domain and in the upstream kinase regulatory domains SH2 (src homology region 2) and SH3 (src homology region 3). The murine v-abl oncogene product has lost the SH3 domain as a consequence of N-terminal fusion of gag sequences. Deletion of the SH3 domain is sufficient to render the murine c-abl proto-oncogene product transforming when myristylated N-terminal membrane localization sequences are also present. In contrast, the human BCR/ABL oncogene of the Philadelphia chromosome translocation has an intact SH3 domain and its product is not myristylated at the N terminus. To analyze the contribution of BCR-encoded sequences to BCR/ABL-mediated transformation, the effects of a series of deletions and substitutions were assessed in fibroblast and hematopoietic-cell transformation assays. BCR first-exon sequences specifically potentiate transformation and tyrosine kinase activation when they are fused to the second exon of otherwise intact c-ABL. This suggests that BCR-encoded sequences specifically interfere with negative regulation of the ABL-encoded tyrosine kinase, which would represent a novel mechanism for the activation of nonreceptor tyrosine kinase-encoding proto-oncogenes
— id: 15163, year: 1991, vol: 11, page: 1785, stat: Journal Article,

Several CD4 domains can play a role in human immunodeficiency virus infection in cells
Poulin L; Evans LA; Tang SB; Barboza A; Legg H; Littman DR; Levy JA
1991 Sep;65(9):4893-4901, Journal of virology
The human immunodefiency virus (HIV) uses the human CD4 glycoprotein as a receptor for infection of susceptible cells. Cells expressing a series of mutated forms of the CD4 gene have shown a variability in their ability to support replication of three HIV type 1 (HIV-1) and three HIV-2 strains. Moreover, when different stages of virus production were examined by a variety of assays, a consistent delay was observed in all cell lines containing CD4 mutants compared with those with intact full-length CD4. Cells expressing the CD4.415 mutant (modified at the serine 415 corresponding to a phosphorylation site of the cytoplasmic domain) showed only a minimal effect on virus replication. Cells expressing CD4.403 and CD4.401 mutants (lacking the whole cytoplasmic domain) manifested a moderate delay in production of virus progeny. The most substantial effect on HIV replication was observed in cells expressing a chimeric hybrid containing sequences corresponding to the first 177 residues of the N-terminal CD4 fused to CD8 sequences encoding the hinge, transmembrane, and cytoplasmic domains of the human CD8. Furthermore, in a cell-to-cell contact assay, fusion was absent when the CD4 proximal membrane domain was replaced by the CD8 counterpart. In addition, a strong correlation between the down-modulation of the surface CD4 and HIV expression was observed. These observations suggest that in addition to the known binding region, other domains of CD4 could play an important role in regulating HIV entry of cells
— id: 15162, year: 1991, vol: 65, page: 4893, stat: Journal Article,

Identification and characterization of a T-cell-specific enhancer adjacent to the murine CD4 gene
Sawada S; Littman DR
1991 Nov;11(11):5506-5515, Molecular & cellular biology
Expression of the CD4 and CD8 glycoproteins is a tightly regulated process tied to the maturation of functionally distinct classes of thymocytes. Therefore, understanding of the mechanism of expression of the genes encoding CD4 and CD8 is likely to yield important insight into regulation of the differentiated functions of T cells. Here, we report the identification of a T-cell-specific enhancer in a DNase I-hypersensitive region about 13 kb 5' of the transcription initiation site of the murine CD4 gene. Within the minimal enhancer element, at least three nuclear protein binding sites were identified by DNase I footprint analysis. One site contains the consensus motif for TCF-1 alpha/LEF-1, a recently identified HMG box transcription factor primarily expressed in pre-B and T cells. By Southwestern (DNA-protein) blotting and binding competition analyses, the protein binding to this site was found to be indistinguishable from TCF-1 alpha/LEF-1. Mutagenesis of this site resulted in loss of factor binding but had a relatively minor effect on enhancer activity. In contrast, mutations in another site, containing two consensus binding motifs for basic helix-loop-helix proteins, abolished factor binding and dramatically reduced enhancer activity. None of the protein binding sites had activity on its own, suggesting that the CD4 enhancer requires the interaction of multiple regulatory sites
— id: 15160, year: 1991, vol: 11, page: 5506, stat: Journal Article,

Participation of CD4 coreceptor molecules in T-cell repertoire selection
Teh HS; Garvin AM; Forbush KA; Carlow DA; Davis CB; Littman DR; Perlmutter RM
1991 Jan 17;349(6306):241-243, Nature
During thymocyte development, progenitor cells bearing both CD4 and CD8 coreceptor molecules mature into functional T lymphocytes that express these proteins in a mutually exclusive way. Although T-cell specificity is determined primarily by the structure of the T-cell antigen receptor (TCR) heterodimer, a developmentally regulated process acts to ensure that cells bearing class II-restricted TCRs are CD4+ and those bearing class I-restricted TCRs express only CD8. To investigate this maturation process, we have engineered transgenic mice in which CD4 is expressed in all thymocyte subsets and in all peripheral T cells. Peripheral CD4+8+ T lymphocytes from these mice react with both class I and class II alloantigens. Moreover, expression of the CD4 transgene disrupts the positive selection of doubly transgenic thymocytes bearing a class I-restricted TCR specific for the male (H-Y) antigen. Hence the CD4 coreceptor participates directly in T-cell repertoire selection
— id: 15165, year: 1991, vol: 349, page: 241, stat: Journal Article,

The thymus leukemia antigen binds human and mouse CD8
Teitell M; Mescher MF; Olson CA; Littman DR; Kronenberg M
1991 Nov 1;174(5):1131-1138, Journal of experimental medicine
The thymus leukemia antigen (TLA) is a class Ib, or 'nonclassical' class I molecule, one of several encoded within the Tla locus of the mouse major histocompatibility complex (MHC). It structurally resembles the H-2K, D, and L class I transplantation antigens, which present processed peptides to cytotoxic T lymphocytes (CTLs). Although their function(s) are unknown, there has been recent speculation concerning the possibility that class Ib molecules may present antigens to T cells that express gamma delta T cell antigen receptors (TCRs). In this report, using both a cell-cell adhesion assay and adhesion of T lymphocyte clones to purified plate-bound TLA, we provide evidence that TLA can bind to both human and mouse CD8. We also show that a chimeric class I molecule containing the peptide antigen binding site of Ld and the alpha 3 domain, transmembrane, and cytoplasmic segments of TLA, can support a CD8-dependent immune response by CTLs. These results demonstrate for the first time binding of a class Ib molecule to CD8 with a functional outcome, as is observed for the class I transplantation antigens. The capacity to interact with CD8 has been conserved despite the extensive sequence divergence of TLA in the peptide antigen binding site, suggesting this interaction is highly significant. TLA is expressed by epithelial cells in the mouse small intestine. As these epithelial cells are in close contact with intestinal intraepithelial lymphocytes that are nearly all CD8+, and many of which express the gamma delta TCR, the data are consistent with the hypothesis that TLA is involved in antigen presentation, perhaps to gamma delta-positive lymphocytes in this site
— id: 15161, year: 1991, vol: 174, page: 1131, stat: Journal Article,

Analysis of the site in CD4 that binds to the HIV envelope glycoprotein
Brodsky MH; Warton M; Myers RM; Littman DR
1990 Apr 15;144(8):3078-3086, Journal of immunology
The first step in infection of human mononuclear cells with HIV involves the high affinity binding of the viral envelope glycoprotein, gp120, to the cell-surface receptor, CD4. To gain a better understanding of the molecular basis of this interaction, we have analyzed the ability of gp120 to bind to a panel of 40 mutant CD4 proteins containing single or double amino acid substitutions. In addition, the binding of several anti-CD4 mAb to the mutant CD4 proteins was measured. These mAb were chosen on the basis of the previous demonstration that they bind to epitopes in CD4 adjacent to the gp120-binding site. This analysis permits discrimination between mutations that probably cause localized conformational changes and those that alter residues likely to make direct contact with gp120 and with the mAb. Our results indicate that gp120 from two different strains of HIV binds to a larger region of the CD4 protein than previously described. The data has also been used to map the epitopes of mAb previously identified as anti-idiotype vaccine candidates. The results have important implications for the development of CD4-based therapies for AIDS
— id: 15170, year: 1990, vol: 144, page: 3078, stat: Journal Article,

Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity
Page KA; Landau NR; Littman DR
1990 Nov;64(11):5270-5276, Journal of virology
We constructed a recombinant human immunodeficiency virus (HIV) vector to facilitate studies of virus infectivity. A drug resistance gene was inserted into a gp160- HIV proviral genome such that it could be packaged into HIV virions. The HIV genome was rendered replication defective by deletion of sequences encoding gp160 and insertion of a gpt gene with a simian virus 40 promoter at the deletion site. Cotransfection of the envelope-deficient genome with a gp160 expression vector resulted in packaging of the defective HIV-gpt genome into infectious virions. The drug resistance gene was transmitted and expressed upon infection of susceptible cells, enabling their selection in mycophenolic acid. This system provides a quantitative measure of HIV infection, since each successful infection event leads to the growth of a drug-resistant colony. The HIV-gpt virus produced was tropic for CD4+ human cells and was blocked by soluble CD4. In the absence of gp160, noninfectious HIV particles were efficiently produced by cells transfected with the HIV-gpt genome. These particles packaged HIV genomic RNA and migrated to the same density as gp160-containing virions in a sucrose gradient. This demonstrates that HIV virion formation is not dependent on the presence of a viral envelope glycoprotein. Expression of a murine leukemia virus amphotropic envelope gene in cells transfected with HIV-gpt resulted in the production of virus capable of infecting both human and murine cells. These results indicate that HIV can incorporate envelope glycoproteins other than gp160 onto particles and that this can lead to altered host range. Like HIV type 1 and vesicular stomatitis virus(HIV) pseudotypes, gp-160+ HIV-gpt did not infect murine NIH 3T3 cells that bear human CD4, confirming that these cells are blocked at an early stage of HIV infection
— id: 15168, year: 1990, vol: 64, page: 5270, stat: Journal Article,

A binding site for the T-cell co-receptor CD8 on the alpha 3 domain of HLA-A2
Salter RD; Benjamin RJ; Wesley PK; Buxton SE; Garrett TP; Clayberger C; Krensky AM; Norment AM; Littman DR; Parham P
1990 May 3;345(6270):41-46, Nature
Adhesion measurements between CD8 and 48 point mutants of HLA-A2.1 show that the CD8 alpha-chain binds to the alpha 3 domain of HLA-A2.1. Three clusters of alpha 3 residues contribute to the binding, with an exposed, negatively charged loop (residues 223-229) playing a dominant role. CD8 binding correlates with cytotoxic T-cell recognition and sensitivity to inhibition by anti-CD8 antibodies. Impaired alloreactive T-cell recognition of an HLA-A2.1 mutant with reduced affinity for CD8 is not restored by functional CD8 binding sites on an antigenically irrelevant class I molecule. Therefore, complexes of CD8 and the T-cell receptor bound to the same class I major histocompatibility complex molecule seem to be necessary for T-cell activation
— id: 15169, year: 1990, vol: 345, page: 41, stat: Journal Article,

Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs
Turner JM; Brodsky MH; Irving BA; Levin SD; Perlmutter RM; Littman DR
1990 Mar 9;60(5):755-765, Cell
p56lck, a lymphocyte-specific member of the src family of cytoplasmic protein-tyrosine kinases, is associated noncovalently with the cell surface glycoproteins CD4 and CD8, which are expressed on functionally distinct subpopulations of T cells. Using transient coexpression of p56lck with CD4 or CD8 alpha in COS-7 cells, we show that the unique N-terminal region of p56lck binds to the membrane-proximal 10 and 28 cytoplasmic residues of CD8 alpha and CD4, respectively. Two cysteine residues in each of the critical sequences in CD4, CD8 alpha, and p56lck are required for association. Our results suggest a novel role for cysteine-mediated interactions between unrelated proteins and provide a model for the association of other src-like cytoplasmic kinases with transmembrane proteins
— id: 15171, year: 1990, vol: 60, page: 755, stat: Journal Article,

Role of cell-to-cell interactions in T lymphocyte development and activation
Littman DR
1989 Oct;1(5):920-928, Current opinion in cell biology
— id: 15172, year: 1989, vol: 1, page: 920, stat: Journal Article,

Alternatively spliced mRNA encodes a secreted form of human CD8 alpha. Characterization of the human CD8 alpha gene
Norment AM; Lonberg N; Lacy E; Littman DR
1989 May 1;142(9):3312-3319, Journal of immunology
We have determined the organization and nucleotide sequence of the gene encoding the human T cell surface glycoprotein CD8 alpha. This gene spans approximately 8 kb and is organized into six exons which encode separate functional domains of the protein. Exon 1 encodes the 5' untranslated region and leader peptide, exon II the Ig V-like region, exon III the hinge-like region, exon IV the transmembrane domain, and exons V and VI the cytoplasmic tail. Alternative splicing that excludes nucleotide sequences from exon IV results in a transcript which encodes a secreted form of the protein. This transcript accounts for approximately 15% of the total CD8 alpha mRNA in human T cell leukemia lines and in normal human tissues. Secreted CD8 alpha protein can be detected in culture supernatants of T cell leukemia lines and PHA-stimulated PBMC by immunoprecipitation with the anti-CD8 alpha mAb OKT8 or with a polyclonal rabbit antiserum specific for the 28 amino acid cytoplasmic domain of CD8 alpha. The secreted CD8 alpha protein forms homodimers; when analyzed by SDS-PAGE, the protein migrates with an apparent molecular mass of 27 or 54 kDa under reducing or non-reducing conditions, respectively. Human secreted CD8 alpha may serve an immunoregulatory role for the interactions of T cells with their targets in vivo
— id: 15173, year: 1989, vol: 142, page: 3312, stat: Journal Article,

Diversity of class I HLA molecules: functional and evolutionary interactions with T cells
Parham P; Benjamin RJ; Chen BP; Clayberger C; Ennis PD; Krensky AM; Lawlor DA; Littman DR; Norment AM; Orr HT; et al
1989 ;54 Pt 1:529-543, Cold Spring Harbor symposia on quantitative biology
— id: 15176, year: 1989, vol: 54 Pt 1, page: 529, stat: Journal Article,

Polymorphism in the alpha 3 domain of HLA-A molecules affects binding to CD8
Salter RD; Norment AM; Chen BP; Clayberger C; Krensky AM; Littman DR; Parham P
1989 Mar 23;338(6213):345-347, Nature
Cytotoxic T lymphocytes (CTL) expressing the CD8 glycoprotein recognize peptide antigens presented by class I major histocompatibility complex (MHC) molecules. This correlation and the absence of CD8 polymorphism led to the hypothesis that CD8 binds to a conserved site of class I MHC molecules. Using a cell-cell binding assay we previously demonstrated specific interaction between human class I MHC (HLA-A,B,C) molecules and CD8. Subsequent analysis of the products of 17 HLA-A,B alleles revealed a natural polymorphism for CD8 binding in the human population. Two molecules, HLA-Aw68.1 and HLA-Aw68.2, which do not bind CD8, have a valine residue at position 245 whereas all other HLA-A,B,C molecules have alanine. Site-directed mutagenesis shows that this single substitution in the alpha 3 domain is responsible for the CD8 binding phenotype and also affects recognition by alloreactive and influenza-specific CTL. Our results indicate that CD8 binds to the alpha 3 domain of class I MHC molecules
— id: 15174, year: 1989, vol: 338, page: 345, stat: Journal Article,

Viral receptors of the immunoglobulin superfamily
White JM; Littman DR
1989 Mar 10;56(5):725-728, Cell
— id: 15175, year: 1989, vol: 56, page: 725, stat: Journal Article,

Internalization of the human immunodeficiency virus does not require the cytoplasmic domain of CD4
Bedinger P; Moriarty A; von Borstel RC 2d; Donovan NJ; Steimer KS; Littman DR
1988 Jul 14;334(6178):162-165, Nature
Binding of the human immunodeficiency virus (HIV) to infectable host cells, such as B and T lymphocytes, monocytes and colorectal cells, is mediated by a high-affinity interaction between the gp120 component of the viral envelope glycoprotein and the CD4 receptor. Upon binding, it is thought that the second component of the envelope, gp41, mediates fusion between the viral envelope and host cell membranes. However, the early steps of HIV infection have not yet been thoroughly elucidated. Viral entry was first reported to be mediated by pH-dependent receptor-mediated endocytosis; subsequent studies have shown entry to be pH-independent. Although direct fusion of virus to plasma membranes of infected cells has been observed by electron microscopy, it is still formally possible that the infectious path of the virus involves receptor-mediated endocytosis. To gain a better understanding of receptor function in viral entry, we have analysed the ability of several altered or truncated forms of CD4 to serve as effective viral receptors. Our results indicate that domains beyond the HIV-binding region of CD4 are not required for viral infection. Some of the altered forms of CD4 that serve as effective HIV receptors are severely impaired in their ability to be endocytosed. These experiments therefore support the notion that viral fusion to the plasma membrane is sufficient for infection
— id: 15181, year: 1988, vol: 334, page: 162, stat: Journal Article,

Nonequivalent effects of PKC activation by PMA on murine CD4 and CD8 cell-surface expression
Kaldjian E; McCarthy SA; Sharrow SO; Littman DR; Klausner RD; Singer A
1988 Sep;2(12):2801-2806, FASEB journal
The membrane glycoproteins CD4 (L3T4) and CD8 (Lyt2) are expressed on distinct populations of mature murine T lymphocytes, and are thought to be receptors for monomorphic determinants expressed on MHC class II and class I molecules, respectively. Although they differ in their ligand specificity, it has been presumed that CD4 and CD8 perform equivalent functions in the T cells that bear them. Since activation of protein kinase C (PKC) is known to cause rapid down-regulation of various receptors, including the T cell receptor complex (TcR complex), we treated cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, to determine whether cell-surface expression of CD4 and CD8 would be similarly affected by this intracellular mediator. Brief or relatively prolonged treatment with PMA induced mature murine T cells to reduce their surface expression of the TcR complex and of CD4, but not of CD8. Similarly, PMA rapidly induced transfected L cells to down-regulate surface CD4 expression, but had no effect on surface CD8 expression. Most significantly, PMA treatment induced CD4+CD8+ immature thymocytes to rapidly reduce their surface CD4 expression, but, again, it had no immediate effect on the surface expression of CD8. These results indicate that CD4 and TcR complex cell-surface expression are both sensitive to PKC activation by brief treatment with PMA, whereas CD8 expression is not, and suggest that CD4 and CD8 surface expression levels are regulated by distinct intracellular mechanisms
— id: 15180, year: 1988, vol: 2, page: 2801, stat: Journal Article,

The envelope glycoprotein of the human immunodeficiency virus binds to the immunoglobulin-like domain of CD4
Landau NR; Warton M; Littman DR
1988 Jul 14;334(6178):159-162, Nature
CD4, a cell-surface glycoprotein expressed on a subset of T-cells and macrophages, serves as the receptor for the human immunodeficiency virus (HIV) (reviewed in ref. 1), binding to the HIV envelope glycoprotein, gp120 with high affinity. Attempts to block infection in vivo by raising antibodies against gp120 have failed, probably because these antibodies have insufficient neutralizing activity. In addition, because of the extensive polymorphism of gp120 in different isolates of HIV, antibodies raised against one HIV isolate are only weakly effective against others. Because interaction with CD4 is essential for infectivity by all isolates of HIV, an agent that could mimic CD4 in its ability to bind to gp120, such as a peptide or monoclonal antibody, might block infection by a wide spectrum of isolates. To aid the identification of such a ligand we have defined regions of CD4 that are required for binding to gp120. Although human CD4 is similar to mouse CD4 in amino-acid sequence (55% identity, ref. 6) and structure, we have found that the murine protein fails to bind detectably to gp120 and have exploited this finding to study binding of gp120 to mouse-human chimaeric CD4 molecules. These studies show that amino-acid residues within the amino-terminal immunoglobulin-like domain of human CD4 are involved in binding to gp120 as well as to many anti-CD4 monoclonal antibodies
— id: 57528, year: 1988, vol: 334, page: 159, stat: Journal Article,

Corrected CD4 sequence
Littman DR; Maddon PJ; Axel R
1988 Nov 18;55(4):541-541, Cell
— id: 15177, year: 1988, vol: 55, page: 541, stat: Journal Article,

Mouse brain CD4 transcripts encode only the COOH-terminal half of the protein
Lonberg N; Gettner SN; Lacy E; Littman DR
1988 May;8(5):2224-2228, Molecular & cellular biology
The T-cell surface glycoprotein CD4 is thought to function as a receptor for class II major histocompatibility complex molecules. Human CD4 is also the lymphoid cell receptor for human immunodeficiency virus, the causative agent of acquired immune deficiency syndrome. The observed infection of the central nervous system in acquired immune deficiency syndrome patients raises the possibility that CD4 is also present in nerve tissue and that a cell surface receptor for class II major histocompatibility complex antigens could play a role in central nervous system function. This possibility is reinforced by the detection of unique CD4-related transcripts in mouse and human brain tissue. In this study, the structure of the mouse brain CD4 transcript was determined. It is identical to the last two-thirds of the CD4 message and is capable of encoding a 217-residue protein that would consist of a truncated, 154-residue, cell surface region, together with the complete CD4 transmembrane and cytoplasmic regions. It would not include an amino-terminal hydrophobic leader peptide
— id: 15182, year: 1988, vol: 8, page: 2224, stat: Journal Article,

Human brain glycogen phosphorylase. Cloning, sequence analysis, chromosomal mapping, tissue expression, and comparison with the human liver and muscle isozymes
Newgard CB; Littman DR; van Genderen C; Smith M; Fletterick RJ
1988 Mar 15;263(8):3850-3857, Journal of biological chemistry
We have cloned the cDNA encoding a new isozyme of glycogen phosphorylase (1,4-D-glucan:orthosphosphate D-glucosyltransferase, EC 2.4.1.1) from a cDNA library prepared from a human brain astrocytoma cell line. Blot-hybridization analysis reveals that this message is preferentially expressed in human brain, but is also found at a low level in human fetal liver and adult liver and muscle tissues. Although previous studies have suggested that the major isozyme of phosphorylase found in all fetal tissues is the brain type, our data show that the predominant mRNA in fetal liver (24-week gestation) is the adult liver form. The protein sequence deduced from the nucleotide sequence of the brain phosphorylase cDNA is 862 amino acids long compared with 846 and 841 amino acids for the liver and muscle isozymes, respectively; the greater length of brain phosphorylase is entirely due to an extension at the far C-terminal portion of the protein. The muscle and brain isozymes share greater identity with regard to nucleotide and deduced amino acid sequences, codon usage, and nucleotide composition than either do with the liver sequence, suggesting a closer evolutionary relationship between them. Spot blot hybridization of the brain phosphorylase cDNA to laser-sorted human chromosome fractions, and Southern blot analysis of hamster/human hybrid cell line DNA reveals that the exact homolog of the newly cloned cDNA maps to chromosome 20, but that a slightly less homologous gene is found on chromosome 10 as well. The liver and muscle genes have previously been localized to chromosomes 14 and 11, respectively. This suggests that the phosphorylase genes evolved by duplication and translocation of a common ancestral gene, leading to divergence of elements controlling gene expression and of structural features of the phosphorylase proteins that confer tissue-specific functional properties
— id: 15183, year: 1988, vol: 263, page: 3850, stat: Journal Article,

A second subunit of CD8 is expressed in human T cells
Norment AM; Littman DR
1988 Nov;7(11):3433-3439, EMBO journal
The CD8 glycoprotein plays important functions in T cell development and in T cell activation. In rodents, CD8 is a heterodimer, consisting of an alpha-chain (Lyt2) and a beta-chain (Lyt3). In humans, only the alpha-chain has been detected, and it has been thought that CD8 consists of homodimers of this protein. We have isolated functional cDNA clones encoding human CD8 beta, and show that the CD8 beta protein is expressed on the surface of CD8+ human T cells. cDNA clones encoding multiple forms of the human CD8 beta-chain have been isolated and characterized. These structural variants, which are likely to arise by alternative splicing, differ in the sequences encoding the cytoplasmic domain, which can consist of 19, 30, or 52 amino acids. One of the cDNAs lacks nucleotide sequences corresponding to a hydrophobic transmembrane domain, and may encode a secreted CD8 beta protein. The protein product of the human CD8 beta gene can be detected by a recently described anti-CD8 monoclonal antibody, 597. Expression of the epitope recognized by this antibody requires co-expression of the CD8 alpha and CD8 beta gene products. About 90% of human CD8 alpha positive thymocytes and peripheral blood lymphocytes express CD8 beta at the cell surface. Expression of the CD8 beta chain is thus conserved between human and rodents, and the variant CD8 beta polypeptides may have distinct roles in T cell function and development
— id: 15179, year: 1988, vol: 7, page: 3433, stat: Journal Article,

Cell-cell adhesion mediated by CD8 and MHC class I molecules
Norment AM; Salter RD; Parham P; Engelhard VH; Littman DR
1988 Nov 3;336(6194):79-81, Nature
CD4 and CD8 are cell-surface glycoproteins expressed on mutually exclusive subsets of peripheral T cells. T cells that express CD4 have T-cell antigen receptors that are specific for antigens presented by major histocompatibility complex class II molecules, whereas T cells that express CD8 have receptors specific for antigens presented by MHC class I molecules (reviewed in ref. 1). Based on this correlation and on the observation that anti-CD4 and anti-CD8 antibodies inhibit T-cell function, it has been suggested that CD4 and CD8 increase the avidity of T cells for their targets by binding to MHC class II or MHC class I molecules respectively. Also, CD4 and CD8 may become physically associated with the T-cell antigen receptor, forming a higher-affinity complex for antigen and MHC molecules, and could be involved in signal transduction. Cell-cell adhesion dependent CD4 and MHC II molecules has recently been demonstrated. To determine whether CD8 can interact with MHC class I molecules in the absence of the T-cell antigen receptor, we have developed a cell-cell binding assay that measures adhesion of human B-cell lines expressing MHC class I molecules to transfected cells expressing high levels of human CD8. In this system, CD8 and class I molecules mediate cell-cell adhesion, showing that CD8 directly binds to MHC class I molecules
— id: 15178, year: 1988, vol: 336, page: 79, stat: Journal Article,

Patterns of T cell receptor gamma gene rearrangement and expression in B and T lymphoid malignancies
Subar M; Giuseppe Pelicci P; Neri A; Allavena P; Littman DR; Knowles DM 2d; Dalla-Favera R
1988 Jan;2(1):19-26, Leukemia
The frequency and pattern of T gamma gene rearrangement and expression was investigated in hematopoietic neoplasms including T and B lymphoid and myeloid malignancies. 39 of 39 T lymphoid neoplasms, including fresh cases and cell lines, were found to display clonal T gamma gene rearrangements. There was heterogeneity with respect to utilization of the two T gamma constant region genes, T gamma C1 and T gamma C2. In 31 cases (80%) T gamma C1 was deleted and T gamma C2 was rearranged, while in the remaining 8 cases (20%) T gamma C1 was rearranged. T gamma gene rearrangements were found in non-T cells, but were restricted to 6/17 (35%) immature B cell neoplasms. All 24 mature B cell and 14 myeloid neoplasms retained the T gamma germ line pattern. T gamma mRNA was found in all T cells tested. However, the majority (16/17) of T cells most likely do not express a T gamma protein since a T alpha/beta heterodimer detected by reactivity with the MoAb WT31 is present on the cell surface together with T3. These data suggest that T gamma gene rearrangements are universal in T cells and frequent in immature B cell neoplastic populations. However, expression of the T gamma protein is extremely infrequent, indicating that T cell neoplasms are very rarely derived from the recently identified T3+T gamma +T alpha/beta- peripheral T cell population
— id: 11261, year: 1988, vol: 2, page: 19, stat: Journal Article,

The structure of the CD4 and CD8 genes
Littman DR
1987 ;5:561-584, Annual review of immunology
— id: 15188, year: 1987, vol: 5, page: 561, stat: Journal Article,

Unusual intron in the immunoglobulin domain of the newly isolated murine CD4 (L3T4) gene
Littman DR; Gettner SN
1987 Jan 29-Feb 4;325(6103):453-455, Nature
The T-cell surface glycoprotein, CD4, is expressed predominantly on helper T cells and is thought to play a major role in cell-cell interactions. Monoclonal antibodies against CD4 have been shown to block numerous T-cell functions; moreover, recent results suggest that the CD4 molecule may be involved in transmembrane signal transduction. The human CD4 glycoprotein has also been shown to form at least part of the receptor for the AIDS virus, HIV-1. Elucidation of the functions of CD4 will be facilitated by the ability to manipulate the protein by genetic means. Because the mouse system is well suited for a variety of functional studies, we have isolated, sequenced and expressed cDNA clones encoding the murine CD4 (L3T4) glycoprotein. Comparison of the mouse and human CD4 sequences reveals striking evolutionary conservation of the cytoplasmic domain, suggesting that this region is essential for CD4 function. In addition, both the human and mouse CD4 gene contain a large intron in the coding region of the V-like domain. As no other members of the immunoglobulin gene superfamily have been shown to contain similarly placed introns, this finding may have important implications regarding the evolution of this gene family in particular and of introns in general
— id: 15187, year: 1987, vol: 325, page: 453, stat: Journal Article,

Characterization of an expressed CD3-associated Ti gamma-chain reveals C gamma domain polymorphism
Littman DR; Newton M; Crommie D; Ang SL; Seidman JG; Gettner SN; Weiss A
1987 Mar 5-11;326(6108):85-88, Nature
The majority of human T cells express an antigen receptor consisting of a disulphide-linked heterodimer (Ti) of relative molecular mass 80,000-90,000 (Mr 80-90K) which is noncovalently associated with a set of at least three proteins of Mr 20-28K termed CD3 (Leu4, T3). Whereas both chains of Ti, an acidic alpha-chain of Mr 48-54K and a more basic beta-chain of Mr 40-44K, contain variable and constant region domains, the component peptides of CD3 are invariant. Several laboratories have more recently reported the expression of CD3 in association with a novel protein. On the surface of long-term T-cell lines and one thymocyte clone this novel structure consists of a 40K protein noncovalently linked to a 55 or 62K protein identified as the protein product of the Ti gamma-chain gene, a T-cell specific gene which like the Ti alpha- and Ti beta-chain genes undergoes rearrangement of variable (V) and joining (J) region gene segments. On the human T-cell leukaemic line PEER we have detected only a single 55K glycoprotein associated with CD3. We here demonstrate that an anti-Ti gamma-peptide antiserum reacts with the 55K CD3-associated protein on PEER. Most previously described human Ti gamma-chain complementary DNA clones encode the products of non-functional rearrangements. One of the Ti gamma cDNAs isolated from PEER, however, represents a functional rearrangement reported for the first time in a cell which expresses a Ti gamma-chain protein product on the cell surface. Interestingly, a 48-base-pair (bp) sequence in the constant (C) region domain of this functional Ti gamma-chain cDNA is triplicated in PEER and duplicated in other cDNAs isolated from PEER and other cell lines
— id: 15186, year: 1987, vol: 326, page: 85, stat: Journal Article,

Identification and sequence of a fourth human T cell antigen receptor chain
Loh EY; Lanier LL; Turck CW; Littman DR; Davis MM; Chien YH; Weiss A
1987 Dec 10-16;330(6148):569-572, Nature
Thymus-derived lymphocytes (T cells) use clonally distributed antigen receptors to recognize peptide fragments associated with products of the major histocompatibility complex (MHC) (refs 1-4). On most murine and human T cells the T cell receptor (TCR) is composed of disulphide-linked alpha and beta chains (TCR alpha/beta), each of which contains constant and variable domains, and which are associated with the invariant chains of the CD3 complex. It has been demonstrated, however, that a distinct CD3-associated TCR is expressed on a small subset of T cells or immature thymocytes which fail to express either CD4 or CD8 (refs 7-14), the molecules associated with class II or class I MHC antigen recognition. Instead of TCR alpha/beta, these cells express heterodimers of gamma and delta chains (TRC gamma/delta). The genes encoding alpha, beta, and gamma have been isolated and characterized. A new murine T cell receptor (Cx) gene which undergoes rearrangement and expression early during T cell ontogeny has recently been identified 5' of the murine J alpha C alpha gene locus. Here we isolate and sequence the homologous transcript from PEER, a human cell line that expresses a TCR gamma/delta, and show that it encodes a protein with characteristic V, D, J, and C segments. Using probes derived from this transcript, we have shown that both PEER and MOLT-13, another TCR gamma/delta-expressing cell line, rearrange this locus and express two sizes of transcripts differing in the 3' untranslated region. Using a synthetic peptide derived from the deduced C region sequence, we have prepared antisera that precipitates the delta chain of the TCR from both PEER and MOLT-13, thus demonstrating that Cx and its human homologue code for the delta chain of the TCR
— id: 15184, year: 1987, vol: 330, page: 569, stat: Journal Article,

Arrangements and rearrangements of the human T-cell receptor gamma gene
Pelicci PG; Neri A; Knowles DM 2d; Littman DR; Dalla-Favera R; Subar M
1987 ;511:232-245, Annals of the New York Academy of Sciences
— id: 11425, year: 1987, vol: 511, page: 232, stat: Journal Article,

Molecular diversity of the human T-gamma constant region genes
Pelicci PG; Subar M; Weiss A; Dalla-Favera R; Littman DR
1987 Aug 28;237(4818):1051-1055, Science
The human T cell antigen-receptor gamma chain, which is expressed on the surface of a subpopulation of CD3+ T lymphocytes, exhibits size polymorphism and varies in its ability to form disulfide bonds with a second polypeptide. Analysis of both genomic and complementary DNA clones encoding the human gamma polypeptide shows differences in lengths of the coding portions of the two constant region genes, C gamma 1 and C gamma 2. A single second-exon segment is always present in the C gamma 1 gene. C gamma 2 alleles containing either duplicated or triplicated second-exon segments are present in the normal human population and are expressed as messenger RNAs. Furthermore, a cysteine residue, encoded by the second exon of C gamma 1 and probably involved in interchain disulfide bridging, is absent in all C gamma 2 second-exon segments. These differences between C gamma 1 and the two alleles of C gamma 2 may explain the variability in molecular weight and disulfide bonding of gamma molecules expressed in different cells
— id: 15185, year: 1987, vol: 237, page: 1051, stat: Journal Article,

The gene encoding the T-cell surface protein T4 is located on human chromosome 12
Isobe M; Huebner K; Maddon PJ; Littman DR; Axel R; Croce CM
1986 Jun;83(12):4399-4402, Proceedings of the National Academy of Sciences of the United States of America
The surface glycoproteins T4 and T8 define functionally distinct populations of T lymphocytes. We have obtained cDNA and genomic clones encoding the T4 molecule and used these as probes to determine the chromosomal location of this gene. Genomic blotting experiments, along with in situ hybridization analyses, indicate that the T4 gene resides on the short arm of human chromosome 12, at region p12-pter. Thus, the T4 gene is not linked to any known member of the immunoglobulin gene family, including its counterpart gene, T8, which resides on human chromosome 2 immediately distal to the immunoglobulin kappa locus
— id: 15189, year: 1986, vol: 83, page: 4399, stat: Journal Article,

The T4 glycoprotein is a cell-surface receptor for the AIDS virus
McDougal JS; Maddon PJ; Dalgleish AG; Clapham PR; Littman DR; Godfrey M; Maddon DE; Chess L; Weiss RA; Axel R
1986 ;51 Pt 2:703-711, Cold Spring Harbor symposia on quantitative biology
Taken together, our studies suggest a mechanism of AIDS virus infection that initially involves the specific association of the AIDS virus with T4 molecules on the cell surface. This association does not require additional T-cell-specific molecules and can be demonstrated on both B lymphocytes and epithelial cell lines. The T4-AIDS virus complex is likely to be internalized in endosomes via receptor-mediated endocytosis. The virus can then fuse with the vacuolar membrane, releasing the viral nucleocapsid into the cytoplasm to undergo uncoating. Viral replication does not appear to require the environment of a T lymphocyte because active infection is also observed in human T4+ B lymphocytes and epithelial cell lines. Moreover, the T4 gene is expressed in the brain as well as in lymphocytes, providing an explanation for the dual neurotropic and lymphotropic character of the virus. In this manner, a T-lymphocyte surface protein thought to be important in mediating effector cell-target cell interactions has been exploited by a human lymphotropic virus to target the AIDS virus specifically to populations of T4+ cells
— id: 15190, year: 1986, vol: 51 Pt 2, page: 703, stat: Journal Article,

The isolation and sequence of the gene encoding T8: a molecule defining functional classes of T lymphocytes
Littman DR; Thomas Y; Maddon PJ; Chess L; Axel R
1985 Feb;40(2):237-246, Cell
The T cell surface glycoproteins T4 and T8 are thought to mediate efficient cell-cell interactions in the immune system and in this way may be responsible for the appropriate targeting of subpopulations of T cells. We have used gene transfer combined with subtractive hybridization to isolate both cDNA and functional genomic clones encoding the T8 protein. The sequence of the cDNA reveals that T8 is a transmembrane protein with an N-terminal domain which shares significant homology to immunoglobulin variable region light chains. This immunoglobulin-like structure is likely to be important in the function of T8 during differentiation and in the course of the immune response
— id: 15192, year: 1985, vol: 40, page: 237, stat: Journal Article,

The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: a new member of the immunoglobulin gene family
Maddon PJ; Littman DR; Godfrey M; Maddon DE; Chess L; Axel R
1985 Aug;42(1):93-104, Cell
The surface glycoproteins T4 and T8 define different functional subsets of T lymphocytes and may act as recognition molecules mediating appropriate interactions between the T cell and its target. Previously we employed gene transfer and subtractive hybridization to isolate a T8 cDNA; now we have isolated and sequenced a cDNA clone encoding the T4 molecule. The deduced protein sequence reveals that T4 is an integral membrane protein that shares significant amino acid and structural homologies with members of the immunoglobulin supergene family. The overall structure of T4 consists of an N-terminal variable (V)-like domain, a joining (J)-like region, a third extracellular domain, a membrane-spanning region homologous to class II MHC beta-chains, and a highly charged cytoplasmic domain. Comparison of the protein sequences deduced from the T4 and T8 cDNAs reveals structural similarities consistent with their postulated role as recognition molecules, as well as differences suggesting that the two proteins recognize different structures on the target cell
— id: 15191, year: 1985, vol: 42, page: 93, stat: Journal Article,

Structural comparison of murine Ia antigens determined by the I-A and I-E subregions
Cullen SE; Kindle CS; Littman DR
1979 Mar;122(3):855-859, Journal of immunology
— id: 15193, year: 1979, vol: 122, page: 855, stat: Journal Article,

Insertion of Ia and H-2 alloantigens into model membranes
Littman DR; Cullen SE; Schwartz BD
1979 Feb;76(2):902-906, Proceedings of the National Academy of Sciences of the United States of America
The study of immune phenomena dependent on the major histocompatibility complex (MHC) would be greatly simplified by the use of MHC antigen-containing liposomes in various functional systems. Towards this end, we have constructed unilamellar phosphatidylcholine liposomes containing H-2 and Ia antigens. These molecules were not simply trapped within the aqueous compartment of the liposome as assessed by their accessibility to papain digestion. They were shown to be integrally inserted in the liposome bilayer because they could not be dissociated from the liposome with high salt and EDTA concentrations but could be solubilized by detergent. A sensitive radioimmunoassay showed that the Ia molecules were antigenically active in the liposome environment. Both Ia and H-2 antigens could be immunoprecipitated from detergent-solubilized liposomes. By comparing liposome-associated Ia activity in the presence and absence of detergent and by showing accessibility of the Ia antigens to papain, it was concluded that the majority of Ia antigens were exposed on the external surface of the liposome. These results suggest that the orientation of MHC antigens in liposomes closely parallels their natural orientation in the cell membrane
— id: 15194, year: 1979, vol: 76, page: 902, stat: Journal Article,

Properties of the depolymerization products of microtubules from mammalian brain
Weingarten MD; Suter MM; Littman DR; Kirschner MW
1974 Dec 31;13(27):5529-5537, Biochemistry
— id: 15195, year: 1974, vol: 13, page: 5529, stat: Journal Article,