Derya Unutmaz, M.D.

Characterization of a new cytotoxin that contributes to Staphylococcus aureus pathogenesis
Dumont, Ashley L; Nygaard, Tyler K; Watkins, Robert L; Smith, Amanda; Kozhaya, Lina; Kreiswirth, Barry N; Shopsin, Bo; Unutmaz, Derya; Voyich, Jovanka M; Torres, Victor J
2011 Feb;79(3):814-825, Molecular microbiology
Staphylococcus aureus is an important pathogen that continues to be a significant global health threat because of the prevalence of methicillin-resistant S. aureus strains (MRSA). The pathogenesis of this organism is partly attributed to the production of a large repertoire of cytotoxins that target and kill innate immune cells, which provide the first line of defence against S. aureus infection. Here we demonstrate that leukocidin A/B (LukAB) is required and sufficient for the ability of S. aureus, including MRSA, to kill human neutrophils, macrophages and dendritic cells. LukAB targets the plasma membrane of host cells resulting in cellular swelling and subsequent cell death. We found that S. aureus lacking lukAB are severely impaired in their ability to kill phagocytes during bacteria-phagocyte interaction, which in turn renders the lukAB-negative staphylococci more susceptible to killing by neutrophils. Notably, we show that lukAB is expressed in vivo within abscesses in a murine infection model and that it contributes significantly to pathogenesis of MRSA in an animal host. Collectively, these results extend our understanding of how S. aureus avoids phagocyte-mediated clearance, and underscore LukAB as an important factor that contributes to staphylococcal pathogenesis
— id: 120726, year: 2011, vol: 79, page: 814, stat: Journal Article,

Revisiting immune exhaustion during HIV infection
Khaitan, Alka; Unutmaz, Derya
2011 Mar;8(1):4-11, Current HIV/AIDS reports
Chronic immune activation is a hallmark of HIV infection, yet the underlying triggers of immune activation remain unclear. Persistent antigenic stimulation during HIV infection may also lead to immune exhaustion, a phenomenon in which effector T cells become dysfunctional and lose effector functions and proliferative capacity. Several markers of immune exhaustion, such as PD-1, LAG-3, Tim-3, and CTLA-4, which are also negative regulators of immune activation, are preferentially upregulated on T cells during HIV infection. It is not yet clear whether accumulation of T cells expressing activation inhibitory molecules is a consequence of general immune or chronic HIV-specific immune activation. Importantly, however, in vitro blockade of PD-1 and Tim-3 restores HIV-specific T-cell responses, indicating potential for immunotherapies. In this review we discuss the evolution of our understanding of immune exhaustion during HIV infection, highlighting novel markers and potential therapeutic targets
— id: 138292, year: 2011, vol: 8, page: 4, stat: Journal Article,

FoxP3 interacts with linker histone H1.5 to modulate gene expression and program Treg cell activity
Mackey-Cushman, S L; Gao, J; Holmes, D A; Nunoya, J-I; Wang, R; Unutmaz, D; Su, L
2011 Oct;12(7):559-567, Genes & immunity
The forkhead box transcription factor FoxP3 controls the development and function of CD4+CD25+ regulatory T (Treg) cell. FoxP3 modulates gene expression in Treg cells by multiple epigenetic mechanisms that are not clearly defined. We identified FoxP3-interacting proteins in human T cells by co-immunoprecipitation/MS. We discovered that FoxP3 interacted with linker histone H1.5 via the leucine zipper (LZ) domain. Two independent IPEX patient-derived single residue mutations in the LZ of FoxP3 both abrogated its interaction with H1.5. Functionally, FoxP3 and H1.5 cooperatively repressed interleukin-2 (IL-2) expression in human T cells; and silencing of H1.5 expression inhibited the ability of FoxP3 to suppress IL-2 expression. We show that FoxP3 specifically enhanced H1.5 association at the IL-2 promoter, but reduce its association at the CTLA4 promoter, correlated with higher or lower histone acetylation of the respective promoters. Finally, silencing of H1.5 expression in human Treg cells impaired the Treg function to suppress target T cells. We conclude that FoxP3 interacts with H1.5 to alter its binding to target genes to modulate their expression and to program Treg function
— id: 141094, year: 2011, vol: 12, page: 559, stat: Journal Article,

Cytokine signals through PI-3 kinase pathway modulate Th17 cytokine production by CCR6+ human memory T cells
Wan, Qi; Kozhaya, Lina; Elhed, Aimee; Ramesh, Radha; Carlson, Thaddeus J; Djuretic, Ivana M; Sundrud, Mark S; Unutmaz, Derya
2011 Aug 29;208(9):1875-1887, Journal of experimental medicine
Human memory T cells (T(M) cells) that produce IL-17 or IL-22 are currently defined as Th17 or Th22 cells, respectively. These T cell lineages are almost exclusively CCR6(+) and are important mediators of chronic inflammation and autoimmunity. However, little is known about the mechanisms controlling IL-17/IL-22 expression in memory Th17/Th22 subsets. We show that common gamma chain (gammac)-using cytokines, namely IL-2, IL-7, and IL-15, potently induce Th17-signature cytokine expression (Il17a, Il17f, Il22, and Il26) in CCR6(+), but not CCR6(-), T(M) cells, even in CCR6(+) cells lacking IL-17 expression ex vivo. Inhibition of phosphoinositide 3-kinase (PI-3K) or Akt signaling selectively prevents Th17 cytokine induction by gammac-cytokines, as does ectopic expression of the transcription factors FOXO1 or KLF2, which are repressed by PI-3K signaling. These results indicate that Th17 cytokines are tuned by PI-3K signaling in CCR6(+) T(M) cells, which may contribute to chronic or autoimmune inflammation. Furthermore, these findings suggest that ex vivo analysis of IL-17 expression may greatly underestimate the frequency and pathogenic potential of the human Th17 compartment
— id: 137001, year: 2011, vol: 208, page: 1875, 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,

Th17 cells and HIV infection
Elhed, Aimee; Unutmaz, Derya
2010 Mar;5(2):146-150, Current Opinion in HIV & AIDS
PURPOSE OF REVIEW: This review summarizes the recent literature about the potential perturbation and role of Th17 cells in HIV pathogenesis. We discuss the recent findings on Th17 deficiency in HIV/simian immunodeficiency virus (SIV) infection and how this deficiency may impact the mucosal host defenses, potentially contributing to chronic immune activation. RECENT FINDINGS: Th17 cells have been implicated in host defense against a variety of pathogens and are involved in the pathogenesis of autoimmune diseases. Recently, Th17 cells were shown to be perturbed during HIV infection in humans and SIV infection in nonhuman primates. Th17 cells were found to be infected in vitro by HIV and SIV and are significantly depleted in the gastrointestinal tract of HIV-infected individuals. In monkeys, Th17 cells are only depleted in the pathogenic SIV infection of rhesus macaques, which correlates with the progression to AIDS in these primates, whereas they remain intact in the nonpathogenic SIV infection of African green monkeys or sooty mangabeys. SUMMARY: Th17 cells appear to be perturbed during HIV and SIV infection. This finding could have important implications in understanding the disruption of mucosal defenses in the gastrointestinal tract and potentially in predicting opportunistic infections during the course of HIV disease
— id: 110105, year: 2010, vol: 5, page: 146, stat: Journal Article,

Perturbation of the P-Body Component Mov10 Inhibits HIV-1 Infectivity
Furtak, Vyacheslav; Mulky, Alok; Rawlings, Stephen A; Kozhaya, Lina; Lee, KyeongEun; Kewalramani, Vineet N; Unutmaz, Derya
2010 ;5(2):e9081-e9081, PLoS ONE
Exogenous retroviruses are obligate cellular parasites that co-opt a number of host proteins and functions to enable their replication and spread. Several host factors that restrict HIV and other retroviral infections have also recently been described. Here we demonstrate that Mov10, a protein associated with P-bodies that has a putative RNA-helicase domain, when overexpressed in cells can inhibit the production of infectious retroviruses. Interestingly, reducing the endogenous Mov10 levels in virus-producing cells through siRNA treatment also modestly suppresses HIV infectivity. The actions of Mov10 are not limited to HIV, however, as ectopic expression of Mov10 restricts the production of other lentiviruses as well as the gammaretrovirus, murine leukemia virus. We found that HIV produced in the presence of high levels of Mov10 is restricted at the pre-reverse transcription stage in target cells. Finally, we show that either helicase mutation or truncation of the C-terminal half of Mov10, where a putative RNA-helicase domain is located, maintained most of its HIV inhibition; whereas removing the N-terminal half of Mov10 completely abolished its activity on HIV. Together these results suggest that Mov10 could be required during the lentiviral lifecycle and that its perturbation disrupts generation of infectious viral particles. Because Mov10 is implicated as part of the P-body complex, these findings point to the potential role of cytoplasmic RNA processing machinery in infectious retroviral production
— id: 107278, year: 2010, vol: 5, page: e9081, 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,

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,

Expression and function of TNF and IL-1 receptors on human regulatory T cells
Mercer, Frances; Kozhaya, Lina; Unutmaz, Derya
2010 ;5(1):e8639-e8639, PLoS ONE
Regulatory T cells (Tregs) suppress immune activation and are critical in preventing autoimmune diseases. While the ability of Tregs to inhibit proliferation of other T cells is well established, it is not yet clear whether Tregs also modulate inflammatory cytokines during an immune response. Here, we show that the expression of inflammatory cytokine receptors IL-1R1 and TNFR2 were higher on resting mature Tregs compared to naive or memory T cells. While upon activation through the T cell receptor (TCR), expression of IL-1R1 and TNFR2 were upregulated on all T cell subsets, IL-1R1 maintained significantly higher expression on activated Tregs as compared to other T cell subsets. The decoy receptor for IL-1 (IL-1R2) was not expressed by any of the resting T cells but was rapidly upregulated and preferentially expressed upon TCR-stimulation on Tregs. In addition, we found that Tregs also expressed high levels of mRNA for IL-1 antagonist, IL-1RA. TCR-stimulation of naive T cells in the presence of TGFbeta, which induces FOXP3 expression, however did not result in upregulation of IL-1R1 or IL-1R2. In addition, ectopic expression of FOXP3 in non-Tregs, while causing significant upregulation of IL-1R1 and IL-1R2, did not achieve the levels seen in bona fide Tregs. We also determined that resting human Tregs expressing IL-1R1 did not have higher suppressive capacity compared to IL-1R1- Tregs, suggesting that IL-1R1 does not discriminate suppressive resting Tregs in healthy individuals. Functionally, activated human Tregs displayed a capacity to neutralize IL-1beta, which suggests a physiological significance for the expression of IL-1 decoy receptor on Tregs. In conclusion, our findings that human Tregs preferentially express receptors for TNF and IL-1 suggest a potential function in sensing and dampening local inflammation
— id: 106210, year: 2010, vol: 5, page: e8639, stat: Journal Article,

Natural Killer T Cells: An Unconventional T-Cell Subset with Diverse Effector and Regulatory Functions
Balato, Anna; Unutmaz, Derya; Gaspari, Anthony A
2009 Jul;129(7):1628-1642, Journal of investigative dermatology
Natural killer T (NKT) cells are a unique subset of lymphocytes that express NK cell markers such as CD161 and CD94, as well as a T-cell receptor (TCR) alpha/beta, with a restricted repertoire, which distinguishes them from NK cells, which lack a TCR. In contrast to conventional T-lymphocytes, the TCR of NKT cells does not interact with that of peptide antigens presented by classical major histocompatibility complex-encoded class I or II molecules. Instead, this TCR recognizes glycolipids presented by CD1d, a non-classical antigen-presenting molecule. The rapid response of NKT cells to their cognate antigens is characteristic of an innate immune response, and allows the polarizing cytokines (IFN-gamma and/or IL-4) to regulate adaptive immunity. NKT cells have been found to be critical in the immune response against viral infections and malaria, as well as in tumor immunity, and certain autoimmune diseases. NKT cells have been assessed to represent the 'trait d'union' between innate and adaptive immunity. They play an active role in skin diseases, such as contact sensitivity, which have been implicated in UV-induced immunosuppression and psoriasis. Thus, NKT-cells are emerging as an important subset of lymphocytes, with a protective role in host defense and a pathogenic role in certain immune-mediated disease states.Journal of Investigative Dermatology advance online publication, 5 March 2009; doi:10.1038/jid.2009.30
— id: 93986, year: 2009, vol: 129, page: 1628, stat: Journal Article,

CCR5 expression levels influence NFAT translocation, IL-2 production, and subsequent signaling events during T lymphocyte activation
Camargo, Jose F; Quinones, Marlon P; Mummidi, Srinivas; Srinivas, Sowmya; Gaitan, Alvaro A; Begum, Kazi; Jimenez, Fabio; VanCompernolle, Scott; Unutmaz, Derya; Ahuja, Seema S; Ahuja, Sunil K
2009 Jan 1;182(1):171-182, Journal of immunology
Ligands of CCR5, the major coreceptor of HIV-1, costimulate T lymphocyte activation. However, the full impact of CCR5 expression on T cell responses remains unknown. Here, we show that compared with CCR5(+/+), T cells from CCR5(-/-) mice secrete lower amounts of IL-2, and a similar phenotype is observed in humans who lack CCR5 expression (CCR5-Delta32/Delta32 homozygotes) as well as after Ab-mediated blockade of CCR5 in human T cells genetically intact for CCR5 expression. Conversely, overexpression of CCR5 in human T cells results in enhanced IL-2 production. CCR5 surface levels correlate positively with IL-2 protein and mRNA abundance, suggesting that CCR5 affects IL-2 gene regulation. Signaling via CCR5 resulted in NFAT transactivation in T cells that was blocked by Abs against CCR5 agonists, suggesting a link between CCR5 and downstream pathways that influence IL-2 expression. Furthermore, murine T cells lacking CCR5 had reduced levels of intranuclear NFAT following activation. Accordingly, CCR5 expression also promoted IL-2-dependent events, including CD25 expression, STAT5 phosphorylation, and T cell proliferation. We therefore suggest that by influencing a NFAT-mediated pathway that regulates IL-2 production and IL-2-dependent events, CCR5 may play a critical role in T cell responses. In accord with our prior inferences from genetic-epidemiologic studies, such CCR5-dependent responses might constitute a viral entry-independent mechanism by which CCR5 may influence HIV-AIDS pathogenesis
— id: 91423, year: 2009, vol: 182, page: 171, stat: Journal Article,

The biology of FoxP3: a key player in immune suppression during infections, autoimmune diseases and cancer
Mercer, Frances; Unutmaz, Derya
2009 ;665:47-59, Advances in experimental medicine & biology
The Transcription factor FoxP3 belongs to the forkhead/winged-helix family of transcriptional regulators and shares general structural features with other FoxP family members. FoxP3 functions as a master of transcription for the development of regulatory T-cells (Treg cells) both in humans and in mice. Natural genetic mutations ofFoxP3 that disrupt its function in humans result in an autoimmune syndrome called Immune Polyendocrinopathy, Enteropathy, X-linked (IPEX) and in mice, its deletion causes the Scurfy phenotype, with similar pathology. The finding that FoxP3 is required for the development and function of Tregs has led to an explosion of research in determining its regulation and function in the immune system. Understanding the biological properties of FoxP3 has a wide range of implications for immune tolerance, autoimmune disorders, inflammation and immune response to infectious diseases and cancer
— id: 109564, year: 2009, vol: 665, page: 47, stat: Journal Article,

Halofuginone inhibits TH17 cell differentiation by activating the amino acid starvation response
Sundrud, Mark S; Koralov, Sergei B; Feuerer, Markus; Calado, Dinis Pedro; Kozhaya, Aimee Elhed; Rhule-Smith, Ava; Lefebvre, Rachel E; Unutmaz, Derya; Mazitschek, Ralph; Waldner, Hanspeter; Whitman, Malcolm; Keller, Tracy; Rao, Anjana
2009 Jun 5;324(5932):1334-1338, Science
A central challenge for improving autoimmune therapy is preventing inflammatory pathology without inducing generalized immunosuppression. T helper 17 (TH17) cells, characterized by their production of interleukin-17, have emerged as important and broad mediators of autoimmunity. Here we show that the small molecule halofuginone (HF) selectively inhibits mouse and human TH17 differentiation by activating a cytoprotective signaling pathway, the amino acid starvation response (AAR). Inhibition of TH17 differentiation by HF is rescued by the addition of excess amino acids and is mimicked by AAR activation after selective amino acid depletion. HF also induces the AAR in vivo and protects mice from TH17-associated experimental autoimmune encephalomyelitis. These results indicate that the AAR pathway is a potent and selective regulator of inflammatory T cell differentiation in vivo
— id: 106016, year: 2009, vol: 324, page: 1334, stat: Journal Article,

GARP (LRRC32) is essential for the surface expression of latent TGF-beta on platelets and activated FOXP3+ regulatory T cells
Tran, Dat Q; Andersson, John; Wang, Rui; Ramsey, Heather; Unutmaz, Derya; Shevach, Ethan M
2009 Aug 11;106(32):13445-13450, Proceedings of the National Academy of Sciences of the United States of America
TGF-beta family members are highly pleiotropic cytokines with diverse regulatory functions. TGF-beta is normally found in the latent form associated with latency-associated peptide (LAP). This latent complex can associate with latent TGFbeta-binding protein (LTBP) to produce a large latent form. Latent TGF-beta is also found on the surface of activated FOXP3(+) regulatory T cells (Tregs), but it is unclear how it is anchored to the cell membrane. We show that GARP or LRRC32, a leucine-rich repeat molecule of unknown function, is critical for tethering TGF-beta to the cell surface. We demonstrate that platelets and activated Tregs co-express latent TGF-beta and GARP on their membranes. The knockdown of GARP mRNA with siRNA prevented surface latent TGF-beta expression on activated Tregs and recombinant latent TGF-beta1 is able to bind directly with GARP. Confocal microscopy and immunoprecipitation strongly support their interactions. The role of TGF-beta on Tregs appears to have dual functions, both for Treg-mediated suppression and infectious tolerance mechanism
— id: 106015, year: 2009, vol: 106, page: 13445, stat: Journal Article,

RORC2: the master of human Th17 cell programming
Unutmaz, Derya
2009 Jun;39(6):1452-1455, European journal of immunology
It is now recognized that Th17 cells constitute the third effector arm of Th cells, complementing the Th1 and Th2 lineages. Although secretion of IL-17 is a primary defining characteristic of Th17 cells, the expression profile of specific chemokine receptors, cell surface molecules and cytokines are also part of the Th17 cell differentiation program. The orphan nuclear receptor ROR gamma t/RORC2 (mice/humans) is the master transcription factor that can drive Th17 cell differentiation in both mice and humans; however, it is not completely clear how much of the human Th17 lineage function and phenotype can be recapitulated by only RORC2 expression. In this issue of the European Journal of Immunology, a study reports that ectopic expression of RORC2 in primary human T cells induces, in addition to IL-17 production, a cytokine and chemokine receptor profile consistent with bona fide human Th17 cells. In addition, programming of human Th17 cells by RORC2 results in partial resistance of these T cells to suppression by Tregs. These findings support the notion that RORC2 is a master switch that initiates a wide range of phenotypic and functional programming during Th17 cell differentiation
— id: 99330, year: 2009, vol: 39, page: 1452, stat: Journal Article,

The gut feeling of Treg cells: IL-10 is the silver lining during colitis
Unutmaz, Derya; Pulendran, Bali
2009 Nov;10(11):1141-1143, Nature immunology
Regulatory T cells that express the transcription factor Foxp3 are pivotal in suppressing autoimmune responses. A report in this issue describes a key role for interleukin 10 produced by lamina propria macrophages in maintaining Foxp3 expression during inflammatory responses in the intestine
— id: 106014, year: 2009, vol: 10, page: 1141, stat: Journal Article,

Differences in APOBEC3G expression in CD4+ T helper lymphocyte subtypes modulate HIV-1 infectivity
Vetter, Michael L; Johnson, Megan E; Antons, Amanda K; Unutmaz, Derya; D'Aquila, Richard T
2009 Feb;5(2):e1000292-e1000292, PLoS pathogens
The cytidine deaminases APOBEC3G and APOBEC3F exert anti-HIV-1 activity that is countered by the HIV-1 vif protein. Based on potential transcription factor binding sites in their putative promoters, we hypothesized that expression of APOBEC3G and APOBEC3F would vary with T helper lymphocyte differentiation. Naive CD4+ T lymphocytes were differentiated to T helper type 1 (Th1) and 2 (Th2) effector cells by expression of transcription factors Tbet and GATA3, respectively, as well as by cytokine polarization. APOBEC3G and APOBEC3F RNA levels, and APOBEC3G protein levels, were higher in Th1 than in Th2 cells. T cell receptor stimulation further increased APOBEC3G and APOBEC3F expression in Tbet- and control-transduced, but not in GATA3-transduced, cells. Neutralizing anti-interferon-gamma antibodies reduced both basal and T cell receptor-stimulated APOBEC3G and APOBEC3F expression in Tbet- and control-transduced cells. HIV-1 produced from Th1 cells had more virion APOBEC3G, and decreased infectivity, compared to virions produced from Th2 cells. These differences between Th1- and Th2-produced virions were greater for viruses lacking functional vif, but also seen with vif-positive viruses. Over-expression of APOBEC3G in Th2 cells decreased the infectivity of virions produced from Th2 cells, and reduction of APOBEC3G in Th1 cells increased infectivity of virions produced from Th1 cells, consistent with a causal role for APOBEC3G in the infectivity difference. These results indicate that APOBEC3G and APOBEC3F levels vary physiologically during CD4+ T lymphocyte differentiation, that interferon-gamma contributes to this modulation, and that this physiological regulation can cause changes in infectivity of progeny virions, even in the presence of HIV-1 vif
— id: 93987, year: 2009, vol: 5, page: e1000292, stat: Journal Article,

Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells
Wang, Rui; Kozhaya, Lina; Mercer, Frances; Khaitan, Alka; Fujii, Hodaka; Unutmaz, Derya
2009 Aug 11;106(32):13439-13444, Proceedings of the National Academy of Sciences of the United States of America
The molecules that define human regulatory T cells (Tregs) phenotypically and functionally remain to be fully characterized. We recently showed that activated human Tregs express mRNA for a transmembrane protein called glycoprotein A repetitions predominant (GARP, or LRRC32). Here, using a GARP-specific mAb, we demonstrate that expression of GARP on activated Tregs correlates with their suppressive capacity. However, GARP was not induced on T cells activated in the presence of TGFbeta, which expressed high levels of FOXP3 and lacked suppressive function. Ectopic expression of FOXP3 in conventional T cells was also insufficient for induction of GARP expression in most donors. Functionally, silencing GARP in Tregs only moderately attenuated their suppressive activity. CD25+ T cells sorted for high GARP expression displayed more potent suppressive activity compared with CD25+GARP- cells. Remarkably, CD25+GARP- T cells expanded in culture contained 3-5 fold higher IL-17-secreting cells compared with either CD25+GARP+ or CD25-GARP- cells, suggesting that high GARP expression can potentially discriminate Tregs from those that have switched to Th17 lineage. We also determined whether GARP expression correlates with FOXP3-expressing T cells in human immunodeficiency virus (HIV) -infected subjects. A subset of HIV+ individuals with high percentages of FOXP3+ T cells did not show proportionate increase in GARP+ T cells. This finding suggests that higher FOXP3 levels observed in these HIV+ individuals is possibly due to immune activation rather than to an increase in Tregs. Our findings highlight the significance of GARP both in dissecting duality of Treg/Th17 cell differentiation and as a marker to identify bona fide Tregs during diseases with chronic immune activation
— id: 101894, year: 2009, vol: 106, page: 13439, stat: Journal Article,

Suppression of HIV-specific and allogeneic T cell activation by human regulatory T cells is dependent on the strength of signals
Antons, Amanda K; Wang, Rui; Kalams, Spyros A; Unutmaz, Derya
2008 ;3(8):e2952-e2952, PLoS ONE
Regulatory T cells (Tregs) suppress immune responses against both self and non-self antigens. Tregs require activation through the T cell receptor (TCR) and IL-2 to exert their suppressive functions. However, how strength of TCR signals modulate the potency of Treg-mediated suppression of antigen-specific T cell activation remain unclear. We found that both strength of TCR signals and ratios of Tregs to target cells, either through superantigen, allogeneic antigens or HIV-specific peptides, modified the suppressive ability of Tregs. While human Tregs were able to mediate suppression in the presence of only autologous antigen-presenting cells, this was much less efficient as compared to when Tregs were activated by allogeneic dendritic cells. In another physiologically relevant system, we show that the strength of peptide stimulation, high frequency of responder CD8+ T cells or presence of high IL-2 can override the suppression of HIV-specific CD8+ T cells by Tregs. These findings suggest that ratios and TCR activation of human Tregs, are important parameters to overcome robust immune responses to pathogens or allogeneic antigens. Modulating the strength of T cell signals and selective enhancement or depletion of antigen-specific Tregs thus may have implications for designing potent vaccines and regulating immune responses during allogeneic transplantation and chronic infections
— id: 93989, year: 2008, vol: 3, page: e2952, stat: Journal Article,

Naive precursors of human regulatory T cells require FoxP3 for suppression and are susceptible to HIV infection
Antons, Amanda K; Wang, Rui; Oswald-Richter, Kyra; Tseng, Michelle; Arendt, Christopher W; Kalams, Spyros A; Unutmaz, Derya
2008 Jan 15;180(2):764-773, Journal of immunology
CD4+CD25+ human regulatory T cells (Treg cells), which express the transcription factor FoxP3, suppress T cell activation. In this study, we sought to define cellular and molecular mechanisms of human Treg cell differentiation. A subset of human naive CD4+ T cells that are CD25+ express high levels of FoxP3. We show that upon activation through the TCR, these FoxP3-expressing naive T cells (termed TNreg cells) greatly expand in vitro. Expanded TNreg cells acquire a full Treg phenotype with potent suppressive activity and display low IL-2 production upon TCR stimulation. TNreg cells in which FoxP3 expression was reduced through RNA interference lost their suppressive activity, but retained their low IL-2 secretion in response to TCR stimulation. Furthermore, in support of the notion that TNreg cells represent a separate lineage of naive cells, we found that they were more susceptible to HIV infection as compared with naive CD4+ T cells. Based on these findings, we propose that TNreg cells are precursors for human Treg cells and that these cells require a high level of FoxP3 expression to maintain their suppressive function. Accordingly, modulation of TNreg cell numbers during infections such as HIV may disrupt human Treg cell development, and contribute to chronic immune activation
— id: 78894, year: 2008, vol: 180, page: 764, stat: Journal Article,

Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel
Faley, Shannon; Seale, Kevin; Hughey, Jacob; Schaffer, David K; VanCompernolle, Scott; McKinney, Brett; Baudenbacher, Franz; Unutmaz, Derya; Wikswo, John P
2008 Oct;8(10):1700-1712, Lab on a chip
Deciphering the signaling pathways that govern stimulation of naive CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of naive CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations
— id: 93988, year: 2008, vol: 8, page: 1700, stat: Journal Article,

Human natural killer T cells infiltrate into the skin at elicitation sites of allergic contact dermatitis
Gober, Michael D; Fishelevich, Rita; Zhao, Yuming; Unutmaz, Derya; Gaspari, Anthony A
2008 Jun;128(6):1460-1469, Journal of investigative dermatology
The purpose of this study is to identify invariant natural killer T cells (NKT cells) in cellular infiltrate of human allergic contact dermatitis (ACD) skin challenge sites. Skin biopsy specimens were taken from positive patch test reactions from 10 different patients (9 different allergens) and studied by immunochemistry, real-time PCR, nested PCR, and in situ hybridization to identify NKT cells and the cytokines associated with this cell type. Invariant NKT cells were identified in all the 10 skin biopsy specimens studied, ranging from 1.72 to 33% of the cellular infiltrate. These NKT cells were activated in all cases, as they expressed cytokine transcripts for IFN-gamma and IL-4. Invariant NKT cells are present in ACD, regardless of the allergen that triggers the reaction, and are in an activated state. We conclude that innate immunity plays a role in late phases of type IV hypersensitivity reactions and may be responding to self-lipids released during allergic inflammation. These data complement the previous work by other investigators that suggest that NKT cells are important in the early cellular response during primary immune responses to allergens. Herein, it is demonstrated that NKT cells are constantly present during the late elicitation phase of human type IV hypersensitivity reactions
— id: 78896, year: 2008, vol: 128, page: 1460, stat: Journal Article,

FoxP3+CD4+ regulatory T cells play an important role in acute HIV-1 infection in humanized Rag2-/-gammaC-/- mice in vivo
Jiang, Qi; Zhang, Liguo; Wang, Rui; Jeffrey, Jerry; Washburn, Michael L; Brouwer, Dedeke; Barbour, Selena; Kovalev, Grigoriy I; Unutmaz, Derya; Su, Lishan
2008 Oct 1;112(7):2858-2868, Blood
The role of FoxP3(+)CD4(+) regulatory T (Treg) cells in HIV-1 disease in vivo is poorly understood due to the lack of a robust model. We report here that CD4(+)FoxP3(+) T cells are developed in all lymphoid organs in humanized Rag2(-/-)gammaC(-/-) (DKO-hu HSC) mice and they display both Treg phenotype and Treg function. These FoxP3(+) Treg cells are preferentially infected and depleted by a pathogenic HIV-1 isolate in HIV-infected DKO-hu HSC mice; and depletion of Treg cells is correlated with induction of their apoptosis in vivo. When CD4(+)CD25(+/hi) Treg cells are depleted with the IL-2-toxin fusion protein (denileukin diftitox), HIV-1 infection is significantly impaired. This is demonstrated by reduced levels of productively infected cells in lymphoid organs and lower plasma viremia. Therefore, FoxP3(+) Treg cells are productively infected and play an important role in acute HIV-1 infection in vivo. The DKO-hu HSC mouse will be a valuable model to study human Treg functions and their role in HIV-1 pathogenesis in vivo
— id: 93990, year: 2008, vol: 112, page: 2858, 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,

IRF1: a deus ex machina in TH1 differentiation
Unutmaz, Derya; Vilcek, Jan
2008 Jan;9(1):9-10, Nature immunology
— id: 78895, year: 2008, vol: 9, page: 9, stat: Journal Article,

Identification of a regulatory T cell specific cell surface molecule that mediates suppressive signals and induces Foxp3 expression
Wang, Rui; Wan, Qi; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya
2008 ;3(7):e2705-e2705, PLoS ONE
Regulatory T (T(reg)) cells control immune activation and maintain tolerance. How T(regs) mediate their suppressive function is unclear. Here we identified a cell surface molecule, called GARP, (or LRRC32), which within T cells is specifically expressed in T(regs) activated through the T cell receptor (TCR). Ectopic expression of GARP in human naive T (T(N)) cells inhibited their proliferation and cytokine secretion upon TCR activation. Remarkably, GARP over-expression in T(N) cells induced expression of T(reg) master transcription factor Foxp3 and endowed them with a partial suppressive function. The extracellular but not the cytoplasmic region of GARP, was necessary for these functions. Silencing Foxp3 in human T(reg) cells reduced expression of GARP and attenuated their suppressive function. However, GARP function was not affected when Foxp3 was downregulated in GARP-overexpressing cells, while silencing GARP in Foxp3-overexpressing cells reduced their suppressive activity. These findings reveal a novel cell surface molecule-mediated regulatory mechanism, with implications for modulating aberrant immune responses
— id: 86551, year: 2008, vol: 3, page: e2705, stat: Journal Article,

Resistance of primary murine CD4+ T cells to Helicobacter pylori vacuolating cytotoxin
Algood, Holly M Scott; Torres, Victor J; Unutmaz, Derya; Cover, Timothy L
2007 Jan;75(1):334-341, Infection & immunity
Persistent colonization of the human stomach by Helicobacter pylori is a risk factor for the development of gastric cancer and peptic ulcer disease. H. pylori secretes a toxin, VacA, that targets human gastric epithelial cells and T lymphocytes and enhances the ability of H. pylori to colonize the stomach in a mouse model. To examine how VacA contributes to H. pylori colonization of the mouse stomach, we investigated whether murine T lymphocytes were susceptible to VacA activity. VacA inhibited interleukin-2 (IL-2) production by a murine T-cell line (LBRM-33), similar to its effects on a human T-cell line (Jurkat), but did not inhibit IL-2 production by primary murine splenocytes or CD4+ T cells. VacA inhibited activation-induced proliferation of primary human CD4+ T cells but did not inhibit the proliferation of primary murine CD4+ T cells. Flow cytometry studies indicated that the levels of VacA binding to primary murine CD4+ T cells were significantly lower than levels of VacA binding to human CD4+ T cells. This suggests that the resistance of primary murine CD4+ T cells to VacA is attributable, at least in part, to impaired VacA binding to these cells
— id: 71091, year: 2007, vol: 75, page: 334, stat: Journal Article,

Production of specific mRNA transcripts, usage of an alternate promoter, and octamer-binding transcription factors influence the surface expression levels of the HIV coreceptor CCR5 on primary T cells
Mummidi, Srinivas; Adams, Lisa M; VanCompernolle, Scott E; Kalkonde, Mrunal; Camargo, Jose F; Kulkarni, Hemant; Bellinger, Adam S; Bonello, Gregory; Tagoh, Hiromi; Ahuja, Seema S; Unutmaz, Derya; Ahuja, Sunil K
2007 May 1;178(9):5668-5681, Journal of immunology
Surface levels of CCR5 on memory CD4(+) T cells influence HIV-1/AIDS susceptibility. Alternative promoter usage results in the generation of CCR5 mRNA isoforms that differ based on whether they contain or lack the untranslated exon 1. The impact of exon 1-containing transcripts on CCR5 surface expression is unknown. In this study, we show that the increased cell surface expression of CCR5 on primary T cells is associated with selective enrichment of exon 1-containing transcripts. The promoter that drives exon 1-containing transcripts is highly active in primary human T cells but not in transformed T cell lines. The transcription factors Oct-1 and -2 inhibit and enhance, respectively, the expression of exon 1-containing transcripts and CCR5 surface levels. However, polymorphisms at homologous octamer-binding sites in the CCR5 promoter of nonhuman primates abrogate the binding of these transcription factors. These results identify exon 1-containing transcripts, and the cis-trans factors that regulate the expression levels of these mRNA isoforms as key parameters that affect CCR5 surface expression levels, and by extension, susceptibility to HIV/AIDS among humans, and possibly, the observed interspecies differences in susceptibility to lentiviral infection
— id: 73847, year: 2007, vol: 178, page: 5668, stat: Journal Article,

Identification of a CCR5-expressing T cell subset that is resistant to R5-tropic HIV infection
Oswald-Richter, Kyra; Grill, Stacy M; Leelawong, Mindy; Tseng, Michelle; Kalams, Spyros A; Hulgan, Todd; Haas, David W; Unutmaz, Derya
2007 Apr;3(4):e58-e58, PLoS pathogens
Infection with HIV-1 perturbs homeostasis of human T cell subsets, leading to accelerated immunologic deterioration. While studying changes in CD4(+) memory and naive T cells during HIV-1 infection, we found that a subset of CD4(+) effector memory T cells that are CCR7(-)CD45RO(-)CD45RA(+) (referred to as TEMRA cells), was significantly increased in some HIV-infected individuals. This T cell subset displayed a differentiated phenotype and skewed Th1-type cytokine production. Despite expressing high levels of CCR5, TEMRA cells were strikingly resistant to infection with CCR5 (R5)-tropic HIV-1, but remained highly susceptible to CXCR4 (X4)-tropic HIV-1. The resistance of TEMRA cells to R5-tropic viruses was determined to be post-entry of the virus and prior to early viral reverse transcription, suggesting a block at the uncoating stage. Remarkably, in a subset of the HIV-infected individuals, the relatively high proportion of TEMRA cells within effector T cells strongly correlated with higher CD4(+) T cell numbers. These data provide compelling evidence for selection of an HIV-1-resistant CD4(+) T cell population during the course of HIV-1 infection. Determining the host factors within TEMRA cells that restrict R5-tropic viruses and endow HIV-1-specific CD4(+) T cells with this ability may result in novel therapeutic strategies against HIV-1 infection
— id: 78898, year: 2007, vol: 3, page: e58, stat: Journal Article,

Histidine phosphorylation of the Ca2+-activated K+ channel KCa3.1 by nucleoside diphosphate kinase B (NDPK-B) is required for KCa3.1 channel activation and the reactivation of CD4 T lymphocytes
Srivastava, S; Li, Z; Ko, K; Choudhury, P; Albaqumi, M; Johnson, AK; Yan, Y; Backer, J; Unutmaz, D; Coetzee, WA; Skolnik, EY
2007 JAN ;108(11):200A-200A, Biophysical journal
— id: 71388, year: 2007, vol: 108, page: 200A, stat: Journal Article,

Helicobacter pylori vacuolating cytotoxin inhibits activation-induced proliferation of human T and B lymphocyte subsets
Torres, Victor J; VanCompernolle, Scott E; Sundrud, Mark S; Unutmaz, Derya; Cover, Timothy L
2007 Oct 15;179(8):5433-5440, Journal of immunology
Helicobacter pylori are Gram-negative bacteria that persistently colonize the human gastric mucosa despite the recruitment of immune cells. The H. pylori vacuolating cytotoxin (VacA) recently has been shown to inhibit stimulation-induced proliferation of primary human CD4(+) T cells. In this study, we investigated effects of VacA on the proliferation of various other types of primary human immune cells. Intoxication of PBMC with VacA inhibited the stimulation-induced proliferation of CD4(+) T cells, CD8(+) T cells, and B cells. VacA also inhibited the proliferation of purified primary human CD4(+) T cells that were stimulated by dendritic cells. VacA inhibited both T cell-induced and PMA/anti-IgM-induced proliferation of purified B cells. Intoxication with VacA did not alter the magnitude of calcium flux that occurred upon stimulation of CD4(+) T cells or B cells, indicating that VacA does not alter early signaling events required for activation and proliferation. VacA reduced the mitochondrial membrane potential of CD4(+) T cells, but did not reduce the mitochondrial membrane potential of B cells. We propose that the immunomodulatory actions of VacA on T and B lymphocytes, the major effectors of the adaptive immune response, may contribute to the ability of H. pylori to establish a persistent infection in the human gastric mucosa
— id: 78897, year: 2007, vol: 179, page: 5433, stat: Journal Article,

Granulocyte-macrophage colony-stimulating factor regulates effector differentiation of invariant natural killer T cells during thymic ontogeny
Bezbradica, Jelena S; Gordy, Laura E; Stanic, Aleksandar K; Dragovic, Srdjan; Hill, Timothy; Hawiger, Jacek; Unutmaz, Derya; Van Kaer, Luc; Joyce, Sebastian
2006 Sep;25(3):487-497, Immunity
Invariant natural killer T (iNKT) cell-derived cytokines have important functions in inflammation, host defense, and immunoregulation. Yet, when and how iNKT cells undergo effector differentiation, which endows them with the capacity to rapidly secrete cytokines upon activation, remains unknown. We discovered that granulocyte-macrophage colony-stimulating factor (Csf-2)-deficient mice developed iNKT cells that failed to respond to the model antigen alpha-galactosylceramide because of an intrinsic defect in the fusion of secretory vesicles with the plasma membrane. Exogenous Csf-2 corrected the functional defect only when supplied during the development of thymic, but not mature, splenic Csf-2-deficient iNKT cells. Thus, we ascribe a unique function to Csf-2, which regulates iNKT cell effector differentiation during development by a mechanism that renders them competent for cytokine secretion
— id: 71094, year: 2006, vol: 25, page: 487, stat: Journal Article,

Yeast zymosan, a stimulus for TLR2 and dectin-1, induces regulatory antigen-presenting cells and immunological tolerance
Dillon, Stephanie; Agrawal, Sudhanshu; Banerjee, Kaustuv; Letterio, John; Denning, Timothy L; Oswald-Richter, Kyra; Kasprowicz, Deborah J; Kellar, Kathryn; Pare, Jeff; van Dyke, Thomas; Ziegler, Steven; Unutmaz, Derya; Pulendran, Bali
2006 Apr;116(4):916-928, Journal of clinical investigation
Emerging evidence suggests critical roles for APCs in suppressing immune responses. Here, we show that zymosan, a stimulus for TLR2 and dectin-1, regulates cytokine secretion in DCs and macrophages to induce immunological tolerance. First, zymosan induces DCs to secrete abundant IL-10 but little IL-6 and IL-12(p70). Induction of IL-10 is dependent on TLR2- and dectin-1-mediated activation of ERK MAPK via a mechanism independent of the activation protein 1 (AP-1) transcription factor c-Fos. Such DCs stimulate antigen-specific CD4+ T cells poorly due to IL-10 and the lack of IL-6. Second, zymosan induces F4-80+ macrophages in the splenic red pulp to secrete TGF-beta. Consistent with these effects on APCs, injection of zymosan plus OVA into mice results in OVA-specific T cells that secrete little or no Th1 or Th2 cytokines, but secrete robust levels of IL-10, and are unresponsive to challenge with OVA plus adjuvant. Finally, coinjection of zymosan with OVA plus LPS suppresses the response to OVA via a mechanism dependent on IL-10, TGF-beta, and lack of IL-6. Together, our data demonstrate that zymosan stimulates IL-10+ IL-12(p70)- IL-6low regulatory DCs and TGF-beta+ macrophages to induce immunological tolerance. These data suggest several targets for pharmacological modulation of immune responses in various clinical settings
— id: 71095, year: 2006, vol: 116, page: 916, stat: Journal Article,

Human natural killer T cells are heterogeneous in their capacity to reprogram their effector functions
Eger, Karla A; Sundrud, Mark S; Motsinger, Alison A; Tseng, Michelle; Van Kaer, Luc; Unutmaz, Derya
2006 ;1:e50-e50, PLoS ONE
BACKGROUND: Natural killer T (NKT) cells are a subset of T cells that help potentiate and regulate immune responses. Although human NKT cell subsets with distinct effector functions have been identified, it is unclear whether the effector functions of these subsets are imprinted during development or can be selectively reprogrammed in the periphery. RESULTS: We found that neonatal NKT cells are predominantly CD4+ and express higher levels of CCR7 and CD62L and lower levels of CD94 and CD161 than adult CD4+ or CD4- NKT cell subsets. Accordingly, neonatal NKT cells were more flexible than adult CD4+ NKT cells in their capacity to acquire Th1- or Th2-like functions upon either cytokine-mediated polarization or ectopic expression of the Th1 or Th2 transcription factors T-bet and GATA-3, respectively. Consistent with their more differentiated phenotype, CD4- NKT cells were predominantly resistant to functional reprogramming and displayed higher cytotoxic function. In contrast to conventional T cells, neither the expression of CXCR3 nor the cytotoxic capacity of neonatal NKT cells could be reprogrammed. CONCLUSIONS AND SIGNIFICANCE: Together, these results suggest that neonatal CD4+, adult CD4+, and adult CD4- NKT may represent unique states of maturation and that some functions of human NKT cells may be developmentally imprinted, while others are acquired similar to conventional T cell subsets during peripheral maturation and differentiation. Given the potent immuno-regulatory functions of NKT cells, these findings have important implications for the development of novel NKT cell-based therapeutics and vaccines
— id: 71090, year: 2006, vol: 1, page: e50, stat: Journal Article,

Immunogenetics of CD4 lymphocyte count recovery during antiretroviral therapy: An AIDS Clinical Trials Group study
Haas, David W; Geraghty, Daniel E; Andersen, Janet; Mar, Jessica; Motsinger, Alison A; D'Aquila, Richard T; Unutmaz, Derya; Benson, Constance A; Ritchie, Marylyn D; Landay, Alan
2006 Oct 15;194(8):1098-1107, Journal of infectious diseases
During antiretroviral therapy, CD4 lymphocyte count increases are modest in some patients despite virologic control. We explored whether polymorphisms in genes important for T cell expansion, survival, and apoptosis are associated with the magnitude of CD4 lymphocyte count recovery during antiretroviral therapy. We studied treatment-naive individuals who achieved sustained control of plasma viremia (<400 HIV-1 RNA copies/mL) for at least 48 weeks after initiation of antiretroviral therapy and compared genotypes among individuals who had an increase of either <200 or > or =200 CD4 cells/mm3 from baseline. A total of 137 single-nucleotide polymorphisms across 17 genes were characterized in 873 study participants. In multivariate analyses that controlled for clinical variables, polymorphisms in genes encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), TNF- alpha , Bcl-2-interacting molecule (Bim), interleukin (IL)-15, and IL-15 receptor alpha chain (IL-15R alpha ) were associated with the magnitude of the increase in CD4 lymphocyte count, as were haplotypes in genes encoding interferon- alpha , IL-2, and IL-15R alpha (P < .05, for each). Multifactor dimensionality reduction identified a gene-gene interaction between IL-2/IL-15 receptor common beta chain and IL-2/IL-7/IL-15 receptor common gamma chain. Immune recovery during antiretroviral therapy is a complex phenotype that is influenced by multiple genetic variants. Future studies should validate these tentative associations and define underlying mechanisms
— id: 71093, year: 2006, vol: 194, page: 1098, stat: Journal Article,

Helicobacter pylori VacA toxin inhibits human immunodeficiency virus infection of primary human T cells
Oswald-Richter, Kyra; Torres, Victor J; Sundrud, Mark S; VanCompernolle, Scott E; Cover, Timothy L; Unutmaz, Derya
2006 Dec;80(23):11767-11775, Journal of virology
Human CD4(+) T cells are major targets for human immunodeficiency virus (HIV) infection. Resting T cells are resistant to HIV infection unless activated through the T-cell receptor (TCR) or by cytokine signals. How T-cell signaling promotes susceptibility of T cells to HIV infection remains poorly understood. Here we demonstrate that the VacA toxin produced by Helicobacter pylori can inhibit HIV infection of primary T cells, stimulated through the TCR or by cytokines alone. This activity of VacA was dependent on its ability to form membrane channels. VacA suppressed HIV infection of T cells at a stage after viral entry, post-reverse transcription and pre-two-long-terminal-repeat circle formation, similar to the cytokine signaling inhibitor rapamycin. Mechanistically, neither VacA nor rapamycin inhibited the activation of cytokine signal transduction components (STAT5, p42/44 mitogen-activated protein kinase, or p38), but both blocked activation of key regulatory proteins required for G(1) cell cycle transition. In contrast to rapamycin, VacA did not suppress phosphorylation of p70 S6 kinase but caused mitochondrial depolarization and ATP depletion within primary T cells. These results suggest that VacA inhibits T-cell activation and HIV infection via a novel mechanism. Identifying the host cell targets of VacA could be useful for elucidating the HIV life cycle within primary T cells
— id: 71092, year: 2006, vol: 80, page: 11767, stat: Journal Article,

Phosphatidylinositol-3 phosphatase myotubularin-related protein 6 negatively regulates CD4 T cells
Srivastava, Shekhar; Ko, Kyung; Choudhury, Papiya; Li, Zhai; Johnson, Amanda K; Nadkarni, Vivek; Unutmaz, Derya; Coetzee, William A; Skolnik, Edward Y
2006 Aug;26(15):5595-5602, Molecular & cellular biology
Intracellular Ca2+ levels rapidly rise following cross-linking of the T-cell receptor (TCR) and function as a critical intracellular second messenger in T-cell activation. It has been relatively under appreciated that K+ channels play an important role in Ca2+ influx into T lymphocytes by helping to maintain a negative membrane potential which provides an electrochemical gradient to drive Ca2+ influx. Here we show that the Ca2+-activated K+ channel, KCa3.1, which is critical for Ca2+ influx in reactivated naive T cells and central memory T cells, requires phosphatidylinositol-3 phosphatase [PI(3)P] for activation and is inhibited by the PI(3)P phosphatase myotubularin-related protein 6 (MTMR6). Moreover, by inhibiting KCa3.1, MTMR6 functions as a negative regulator of Ca2+ influx and proliferation of reactivated human CD4 T cells. These findings point to a new and unexpected role for PI(3)P and the PI(3)P phosphatase MTMR6 in the regulation of Ca2+ influx in activated CD4 T cells and suggest that MTMR6 plays a critical role in setting a minimum threshold for a stimulus to activate a T cell
— id: 68660, year: 2006, vol: 26, page: 5595, stat: Journal Article,

Histidine phosphorylation of the potassium channel KCa3.1 by nucleoside diphosphate kinase B is required for activation of KCa3.1 and CD4 T cells
Srivastava, Shekhar; Li, Zhai; Ko, Kyung; Choudhury, Papiya; Albaqumi, Mamdouh; Johnson, Amanda K; Yan, Ying; Backer, Jonathan M; Unutmaz, Derya; Coetzee, William A; Skolnik, Edward Y
2006 Dec 8;24(5):665-675, Molecular cell
The Ca2+ -activated K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of B and T cells. Inhibitors of KCa3.1 are in development to treat autoimmune diseases and transplant rejection, underscoring the importance in understanding how these channels are regulated. We show that nucleoside diphosphate kinase B (NDPK-B), a mammalian histidine kinase, functions downstream of PI(3)P to activate KCa3.1. NDPK-B directly binds and activates KCa3.1 by phosphorylating histidine 358 in the carboxyl terminus of KCa3.1. Endogenous NDPK-B is also critical for KCa3.1 channel activity and the subsequent activation of CD4 T cells. These findings provide one of the best examples whereby histidine phosphorylation regulates a biological process in mammals, and provide an example whereby a channel is regulated by histidine phosphorylation. The critical role for NDPK-B in the reactivation of CD4 T cells indicates that understanding NDPK-B regulation should uncover novel pathways required for T cell activation
— id: 69707, year: 2006, vol: 24, page: 665, stat: Journal Article,

Distinct roles of dendritic cells and B cells in Va14Ja18 natural T cell activation in vivo
Bezbradica, Jelena S; Stanic, Aleksandar K; Matsuki, Naoto; Bour-Jordan, Helene; Bluestone, Jeffrey A; Thomas, James W; Unutmaz, Derya; Van Kaer, Luc; Joyce, Sebastian
2005 Apr 15;174(8):4696-4705, Journal of immunology
Va14Ja18 natural T (iNKT) cells are innate, immunoregulatory lymphocytes that recognize CD1d-restricted lipid Ags such as alpha-galactosylceramide (alpha GalCer). The immunoregulatory functions of iNKT cells are dependent upon either IFN-gamma or IL-4 production by these cells. We hypothesized that alpha GalCer presentation by different CD1d-positive cell types elicits distinct iNKT cell functions. In this study we report that dendritic cells (DC) play a critical role in alpha GalCer-mediated activation of iNKT cells and subsequent transactivation of NK cells. Remarkably, B lymphocytes suppress DC-mediated iNKT and NK cell activation. Nevertheless, alpha GalCer presentation by B cells elicits low IL-4 responses from iNKT cells. This finding is particularly interesting because we demonstrate that NOD DC are defective in eliciting iNKT cell function, but their B cells preferentially activate this T cell subset to secrete low levels of IL-4. Thus, the differential immune outcome based on the type of APC that displays glycolipid Ags in vivo has implications for the design of therapies that harness the immunoregulatory functions of iNKT cells
— id: 71100, year: 2005, vol: 174, page: 4696, stat: Journal Article,

Nanoparticulate system for efficient gene transfer into refractory cell targets
Carlesso, Gianluca; Kozlov, Eugene; Prokop, Ales; Unutmaz, Derya; Davidson, Jeffrey M
2005 May-Jun;6(3):1185-1192, Biomacromolecules
A biocompatible, nanoparticulate formulation has been designed to retain, protect, and deliver adenoviral gene constructs over an extended time course. Such devices can be administered locally or systemically with low toxicity. A multipolymeric nanoparticulate system, featuring very high stability in physiologic media, was designed to allow efficient in vitro gene transfer. The efficacy of nanoparticulate delivery is effective in cell systems that are normally refractory to gene transfer, such as pancreatic islets and antigen-presenting cells. The findings suggest a nonspecific uptake system that permits adenoviral particle release within the transfected cells. A comparison with literature data revealed that our system is efficient at much lower levels (at least three orders of magnitude) of infectious viral particles
— id: 71099, year: 2005, vol: 6, page: 1185, stat: Journal Article,

Mechanisms of nonrandom human immunodeficiency virus type 1 infection and double infection: preference in virus entry is important but is not the sole factor
Chen, Jianbo; Dang, Que; Unutmaz, Derya; Pathak, Vinay K; Maldarelli, Frank; Powell, Douglas; Hu, Wei-Shau
2005 Apr;79(7):4140-4149, Journal of virology
We previously demonstrated that human immunodeficiency virus type 1 (HIV-1) infection is nonrandom and that double infection occurs more frequently than predicted from random events. To probe the possible mechanisms for nonrandom infection, we examined the role of HIV-1 entry pathways by using viruses pseudotyped with either CCR5-tropic HIV-1 Env or vesicular stomatitis virus G protein (VSV G). These two proteins use different receptors and entry pathways. We found that regardless of the protein used, double infection occurred more frequently than random events, indicating nonrandom HIV-1 infection in both entry pathways. However, the frequency of double infection differed significantly, depending on the envelope protein. In primary CD4(+) T cells, double infection occurred most frequently when both viruses had CCR5-tropic HIV-1 Env and least frequently when the two viruses had different envelopes. These results indicated that the preference in virus entry was a significant but not the only factor contributing to nonrandom double infection. Furthermore, we demonstrated that the CD4 expression level in primary T cells affects their susceptibility to CCR5-tropic HIV-1 infection but not VSV G-pseudotyped HIV-1 infection. We have also examined infection with two viruses pseudotyped with CCR5- or CXCR4-tropic HIV-1 Env and have found that double infection occurred more frequently than random events. These results indicate that coreceptor usage is not a barrier to recombination between the two virus populations. In our previous study, we also demonstrated nonrandom double infection via dendritic cell (DC)-mediated HIV-1 transmission. To test our hypothesis that multiple HIV-1 virions are transmitted during DC-T-cell contact, we used two populations of DCs, each capturing one vector virus, and added both DC populations to T cells. We observed a decreased frequency of double infection compared with experiments in which DCs captured both viruses simultaneously. Therefore, these results support our hypothesis that multiple virions are transmitted from DCs to T cells during cell-mediated HIV-1 transmission
— id: 71101, year: 2005, vol: 79, page: 4140, stat: Journal Article,

The innate immune system and HIV pathogenesis
Eger, Karla A; Unutmaz, Derya
2005 Feb;2(1):10-15, Current HIV/AIDS reports
Innate immunity represents the first line of defense against microbial infections. The innate immune system is activated by conserved structures present on most pathogens and profoundly regulates subsequent adaptive immune responses. HIV is notorious for evading and disrupting the immune system. Although HIV directly targets and gradually destroys the adaptive immunity, it has become clear that the virus also perturbs the components of the innate immune system. In this paper, we review the role of two innate lymphocyte subsets, natural killer and natural killer T cells, that are disrupted during HIV infection
— id: 71098, year: 2005, vol: 2, page: 10, stat: Journal Article,

Transcription factor GATA-1 potently represses the expression of the HIV-1 coreceptor CCR5 in human T cells and dendritic cells
Sundrud, Mark S; Vancompernolle, Scott E; Eger, Karla A; Bruno, Tullia C; Subramaniam, Arun; Mummidi, Srinivas; Ahuja, Sunil K; Unutmaz, Derya
2005 Nov 15;106(10):3440-3448, Blood
CC chemokine receptor 5 (CCR5) is the major HIV-1 coreceptor and its expression levels are a critical determinant of HIV-1 infection. However, the molecular mechanisms of CCR5 regulation in primary targets of HIV-1 remain unknown. Despite binding to conserved DNA elements, we show that the transcription factors GATA binding protein 1 (GATA-1) and GATA-3 differentially suppress the expression of CCR5 in stem-cell-derived dendritic cells and primary human T-cell subsets. In addition, GATA-1 expression was also more potent than GATA-3 in suppressing T helper 1 (Th1)-associated genes, interferon-gamma (IFNgamma), and CXC chemokine receptor-3 (CXCR3). GATA-1, but not GATA-3, potently suppressed CCR5 transcription, thereby rendering human T cells resistant to CCR5-tropic HIV-1 infection. However, GATA-1 could also serve as a surrogate for GATA-3 in its canonic role of programming Th2 gene expression. These findings provide insight into GATA-3-mediated gene regulation during T-cell differentiation. Importantly, decoding the mechanisms of GATA-1-mediated repression of CCR5 may offer an opportunity to develop novel approaches to inhibit CCR5 expression in T cells
— id: 71097, year: 2005, vol: 106, page: 3440, stat: Journal Article,

Antimicrobial peptides from amphibian skin potently inhibit human immunodeficiency virus infection and transfer of virus from dendritic cells to T cells
VanCompernolle, Scott E; Taylor, R Jeffery; Oswald-Richter, Kyra; Jiang, Jiyang; Youree, Bryan E; Bowie, John H; Tyler, Michael J; Conlon, J Michael; Wade, David; Aiken, Christopher; Dermody, Terence S; KewalRamani, Vineet N; Rollins-Smith, Louise A; Unutmaz, Derya
2005 Sep;79(18):11598-11606, Journal of virology
Topical antimicrobicides hold great promise in reducing human immunodeficiency virus (HIV) transmission. Amphibian skin provides a rich source of broad-spectrum antimicrobial peptides including some that have antiviral activity. We tested 14 peptides derived from diverse amphibian species for the capacity to inhibit HIV infection. Three peptides (caerin 1.1, caerin 1.9, and maculatin 1.1) completely inhibited HIV infection of T cells within minutes of exposure to virus at concentrations that were not toxic to target cells. These peptides also suppressed infection by murine leukemia virus but not by reovirus, a structurally unrelated nonenveloped virus. Preincubation with peptides prevented viral fusion to target cells and disrupted the HIV envelope. Remarkably, these amphibian peptides also were highly effective in inhibiting the transfer of HIV by dendritic cells (DCs) to T cells, even when DCs were transiently exposed to peptides 8 h after virus capture. These data suggest that amphibian-derived peptides can access DC-sequestered HIV and destroy the virus before it can be transferred to T cells. Thus, amphibian-derived antimicrobial peptides show promise as topical inhibitors of mucosal HIV transmission and provide novel tools to understand the complex biology of HIV capture by DCs
— id: 71096, year: 2005, vol: 79, page: 11598, 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,

Nonrandom HIV-1 infection and double infection via direct and cell-mediated pathways
Dang, Que; Chen, Jianbo; Unutmaz, Derya; Coffin, John M; Pathak, Vinay K; Powell, Douglas; KewalRamani, Vineet N; Maldarelli, Frank; Hu, Wei-Shau
2004 Jan 13;101(2):632-637, Proceedings of the National Academy of Sciences of the United States of America
Cells infected with two related retroviruses can generate heterozygous virions, which are the precursors of recombinant proviruses. Although many studies have focused on the frequencies and mechanisms of retroviral recombination, little is known about the dynamics of double infection. To examine this issue, viruses generated from two HIV-1 vectors containing different markers were mixed together, and were used to infect target cells. The numbers of cells expressing none, one, or both markers were measured and were used to calculate whether double infection occurred at frequencies expected from random infection events. We found that double infection occurred significantly more frequently than predicted from random distribution; increased rates of double infection were observed in both a T cell line and primary activated CD4(+) T cells. In addition to direct virus infection, we also examined the nature of cell-mediated HIV-1 double infection. Increased double infection was observed in all experiments regardless of whether a cell line or primary human dendritic cells were used for capture and transmission of HIV-1. Therefore, our results indicate that HIV-1 double infection occurs more frequently than it would at random in both direct and cell-mediated HIV-1 infections. To our knowledge, this is the first direct evidence of nonrandom double infection in HIV-1. Frequent double HIV-1 infections in infected individuals would allow the generation of recombinant viruses that could then affect their pathogenesis and evolution
— id: 71106, year: 2004, vol: 101, page: 632, stat: Journal Article,

Perturbation of natural killer cell function and receptors during HIV infection
Eger, Karla A; Unutmaz, Derya
2004 Jul;12(7):301-303, Trends in microbiology
Natural killer (NK) cells play an important role in innate host defenses against a variety of pathogens. A recent report described striking perturbations in the expression of NK cell inhibitory and activating receptors in viremic human immunodeficiency virus (HIV)-infected patients, leading to functional abnormalities in these cells. This finding provides a mechanistic insight into NK cell dysfunction and its possible contribution to the impairment of innate host defenses in HIV-infected individuals
— id: 71103, year: 2004, vol: 12, page: 301, stat: Journal Article,

HIV infection of primary human T cells is determined by tunable thresholds of T cell activation
Oswald-Richter, Kyra; Grill, Stacy M; Leelawong, Mindy; Unutmaz, Derya
2004 Jun;34(6):1705-1714, European journal of immunology
HIV infection of primary human T cells requires T cell activation signals. However, how strength, duration, and quality of TCR signals affect susceptibility of resting human T cells to HIV infection remains poorly understood. We found that the same threshold and duration of antigen signals that lead to optimal T cell activation are required for HIV to progress beyond the level of reverse transcription within resting T cells. Remarkably, sustained cytokine signaling from the IL-2 receptor following TCR triggering was critical in establishing productive infection. While blockade of TCR signaling pathways with inhibitors of the phosphatidylinositol 3-kinase pathway caused a partial pre-integration block, another inhibitor, rapamycin, completely suppressed the infection. In contrast, cyclosporin A or FK506, inhibitors of NFAT, failed to block infection if the T cells were pre-activated. Collectively, these results bring to light significant parallels between successful HIV infection and optimal thresholds of T cell activation. Furthermore, our results underscore the critical role of IL-2 signaling in establishing productive HIV infection. These findings have important implications for our understanding of the complex interplay of HIV with host factors induced upon T cell activation
— id: 71104, year: 2004, vol: 34, page: 1705, stat: Journal Article,

HIV infection of naturally occurring and genetically reprogrammed human regulatory T-cells
Oswald-Richter, Kyra; Grill, Stacy M; Shariat, Nikki; Leelawong, Mindy; Sundrud, Mark S; Haas, David W; Unutmaz, Derya
2004 Jul;2(7):E198-E198, PLoS biology
A T-cell subset, defined as CD4(+)CD25(hi) (regulatory T-cells [Treg cells]), was recently shown to suppress T-cell activation. We demonstrate that human Treg cells isolated from healthy donors express the HIV-coreceptor CCR5 and are highly susceptible to HIV infection and replication. Because Treg cells are present in very few numbers and are difficult to expand in vitro, we genetically modified conventional human T-cells to generate Treg cells in vitro by ectopic expression of FoxP3, a transcription factor associated with reprogramming T-cells into a Treg subset. Overexpression of FoxP3 in naive human CD4(+) T-cells recapitulated the hyporesponsiveness and suppressive function of naturally occurring Treg cells. However, FoxP3 was less efficient in reprogramming memory T-cell subset into regulatory cells. In addition, FoxP3-transduced T-cells also became more susceptible to HIV infection. Remarkably, a portion of HIV-positive individuals with a low percentage of CD4(+) and higher levels of activated T-cells have greatly reduced levels of FoxP3(+)CD4(+)CD25(hi) T-cells, suggesting disruption of the Treg cells during HIV infection. Targeting and disruption of the T-cell regulatory system by HIV may contribute to hyperactivation of conventional T-cells, a characteristic of HIV disease progression. Moreover, the ability to reprogram human T-cells into Treg cells in vitro will greatly aid in decoding their mechanism of suppression, their enhanced susceptibility to HIV infection, and the unique markers expressed by this subset
— id: 71102, year: 2004, vol: 2, page: E198, stat: Journal Article,

Inhibition of primary human T cell proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects on IL-2 secretion
Sundrud, Mark S; Torres, Victor J; Unutmaz, Derya; Cover, Timothy L
2004 May 18;101(20):7727-7732, Proceedings of the National Academy of Sciences of the United States of America
Recent evidence indicates that the secreted Helicobacter pylori vacuolating toxin (VacA) inhibits the activation of T cells. VacA blocks IL-2 secretion in transformed T cell lines by suppressing the activation of nuclear factor of activated T cells (NFAT). In this study, we investigated the effects of VacA on primary human CD4(+) T cells. VacA inhibited the proliferation of primary human T cells activated through the T cell receptor (TCR) and CD28. VacA-treated Jurkat T cells secreted markedly diminished levels of IL-2 compared with untreated cells, whereas VacA-treated primary human T cells continued to secrete high levels of IL-2. Further experiments indicated that the VacA-induced inhibition of primary human T cell proliferation was not attributable to VacA effects on NFAT activation or IL-2 secretion. We show here that VacA suppresses IL-2-induced cell-cycle progression and proliferation of primary human T cells without affecting IL-2-dependent survival. Through the analysis of a panel of mutant VacA proteins, we demonstrate that VacA-mediated inhibition of T cell proliferation requires an intact N-terminal hydrophobic region necessary for the formation of anion-selective membrane channels. Remarkably, we demonstrate that one of these mutant VacA proteins [VacA-Delta(6-27)] abrogates the immunosuppressive actions of wild-type VacA in a dominant-negative fashion. We suggest that VacA may inhibit the clonal expansion of T cells that have already been activated by H. pylori antigens, thereby allowing H. pylori to evade the adaptive immune response and establish chronic infection
— id: 71105, year: 2004, vol: 101, page: 7727, stat: Journal Article,

APOBEC3B and APOBEC3C are potent inhibitors of simian immunodeficiency virus replication
Yu, Qin; Chen, Darlene; Konig, Renate; Mariani, Roberto; Unutmaz, Derya; Landau, Nathaniel R
2004 Dec 17;279(51):53379-53386, Journal of biological chemistry
In the human genome the apolipoprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC)3 gene has expanded into a tandem array of genes termed APOBEC3A-G. Two members of this family, APOBEC3G and APOBEC3F, have been found to have potent activity against virion infectivity factor deficient (Deltavif) human immunodeficiency virus 1 (HIV-1). These enzymes become encapsidated in Deltavif HIV-1 virions and in the next round of infection deaminate the newly synthesized reverse transcripts. The lentiviral Vif protein prevents the deamination by inducing the degradation of APOBEC3G and APOBEC3F. We report here that two additional APOBEC3 family members, APOBEC3B and APOBEC3C, have potent antiviral activity against simian immuno-deficiency virus (SIV), but not HIV-1. Both enzymes were encapsidated in HIV-1 and SIV virions and were active against Deltavif SIV(mac) and SIV(agm). SIV Vif neutralized the antiviral activity of APOBEC3C, but not that of APOBEC3B. APOBEC3B induced abundant G --> A mutations in both wild-type and Deltavif SIV reverse transcripts. APOBEC3C induced substantially fewer mutations. APOBEC3F was found to be active against SIV and sensitive to SIV(mac) Vif. These findings raise the possibility that the different APOBEC3 family members function to neutralize specific lentiviruses
— id: 68239, year: 2004, vol: 279, page: 53379, stat: Journal Article,

Effects of nelfinavir and its M8 metabolite on lymphocyte P-glycoprotein activity during antiretroviral therapy
Donahue, John P; Dowdy, David; Ratnam, Krishna K; Hulgan, Todd; Price, James; Unutmaz, Derya; Nicotera, Janet; Raffanti, Steven; Becker, Mark; Haas, David W
2003 Jan;73(1):78-86, Clinical pharmacology & therapeutics
The efflux pump P-glycoprotein decreases drug penetration into cells and tissues. To determine whether nelfinavir or its metabolites inhibit P-glycoprotein in lymphocytes from a healthy volunteer, whole blood cells from human immunodeficiency virus-negative donors were incubated either in human plasma to which nelfinavir or its M8 metabolite were added ex vivo or in plasma from human immunodeficiency virus-positive patients receiving nelfinavir. The 50% P-glycoprotein inhibitory concentrations of purified nelfinavir and M8 were 10.9 micromol/L and 29.5 micromol/L, respectively, for CD4(+) T cells and 19.3 micromol/L and >48 micromol/L, respectively, for CD8(+) T cells. Significant inhibitory activity was present in plasma from 27 of 46 patients (59%) receiving nelfinavir. Plasma nelfinavir concentrations correlated with percent inhibition on CD4(+) (rho = 0.85, P <.0001) and CD8(+) (rho = 0.83, P <.0001) T cells. The M8 concentrations correlated weakly with both inhibition and nelfinavir concentrations. On the basis of our findings in lymphocytes from a healthy volunteer exposed to plasma from human immunodeficiency virus-positive patients, we believe it is likely that CD4(+) and CD8(+) lymphocytes in patients receiving nelfinavir as therapy for human immunodeficiency virus may have P-glycoprotein inhibited by plasma concentrations of nelfinavir
— id: 71113, year: 2003, vol: 73, page: 78, stat: Journal Article,

Implications of T-cell P-glycoprotein activity during HIV-1 infection and its therapy
Hulgan, Todd; Donahue, John P; Hawkins, Charlene; Unutmaz, Derya; D'Aquila, Richard T; Raffanti, Stephen; Nicotera, Fred; Rebeiro, Peter; Erdem, Husamettin; Rueff, Melissa; Haas, David W
2003 Oct 1;34(2):119-126, Journal of acquired immune deficiency syndromes. JAIDS
OBJECTIVES: P-glycoprotein (P-gp) may reduce antiretroviral efficacy by decreasing disposition of HIV-1 protease inhibitors into tissues and cells. In contrast, P-gp overexpression in vitro can inhibit HIV-1 replication, and some drugs induce P-gp expression. To explore which of these mechanisms predominate in vivo, this study characterized relationships between T-cell P-gp activity and clinical parameters in HIV-infected adults. METHODS: P-gp activity was quantified in total and naive CD4+ and CD8+ T cells of HIV-infected adults by flow cytometry using the substrate dye DiOC2(3). Demographic, virologic, immunologic, and treatment factors were obtained from medical records. Factors associated with P-gp activity were identified using multivariate linear regression. RESULTS: A total of 185 subjects (22% female; 34% African American) were studied, of whom 131 (71%) were receiving antiretroviral treatment. There was marked interindividual variability in P-gp activity. By multivariate analysis, higher CD4+ T-cell P-gp activity was associated with lower log10 HIV-1 RNA (P = 0.005), but not treatment or demographic factors. P-gp activity was correlated across T-cell subsets. CONCLUSIONS: The inverse relationship between P-gp activity and plasma HIV-1 RNA is most consistent with an inhibitory effect on viral replication rather than drug disposition. Antiretroviral drug class did not independently predict P-gp activity
— id: 71107, year: 2003, vol: 34, page: 119, stat: Journal Article,

Cell cycle-dependent transduction of cell-permeant Cre recombinase proteins
Jo, Daewoong; Lin, Qing; Nashabi, Abudi; Mays, Deborah J; Unutmaz, Derya; Pietenpol, Jennifer A; Ruley, H Earl
2003 Jul 1;89(4):674-687, Journal of cellular biochemistry
Protein transduction has been widely used to analyze biochemical processes in living cells quantitatively and under non-steady-state conditions. The present study analyzed the effects of cell cycle on the uptake and activity of cell-permeant Cre recombinase proteins. Previous studies had suggested that the efficiency of recombination and/or protein transduction varied among individual cells, even within a clonal population. We report here that cells in the G1 phase of the cell cycle undergo recombination at a lower rate than cells at other phases of the cell cycle, and that this variation results largely from differences in protein uptake, associated with differences in cell size. These results have implications regarding the mechanism of protein transduction and identify a source of heterogeneity that can influence the response of individual cells to cell-permeant proteins
— id: 71110, year: 2003, vol: 89, page: 674, stat: Journal Article,

Recruitment of HIV and its receptors to dendritic cell-T cell junctions
McDonald, David; Wu, Li; Bohks, Stacy M; KewalRamani, Vineet N; Unutmaz, Derya; Hope, Thomas J
2003 May 23;300(5623):1295-1297, Science
Monocyte-derived dendritic cells (MDDCs) can efficiently bind and transfer HIV infectivity without themselves becoming infected. Using live-cell microscopy, we found that HIV was recruited to sites of cell contact in MDDCs. Analysis of conjugates between MDDCs and T cells revealed that, in the absence of antigen-specific signaling, the HIV receptors CD4, CCR5, and CXCR4 on the T cell were recruited to the interface while the MDDCs concentrated HIV to the same region. We propose that contact between dendritic cells and T cells facilitates transmission of HIV by locally concentrating virus, receptor, and coreceptor during the formation of an infectious synapse
— id: 71112, year: 2003, vol: 300, page: 1295, stat: Journal Article,

Identification and simian immunodeficiency virus infection of CD1d-restricted macaque natural killer T cells
Motsinger, Alison; Azimzadeh, Agnes; Stanic, Aleksandar K; Johnson, R Paul; Van Kaer, Luc; Joyce, Sebastian; Unutmaz, Derya
2003 Jul;77(14):8153-8158, Journal of virology
Natural killer T (NKT) cells express a highly conserved T-cell receptor (TCR) and recognize glycolipids in the context of CD1d molecules. We recently demonstrated that CD4+ NKT cells are highly susceptible to human immunodeficiency virus type 1 (HIV-1) infection and are selectively depleted in HIV-infected individuals. Here, we identified macaque NKT cells using CD1d tetramers and human Valpha24 antibodies. Similar to human NKT cells, alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells activate and expand macaque NKT cells. Upon restimulation with alpha-GalCer-pulsed CD1d(+) cells, macaque NKT cells secreted high levels of cytokines, a characteristic of these T cells. Remarkably, the majority of resting and activated macaque NKT cells expressed CD8, and a smaller portion expressed CD4. Macaque NKT cells also expressed the HIV-1/simian immunodeficiency virus (SIV) coreceptor CCR5, and the CD4+ subset was susceptible to SIV infection. Identification of macaque NKT cells has major implications for delineating the role of these cells in nonhuman primate disease models of HIV as well as other pathological conditions, such as allograft rejection and autoimmunity
— id: 71111, year: 2003, vol: 77, page: 8153, stat: Journal Article,

Genetic reprogramming of primary human T cells reveals functional plasticity in Th cell differentiation
Sundrud, Mark S; Grill, Stacy M; Ni, Donghui; Nagata, Kinya; Alkan, Sefik S; Subramaniam, Arun; Unutmaz, Derya
2003 Oct 1;171(7):3542-3549, Journal of immunology
Activation of naive T cells through the TCR and cytokine signals directs their differentiation into effector or memory subsets with different cytokine profiles. Here, we tested the flexibility of human Th1 or Th2 differentiation by forced expression of transcription factors T-bet and GATA-3. Ectopic expression of T-bet and GATA-3 in freshly isolated human T(N) cells resulted in their differentiation to a Th1 and Th2 phenotype, respectively, in the absence of polarizing cytokines. Introduction of GATA-3 into lineage-committed Th1 cells induced the expression of Th2-specific cytokines (IL-4 and IL-5) and chemotactic receptors (CCR4, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). However, these cells partially maintained their Th1-specific profile (IFN-gamma and IL-12Rbeta2 expression). Conversely, expression of T-bet in lineage-committed Th2 cells caused a more profound switch to the Th1 phenotype, including the up-regulation of CXCR3 and down-regulation of CCR4 and CRTH2. Interestingly, similar to the naive T cell subset, central memory T cells were also largely programmed toward Th1 or Th2 effector cells upon expression of T-bet and GATA-3, respectively. However, expression of these transcription factors in effector memory T cells was much less influential on cytokine and chemokine receptor expression profiles. Our results reveal remarkable plasticity in the differentiation programs of human memory T cells. This flexibility is progressively diminished as cells mature from naive to effector T cells. These findings have important implications in understanding the molecular mechanisms of human T cell differentiation and for devising novel therapeutic strategies aimed at immunomodulation of skewed effector T cell responses
— id: 71108, year: 2003, vol: 171, page: 3542, stat: Journal Article,

NKT cells and HIV infection
Unutmaz, Derya
2003 Sep;5(11):1041-1047, Microbes & infection
Natural killer T (NKT) cells are a subset of lymphocytes that express a semi-invariant T cell receptor (TCR) that recognizes glycolipids presented by the non-polymorphic MHC class I-like molecule CD1d. NKT cells regulate a wide variety of immune functions against autoantigens and pathogens. Recently, it was shown that NKT cells are targeted by HIV-1 and selectively lost in HIV-infected individuals. This review will focus on the mechanisms, consequences and therapeutic implications of these findings
— id: 71109, year: 2003, vol: 5, page: 1041, 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,

Nef-mediated downregulation of CD4 enhances human immunodeficiency virus type 1 replication in primary T lymphocytes
Lundquist, Christopher A; Tobiume, Minoru; Zhou, Jing; Unutmaz, Derya; Aiken, Christopher
2002 May;76(9):4625-4633, Journal of virology
The accessory protein Nef plays a crucial role in primate lentivirus pathogenesis. Nef enhances human immunodeficiency virus type 1 (HIV-1) infectivity in culture and stimulates viral replication in primary T cells. In this study, we investigated the relationship between HIV-1 replication efficiency in CD4(+) T cells purified from human blood and two various known activities of Nef, CD4 downregulation and single-cycle infectivity enhancement. Using a battery of reporter viruses containing point mutations in nef, we observed a strong genetic correlation between CD4 downregulation by Nef during acute HIV-1 infection of activated T cells and HIV-1 replication efficiency in T cells. In contrast, HIV-1 replication ability was not significantly correlated with the ability of Nef to enhance single-cycle virion infectivity, as determined by using viruses produced in cells lacking CD4. These results demonstrate that CD4 downregulation by Nef plays a crucial role in HIV-1 replication in activated T cells and underscore the potential for the development of therapies targeting this conserved activity of Nef
— id: 71115, year: 2002, vol: 76, page: 4625, stat: Journal Article,

CD1d-restricted human natural killer T cells are highly susceptible to human immunodeficiency virus 1 infection
Motsinger, Alison; Haas, David W; Stanic, Aleksandar K; Van Kaer, Luc; Joyce, Sebastian; Unutmaz, Derya
2002 Apr 1;195(7):869-879, Journal of experimental medicine
Human natural killer (NK) T cells are unique T lymphocytes that express an invariant T cell receptor (TCR) Valpha24-Vbeta11 and have been implicated to play a role in various diseases. A subset of NKT cells express CD4 and hence are potential targets for human immunodeficiency virus (HIV)-1 infection. We demonstrate that both resting and activated human Valpha24(+) T cells express high levels of the HIV-1 coreceptors CCR5 and Bonzo (CXCR6), but low levels of CCR7, as compared with conventional T cells. Remarkably NKT cells activated with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells were profoundly more susceptible to infection with R5-tropic, but not X4-tropic, strains of HIV-1, compared with conventional CD4(+) T cells. Furthermore, resting CD4(+) NKT cells were also more susceptible to infection. After initial infection, HIV-1 rapidly replicated and depleted the CD4(+) subset of NKT cells. In addition, peripheral blood NKT cells were markedly and selectively depleted in HIV-1 infected individuals. Although the mechanisms of this decline are not clear, low numbers or absence of NKT cells may affect the course of HIV-1 infection. Taken together, our findings indicate that CD4(+) NKT cells are directly targeted by HIV-1 and may have a potential role during viral transmission and spread in vivo
— id: 71116, year: 2002, vol: 195, page: 869, stat: Journal Article,

Rhesus macaque dendritic cells efficiently transmit primate lentiviruses independently of DC-SIGN
Wu, Li; Bashirova, Arman A; Martin, Thomas D; Villamide, Loreley; Mehlhop, Erin; Chertov, Andrei O; Unutmaz, Derya; Pope, Melissa; Carrington, Mary; KewalRamani, Vineet N
2002 Feb 5;99(3):1568-1573, Proceedings of the National Academy of Sciences of the United States of America
Here, we describe the isolation and characterization of the rhesus macaque homolog for human DC-SIGN, a dendritic cell-specific C-type lectin. mac-DC-SIGN is 92% identical to hu-DC-SIGN. mac-DC-SIGN preserves the virus transmission function of hu-DC-SIGN, capturing and efficiently transducing simian and human immunodeficiency virus to target CD4(+) T cells. Surprisingly, however, mac-DC-SIGN plays no discernable role in the ability of rhesus macaque dendritic cells to capture and transmit primate lentiviruses. Expression and neutralization analyses suggest that this process is DC-SIGN independent in macaque, although the participation of other lectin molecules cannot be ruled out. The ability of primate lentiviruses to effectively use human and rhesus dendritic cells in virus transmission without the cells becoming directly infected suggests that these viruses have taken advantage of a conserved dendritic cell mechanism in which DC-SIGN family molecules are significant contributors but not the only participants
— id: 71117, year: 2002, vol: 99, page: 1568, stat: Journal Article,

Functional evaluation of DC-SIGN monoclonal antibodies reveals DC-SIGN interactions with ICAM-3 do not promote human immunodeficiency virus type 1 transmission
Wu, Li; Martin, Thomas D; Vazeux, Rosemay; Unutmaz, Derya; KewalRamani, Vineet N
2002 Jun;76(12):5905-5914, Journal of virology
DC-SIGN, a type II membrane-spanning C-type lectin that is expressed on the surface of dendritic cells (DC), captures and promotes human and simian immunodeficiency virus (HIV and SIV) infection of CD4(+) T cells in trans. To better understand the mechanism of DC-SIGN-mediated virus transmission, we generated and functionally evaluated a panel of seven monoclonal antibodies (MAbs) against DC-SIGN family molecules. Six of the MAbs reacted with myeloid-lineage DC, whereas one MAb preferentially bound DC-SIGNR/L-SIGN, a homolog of DC-SIGN. Characterization of hematopoietic cells also revealed that stimulation of monocytes with interleukin-4 (IL-4) or IL-13 was sufficient to induce expression of DC-SIGN. All DC-SIGN-reactive MAbs competed with intercellular adhesion molecule 3 (ICAM-3) for adhesion to DC-SIGN and blocked HIV-1 transmission to T cells that was mediated by THP-1 cells expressing DC-SIGN. Similar but less efficient MAb blocking of DC-mediated HIV-1 transmission was observed, indicating that HIV-1 transmission to target cells via DC may not be dependent solely on DC-SIGN. Attempts to neutralize DC-SIGN capture and transmission of HIV-1 with soluble ICAM-3 prophylaxis were limited in success, with a maximal inhibition of 60%. In addition, disrupting DC-SIGN/ICAM-3 interactions between cells with MAbs did not impair DC-SIGN-mediated HIV-1 transmission. Finally, forced expression of ICAM-3 on target cells did not increase their susceptibility to HIV-1 transmission mediated by DC-SIGN. While these findings do not discount the role of intercellular contact in facilitating HIV-1 transmission, our in vitro data indicate that DC-SIGN interactions with ICAM-3 do not promote DC-SIGN-mediated virus transmission
— id: 71114, year: 2002, vol: 76, page: 5905, stat: Journal Article,

Unique subpopulations of CD56+ NK and NK-T peripheral blood lymphocytes identified by chemokine receptor expression repertoire
Campbell, J J; Qin, S; Unutmaz, D; Soler, D; Murphy, K E; Hodge, M R; Wu, L; Butcher, E C
2001 Jun 1;166(11):6477-6482, Journal of immunology
CD56, an adhesion molecule closely related to neural cell adhesion molecule, is an immunophenotypic marker for several unique populations of PBLS: Although CD56(+) cells derive from multiple lymphocyte lineages, they share a role in immunosurveillance and antitumor responses. We have studied the chemokine receptor expression patterns and functional migratory responses of three distinct CD56(+) populations from human peripheral blood. NK-T cells were found to differ greatly from NK cells, and CD16(+) NK cells from CD16(-) NK cells. CD16(+) NK cells were the predominant population responding to IL-8 and fractalkine, whereas NK-T cells were the predominant population responding to the CCR5 ligand macrophage-inflammatory protein-1beta. CD16(-) NK cells were the only CD56(+) population that uniformly expressed trafficking molecules necessary for homing into secondary lymphoid organs through high endothelial venule. These findings describe a diverse population of cells that may have trafficking patterns entirely different from each other, and from other lymphocyte types
— id: 106019, year: 2001, vol: 166, page: 6477, stat: Journal Article,

Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase
Jo, D; Nashabi, A; Doxsee, C; Lin, Q; Unutmaz, D; Chen, J; Ruley, H E
2001 Oct;19(10):929-933, Nature biotechnology
Studies of mammalian gene function are hampered by temporal limitations in which phenotypes occurring at one stage of development interfere with analysis at later stages. Moreover, phenotypes resulting from altered gene activity include both direct and indirect effects that may be difficult to distinguish. In the present study, recombinant fusion proteins bearing the 12 amino acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF-4) were used to transduce enzymatically active Cre proteins directly into mammalian cells. High levels of recombination were observed in a variety of cultured cell types and in all tissues examined in mice following intraperitoneal administration. This represents the first use of protein transduction to induce the enzymatic conversion of a substrate in living cells and animals and provides a rapid and efficient means to manipulate mammalian gene structure and function
— id: 106017, year: 2001, vol: 19, page: 929, stat: Journal Article,

T cell signaling mechanisms that regulate HIV-1 infection
Unutmaz, D
2001 ;23(2-3):167-177, Immunologic research
The ability of human immunodeficiency virus type-1 (HIV-1) to establish a persistent infection is critically dependent on the cellular signals that regulate HIV-1 replication within target cells. The balance between numerous host factors that either enhance or suppress viral infection determines the clinical outcome. Perturbation of the steady-state level of viral replication can significantly influence the course and the speed at which the infection develops into clinical disease. Activation signals delivered to T cells by cytokines and antigen-presenting cells (APC), are key modulators of viral replication. Our laboratory seeks to decipher how HIV-1 exploits T cell signaling mechanisms and host factors that regulate viral replication. Elucidation of the molecular mechanisms by which cellular signals regulate the HIV-1 life cycle within target cells will significantly advance our understanding of host-virus interactions
— id: 106018, year: 2001, vol: 23, page: 167, 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,

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,

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,

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,

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,

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,

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,

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,

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,

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,

Microbial pathogenesis--an interdisciplinary point of view
Rappuoli, R; Unutmaz, D
1995 Apr;13(4):128-129, Trends in biotechnology
— id: 106021, year: 1995, vol: 13, page: 128, stat: Journal Article,

Human naive T cells activated by cytokines differentiate into a split phenotype with functional features intermediate between naive and memory T cells
Unutmaz, D; Baldoni, F; Abrignani, S
1995 Sep;7(9):1417-1424, International immunology
We have recently shown that CD45RA+CD4+ naive T cells can be activated to proliferate by a combination of IL-2, TNF-alpha and IL-6, but, at variance with TCR-mediated activation, they do not acquire the CD45RO molecule. This prompted us to investigate the phenotype of these cells and the functional features they display upon TCR stimulation. Naive T cells expanded by cytokines, though remaining CD45RA+, express a variety of activation and adhesion molecules which are peculiar to effector or memory T cells. Naive cells primed by cytokines, when activated with anti-CD3 mAb, produce a broad spectrum of cytokines, express CD40 ligand, but are unable to help B cells for Ig synthesis. A subset of CD4+CD45RA+RO-T cells with a phenotype (HLA-DR-, VLA-2+ or IL-2R+) similar to that of cells activated by cytokines in vitro can be found in vivo. These results demonstrate that activation signals delivered by cytokines, in the absence of TCR stimulation, can activate naive T cells to proliferate and differentiate into a 'split phenotype' with elements common to both naive and memory T cells. This novel antigen-independent activation may help to maintain the naive T cell repertoire and facilitate the antigen-responsiveness of naive T cells
— id: 106020, year: 1995, vol: 7, page: 1417, stat: Journal Article,

Antigen-independent activation of naive and memory resting T cells by a cytokine combination
Unutmaz, D; Pileri, P; Abrignani, S
1994 Sep 1;180(3):1159-1164, Journal of experimental medicine
We investigated whether human resting T cells could be activated to proliferate and display effector function in the absence of T cell receptor occupancy. We report that combination of interleukin 2 (IL-2), tumor necrosis factor alpha, and IL-6 activated highly purified naive (CD45RA+) and memory (CD45RO+) resting CD4+ T cells to proliferate. Under this condition, memory resting T cells could also display effector function as measured by lymphokine synthesis and help for immunoglobulin production by B cells. This novel Ag-independent pathway of T cell activation may play an important role in vivo in recruiting effector T cells at the site of immune response and in maintaining the clonal size of memory T cells in the absence of antigenic stimulation. Moreover, cytokines can induce proliferation of naive T cells without switch to memory phenotype and this may help the maintenance of the peripheral pool of naive T cells
— id: 106022, year: 1994, vol: 180, page: 1159, stat: Journal Article,

T-lymphocyte response to hepatitis C virus in different clinical courses of infection
Botarelli, P; Brunetto, M R; Minutello, M A; Calvo, P; Unutmaz, D; Weiner, A J; Choo, Q L; Shuster, J R; Kuo, G; Bonino, F
1993 Feb;104(2):580-587, Gastroenterology
BACKGROUND: To assess the role played by the immune response in the outcome of hepatitis C virus infection, the CD4+ T-lymphocyte response to viral antigens was studied in infected individuals with different clinical courses. METHODS: Using six recombinant proteins of hepatitis C virus, the study assessed the proliferative responses of peripheral blood mononuclear cells from 41 patients with chronic hepatitis C, 11 patients whose chronic hepatitis was successfully treated with interferon alfa and 11 healthy HCV seropositive individuals. RESULTS: (1) Sixty-five percent of hepatitis C virus-seropositive individuals had CD4+ T-cell responses to viral proteins. (2) All viral proteins were immunogenic for T cells, although NS4 was the most immunogenic. (3) There was a significant correlation between the presence of CD4+ T cell responses to Core and a benign course of infection in healthy seropositives, most of whom were viremic. CONCLUSIONS: CD4+ T-cell responses to Core, although they do not coincide with virus clearance, are associated with a benign course of infection and may be required to maintain humoral and cellular responses protective against the disease
— id: 106024, year: 1993, vol: 104, page: 580, stat: Journal Article,

Compartmentalization of T lymphocytes to the site of disease: intrahepatic CD4+ T cells specific for the protein NS4 of hepatitis C virus in patients with chronic hepatitis C
Minutello, M A; Pileri, P; Unutmaz, D; Censini, S; Kuo, G; Houghton, M; Brunetto, M R; Bonino, F; Abrignani, S
1993 Jul 1;178(1):17-25, Journal of experimental medicine
The adult liver is an organ without constitutive lymphoid components. Therefore, any intrahepatic T cell found in chronic hepatitis should have migrated to the liver after infection and inflammation. Because of the little information available on the differences between intrahepatic and peripheral T cells, we used recombinant proteins of the hepatitis C virus (HCV) to establish specific T cell lines and clones from liver biopsies of patients with chronic hepatitis C and compared them with those present in peripheral blood mononuclear cells (PBMC). We found that the protein nonstructural 4 (NS4) was able to stimulate CD4+ T cells isolated from liver biopsies, whereas with all the other HCV proteins we consistently failed to establish liver-derived T cell lines from 16 biopsies. We then compared NS4-specific T cell clones obtained on the same day from PBMC and liver of the same patient. We found that the 22 PBMC-derived T cell clones represent, at least, six distinct clonal populations that differ in major histocompatibility complex restriction and response to superantigens, whereas the 27 liver-derived T cell clones appear all identical, as further confirmed by cloning and sequencing of the T cell receptor (TCR) variable and hypervariable regions. Remarkably, none of the PBMC-derived clones has a TCR identical to the liver-derived clone, and even with polymerase chain reaction oligotyping we did not find the liver-derived clonotypic TCR transcript in the PBMC, indicating a preferential intrahepatic localization of these T cells. Functionally, the liver-derived T cells provided help for polyclonal immunoglobulin (Ig)A production by B cells in vitro that is 10-fold more effective than that provided by the PBMC-derived clones, whereas there is no difference in the help provided for IgM and IgG production. Altogether these results demonstrate that the protein NS4 is highly immunogenic for intrahepatic CD4+ T cells primed by HCV in vivo, and that there can be compartmentalization of some NS4-specific CD4+ T cells to the liver of patients with chronic hepatitis C
— id: 106023, year: 1993, vol: 178, page: 17, stat: Journal Article,