Michael L Dustin, Ph.D.

Distinct influences of peptide-MHC quality and quantity on in vivo T-cell responses
Gottschalk, Rachel A.; Hathorn, Matthew M.; Beuneu, Helene; Corse, Emily; Dustin, Michael L.; Altan-Bonnet, Gregoire; Allison, James P.
2012 JAN 17 ;109(3):881-886, Proceedings of the National Academy of Sciences of the United States of America
The strength of T-cell receptor (TCR) stimulation and subsequent T-cell response depend on a combination of peptide-major histocompatibility complex (pMHC) density and potency. By comparing two different pMHC at doses yielding similar proliferation in vivo, we have highlighted unexpected differences in the qualitative and quantitative effects of TCR ligand. Measurements of cytokine sensitivity and two-photon imaging of T cell-dendritic cell (T-DC) interactions reveal discrimination between comparably weak stimuli resulting from either decreased pMHC potency or pMHC density. In addition, TCR-induced genes in broad gene expression profiles segregate into two groups: one that responds to cumulative TCR signal and another that responds to pMHC quality, independent of quantity. These observations suggest that models of TCR ligand discrimination must account for disparate sensitivity of downstream responses to specific influences of pMHC potency
— id: 150787, year: 2012, vol: 109, page: 881, stat: Journal Article,

Flt3L controls the development of radiosensitive dendritic cells in the meninges and choroid plexus of the steady-state mouse brain
Anandasabapathy, Niroshana; Victora, Gabriel D; Meredith, Matthew; Feder, Rachel; Dong, Baojun; Kluger, Courtney; Yao, Kaihui; Dustin, Michael L; Nussenzweig, Michel C; Steinman, Ralph M; Liu, Kang
2011 Aug 1;208(8):1695-1705, Journal of experimental medicine
Antigen-presenting cells in the disease-free brain have been identified primarily by expression of antigens such as CD11b, CD11c, and MHC II, which can be shared by dendritic cells (DCs), microglia, and monocytes. In this study, starting with the criterion of Flt3 (FMS-like receptor tyrosine kinase 3)-dependent development, we characterize the features of authentic DCs within the meninges and choroid plexus in healthy mouse brains. Analyses of morphology, gene expression, and antigen-presenting function established a close relationship between meningeal and choroid plexus DCs (m/chDCs) and spleen DCs. DCs in both sites shared an intrinsic requirement for Flt3 ligand. Microarrays revealed differences in expression of transcripts encoding surface molecules, transcription factors, pattern recognition receptors, and other genes in m/chDCs compared with monocytes and microglia. Migrating pre-DC progenitors from bone marrow gave rise to m/chDCs that had a 5-7-d half-life. In contrast to microglia, DCs actively present self-antigens and stimulate T cells. Therefore, the meninges and choroid plexus of a steady-state brain contain DCs that derive from local precursors and exhibit a differentiation and antigen-presenting program similar to spleen DCs and distinct from microglia
— id: 137986, year: 2011, vol: 208, page: 1695, stat: Journal Article,

Boltzmann energy-based image analysis demonstrates that extracellular domain size differences explain protein segregation at immune synapses
Burroughs, Nigel J; Kohler, Karsten; Miloserdov, Vladimir; Dustin, Michael L; van der Merwe, P Anton; Davis, Daniel M
2011 Aug;7(8):e1002076-e1002076, PLoS Computational Biology
Immune synapses formed by T and NK cells both show segregation of the integrin ICAM1 from other proteins such as CD2 (T cell) or KIR (NK cell). However, the mechanism by which these proteins segregate remains unclear; one key hypothesis is a redistribution based on protein size. Simulations of this mechanism qualitatively reproduce observed segregation patterns, but only in certain parameter regimes. Verifying that these parameter constraints in fact hold has not been possible to date, this requiring a quantitative coupling of theory to experimental data. Here, we address this challenge, developing a new methodology for analysing and quantifying image data and its integration with biophysical models. Specifically we fit a binding kinetics model to 2 colour fluorescence data for cytoskeleton independent synapses (2 and 3D) and test whether the observed inverse correlation between fluorophores conforms to size dependent exclusion, and further, whether patterned states are predicted when model parameters are estimated on individual synapses. All synapses analysed satisfy these conditions demonstrating that the mechanisms of protein redistribution have identifiable signatures in their spatial patterns. We conclude that energy processes implicit in protein size based segregation can drive the patternation observed in individual synapses, at least for the specific examples tested, such that no additional processes need to be invoked. This implies that biophysical processes within the membrane interface have a crucial impact on cell:cell communication and cell signalling, governing protein interactions and protein aggregation
— id: 139619, year: 2011, vol: 7, page: e1002076, stat: Journal Article,

Visualization of cell-cell interaction contacts: Synapses and kinapses
Dustin M.L.
2011 ;2(2):85-97, Self/Nonself : Immune Recognition & Signaling
T-cell activation requires interactions of T-cell antigen receptors (TCR) and peptides presented by major histocompatibility complex molecules (MHCp) in an adhesive junction between the T-cell and antigen-presenting cell (APC). Stable junctions with bull's eye supramolecular activation clusters (SMACs) have been defned as immunological synapses. The term synapse works in this case because it joins roots for 'same' and 'fasten,' which could be translated as 'fasten in the same place.' These structures maintain T-cell-APC interaction and allow directed secretion. We have proposed that SMACs are not really clusters, but are analogous to higher order membrane-cytoskeleton zones involved in amoeboid locomotion including a substrate testing lamellipodium, an adhesive lamella and anti-adhesive uropod. Since T-cells can also integrate signaling during locomotion over antigen presenting cells, it is important to consider adhesive junctions maintained as cells move past each other. This combination of movement (kine-) and fastening (-apse) can be described as a kinapse or moving junction. Synapses and kinapses operate in diferent stages of T-cell priming. Optimal efector functions may also depend upon cyclical use of synapses and kinapses. Visualization of these structures in vitro and in vivo presents many distinct challenges that will be discussed in this paper. 2011 Landes Bioscience
— id: 147754, year: 2011, vol: 2, page: 85, stat: Journal Article,

PKC-theta: hitting the bull's eye
Dustin, Michael L
2011 ;12(11):1031-1032, Nature immunology
— id: 139484, year: 2011, vol: 12, page: 1031, stat: Journal Article,

New insights into the T cell synapse from single molecule techniques
Dustin, Michael L; Depoil, David
2011 ;11(10):672-684, Nature reviews. Immunology
T cell activation depends on extracellular ligation of the T cell receptor (TCR) by peptide-MHC complexes in a synapse between the T cell and an antigen-presenting cell. The process then requires the assembly of signalling complexes between the TCR and the adaptor protein linker for activation of T cells (LAT), and subsequent filamentous actin (F-actin)-dependent TCR cluster formation. Recent progress in each of these areas, made possible by the emergence of new techniques, has forced us to rethink our assumptions and consider some radical new models. These describe the receptor interaction parameters that control T cell responses and the mechanism by which LAT is recruited to the TCR signalling machinery. This is an exciting time in T cell biology, and further innovation in imaging and genomics is likely to lead to a greater understanding of how T cells are activated
— id: 137887, year: 2011, vol: 11, page: 672, stat: Journal Article,

Role of T-lymphocytes for tumour response to radiotherapy
Formenti S.; Encouse G.; Adams S.; Pilones K.; Grazia Ruocco M.; Dustin M.; Demaria S.
2011 ;47:S11-S11, European journal of cancer
Over the past ten years we have developed a clinical translational program based on the rationale of immunizing patients against their own tumour by concomitantly: 1) removing existing 'breaks' in their immune system and 2) harnessing local ionizing radiation (IR) to induce physical and biological perturbations at the irradiated tumour site, to achieve the successful conversion of the original tumour into an immunogenic hub (Formenti, Lancet Oncology 2009). Preclinical investigations have shed some light on the specific role of T cells in these processes. For instance, in the 4T1 syngeneic murine model of metastatic breast cancer targeting regulatory receptors or cells (Treg) by anti-CTLA-4 and anti-CD25 antibodies, respectively, synergized with IR and reduced the number of metastases to the lung (an abscopal effect, defined as a significant growth inhibition of the tumour outside the irradiated field) in a CD8+ T cells dependent way. In the same model IR increased the migration of CD8 CXCR6 activated T cells to tumours. This effect was mediated by IRenhanced secretion by cancer cells of CXCL16, a chemokine that binds to CXCR6 on Th1 and activated CD8 effector T cells. CXCR6-deficient mice showed reduced infiltration of tumours by activated CD8+ T cells and impaired tumour regression following treatment with local IR + CTLA-4 blockade. Interestingly, an abscopal effect, occurred only in mice treated with the combination of 9H10 and fractionated radiotherapy, but not when a single dose of 20 Gy was administered (P < 0.01), as reflected by the frequency of CD8+ T cells showing tumour-specific IFN-gamma production. The contribution of invariant natural killer T (iNKT) cells, a subset with unique regulatory functions, in the response to IR and CTLA-4 blockade was also studied. Growth of 4T1 primary tumours and lung metastases was compared in wild type (WT) and iNKT cells-deficient (iNKT-/-) mice. The response to IR+CTLA-4 blockade was markedly enhanced in the absence of iNKT cells: 50% of iNKT-/- compared to none of the WT mice had complete tumour regression, long-term survival, and resistance to a challenge with 4T1 cells. Finally, intravital microscopy demonstrated that while both IR and CTLA-4 blockade given as monotherapy enhanced the motility of activated CD8 T cells infiltrating 4T1 tumours, IR with anti-CTLA-4 increased the arrest of T cells in contact with tumour cells. The latter required interaction of NKG2D on CD8+ T cells with its ligand retinoic acid early inducible-1 (Rae-1) on the tumour cells, which was up-regulated by IR. Blocking NKG2D-Rae-1 interactions increased markedly the motility of anti-CTLA-4 treated T cells within irradiated tumours inhibiting their contact with tumour cells, and abrogated immune-mediated tumour rejection, suggesting a critical role of radiation-induced NKG2D ligands for the antitumour effects of anti-CTLA-4 in the setting of a poorly immunogenic tumour
— id: 138729, year: 2011, vol: 47, page: S11, stat: Journal Article,

A biophysical model of cell adhesion mediated by immunoadhesin drugs and antibodies
Gutenkunst, Ryan N; Coombs, Daniel; Starr, Toby; Dustin, Michael L; Goldstein, Byron
2011 ;6(5):e19701-e19701, PLoS ONE
A promising direction in drug development is to exploit the ability of natural killer cells to kill antibody-labeled target cells. Monoclonal antibodies and drugs designed to elicit this effect typically bind cell-surface epitopes that are overexpressed on target cells but also present on other cells. Thus it is important to understand adhesion of cells by antibodies and similar molecules. We present an equilibrium model of such adhesion, incorporating heterogeneity in target cell epitope density, nonspecific adhesion forces, and epitope immobility. We compare with experiments on the adhesion of Jurkat T cells to bilayers containing the relevant natural killer cell receptor, with adhesion mediated by the drug alefacept. We show that a model in which all target cell epitopes are mobile and available is inconsistent with the data, suggesting that more complex mechanisms are at work. We hypothesize that the immobile epitope fraction may change with cell adhesion, and we find that such a model is more consistent with the data, although discrepancies remain. We also quantitatively describe the parameter space in which binding occurs. Our model elaborates substantially on previous work, and our results offer guidance for the refinement of therapeutic immunoadhesins. Furthermore, our comparison with data from Jurkat T cells also points toward mechanisms relating epitope immobility to cell adhesion
— id: 134331, year: 2011, vol: 6, page: e19701, stat: Journal Article,

HIV Envelope gp120 Activates LFA-1 on CD4 T-Lymphocytes and Increases Cell Susceptibility to LFA-1-Targeting Leukotoxin (LtxA)
Hioe, Catarina E; Tuen, Michael; Vasiliver-Shamis, Gaia; Alvarez, Yelina; Prins, Kathleen C; Banerjee, Sagarika; Nadas, Arthur; Cho, Michael W; Dustin, Michael L; Kachlany, Scott C
2011 ;6(8):e23202-e23202, PLoS ONE
The cellular adhesion molecule LFA-1 and its ICAM-1 ligand play an important role in promoting HIV-1 infectivity and transmission. These molecules are present on the envelope of HIV-1 virions and are integral components of the HIV virological synapse. However, cellular activation is required to convert LFA-1 to the active conformation that has high affinity binding for ICAM-1. This study evaluates whether such activation can be induced by HIV itself. The data show that HIV-1 gp120 was sufficient to trigger LFA-1 activation in fully quiescent naive CD4 T cells in a CD4-dependent manner, and these CD4 T cells became more susceptible to killing by LtxA, a bacterial leukotoxin that preferentially targets leukocytes expressing high levels of the active LFA-1. Moreover, virus p24-expressing CD4 T cells in the peripheral blood of HIV-infected subjects were found to have higher levels of surface LFA-1, and LtxA treatment led to significant reduction of the viral DNA burden. These results demonstrate for the first time the ability of HIV to directly induce LFA-1 activation on CD4 T cells. Although LFA-1 activation may enhance HIV infectivity and transmission, it also renders the cells more susceptible to an LFA-1-targeting bacterial toxin, which may be harnessed as a novel therapeutic strategy to deplete virus reservoir in HIV-infected individuals
— id: 136651, year: 2011, vol: 6, page: e23202, stat: Journal Article,

Migration of cytotoxic lymphocytes in cell cycle permits local MHC I-dependent control of division at sites of viral infection
Kang, Silvia S; Herz, Jasmin; Kim, Jiyun V; Nayak, Debasis; Stewart-Hutchinson, Phillip; Dustin, Michael L; McGavern, Dorian B
2011 Apr 11;208(4):747-759, Journal of experimental medicine
After virus infection, cytotoxic T lymphocytes (CTLs) divide rapidly to eradicate the pathogen and prevent the establishment of persistence. The magnitude of an antiviral CTL response is thought to be controlled by the initiation of a cell cycle program within lymphoid tissues. However, it is presently not known whether this division program proceeds during migration or is influenced locally at sites of viral infection. We demonstrate that antiviral CTLs remain in cell cycle while transiting to infected tissues. Up to one third of virus-specific CTLs within blood were found to be in cell cycle after infection with lymphocytic choriomeningitis virus or vesicular stomatitis virus. Using two-photon microscopy, we found that effector CTL divided rapidly upon arrest in the virus-infected central nervous system as well as in meningeal blood vessels. We also observed that MHC I-dependent interactions, but not costimulation, influenced the division program by advancing effector CTL through stages of the cell cycle. These results demonstrate that CTLs are poised to divide in transit and that their numbers can be influenced locally at the site of infection through interactions with cells displaying cognate antigen
— id: 134242, year: 2011, vol: 208, page: 747, stat: Journal Article,

Intravital two-photon microscopy of Staphylococcus aureus skin infections
Liese J.; Novick R.P.; Dustin M.L.
2011 ;301:47-47, International journal of medical microbiology : IJMM
Vereinigte Staaten von Amerika Staphylococcus (S.) aureus is a frequent cause of severe skin and soft tissue infections in humans, the treatment of which is complicated by the emergence of multi-resistant and hyper-virulent strains. The ability to control the infection is largely dependent on the rapid recruitment of polymorphonuclear neutrophilic granulocytes (PMN). To gain more insight into PMN migration and host-pathogen interactions in vivo, we investigated a mouse model of S. aureus flank skin infection using intravital two-photon microscopy. For this purpose we generated S. aureus reporter strains, which expressed codon-adapted fluorescent proteins. PMN migration in response to infection in vivo was then visualized using LysM-EGFP transgenic mice. After injection of the bacteria, we observed the rapid appearance of PMN in the extravascular space of the dermis and their directed movement towards the focus of infection. This resulted in the accumulation of large numbers of cells, which led to the delineation of an abscess within one day. Surprisingly, the direct encounter of bacterial cells by PMN did not always lead to an uptake of the pathogen. Depletion of PMN or blocking G-protein coupled receptors (GPCR) lead to an uncontrolled proliferation of the bacteria. Moreover, tracking of transferred labeled bone-marrow derived neutrophils showed that PMN recruitment to the site of infection is dependent on GPCR on the cells themselves, whereas IL-1-receptor was required on host cells other than PMN. Finally, by using a reporter of the staphylococcal Agr quorum-sensing system, we were able to follow bacterial gene regulation in vivo on a single cell level. Here, we observed that Agr activity could be localized to small areas at the margin of the infection focus. Our results establish that two-photon microscopy is a powerful tool to investigate the dynamics of the immune response, bacterial cell location, and gene expression in vivo on a single cell level during S. aureus infections
— id: 149831, year: 2011, vol: 301, page: 47, stat: Journal Article,

T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters
Manz, Boryana N; Jackson, Bryan L; Petit, Rebecca S; Dustin, Michael L; Groves, Jay
2011 May 31;108(22):9089-9094, Proceedings of the National Academy of Sciences of the United States of America
T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. Here we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC content within individual TCR clusters can decrease T-cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Results from a series of experiments in which the overall agonist density and the maximum number of agonist per TCR cluster are independently varied in primary T cells indicate that the most probable minimal triggering unit for calcium signaling is at least four pMHC in a single cluster for this system. This threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower
— id: 134110, year: 2011, vol: 108, page: 9089, stat: Journal Article,

The critical role of agrin in the hematopoietic stem cell niche
Mazzon, Cristina; Anselmo, Achille; Cibella, Javier; Soldani, Cristiana; Destro, Annarita; Kim, Natalie; Roncalli, Massimo; Burden, Steven J; Dustin, Michael L; Sarukhan, Adelaida; Viola, Antonella
2011 Sep 8;118(10):2733-2742, Blood
Hematopoiesis is the process leading to the sustained production of blood cells by hematopoietic stem cells (HSCs). Growth, survival, and differentiation of HSCs occur in specialized microenvironments called 'hematopoietic niches,' through molecular cues that are only partially understood. Here we show that agrin, a proteoglycan involved in the neuromuscular junction, is a critical niche-derived signal that controls survival and proliferation of HSCs. Agrin is expressed by multipotent nonhematopoietic mesenchymal stem cells (MSCs) and by differentiated osteoblasts lining the endosteal bone surface, whereas Lin(-)Sca1(+)c-Kit(+) (LSK) cells express the alpha-dystroglycan receptor for agrin. In vitro, agrin-deficient MSCs were less efficient in supporting proliferation of mouse Lin(-)c-Kit(+) cells, suggesting that agrin plays a role in the hematopoietic cell development. These results were indeed confirmed in vivo through the analysis of agrin knockout mice (Musk-L;Agrn(-/-)). Agrin-deficient mice displayed in vivo apoptosis of CD34(+)CD135(-) LSK cells and impaired hematopoiesis, both of which were reverted by an agrin-sufficient stroma. These data unveil a crucial role of agrin in the hematopoietic niches and in the cross-talk between stromal and hematopoietic stem cells
— id: 137976, year: 2011, vol: 118, page: 2733, stat: Journal Article,

CTLs respond with activation and granule secretion when serving as targets for T-cell recognition
Milstein, Oren; Hagin, David; Lask, Assaf; Reich-Zeliger, Shlomit; Shezen, Elias; Ophir, Eran; Eidelstein, Yaki; Afik, Ran; Antebi, Yaron E; Dustin, Michael L; Reisner, Yair
2011 Jan 20;117(3):1042-1052, Blood
Cytotoxic T lymphocytes (CTLs) suppress T cell responses directed against their antigens regardless of their own T cell receptor (TCR) specificity. This makes the use of CTLs promising for tolerance induction in autoimmunity and transplantation. It has been established that binding of the CTL CD8 molecule to the major histocompatibility complex (MHC) class I alpha3 domain of the recognizing T cell must be permitted for death of the latter cell to ensue. However, the signaling events triggered in the CTL by this molecular interaction in the absence of TCR recognition have never been clarified. Here we use single-cell imaging to study the events occurring in CTLs serving as targets for recognition by specific T cells. We demonstrate that CTLs actively respond to recognition by polarizing their cytotoxic granules to the contact area, releasing their lethal cargo, and vigorously proliferating. Using CTLs from perforin knockout (KO) mice and lymphocyte specific kinase (Lck) knockdown with specific small interfering RNA (siRNA), we show that the killing of the recognizing CD8 T cell is perforin dependent and is initiated by Lck signaling in the CTL. Collectively, these data suggest a novel mechanism in which the entire cascade generally triggered by TCR engagement is 'hijacked' in CTLs serving as targets for T cell recognition without TCR ligation
— id: 133313, year: 2011, vol: 117, page: 1042, stat: Journal Article,

A dynamic T cell-limited checkpoint regulates affinity-dependent B cell entry into the germinal center
Schwickert, Tanja A.; Victora, Gabriel D.; Fooksman, David R.; Kamphorst, Alice O.; Mugnier, Monica R.; Gitlin, Alexander D.; Dustin, Michael L.; Nussenzweig, Michel C.
2011 JUN 6 ;208(6):1243-1252, Journal of experimental medicine
The germinal center (GC) reaction is essential for the generation of the somatically hypermutated, high-affinity antibodies that mediate adaptive immunity. Entry into the GC is limited to a small number of B cell clones; however, the process by which this limited number of clones is selected is unclear. In this study, we demonstrate that low-affinity B cells intrinsically capable of seeding a GC reaction fail to expand and become activated in the presence of higher-affinity B cells even before GC coalescence. Live multiphoton imaging shows that selection is based on the amount of peptide-major histocompatibility complex (pMHC) presented to cognate T cells within clusters of responding B and T cells at the T-B border. We propose a model in which T cell help is restricted to the B cells with the highest amounts of pMHC, thus allowing for a dynamic affinity threshold to be imposed on antigen-binding B cells
— id: 134492, year: 2011, vol: 208, page: 1243, stat: Journal Article,

The Growth Factor Progranulin Binds to TNF Receptors and Is Therapeutic Against Inflammatory Arthritis in Mice
Tang W; Lu Y; Tian QY; Zhang Y; Guo FJ; Liu GY; Syed NM; Lai Y; Lin EA; Kong L; Su J; Yin F; Ding AH; Zanin-Zhorov A; Dustin ML; Tao J; Craft J; Yin Z; Feng JQ; Abramson SB; Yu XP; Liu CJ
2011 Apr 22;332(6028):478-484, Science
The growth factor progranulin (PGRN) has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation, but its receptors remain unidentified. We report that PGRN bound directly to tumor necrosis factor receptors (TNFR) and disturbed the TNFalpha/TNFR interaction. PGRN-deficient mice were susceptible to collagen-induced arthritis, and administration of PGRN reversed inflammatory arthritis. Atsttrin, an engineered protein composed of three PGRN fragments, exhibited selective TNFR binding. PGRN and Atsttrin prevented inflammation in multiple arthritis mouse models and inhibited TNFalpha-activated intracellular signaling. Collectively, these findings demonstrate that PGRN is a ligand of TNFR, an antagonist of TNFalpha signaling and plays a critical role in the pathogenesis of inflammatory arthritis in mice. They also suggest new potential therapeutic interventions for various TNFalpha-mediated pathologies and conditions, including rheumatoid arthritis
— id: 126500, year: 2011, vol: 332, page: 478, stat: Journal Article,

Dynamic imaging of the effector immune response to listeria infection in vivo
Waite, Janelle C; Leiner, Ingrid; Lauer, Peter; Rae, Chris S; Barbet, Gaetan; Zheng, Huan; Portnoy, Daniel A; Pamer, Eric G; Dustin, Michael L
2011 Mar;7(3):e1001326-e1001326, PLoS pathogens
Host defense against the intracellular pathogen Listeria monocytogenes (Lm) requires innate and adaptive immunity. Here, we directly imaged immune cell dynamics at Lm foci established by dendritic cells in the subcapsular red pulp (scDC) using intravital microscopy. Blood borne Lm rapidly associated with scDC. Myelomonocytic cells (MMC) swarmed around non-motile scDC forming foci from which blood flow was excluded. The depletion of scDC after foci were established resulted in a 10-fold reduction in viable Lm, while graded depletion of MMC resulted in 30-1000 fold increase in viable Lm in foci with enhanced blood flow. Effector CD8(+) T cells at sites of infection displayed a two-tiered reduction in motility with antigen independent and antigen dependent components, including stable interactions with infected and non-infected scDC. Thus, swarming MMC contribute to control of Lm prior to development of T cell immunity by direct killing and sequestration from blood flow, while scDC appear to promote Lm survival while preferentially interacting with CD8(+) T cells in effector sites
— id: 130307, year: 2011, vol: 7, page: e1001326, stat: Journal Article,

PKC-theta function at the immunological synapse: prospects for therapeutic targeting
Zanin-Zhorov, Alexandra; Dustin, Michael L; Blazar, Bruce R
2011 Aug;32(8):358-363, Trends in immunology
Protein kinase C (PKC)-theta regulates conventional effector T (Teff) cell function. Since this initial finding, it has become clear that the role of PKC-theta in T cells is complex. PKC-theta plays a central role in Teff cell activation and survival, and negatively regulates stability of the immunological synapse (IS). Recent studies demonstrated that PKC-theta is required for the development of natural CD4(+)Foxp3(+) regulatory T (Treg) cells, and mediates negative regulation of Treg cell function. Here, we examine the role of PKC-theta in the IS, evidence for its distinct localization in Treg cells and the therapeutic implications of inhibiting PKC-theta in Teff cells, to reduce effector function, and in Treg cells, to increase suppressor function, for the prevention and treatment of autoimmune and alloimmune disease states
— id: 136515, year: 2011, vol: 32, page: 358, stat: Journal Article,

Signaling microdomains in T cells
Choudhuri, Kaushik; Dustin, Michael L
2010 Dec 15;584(24):4823-4831, FEBS letters
Sub-micron scale signaling domains induced in the plasma membrane of cells are thought to play important roles in signal transduction. In T cells, agonist MHC-peptide complexes induce small diffraction-limited domains enriched in T cell receptor (TCR) and signaling molecules. These microclusters serve as transient platforms for signal initiation and are required for sustained signaling in T cells, although each microcluster functions for only a couple of minutes. How they are formed, and what mechanisms promote and regulate signaling within TCR microclusters is largely unknown, although it is clear that TCR engagement and dynamic reorganization of cortical actin are involved. Here, we review current understanding of signaling within microclusters in T cells, and speculate on how these structures may form, initiate biochemical signals, and serve as sites of both signal integration and amplification, while also facilitating appropriate termination of TCR and related signaling
— id: 134410, year: 2010, vol: 584, page: 4823, stat: Journal Article,

Insights into function of the immunological synapse from studies with supported planar bilayers
Dustin, Michael L
2010 ;340:1-24, Current topics in microbiology & immunology
Innate and adaptive immunity is dependent upon reliable cell-cell communication mediated by direct interactions of cell surface receptors with ligands integrated into the surface of apposing cells or bound directly to the surface as in complement deposition or antibody mediated recognition through Fc receptors. Supported lipid bilayers formed on glass surfaces offer a useful model system in which to explore some basic features of molecular interactions in immunological relevant contacts, which include signal integration and effector functions through immunological synapses and kinapses. We have exploited that lateral mobility of molecules in the supported planar bilayers and fluorescence microscopy to develop a system for measurement of two-dimensional affinities and kinetic rates in the contact area, which is of immunological interest. Affinity measurements are based on a modified Scatchard analysis. Measurements of kinetic rates are based on fluorescence photo bleaching after recovery at the level of the entire contact area. This has been coupled to a reaction-diffusion equation that allows calculation of on- and off-rates. We have found that mixtures of ligands in supported planar bilayers can effectively activate T lymphocytes and simultaneously allow monitoring of the immunological synapse. Recent studies in planar bilayers have provided additional insights into organization principles of cell-cell interfaces. Perennial problems in understanding cell-cell communication are yielding quantitative measurements based on planar bilayers in areas of ligand-driven receptor clustering and the role of the actin cytoskeleton in immune cell activation. A major goal for the field is determining quantitative rules involved in signaling complex formation by innate and adaptive receptor systems
— id: 105655, year: 2010, vol: 340, page: 1, stat: Journal Article,

Understanding the structure and function of the immunological synapse
Dustin, Michael L; Chakraborty, Arup K; Shaw, Andrey S
2010 Oct;2(10):a002311-a002311, Cold Spring Harbor perspectives in biology
The immunological synapse has been an area of very active scientific interest over the last decade. Surprisingly, much about the synapse remains unknown or is controversial. Here we review some of these current issues in the field: how the synapse is defined, its potential role in T-cell function, and our current understanding about how the synapse is formed
— id: 140032, year: 2010, vol: 2, page: a002311, stat: Journal Article,

Cytotoxic immunological synapses
Dustin, Michael L; Long, Eric O
2010 May;235(1):24-34, Immunological reviews
One of the most fundamental activities of the adaptive immune system is to kill infected cells and tumor cells. Two distinct pathways mediate this process, both of which are facilitated by a cytotoxic immunological synapse. While traditionally thought of as innate immune cells, natural killer (NK) cells are now appreciated to have the capacity for long-term adaptation to chemical and viral insults. These cells integrate multiple positive and negative signals through NK cell cytotoxic or inhibitory synapses. The traditional CD8(+)alphabeta T-cell receptor-positive cells are among the best models for the concept of an immunological synapse, in which vectoral signaling is linked to directed secretion in a stable interface to induce apoptotic cell death in an infected cell. Large-scale molecular organization in synapses generated a number of hypotheses. Studies in the past 5 years have started to provide clear answers regarding the validity of these models. In vivo imaging approaches have provided some hints as to the physiologic relevance of these processes with great promise for the future. This review provides an overview of work on cytotoxic immunological synapses and suggests pathways forward in applying this information to the development of therapeutic agents
— id: 110101, year: 2010, vol: 235, page: 24, stat: Journal Article,

Affinity measured by microcluster
Fooksman, David R; Dustin, Michael L
2010 May 10;207(5):907-909, Journal of experimental medicine
Like T cell activation, B cell activation is driven by aggregation of B cell receptors (BCRs) into microclusters. New work suggests that the early dynamics of BCR mobility and microcluster formation 'translate' BCR affinity for antigen into B cell responsiveness
— id: 109677, year: 2010, vol: 207, page: 907, stat: Journal Article,

Development and migration of plasma cells in the mouse lymph node
Fooksman, David R; Schwickert, Tanja A; Victora, Gabriel D; Dustin, Michael L; Nussenzweig, Michel C; Skokos, Dimitris
2010 Jul 23;33(1):118-127, Immunity
In this study, we imaged the differentiation and migratory behavior of nascent plasma cells (PCs) in mouse lymph nodes by intravital microscopy. Pre-PCs exhibited a unique migration pattern characterized by long, linear paths that were randomly oriented. Although chemotaxis via Galphai coupled-receptors has been implicated in PC migration, treatment with Pertussis toxin (Ptx), which ablates these signals, did not prevent movement of pre-PCs while it arrested other lymphocytes. In vitro, pre-PCs displayed processive amoeboid locomotion on surfaces coated with integrin ligand, whereas fully differentiated PCs moved slowly or were arrested. Both PC arrest and differentiation occurred in the medullary cords. Ptx treatment before PC differentiation blocked their accumulation in the medullary cords but pre-PCs still differentiated in other lymph node regions. Taken together, we suggest pre-PCs undergo a persistent random walk to find the medullary cords, where localized chemokines help retain these cells until they undergo differentiation and arrest in situ
— id: 111358, year: 2010, vol: 33, page: 118, stat: Journal Article,

Functional anatomy of T cell activation and synapse formation
Fooksman, David R; Vardhana, Santosh; Vasiliver-Shamis, Gaia; Liese, Jan; Blair, David A; Waite, Janelle; Sacristan, Catarina; Victora, Gabriel D; Zanin-Zhorov, Alexandra; Dustin, Michael L
2010 Apr 23;28:79-105, Annual review of immunology
T cell activation and function require a structured engagement of antigen-presenting cells. These cell contacts are characterized by two distinct dynamics in vivo: transient contacts resulting from promigratory junctions called immunological kinapses or prolonged contacts from stable junctions called immunological synapses. Kinapses operate in the steady state to allow referencing to self-peptide-MHC (pMHC) and searching for pathogen-derived pMHC. Synapses are induced by T cell receptor (TCR) interactions with agonist pMHC under specific conditions and correlate with robust immune responses that generate effector and memory T cells. High-resolution imaging has revealed that the synapse is highly coordinated, integrating cell adhesion, TCR recognition of pMHC complexes, and an array of activating and inhibitory ligands to promote or prevent T cell signaling. In this review, we examine the molecular components, geometry, and timing underlying kinapses and synapses. We integrate recent molecular and physiological data to provide a synthesis and suggest ways forward
— id: 108785, year: 2010, vol: 28, page: 79, stat: Journal Article,

Efficient Activation of Self-reactive T Cells from MS Patients with Altered Synapse Formation
Gordo, S; Schubert, D; Vardhana, S; Seth, N; Pyrdol, J; Raddassi, K; Hafler, D; Dustin, M; Wucherpfennig, K
2010 MAY ;135(2):S8-S8, Clinical immunology
— id: 111942, year: 2010, vol: 135, page: S8, stat: Journal Article,

Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites
Kim, Jiyun V; Jiang, Ning; Tadokoro, Carlos E; Liu, Liping; Ransohoff, Richard M; Lafaille, Juan J; Dustin, Michael L
2010 Jan 31;352(1-2):89-100, Journal of immunological methods
The mouse spinal cord is an important site for autoimmune and injury models. Skull thinning surgery provides a minimally invasive window for microscopy of the mouse cerebral cortex, but there are no parallel methods for the spinal cord. We introduce a novel, facile and inexpensive method for two-photon laser scanning microscopy of the intact spinal cord in the mouse by taking advantage of the naturally accessible intervertebral space. These are powerful methods when combined with gene-targeted mice in which endogenous immune cells are labeled with green fluorescent protein (GFP). We first demonstrate that generation of the intervertebral window does not elicit a reaction of GFP(+) microglial cells in CX3CR1(gfp/+) mice. We next demonstrate a distinct rostrocaudal migration of GFP(+) immune cells in the spinal cord of CXCR6(gfp/+) mice during active experimental autoimmune encephalomyelitis (EAE). Interestingly, infiltration of the cerebral cortex by GFP(+) cells in these mice required three conditions: EAE induction, cortical injury and expression of CXCR6 on immune cells
— id: 106273, year: 2010, vol: 352, page: 89, stat: Journal Article,

Agrin is essential for the fitness of hematopoietic niches and myeloid cells
Mazzon, C; Anselmo, A; Destro, A; Cibella, J; Soldani, C; D'Amico, G; Kim, N; Roncalli, M; Biondi, A; Burden, S; Dustin, ML; Sarukhan, A; Viola, A
2010 MAR ;40(11):53-53, European journal of clinical investigation
— id: 109834, year: 2010, vol: 40, page: 53, stat: Journal Article,

High plasma membrane lipid order imaged at the immunological synapse periphery in live T cells
Owen, Dylan M; Oddos, Stephane; Kumar, Sunil; Davis, Daniel M; Neil, Mark A A; French, Paul M W; Dustin, Michael L; Magee, Anthony I; Cebecauer, Marek
2010 Aug;27(4-6):178-189, Molecular Membrane Biology
Cholesterol- and glycosphingolipid-enriched membrane lipid microdomains, frequently called lipid rafts, are thought to play an important role in the spatial and temporal organization of immunological synapses. Higher ordering of lipid acyl chains was suggested for these entities and imaging of membrane order in living cells during activation can therefore help to understand the mechanisms responsible for the supramolecular organization of molecules involved in the activation of T cells. Here, we employ the phase-sensitive membrane dye di-4-ANEPPDHQ together with a variety of spectrally-resolved microscopy techniques, including 2-channel ratiometric TIRF microscopy and fluorescence lifetime imaging, to characterize membrane order at the T cell immunological synapse at high spatial and temporal resolution in live cells at physiological temperature. We find that higher membrane order resides at the immunological synapse periphery where proximal signalling through the immunoreceptors and accessory proteins in microclusters has previously been shown to take place. The observed spatial patterning of membrane order in the immunological synapse depends on active receptor signalling
— id: 133516, year: 2010, vol: 27, page: 178, stat: Journal Article,

Monocyte Trafficking to Hepatic Sites of Bacterial Infection Is Chemokine Independent and Directed by Focal Intercellular Adhesion Molecule-1 Expression
Shi, C; Velazquez, P; Hohl, TM; Leiner, I; Dustin, ML; Pamer, EG
2010 JUN 1 ;184(11):6266-6274, Journal of immunology
Recruitment of CCR2(+)Ly6C(high) monocytes to sites of infection is essential for efficient clearance of microbial pathogens. Although CCR2-mediated signals promote monocyte emigration from bone marrow, the contribution of CCR2 to later stages of monocyte recruitment remains unresolved. In this article, we show that CCR2 deficiency markedly worsens hepatic Listeria monocytogenes infection because Ly6C(high) monocytes are retained in the bone marrow. Intravenously transferred, CCR2-deficient Ly6C(high) monocytes traffic normally to hepatic foci of infection and contribute to bacterial clearance. Pertussis toxin treatment of adoptively transferred monocytes does not impair their intrahepatic trafficking, suggesting that chemokine signaling, once CCR2(+)Ly6C(high) monocytes emigrate from the bone marrow, is not required for monocyte localization to sites of bacterial infection in the liver. Expression of ICAM-1 is induced in close proximity to foci of bacterial infection in the liver, including on CD31(+) endothelial cells, and blockade of CD11b and CD44 diminishes monocyte localization to these hepatic foci. Our studies demonstrated that Ly6C(high) monocyte recruitment from the bloodstream to the L. monocytogenes-infected liver does not require chemokine receptor-mediated signals but instead is principally dependent on integrin-and extracellular matrix-mediated monocyte adhesion. The Journal of Immunology, 2010, 184: 6266-6274
— id: 110148, year: 2010, vol: 184, page: 6266, stat: Journal Article,

A bacterial leukotoxin for the prevention of HIV infection by selective killing of CD4 T cells targeted by HIV
Tuen, M.; Vasiliver-Shamis, G.; Dustin, M. L.; Barengolts, D.; Kumar, R.; Liu, J.; Kachlany, S. C.; Hioe, C. E.
2010 OCT ;26(10):A94-A94, AIDS research & human retroviruses
— id: 117319, year: 2010, vol: 26, page: A94, stat: Journal Article,

Essential Role of Ubiquitin and TSG101 Protein in Formation and Function of the Central Supramolecular Activation Cluster
Vardhana, S; Choudhuri, K; Varma, R; Dustin, ML
2010 APR 23 ;32(4):531-540, Immunity
Agonist MHC-peptide complexes in the immunological synapse (IS) signal through T cell receptor (TCR) microclusters (MCs) that converge into a central supramolecular activation cluster (cSMAC). The determinants and function of the cSMAC remain unknown. We demonstrate an essential role for ubiquitin (Ub) and TSG101, but less so for HRS, in signal processing events at the cSMAC. Using siRNA in primary T cells, we show that Ub recognition by TSG101 is required for cSMAC formation, TCR MC signal termination, TCR downregulation, and segregation of TCR-MHC-peptide from PKC-0-enriched signaling complexes. Weak agonist MHC-peptide induced CD80-dependent TCR MCs that dissociated in the center of the IS without recruiting TSG101. These results support TSG101-dependent recognition of CD80-independent TCR MCs as a molecular checkpoint for TCR downregulation
— id: 109687, year: 2010, vol: 32, page: 531, stat: Journal Article,

HIV-1 Virological Synapse is not Simply a Copycat of the Immunological Synapse
Vasiliver-Shamis G; Dustin ML; Hioe CE
2010 May 1;2(5):1239-1260, Viruses
The virological synapse (VS) is a tight adhesive junction between an HIV-infected cell and an uninfected target cell, across which virus can be efficiently transferred from cell to cell in the absence of cell-cell fusion. The VS has been postulated to resemble, in its morphology, the well-studied immunological synapse (IS). This review article discusses the structural similarities between IS and VS and the shared T cell receptor (TCR) signaling components that are found in the VS. However, the IS and the VS display distinct kinetics in disassembly and intracellular signaling events, possibly leading to different biological outcomes. Hence, HIV-1 exploits molecular components of IS and TCR signaling machinery to trigger unique changes in cellular morphology, migration, and activation that facilitate its transmission and cell-to-cell spread
— id: 138346, year: 2010, vol: 2, page: 1239, stat: Journal Article,

Germinal Center Dynamics Revealed by Multiphoton Microscopy with a Photoactivatable Fluorescent Reporter
Victora, Gabriel D; Schwickert, Tanja A; Fooksman, David R; Kamphorst, Alice O; Meyer-Hermann, Michael; Dustin, Michael L; Nussenzweig, Michel C
2010 Nov 12;143(4):592-605, Cell
The germinal center (GC) reaction produces high-affinity antibodies by random mutation and selective clonal expansion of B cells with high-affinity receptors. The mechanism by which B cells are selected remains unclear, as does the role of the two anatomically defined areas of the GC, light zone (LZ) and dark zone (DZ). We combined a transgenic photoactivatable fluorescent protein tracer with multiphoton laser-scanning microscopy and flow cytometry to examine anatomically defined LZ and DZ B cells and GC selection. We find that B cell division is restricted to the DZ, with a net vector of B cell movement from the DZ to the LZ. The decision to return to the DZ and undergo clonal expansion is controlled by T helper cells in the GC LZ, which discern between LZ B cells based on the amount of antigen captured and presented. Thus, T cell help, and not direct competition for antigen, is the limiting factor in GC selection. PAPERCLIP:
— id: 114514, year: 2010, vol: 143, page: 592, stat: Journal Article,

Protein kinase C-theta mediates negative feedback on regulatory T cell function
Zanin-Zhorov, Alexandra; Ding, Yi; Kumari, Sudha; Attur, Mukundan; Hippen, Keli L; Brown, Maryanne; Blazar, Bruce R; Abramson, Steven B; Lafaille, Juan J; Dustin, Michael L
2010 Apr 16;328(5976):372-376, Science
T cell receptor (TCR)-dependent regulatory T cell (Treg) activity controls effector T cell (Teff) function and is inhibited by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Protein kinase C-theta (PKC-theta) recruitment to the immunological synapse is required for full Teff activation. In contrast, PKC-theta was sequestered away from the Treg immunological synapse. Furthermore, PKC-theta blockade enhanced Treg function, demonstrating PKC-theta inhibits Treg-mediated suppression. Inhibition of PKC-theta protected Treg from inactivation by TNF-alpha, restored activity of defective Treg from rheumatoid arthritis patients, and enhanced protection of mice from inflammatory colitis. Treg freed of PKC-theta-mediated inhibition can function in the presence of inflammatory cytokines and thus have therapeutic potential in control of inflammatory diseases
— id: 109214, year: 2010, vol: 328, page: 372, stat: Journal Article,

Kinetics of early T cell receptor signaling regulate the pathway of lytic granule delivery to the secretory domain
Beal, Allison M; Anikeeva, Nadia; Varma, Rajat; Cameron, Thomas O; Vasiliver-Shamis, Gaia; Norris, Philip J; Dustin, Michael L; Sykulev, Yuri
2009 Oct 16;31(4):632-642, Immunity
Cytolytic granules mediate killing of virus-infected cells by cytotoxic T lymphocytes. We show here that the granules can take long or short paths to the secretory domain. Both paths utilized the same intracellular molecular events, which have different spatial and temporal arrangements and are regulated by the kinetics of Ca(2+)-mediated signaling. Rapid signaling caused swift granule concentration near the microtubule-organizing center (MTOC) and subsequent delivery by the polarized MTOC directly to the secretory domain-the shortest path. Indolent signaling led to late recruitment of granules that moved along microtubules to the periphery of the synapse and then moved tangentially to fuse at the outer edge of the secretory domain-a longer path. The short pathway is associated with faster granule release and more efficient killing than the long pathway. Thus, the kinetics of early signaling regulates the quality of the T cell cytolytic response
— id: 133734, year: 2009, vol: 31, page: 632, stat: Journal Article,

CCR7 signalling as an essential regulator of CNS infiltration in T-cell leukaemia
Buonamici, Silvia; Trimarchi, Thomas; Ruocco, Maria Grazia; Reavie, Linsey; Cathelin, Severine; Mar, Brenton G; Klinakis, Apostolos; Lukyanov, Yevgeniy; Tseng, Jen-Chieh; Sen, Filiz; Gehrie, Eric; Li, Mengling; Newcomb, Elizabeth; Zavadil, Jiri; Meruelo, Daniel; Lipp, Martin; Ibrahim, Sherif; Efstratiadis, Argiris; Zagzag, David; Bromberg, Jonathan S; Dustin, Michael L; Aifantis, Iannis
2009 Jun 18;459(7249):1000-1004, Nature
T-cell acute lymphoblastic leukaemia (T-ALL) is a blood malignancy afflicting mainly children and adolescents. T-ALL patients present at diagnosis with increased white cell counts and hepatosplenomegaly, and are at an increased risk of central nervous system (CNS) relapse. For that reason, T-ALL patients usually receive cranial irradiation in addition to intensified intrathecal chemotherapy. The marked increase in survival is thought to be worth the considerable side-effects associated with this therapy. Such complications include secondary tumours, neurocognitive deficits, endocrine disorders and growth impairment. Little is known about the mechanism of leukaemic cell infiltration of the CNS, despite its clinical importance. Here we show, using T-ALL animal modelling and gene-expression profiling, that the chemokine receptor CCR7 (ref. 5) is the essential adhesion signal required for the targeting of leukaemic T-cells into the CNS. Ccr7 gene expression is controlled by the activity of the T-ALL oncogene Notch1 and is expressed in human tumours carrying Notch1-activating mutations. Silencing of either CCR7 or its chemokine ligand CCL19 (ref. 6) in an animal model of T-ALL specifically inhibits CNS infiltration. Furthermore, murine CNS-targeting by human T-ALL cells depends on their ability to express CCR7. These studies identify a single chemokine-receptor interaction as a CNS 'entry' signal, and open the way for future pharmacological targeting. Targeted inhibition of CNS involvement in T-ALL could potentially decrease the intensity of CNS-targeted therapy, thus reducing its associated short- and long-term complications
— id: 105354, year: 2009, vol: 459, page: 1000, stat: Journal Article,

Galectin-3 negatively regulates TCR-mediated CD4+T cell activation at the immunological synapse
Chen, H; Fermin, A; Vardhana, S; Weng, I; Lo, K; Chang, E; Yang, R; Hsu, D; Dustin, M; Liu, F
2009 APR ;129(5):S18-S18, Journal of investigative dermatology
— id: 97874, year: 2009, vol: 129, page: S18, stat: Journal Article,

Galectin-3 negatively regulates TCR-mediated CD4(+) T-cell activation at the immunological synapse
Chen, HY; Fermin, A; Vardhana, S; Weng, IC; Lo, KFR; Chang, EY; Maverakis, E; Yang, RY; Hsu, DK; Dustin, ML; Liu, FT
2009 AUG 25 ;106(34):14496-14501, Proceedings of the National Academy of Sciences of the United States of America
We have investigated the function of endogenous galectin-3 in T cells. Galectin-3-deficient (gal3(-/-)) CD4(+) T cells secreted more IFN-gamma and IL-4 than gal3(+/+) CD4(+) T cells after T-cell receptor (TCR) engagement. Galectin-3 was recruited to the cytoplasmic side of the immunological synapse (IS) in activated T cells. In T cells stimulated on supported lipid bilayers, galectin-3 was primarily located at the peripheral supramolecular activation cluster (pSMAC). Gal3(+/+) T cells formed central SMAC on lipid bilayers less effectively and adhered to antigen-presenting cells less firmly than gal3(-/-) T cells, suggesting that galectin-3 destabilizes the IS. Galectin-3 expression was associated with lower levels of early signaling events and phosphotyrosine signals at the pSMAC. Additional data suggest that galectin-3 potentiates down-regulation of TCR in T cells. By yeast two-hybrid screening, we identified as a galectin-3-binding partner, Alix, which is known to be involved in protein transport and regulation of cell surface expression of certain receptors. Co-immunoprecipitation confirmed galectin-3-Alix association and immunofluorescence analysis demonstrated the translocation of Alix to the IS in activated T cells. We conclude that galectin-3 is an inhibitory regulator of T-cell activation and functions intracellularly by promoting TCR down-regulation, possibly through modulating Alix's function at the IS
— id: 102138, year: 2009, vol: 106, page: 14496, stat: Journal Article,

Feedback control of regulatory T cell homeostasis by dendritic cells in vivo
Darrasse-Jeze, G; Deroubaix, S; Mouquet, H; Victora, GD; Eisenreich, T; Yao, KH; Masilamani, RF; Dustin, ML; Rudensky, A; Liu, K; Nussenzweig, MC
2009 AUG 31 ;206(9):1853-1862, Journal of experimental medicine
CD4(+)CD25(+)Foxp3(+) natural regulatory T cells (T reg cells) maintain self-tolerance and suppress autoimmune diseases such as type 1 diabetes and inflammatory bowel disease (IBD). In addition to their effects on T cells, T reg cells are essential for maintaining normal numbers of dendritic cells (DCs): when T reg cells are depleted, there is a compensatory Flt3-dependent increase in DCs. However, little is known about how T reg cell homeostasis is maintained in vivo. We demonstrate the existence of a feedback regulatory loop between DCs and T reg cells. We find that loss of DCs leads to a loss of T reg cells, and that the remaining T reg cells exhibit decreased Foxp3 expression. The DC-dependent loss in T reg cells leads to an increase in the number of T cells producing inflammatory cytokines, such as interferon. and interleukin 17. Conversely, increasing the number of DCs leads to increased T reg cell division and accumulation by a mechanism that requires major histocompatibility complex II expression on DCs. The increase in T reg cells induced by DC expansion is sufficient to prevent type 1 autoimmune diabetes and IBD, which suggests that interference with this feedback loop will create new opportunities for immune-based therapies
— id: 102300, year: 2009, vol: 206, page: 1853, stat: Journal Article,

betaTrCP- and Rsk1/2-mediated degradation of BimEL inhibits apoptosis
Dehan, Elinor; Bassermann, Florian; Guardavaccaro, Daniele; Vasiliver-Shamis, Gaia; Cohen, Michael; Lowes, Kym N; Dustin, Michael; Huang, David C S; Taunton, Jack; Pagano, Michele
2009 Jan 16;33(1):109-116, Molecular cell
The BimEL tumor suppressor is a potent proapoptotic BH3-only protein. We found that, in response to survival signals, BimEL was rapidly phosphorylated on three serine residues in a conserved degron, facilitating binding and degradation via the F box protein betaTrCP. Phosphorylation of the BimEL degron was executed by Rsk1/2 and promoted by the Erk1/2-mediated phosphorylation of BimEL on Ser69. Compared to wild-type BimEL, a BimEL phosphorylation mutant unable to bind betaTrCP was stabilized and consequently potent at inducing apoptosis by the intrinsic mitochondrial pathway. Moreover, although non-small cell lung cancer (NSCLC) cells often become resistant to gefitinib (a clinically relevant tyrosine kinase inhibitor that induces apoptosis through BimEL), silencing of either betaTrCP or Rsk1/2 resulted in BimEL-mediated apoptosis of both gefitinib-sensitive and gefitinib-insensitive NSCLC cells. Our findings reveal that betaTrCP promotes cell survival in cooperation with the ERK-RSK pathway by targeting BimEL for degradation
— id: 92189, year: 2009, vol: 33, page: 109, stat: Journal Article,

Modular design of immunological synapses and kinapses
Dustin, Michael L
2009 Jul;1(1):a002873-a002873, Cold Spring Harbor perspectives in biology
The concept of an immunological synapse goes back to the early 1980s with the discovery of the relationship between T-cell antigen receptor mediated Ca(2+) signaling, adhesion, and directed secretion. However, this concept did not gain traction until images were published starting in 1998 that revealed a specific molecular pattern in the interface between T cells and model antigen-presenting cells or supported planar bilayers. The dominant pattern, a ring of adhesion molecules surrounding a central cluster of antigen receptors, was observed in both model systems. Analysis of the origins of this pattern over the past 10 years has presented a solution for a difficult problem in lymphocyte biology-how a highly motile cell can suddenly stop when it encounters a signal delivered by just a few antigenic ligands on the surface of another cell without disabling the sensory machinery of the motile cell. The T lymphocyte actively assembles the immunological synapse pattern following a modular design with roots in actin-myosin-based motility
— id: 106209, year: 2009, vol: 1, page: a002873, stat: Journal Article,

Multiscale analysis of T cell activation: correlating in vitro and in vivo analysis of the immunological synapse
Dustin, Michael L
2009 ;334:47-70, Current topics in microbiology & immunology
— id: 100192, year: 2009, vol: 334, page: 47, stat: Journal Article,

Supported bilayers at the vanguard of immune cell activation studies
Dustin, Michael L
2009 Oct;168(1):152-160, Journal of structural biology
Biological adhesion between cells is critical for development of multicellular organisms and for the function of the adaptive immune system of vertebrates. A gap in understanding of adhesion systems arises from the difficulty of collecting quantitative data on the molecular interactions underlying adhesion, which is typically studied by population statistics such as percent adhesion in the presence of empirically defined forces to separate less adherent cells. Supported lipid bilayers formed on glass surfaces offer a useful model system in which to explore some basic features of molecular interactions in adhesive contacts. We have exploited the lateral mobility of molecules in the supported planar bilayers and fluorescence microscopy to develop a system for measurement of two-dimensional affinities and kinetic rates in contact areas. Affinity measurements are based on a modified Scatchard analysis. Measurements of kinetic rates are based on fluorescence photobleaching after recovery at the level of the entire contact area. This has been coupled to a reaction-diffusion equation that allows calculation of on- and off-rates. We have found that mixtures of ligands in supported planar bilayers can effectively activate T lymphocytes and simultaneously allow monitoring of the immunological synapse. Recent studies in planar bilayers have provided additional insights into organization principles of cell-cell interfaces. Perennial problems in understanding cell-cell communication are yielding to quantitative measurements based on planar bilayers in areas of ligand driven receptor clustering and the role of the actin cytoskeleton in immune cell activation. A major goal for the field is determining quantitative rules involved in signaling complex formation
— id: 101950, year: 2009, vol: 168, page: 152, stat: Journal Article,

The cellular context of T cell signaling
Dustin, Michael L
2009 Apr;30(4):482-492, Immunity
Classical alphabeta T cells protect the host by monitoring intracellular and extracellular proteins in a two-step process. The first step is protein degradation and combination with a major histocompatibility complex (MHC) molecule, leading to surface expression of this amalgam (antigen processing). The second step is the interaction of the T cell receptor with the MHC-peptide complex, leading to signaling in the T cells (antigen recognition). The context for this interaction is a T cell-antigen presenting cell junction, known as an immunological synapse if symmetric and stable and as a kinapse if asymmetric and mobile. The physiological recognition of a ligand takes place most efficiently in the F-actin-rich lamellipodium and is F-actin dependent in stages of formation and triggering and myosin II dependent for signal amplification. This review discusses how these concepts emerged from early studies on adhesion, signaling, and cell biology of T cells
— id: 98993, year: 2009, vol: 30, page: 482, stat: Journal Article,

Visualizing immune system complexity
Dustin, Michael L
2009 ;2(66):mr4-mr4, Science signaling
The European Molecular Biology Organization (EMBO) meeting Visualizing Immune System Complexity, held in January 2009, covered multiple scales, from imaging single molecules to imaging whole animals. In addition to experimental details, there was an emphasis on modeling both for data analysis and as a predictive tool to support experimental design. Imaging technologies discussed included total internal reflection fluorescence microscopy, fluorescence correlation spectroscopy, two-photon laser scanning microscopy, and magnetic resonance imaging. The biological systems included basic aspects of adaptive and innate immunity. The type 1 diabetes model was used to illustrate how a human disease was dissected at all the scales, from single-molecule analysis of the interactions of T cell receptors with peptide-loaded major histocompatibility complexes to dynamics of immune cell infiltrates by intravital microscopy, as well as the application of imaging diagnostics in humans
— id: 111662, year: 2009, vol: 2, page: mr4, stat: Journal Article,

T cell antigen receptor signaling and immunological synapse stability require myosin IIA
Ilani, T; Vasiliver-Shamis, G; Vardhana, S; Bretscher, A; Dustin, ML
2009 MAY ;10(5):531-539, Nature immunology
Immunological synapses are initiated by signaling in discrete T cell antigen receptor microclusters and are important for the differentiation and effector functions of T cells. Synapse formation involves the orchestrated movement of microclusters toward the center of the contact area with the antigen-presenting cell. Microcluster movement is associated with centripetal actin flow, but the function of motor proteins is unknown. Here we show that myosin IIA was necessary for complete assembly and movement of T cell antigen receptor microclusters. In the absence of myosin IIA or its ATPase activity, T cell signaling was interrupted 'downstream' of the kinase Lck and the synapse was destabilized. Thus, T cell antigen receptor signaling and the subsequent formation of immunological synapses are active processes dependent on myosin IIA
— id: 97971, year: 2009, vol: 10, page: 531, stat: Journal Article,

The coreceptor CD2 uses plasma membrane microdomains to transduce signals in T cells
Kaizuka, Y; Douglass, AD; Vardhana, S; Dustin, ML; Vale, RD
2009 MAY 4 ;185(3):521-534, Journal of cell biology
The interaction between a T cell and an antigen-presenting cell (APC) can trigger a signaling response that leads to T cell activation. Prior studies have shown that ligation of the T cell receptor (TCR) triggers a signaling cascade that proceeds through the coalescence of TCR and various signaling molecules (e.g., the kinase Lck and adaptor protein LAT [linker for T cell activation]) into microdomains on the plasma membrane. In this study, we investigated another ligand-receptor interaction (CD58-CD2) that facilities T cell activation using a model system consisting of Jurkat T cells interacting with a planar lipid bilayer that mimics an APC. We show that the binding of CD58 to CD2, in the absence of TCR activation, also induces signaling through the actin-dependent coalescence of signaling molecules (including TCR-zeta chain, Lck, and LAT) into microdomains. When simultaneously activated, TCR and CD2 initially colocalize in small microdomains but then partition into separate zones; this spatial segregation may enable the two receptors to enhance signaling synergistically. Our results show that two structurally distinct receptors both induce a rapid spatial reorganization of molecules in the plasma membrane, suggesting a model for how local increases in the concentration of signaling molecules can trigger T cell signaling
— id: 99177, year: 2009, vol: 185, page: 521, stat: Journal Article,

Myelomonocytic cell recruitment causes fatal CNS vascular injury during acute viral meningitis
Kim, Jiyun V; Kang, Silvia S; Dustin, Michael L; McGavern, Dorian B
2009 Jan 8;457(7226):191-195, Nature
Lymphocytic choriomeningitis virus infection of the mouse central nervous system (CNS) elicits fatal immunopathology through blood-brain barrier breakdown and convulsive seizures. Although lymphocytic-choriomeningitis-virus-specific cytotoxic T lymphocytes (CTLs) are essential for disease, their mechanism of action is not known. To gain insights into disease pathogenesis, we observed the dynamics of immune cells in the meninges by two-photon microscopy. Here we report visualization of motile CTLs and massive secondary recruitment of pathogenic monocytes and neutrophils that were required for vascular leakage and acute lethality. CTLs expressed multiple chemoattractants capable of recruiting myelomonocytic cells. We conclude that a CD8(+) T-cell-dependent disorder can proceed in the absence of direct T-cell effector mechanisms and rely instead on CTL-recruited myelomonocytic cells
— id: 91980, year: 2009, vol: 457, page: 191, stat: Journal Article,

Intravital two-photon microscopy as a method to analyze the cellular immune response during staphylococcus aureus induced abscess development
Liese J.; Novick R.P.; Dustin M.L.
2009 ;39:S626-S626, European journal of immunology
Staphylococcus (S.) aureus is a gram-positive bacterium, which causes local and systemic infections in humans and animals. The immune response to this pathogen typically leads to the development of abscesses in the skin and in inner organs. The cavity of the abscess is filled with neutrophils and is surrounded by macrophages, T cells and a fibrinous capsule. Little is known about the cell migration that takes place during abscess formation after S. aureus infection. Here, we describe an experimental system of intravital two-photon microscopy that allows for studying host-pathogen interactions during abscess development in the skin and in the kidney. Different fluorescent proteins (FP) were expressed in S. aureus in order to visualize the microorganisms. The corresponding genes were adapted to the codon usage of gram-positive bacteria and expressed from a plasmid under the control of the agr P3 or the sarA P1 promoter. Furthermore, the genes and the promoters could be stably integrated into the bacterial chromosome at the phage-related pathogenicity island SaPI1 site. The reporter constructs could then be transferred successfully via phage transduction into the commonly used virulent S. aureus strains RN6734 and RN9130. In addition, an ovalbumin expressing S. aureus strain was generated, which will be used to visualize and elucidate the role of antigen-specific T cells during abscess development. LysM-eGFP knock-in mice, CD11c-YFP and OTII transgenic mice will be used to visualize the recruitment of neutrophils, dendritic cells, and T cells to the site of abscess formation. Initial studies in C57BL/6 wild type mice revealed the presence of FP+ S. aureus in the subcapsular blood vessels of the kidney immediately after injection, where some bacteria were able to adhere to the endothelium. Interestingly, accumulation of pathogens during abscess development was also visible predominantly in the capsular and subcapsular area. Altogether, the described system of intravital microscopy using fluorescent S. aureus strains and reporter mice is a powerful tool, which allows us to follow the immune response during abscess formation in real-time and will provide new insights into the pathogenesis of this clinically highly relevant infection
— id: 125474, year: 2009, vol: 39, page: S626, stat: Journal Article,

Integrin-Dependent Organization and Bidirectional Vesicular Traffic at Cytotoxic Immune Synapses
Liu, DF; Bryceson, YT; Meckel, T; Vasiliver-Shamis, G; Dustin, ML; Long, EO
2009 JUL 17 ;31(1):99-109, Immunity
Cytotoxic lymphocytes kill target cells by releasing the content of secretory lysosomes at the immune synapse. To understand the dynamics and control of cytotoxic immune synapses, we imaged human primary, live natural killer cells on lipid bilayers carrying ligands of activation receptors. Formation of an organized synapse was dependent on the presence of the beta 2 integrin ligand ICAM-1. Ligands of coactivation receptors 2B4 and NKG2D segregated into central and peripheral regions, respectively. Lysosomal protein LAMP-1 that was exocytosed during degranulation accumulated in a large and spatially stable cluster, which overlapped with a site of membrane internalization. Lysosomal compartments reached the plasma membrane at focal points adjacent to centrally accumulated LAMP-1. Imaging of fixed cells revealed that perforin-containing granules were juxtaposed to an intracellular compartment where exocytosed LAMP-1 was retrieved. Thus, cytotoxic immune synapses include a central region of bidirectional vesicular traffic, which is controlled by integrin signaling
— id: 101253, year: 2009, vol: 31, page: 99, stat: Journal Article,

Phospholipase D1 regulates lymphocyte adhesion via upregulation of Rap1 at the plasma membrane
Mor, Adam; Wynne, Joseph P; Ahearn, Ian M; Dustin, Michael L; Du, Guangwei; Philips, Mark R
2009 Jun;29(12):3297-3306, Molecular & cellular biology
Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane
— id: 99231, year: 2009, vol: 29, page: 3297, stat: Journal Article,

Synergy of Radiation and Immune Therapy in Tumor Eradication
Ruocco, MG; Kawashima, N; Huang, J; Formenti, S; Dustin, ML; Demaria, S
2009 NOV-DEC ;32(9):995-995, Journal of immunotherapy (Hagerstown)
— id: 105633, year: 2009, vol: 32, page: 995, stat: Journal Article,

The class II phosphatidylinositol 3 kinase C2beta is required for the activation of the K+ channel KCa3.1 and CD4 T-cells
Srivastava, Shekhar; Di, Lie; Zhdanova, Olga; Li, Zhai; Vardhana, Santosha; Wan, Qi; Yan, Ying; Varma, Rajat; Backer, Jonathan; Wulff, Heike; Dustin, Michael L; Skolnik, Edward Y
2009 Sep;20(17):3783-3791, Molecular biology of the cell
The Ca(2+)-activated K(+) channel KCa3.1 is required for Ca(2+) influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2beta (PI3K-C2beta) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2beta by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2beta siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2beta in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca(2+) influx, whereas silencing of PI3K-C2beta inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2beta colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation
— id: 101953, year: 2009, vol: 20, page: 3783, stat: Journal Article,

HIV gp120 interaction with CD4+T cells induces local intracellular signaling and creates an F-actin depleted zone in the virological synapse
Vasiliver-Shamis, G; Cho, MW; Dustin, ML; Hioe, CE
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105705, year: 2009, vol: 6, page: 115, stat: Journal Article,

Human immunodeficiency virus type 1 envelope gp120-induced partial T-cell receptor signaling creates an F-actin-depleted zone in the virological synapse
Vasiliver-Shamis, Gaia; Cho, Michael W; Hioe, Catarina E; Dustin, Michael L
2009 Nov;83(21):11341-11355, Journal of virology
Cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1) occurs via a virological synapse (VS), a tight cell-cell junction formed between HIV-infected cells and target cells in which the HIV-1-infected cell polarizes and releases virions toward the noninfected target cell in a gp120- and intercellular adhesion molecule 1 (ICAM-1)-dependent process. The response of the target cell has been less studied. We utilized supported planar bilayers presenting gp120 and ICAM-1 as a reductionist model for the infected-cell membrane and investigated its effect on the target CD4 T cell. This study shows that HIV-1 gp120 interaction with its receptors is initially organized into microclusters that undergo F-actin-dependent consolidation into a central supramolecular activation complex (cSMAC). Src kinases are active in both gp120 microclusters and in the VS cSMAC. The early T-cell receptor (TCR) signaling machinery is partially activated at the VS, and signaling does not propagate to trigger Ca(2+) elevation or increase CD69 expression. However, these partial TCR signals act locally to create an F-actin-depleted zone. We propose a model in which the F-actin-depleted zone formed within the target CD4 T cell enhances the reception of virions by releasing the physical barrier for HIV-1 entry and facilitating postentry events
— id: 104347, year: 2009, vol: 83, page: 11341, stat: Journal Article,

HIV-1 envelope gp120 induces a stop signal and virological synapse formation in non-infected CD4+T cells
Vasiliver-Shamis, Gaia; Cho, Michael; Dustin, Michael L.; Hioe, Catarina E.
2009 JUN ;51(10):42-42, Journal of acquired immune deficiency syndromes. JAIDS
— id: 113757, year: 2009, vol: 51, page: 42, stat: Journal Article,

Renal Dendritic Cells: An Update
Velazquez, Peter; Dustin, Michael L; Nelson, Peter J
2009 Mar 10;111(3):e67-e71, Nephron. Experimental nephrology (Online)
Discovery into the role of renal dendritic cells (rDCs) in health and disease of the kidney is rapidly accelerating. Progress in deciphering DC precursors and the heterogeneity of monocyte subsets in mice and humans is providing insight into the biology of rDCs. Recent findings have extended knowledge of the origins, anatomy and function of the rDC network at steady state and during periods of injury to the renal parenchyma. This brief review highlights these new findings and provides an update on the study of rDCs
— id: 96436, year: 2009, vol: 111, page: e67, stat: Journal Article,

Protein kinase C theta regulates stability of the peripheral adhesion ring junction and contributes to the sensitivity of target cell lysis by CTL
Beal, Allison M; Anikeeva, Nadia; Varma, Rajat; Cameron, Thomas O; Norris, Philip J; Dustin, Michael L; Sykulev, Yuri
2008 Oct 1;181(7):4815-4824, Journal of immunology
Destruction of virus-infected cells by CTL is an extremely sensitive and efficient process. Our previous data suggest that LFA-1-ICAM-1 interactions in the peripheral supramolecular activation cluster (pSMAC) of the immunological synapse mediate formation of a tight adhesion junction that might contribute to the sensitivity of target cell lysis by CTL. Herein, we compared more (CD8(+)) and less (CD4(+)) effective CTL to understand the molecular events that promote efficient target cell lysis. We found that abrogation of the pSMAC formation significantly impaired the ability of CD8(+) but not CD4(+) CTL to lyse target cells despite having no effect of the amount of released granules by both CD8(+) and CD4(+) CTL. Consistent with this, CD4(+) CTL break their synapses more often than do CD8(+) CTL, which leads to the escape of the cytolytic molecules from the interface. CD4(+) CTL treatment with a protein kinase Ctheta inhibitor increases synapse stability and sensitivity of specific target cell lysis. Thus, formation of a stable pSMAC, which is partially controlled by protein kinase Ctheta, functions to confine the released lytic molecules at the synaptic interface and to enhance the effectiveness of target cell lysis
— id: 96438, year: 2008, vol: 181, page: 4815, stat: Journal Article,

T cell receptor microcluster transport through molecular mazes reveals mechanism of translocation
DeMond, Andrew L; Mossman, Kaspar D; Starr, Toby; Dustin, Michael L; Groves, Jay T
2008 Apr 15;94(8):3286-3292, Biophysical journal
Recognition of peptide antigen by T cells involves coordinated movement of T cell receptors (TCRs) along with other costimulatory and signaling molecules. The spatially organized configurations that result are collectively referred to as the immunological synapse. Experimental investigation of the role of spatial organization in TCR signaling has been facilitated by the use of nanopatterned-supported membranes to direct TCR into alternative patterns. Here we study the mechanism by which substrate structures redirect TCR transport. Using a flow-tracking algorithm, the ensemble of TCR clusters within each cell was tracked during synapse formation under various constraint geometries. Shortly after initial cluster formation, a coordinated centripetal flow of approximately 20 nm/s develops. Clusters that encounter substrate-imposed constraint are deflected and move parallel to the constraint at speeds that scale with the relative angle of motion to the preferred centripetal direction. TCR transport is driven by actin polymerization, and the distribution of F-actin was imaged at various time points during the synapse formation process. At early time points, there is no significant effect on actin distribution produced by substrate constraints. At later time points, modest differences were observed. These data are consistent with a frictional model of TCR coupling to cytoskeletal flow, which allows slip. Implications of this model regarding spatial sorting of cell-surface molecules are discussed
— id: 78779, year: 2008, vol: 94, page: 3286, stat: Journal Article,

Hunter to gatherer and back: immunological synapses and kinapses as variations on the theme of amoeboid locomotion
Dustin, Michael L
2008 ;24:577-596, Annual review of cell & developmental biology
The immunological synapse was initially defined as a stable cell-cell junction composed of three concentric supramolecular activation clusters (SMACs) enriched in particular components: a central SMAC with clustered antigen receptors and kinases, a peripheral SMAC rich in beta2 integrin adhesion molecule LFA-1, and a distal SMAC marked by a critical tyrosine phosphatase. In the past year the SMACs have each been identified with functional modules of amoeboid motility, and the stability of the immunological synapse has been revealed as a reconfiguration of the motile apparatus from an asymmetric hunting mode, a kinapse, to a symmetric gathering mode, the synapse. The genetic control of this process involves actinomyosin regulators PKCtheta and WASp. Crtam is involved in postsynaptic polarity in early kinapses prior to cell division. It is unlikely that the immune system is unique in using symmetrization to stop migration without inactivating motile machinery
— id: 93329, year: 2008, vol: 24, page: 577, stat: Journal Article,

Synaptic asymmetry to go
Dustin, Michael L
2008 Mar 7;132(5):733-734, Cell
Cell polarity is critical for T lymphocyte movement during their hunt for antigen-bearing cells and for infected target cells. In this issue of Cell, Yeh et al. (2008) now reveal a direct link between T cell polarity and the production of proinflammatory cytokines in mice lacking the class I MHC-restricted T cell-associated molecule (Crtam)
— id: 76472, year: 2008, vol: 132, page: 733, stat: Journal Article,

T-cell activation through immunological synapses and kinapses
Dustin, Michael L
2008 Feb;221:77-89, Immunological reviews
T-cell activation requires 'contact' with antigen-presenting cells (APCs) to bring the T-cell receptor (TCR) and antigenic major histocompatibility complex (MHC)-peptide complex together. Contact is defined by the size of the TCR and MHC-peptide complex, which at approximately 13 nm requires extensive interdigitation of the glycocalyx of the T cell and APC. T cells may be activated through formation of a stable T cell-APC junction, referred to as an immunological synapse. It has also been shown in vitro that T cells can integrate signals from APCs without a stable interaction. In vivo imaging studies supported the importance of both motile and stable T cell-APC interactions in T-cell priming. We have found that stability depends not upon turning off motile machinery but by symmetrization of force-generating structures to balance forces and hold the cell in place. Motility is induced by breaking this symmetry, which may be necessary to maintain the differentiation potential of the T cell. Recently, we also discovered a mode of T-cell signaling leading to tolerance in vivo based purely on motile interactions. Because this entire process takes place in a state of continuous T-cell kinesis, I propose the term 'kinapse' for motile T cell-APC contacts leading to signaling. Synapses and kinapses are inter-convertible by symmetrization/symmetry breaking processes, and both modes appear to be involved in normal T-cell priming. Imbalance of synapse/kinapse states may lead to immunopathology
— id: 78358, year: 2008, vol: 221, page: 77, stat: Journal Article,

Visualization of cell-cell interaction contacts-synapses and kinapses
Dustin, Michael L
2008 ;640:164-182, Advances in experimental medicine & biology
T-cell activation requires interactions of T-cell antigen receptors (TCR) and peptides presented by major histocompatibility complex molecules (MHCp) in an adhesive junction between the T-cell and antigen-presenting cell (APC). Stable junctions with bull's eye supramolecular activation clusters (SMACs) have been defined as immunological synapses. The term synapse works in this case because it joins roots for 'same' and 'fasten', which could be translated as 'fasten in the same place'. These structures maintain T-cell-APC interaction and allow directed secretion. We have proposed that SMACs are not really clusters, but are analogous to higher order membrane-cytoskeleton zones involved in amoeboid locomotion including a substrate testing lamellipodium, an adhesive lamella and anti-adhesive uropod. Since T-cells can also integrate signaling during locomotion over antigen presenting cells, it is important to consider adhesive junctions maintained as cells move past each other. This combination of movement (kine-) and fastening (-apse) can be described as a kinapse or moving junction. Synapses and kinapses operate in different stages of T-cell priming. Optimal effector functions may also depend upon cyclical use of synapses and kinapses. Visualization of these structures in vitro and in vivo presents many distinct challenges that will be discussed in this chapter
— id: 91462, year: 2008, vol: 640, page: 164, stat: Journal Article,

Tug of war at the exit door
Dustin, Michael L; Chakraborty, Arup K
2008 Jan;28(1):15-17, Immunity
The lipid sphingosine-1-phosphate has been identified as a key exit signal for lymph nodes. In this issue of Immunity, Pham et al. (2008) show that its action can only be understood in the context of retention signals transduced by CCR7
— id: 76340, year: 2008, vol: 28, page: 15, stat: Journal Article,

CXCR6 is required for T cell recruitment into injured gray matter in the context of experimental autoimmune encephalomyelitis
Kim, JY; Tadokoro, C; Shen, SQ; Ning, J; Lafaille, J; Dustin, M
2008 OCT 25 ;203(2):165-166, Journal of neuroimmunology
— id: 91493, year: 2008, vol: 203, page: 165, stat: Journal Article,

Lrp4 is a receptor for Agrin and forms a complex with MuSK
Kim, Natalie; Stiegler, Amy L; Cameron, Thomas O; Hallock, Peter T; Gomez, Andrea M; Huang, Julie H; Hubbard, Stevan R; Dustin, Michael L; Burden, Steven J
2008 Oct 17;135(2):334-342, Cell
Neuromuscular synapse formation requires a complex exchange of signals between motor neurons and skeletal muscle fibers, leading to the accumulation of postsynaptic proteins, including acetylcholine receptors in the muscle membrane and specialized release sites, or active zones in the presynaptic nerve terminal. MuSK, a receptor tyrosine kinase that is expressed in skeletal muscle, and Agrin, a motor neuron-derived ligand that stimulates MuSK phosphorylation, play critical roles in synaptic differentiation, as synapses do not form in their absence, and mutations in MuSK or downstream effectors are a major cause of a group of neuromuscular disorders, termed congenital myasthenic syndromes (CMS). How Agrin activates MuSK and stimulates synaptic differentiation is not known and remains a fundamental gap in our understanding of signaling at neuromuscular synapses. Here, we report that Lrp4, a member of the LDLR family, is a receptor for Agrin, forms a complex with MuSK, and mediates MuSK activation by Agrin
— id: 93378, year: 2008, vol: 135, page: 334, stat: Journal Article,

Modulation of T cell activation by stomatin-like protein 2
Kirchhof, Mark G; Chau, Luan A; Lemke, Caitlin D; Vardhana, Santosh; Darlington, Peter J; Marquez, Maria E; Taylor, Roy; Rizkalla, Kamilia; Blanca, Isaac; Dustin, Michael L; Madrenas, Joaquin
2008 Aug 1;181(3):1927-1936, Journal of immunology
T cell activation through the Ag receptor (TCR) requires sustained signaling from signalosomes within lipid raft microdomains in the plasma membrane. In a proteomic analysis of lipid rafts from human T cells, we identified stomatin-like protein (SLP)-2 as a candidate molecule involved in T cell activation through the Ag receptor. In this study, we show that SLP-2 expression in human primary lymphocytes is up-regulated following in vivo and ex vivo activation. In activated T cells, SLP-2 interacts with components of TCR signalosomes and with polymerized actin. More importantly, up-regulation of SLP-2 expression in human T cell lines and primary peripheral blood T cells increases effector responses, whereas down-regulation of SLP-2 expression correlates with loss of sustained TCR signaling and decreased T cell activation. Our data suggest that SLP-2 is an important player in T cell activation by ensuring sustained TCR signaling, which is required for full effector T cell differentiation, and point to SLP-2 as a potential target for immunomodulation
— id: 96439, year: 2008, vol: 181, page: 1927, stat: Journal Article,

Radiation-induced CXCL16 release by breast cancer cells attracts effector T cells
Matsumura, Satoko; Wang, Baomei; Kawashima, Noriko; Braunstein, Steve; Badura, Michelle; Cameron, Thomas O; Babb, James S; Schneider, Robert J; Formenti, Silvia C; Dustin, Michael L; Demaria, Sandra
2008 Sep 1;181(5):3099-3107, Journal of immunology
Recruitment of effector T cells to inflamed peripheral tissues is regulated by chemokines and their receptors, but the factors regulating recruitment to tumors remain largely undefined. Ionizing radiation (IR) therapy is a common treatment modality for breast and other cancers. Used as a cytocidal agent for proliferating cancer cells, IR in combination with immunotherapy has been shown to promote immune-mediated tumor destruction in preclinical studies. In this study we demonstrate that IR markedly enhanced the secretion by mouse and human breast cancer cells of CXCL16, a chemokine that binds to CXCR6 on Th1 and activated CD8 effector T cells, and plays an important role in their recruitment to sites of inflammation. Using a poorly immunogenic mouse model of breast cancer, we found that irradiation increased the migration of CD8(+)CXCR6(+) activated T cells to tumors in vitro and in vivo. CXCR6-deficient mice showed reduced infiltration of tumors by activated CD8 T cells and impaired tumor regression following treatment with local IR to the tumor and Abs blocking the negative regulator of T cell activation, CTLA-4. These results provide the first evidence that IR can induce the secretion by cancer cells of proinflammatory chemotactic factors that recruit antitumor effector T cells. The ability of IR to convert tumors into 'inflamed' peripheral tissues could be exploited to overcome obstacles at the effector phase of the antitumor immune response and improve the therapeutic efficacy of immunotherapy
— id: 81352, year: 2008, vol: 181, page: 3099, stat: Journal Article,

Nanoscale increases in CD2-CD48-mediated intermembrane spacing decrease adhesion and reorganize the immunological synapse
Milstein, Oren; Tseng, Su-Yi; Starr, Toby; Llodra, Jaime; Nans, Andrea; Liu, Mengling; Wild, Martin K; van der Merwe, P Anton; Stokes, David L; Reisner, Yair; Dustin, Michael L
2008 Dec 5;283(49):34414-34422, Journal of biological chemistry
The relationship between intermembrane spacing, adhesion efficiency, and lateral organization of adhesion receptors has not been established for any adhesion system. We have utilized the CD2 ligand CD48 with two (wild type CD48 (CD48-WT)), four (CD48-CD2), or five (CD48-CD22) Ig-like domains. CD48-WT was 10-fold more efficient in mediating adhesion than CD48-CD2 or CD48-CD22. Electron tomography of contact areas with planar bilayers demonstrated average intermembrane spacing of 12.8 nm with CD48-WT, 14.7 nm with CD48-CD2, and 15.6 nm with CD48-CD22. Both CD48-CD2 and CD48-CD22 chimeras segregated completely from CD48-WT in mixed contact areas. In contrast, CD48-CD2 and CD48-CD22 co-localized when mixed contacts were formed. Confocal imaging of immunological synapses formed between primary T lymphocytes and Chinese hamster ovary cells presenting major histocompatibility complex-peptide complexes, and different forms of CD48 demonstrated that CD48-CD2 and CD48-CD22 induce an eccentric CD2/T cell antigen receptor cluster. We propose that this reorganization of the immunological synapse sequesters the T cell antigen receptor in a location where it cannot interact with its ligand and dramatically reduces T cell sensitivity
— id: 92165, year: 2008, vol: 283, page: 34414, stat: Journal Article,

Micropatterning of costimulatory ligands enhances CD4+ T cell function
Shen, Keyue; Thomas, V Kaye; Dustin, Michael L; Kam, Lance C
2008 Jun 3;105(22):7791-7796, Proceedings of the National Academy of Sciences of the United States of America
Spatial organization of signaling complexes is a defining characteristic of the immunological synapse (IS), but its impact on cell communication is unclear. In T cell-APC pairs, more IL-2 is produced when CD28 clusters are segregated from central supramolecular activation cluster (cSMAC)-localized CD3 and into the IS periphery. However, it is not clear in these cellular experiments whether the increased IL-2 is driven by the pattern itself or by upstream events that precipitate the patterns. In this article, we recapitulate key features of physiological synapses using planar costimulation arrays containing antibodies against CD3 and CD28, surrounded by ICAM-1, created by combining multiple rounds of microcontact printing on a single surface. Naive T cells traverse these arrays, stopping at features of anti-CD3 antibodies and forming a stable synapse. We directly demonstrate that presenting anti-CD28 in the cell periphery, surrounding an anti-CD3 feature, enhances IL-2 secretion by naive CD4(+) T cells compared with having these signals combined in the center of the IS. This increased cytokine production correlates with NF-kappaB translocation and requires PKB/Akt signaling. The ability to arbitrarily and independently control the locations of anti-CD3 and anti-CD28 offered the opportunity to examine patterns not precisely attainable in cell-cell interfaces. With these patterns, we show that the peripheral presentation of CD28 has a larger impact on IL-2 secretion than CD3 colocalization/segregation
— id: 96441, year: 2008, vol: 105, page: 7791, stat: Journal Article,

Th1 and Th2 cells form morphologically distinct immunological synapses
Thauland, Timothy J; Koguchi, Yoshinobu; Wetzel, Scott A; Dustin, Michael L; Parker, David C
2008 Jul 1;181(1):393-399, Journal of immunology
The arrangement of molecules at the interface between T cells and APCs is known as the immunological synapse (IS). We conducted experiments with supported planar bilayers and transfected fibroblast APC to examine the IS formed by polarized Th1 and Th2 cells. Th1 cells formed typical 'bull's-eye' IS with a ring of adhesion molecules surrounding MHC/TCR interactions at all Ag concentrations tested, while Th2 cells formed multifocal IS at high concentrations of Ag. At low Ag concentrations, the majority of Th2 cells formed IS with a compact, central accumulation of MHC/TCR, but ICAM-1 was not excluded from the center of the IS. Additionally, CD45 was excluded from the center of the interface between Th1 cells and APC, while CD45 was found at the center of the multifocal IS formed by Th2 cells. Finally, phosphorylated signaling molecules colocalized with MHC/TCR to a greater extent in Th2 IS. Together, our results indicate that the IS formed by Th1 and Th2 cells are distinct in structure, with Th2 cells failing to form bull's-eye IS
— id: 96440, year: 2008, vol: 181, page: 393, stat: Journal Article,

Measuring Diffusion and Binding Kinetics by Contact Area FRAP
Tolentino, Timothy P; Wu, Jianhua; Zarnitsyna, Veronika I; Fang, Ying; Dustin, Michael L; Zhu, Cheng
2008 Jul;95(2):920-930, Biophysical journal
The immunological synapse is a stable intercellular structure that specializes in substance and signal transfer from one immune cell to another. Its formation is regulated in part by the diffusion of adhesion and signaling molecules into, and their binding of counter-molecules in the contact area. The stability of immunological synapses allows receptor-ligand interactions to approximate chemical equilibrium despite other dynamic aspects. We have developed a mathematical model that describes the coupled reaction-diffusion process in an established immunological synapse (Wu et al., 2008, Biophys. J. vol:pp-pp). Here we extend a previously described contact area fluorescence recovery after photobleaching (FRAP) experiment (Dustin, 1997, J. Biol. Chem. 272:15782-15788) to test the validity of the model. The receptor binding and lateral mobility of fluorescently-labeled, lipid-anchored ligands in the bilayer resulted in their accumulation, as revealed by the much higher fluorescence intensity inside the contact area than outside. After complete photobleaching of the synapse, fluorescence recovery requires ligands to dissociate and rebind as well as to diffuse in and out of the contact area. Such a FRAP time course thus contains information on both reaction and diffusion, which can be extracted by fitting the model solution to the data. Surprisingly, reverse-rates in the 2D contact area were at least 100-fold slower than in 3D solution. As previously reported in immunological synapses, a significant nonrecoverable fraction of fluorescence was observed with one of two systems studied, suggesting some ligands either dissociated much more slowly or diffused much more slowly, compared to other ligands in the same synapse. Thus, the combined theory and experiment provides a new method for in situ measurements of kinetic rates, diffusion coefficient, and nonrecoverable fraction of interacting molecules in immunological synapses and other stable cell-bilayer junctions
— id: 78777, year: 2008, vol: 95, page: 920, stat: Journal Article,

T cell-dendritic cell immunological synapses contain TCR-dependent CD28-CD80 clusters that recruit protein kinase C theta
Tseng, Su-Yi; Waite, Janelle C; Liu, Mengling; Vardhana, Santosha; Dustin, Michael L
2008 Oct 1;181(7):4852-4863, Journal of immunology
Short-lived TCR microclusters and a longer-lived protein kinase Ctheta-focusing central supramolecular activation cluster (cSMAC) have been defined in model immunological synapses (IS). In different model systems, CD28-mediated costimulatory interactions have been detected in microclusters, the cSMAC, or segregated from the TCR forming multiple distinct foci. The relationship between TCR and costimulatory molecules in the physiological IS of T cell-dendritic cell (DC) is obscure. To study the dynamic relationship of CD28-CD80 and TCR interactions in the T cell-DC IS during Ag-specific T cell activation, we generated CD80-eCFP mice using bacterial artificial chromosome transgenic technology. In splenic DCs, endogenous CD80 and CD80-eCFP localized to plasma membrane and Golgi apparatus, and CD80-eCFP was functional in vivo. In the OT-II T cell-DC IS, multiple segregated TCR, CD80, and LFA-1 clusters were detected. In the T cell-DC synapse CD80 clusters were colocalized with CD28 and PKCtheta, a characteristic of the cSMAC. Acute blockade of TCR signaling with anti-MHC Ab resulted in a rapid reduction in Ca(2+) signaling and the number and size of the CD80 clusters, a characteristic of TCR microclusters. Thus, the T cell-DC interface contains dynamic costimulatory foci that share characteristics of microclusters and cSMACs
— id: 91440, year: 2008, vol: 181, page: 4852, stat: Journal Article,

Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells
Vasiliver-Shamis, Gaia; Tuen, Michael; Wu, Teresa W; Starr, Toby; Cameron, Thomas O; Thomson, Russell; Kaur, Gurvinder; Liu, Jianping; Visciano, Maria Luisa; Li, Hualin; Kumar, Rajnish; Ansari, Rais; Han, Dong P; Cho, Michael W; Dustin, Michael L; Hioe, Catarina E
2008 Oct;82(19):9445-9457, Journal of virology
Human immunodeficiency virus type 1 (HIV-1)-infected T cells form a virological synapse with noninfected CD4(+) T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T-cell activation and effector functions mediated by the T-cell antigen receptor. The immunological synapse stops T-cell migration to allow a sustained interaction between T-cells and antigen-presenting cells. Here, we have asked whether HIV-1 envelope gp120 presented on a surface to mimic an HIV-1-infected cell also delivers a stop signal and if this is sufficient to induce a virological synapse. We demonstrate that HIV-1 gp120-presenting surfaces arrested the migration of primary activated CD4 T cells that occurs spontaneously in the presence of ICAM-1 and induced the formation of a virological synapse, which was characterized by segregated supramolecular structures with a central cluster of envelope surrounded by a ring of ICAM-1. The virological synapse was formed transiently, with the initiation of migration within 30 min. Thus, HIV-1 gp120-presenting surfaces induce a transient stop signal and supramolecular segregation in noninfected CD4(+) T cells
— id: 87804, year: 2008, vol: 82, page: 9445, stat: Journal Article,

Cutting edge: activation by innate cytokines or microbial antigens can cause arrest of natural killer T cell patrolling of liver sinusoids
Velazquez, Peter; Cameron, Thomas O; Kinjo, Yuki; Nagarajan, Niranjana; Kronenberg, Mitchell; Dustin, Michael L
2008 Feb 15;180(4):2024-2028, Journal of immunology
Natural killer T (NKT) cells are innate-like lymphocytes that rapidly secrete large amounts of effector cytokines upon activation. Recognition of alpha-linked glycolipids presented by CD1d leads to the production of IL-4, IFN-gamma, or both, while direct activation by the synergistic action of IL-12 and IL-18 leads to IFN-gamma production only. We previously reported that in vitro cultured dendritic cells can modulate NKT cell activation and, using intravital fluorescence laser scanning microscopy, we reported that the potent stimulation of NKT cells results in arrest within hepatic sinusoids. In this study, we examine the relationship between murine NKT cell patrolling and activation. We report that NKT cell arrest results from activation driven by limiting doses of a bacteria-derived weak agonist, galacturonic acid-containing glycosphingolipid, or a synthetic agonist, alpha-galactosyl ceramide. Interestingly, NKT cell arrest also results from IL-12 and IL-18 synergistic activation. Thus, innate cytokines and natural microbial TCR agonists trigger sinusoidal NKT cell arrest and an effector response
— id: 78778, year: 2008, vol: 180, page: 2024, stat: Journal Article,

A Coupled Diffusion-Kinetics Model for Analysis of Contact Area FRAP Experiment
Wu, Jianhua; Fang, Ying; Zarnitsyna, Veronika I; Tolentino, Timothy P; Dustin, Michael L; Zhu, Cheng
2008 Jul;95(2):910-919, Biophysical journal
Kinetic rates and binding affinity of receptor-ligand interactions are important determinants of cell adhesion. Measurements of these parameters in fluid phase using soluble molecules (i.e., 3D parameters) do not necessarily correlate with their counterparts measured when both binding partners are respectively anchored to two apposing surfaces (i.e., 2D parameters). Moreover, 2D affinities measured by different methods can differ by orders of magnitude (Dustin et al., 2001, Annu. Rev. Cell Dev. Biol., 17: 133-157). Here we describe a coupled diffusion-reaction model for the fluorescence recovery after photobleaching (FRAP) experiment previously used to demonstrate the dynamics of adhesive bonds in the contact area (Dustin, J Biol Chem 272:15782-15788). Applying the mathematical model to the contact area FRAP experiment enables in situ measurements of 2D kinetic rates of the adhesion molecules and their retarded diffusion in a stable contact area. The mathematical properties of model are characterized in this paper and its experimental validation will be presented in the companion paper (Tolentino et al., 2008, Biophys. J. vol:pp-pp)
— id: 78776, year: 2008, vol: 95, page: 910, stat: Journal Article,

Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C theta translocation
Yokosuka, Tadashi; Kobayashi, Wakana; Sakata-Sogawa, Kumiko; Takamatsu, Masako; Hashimoto-Tane, Akiko; Dustin, Michael L; Tokunaga, Makio; Saito, Takashi
2008 Oct;29(4):589-601, Immunity
T cell activation is mediated by microclusters (MCs) containing T cell receptors (TCRs), kinases, and adaptors. Although TCR MCs translocate to form a central supramolecular activation cluster (cSMAC) of the immunological synapse at the interface of a T cell and an antigen-presenting cell, the role of MC translocation in T cell signaling remains unclear. Here, we found that the accumulation of MCs at cSMAC was important for T cell costimulation. Costimulatory receptor CD28 was initially recruited coordinately with TCR to MCs, and its signals were mediated through the assembly with the kinase PKCtheta. The accumulation of MCs at the cSMAC was accompanied by the segregation of CD28 from the TCR, which resulted in the translocation of both CD28 and PKCtheta to a spatially unique subregion of cSMAC. Thus, costimulation is mediated by the generation of a unique costimulatory compartment in the cSMAC via the dynamic regulation of MC translocation
— id: 96437, year: 2008, vol: 29, page: 589, stat: Journal Article,

Force as a facilitator of integrin conformational changes during leukocyte arrest on blood vessels and antigen-presenting cells
Alon, Ronen; Dustin, Michael L
2007 Jan;26(1):17-27, Immunity
Integrins comprise a large family of cell-cell and cell-matrix adhesion receptors that rapidly modulate their adhesiveness. The arrest of leukocyte integrins on target vascular beds involves instantaneous conformational switches generating shear-resistant adhesions. Structural data suggest that these integrins are maintained in low-affinity conformations and must rapidly undergo conformational switches transduced via cytoplasmic changes ('inside-out' signaling) and simultaneous ligand-induced rearrangements ('outside-in'). This bidirectional activation is accelerated by signals from endothelial chemoattractants (chemokines). Recent studies predict that shear forces in the piconewton (pN) range per integrin can facilitate these biochemical switches. After extravasation, antigen recognition involves smaller internal forces from cytoskeletal motors and actin polymers forming the immune synapse. In this review, we address how forces facilitate allosteric integrin activation by biochemical signals. Evidence suggests that preformed cytoskeletal anchorage rather than free integrin mobility is key for force-enhanced integrin activation by chemokines and TCR signals
— id: 78782, year: 2007, vol: 26, page: 17, stat: Journal Article,

Exploiting breast cancer cells stress response to ionizing radiation to improve the effectiveness of immunotherapy
Demaria, S; Wang, B; Badura, M; Matsumura, S; Kawashima, N; Cameron, T; Dustin, M; Schneider, RJ; Formenti, SC
2007 JAN ;69(3):S597-S597, International journal of radiation oncology biology physics
— id: 87199, year: 2007, vol: 69, page: S597, stat: Journal Article,

Cell adhesion molecules and actin cytoskeleton at immune synapses and kinapses
Dustin, Michael L
2007 Oct;19(5):529-533, Current opinion in cell biology
The immunological synapse is a stable adhesive junction between a polarized immune effector cell and an antigen-bearing cell. Immunological synapses are often observed to have a striking radial symmetry in the plane of contact with a prominent central cluster of antigen receptors surrounded by concentric rings of adhesion molecules and actin-rich projections. There is a striking similarity between the radial zones of the immunological synapse and the dynamic actinomyosin modules employed by migrating cells. Breaking the symmetry of an immunological synapse generates a moving adhesive junction that can be defined as a kinapse, which facilitates signal integration by immune cells while moving over the surface of antigen-presenting cells
— id: 75665, year: 2007, vol: 19, page: 529, stat: Journal Article,

Quantification and modeling of tripartite CD2-, CD58FC chimera (alefacept)-, and CD16-mediated cell adhesion
Dustin, Michael L; Starr, Toby; Coombs, Daniel; Majeau, Gerard R; Meier, Werner; Hochman, Paula S; Douglass, Adam; Vale, Ron; Goldstein, Byron; Whitty, Adrian
2007 Nov 30;282(48):34748-34757, Journal of biological chemistry
Alefacept is a chimeric protein combining CD58 immunoglobulin-like domain 1 with human IgG1 Fc. Alefacept mediates adhesion by bridging CD2 on T cells to activating Fc receptors on effector cells, but the equilibrium binding parameters have not been determined. Alefacept mediated T cell killing by NK cells and adhesion between CD2- and CD16-expressing cells at an optimum concentration of 100 nM. We introduce novel measurements with supported planer bilayers, from which key two-dimensional and three-dimensional parameters can be determined by data fitting. Alefacept competitively inhibited cell bilayer adhesion mediated by the CD2-CD58 interaction. Alefacept mediated maximal adhesion of CD2(+) T cells to CD16B, an Fc receptor, in planar bilayers at 500 nM. A mechanistic model for alefacept-mediated cell-bilayer adhesion allowed fitting of the data and determination of two-dimensional binding parameters. These included the density of bonds in the adhesion area, which grew to maintain a consistent average bond density of 200 molecules/microm(2) and two-dimensional association constants of 3.1 and 630 microm(2) for bivalently and monovalently bound forms of alefacept, respectively. The maximum number of CD16 bound and the fit value of 4,350 CD2 per cell are much lower than the 40,000 CD2 per cell measured with anti-CD2 Fab. These results suggest that additional information is needed to correctly predict Alefacept-mediated bridge formation
— id: 75766, year: 2007, vol: 282, page: 34748, stat: Journal Article,

Supported planar bilayers for study of the immunological synapse
Dustin, Michael L; Starr, Toby; Varma, Rajat; Thomas, V Kaye
2007 Feb;Chapter 18:Unit 18.13-Unit 18.13, Current protocols in immunology
Supported planar bilayers have been used in immunology research for over 25 years, including in the initial demonstrations of MHC-peptide complex functional activity and adhesion molecule activity. More recent modifications of the method have been used to measure two-dimensional affinities and to study the formation of the immunological synapse. This unit covers the incorporation of glycolipid-anchored membrane proteins, 6-histidine-tagged soluble proteins, and monobiotinylated soluble proteins into supported planar bilayers. Reagents developed for the MHC-peptide tetramer staining method (UNIT 17.3) can readily be adapted to presentation on planar bilayers. The unique advantage of this approach is that the proteins presented on the surface of the supported bilayer are laterally mobile. This provides a more physiological presentation of cell-surface molecules and supports visualization of protein rearrangement on the bilayer by live cells
— id: 78775, year: 2007, vol: Chapter 18, page: Unit 18.13, stat: Journal Article,

Cell Biology of Signal Integration by T cells: Synapses and Kinapses
Dustin, Mike
[S.l.] : NIH, 2007,
— id: 1430, year: 2007, vol: , page: , stat: ,

Requirements for T lymphocyte migration in explanted lymph nodes
Huang, Julie H; Cardenas-Navia, L Isabel; Caldwell, Charles C; Plumb, Troy J; Radu, Caius G; Rocha, Paulo N; Wilder, Tuere; Bromberg, Jonathan S; Cronstein, Bruce N; Sitkovsky, Michail; Dewhirst, Mark W; Dustin, Michael L
2007 Jun 15;178(12):7747-7755, Journal of immunology
Although the requirements for T lymphocyte homing to lymph nodes (LNs) are well studied, much less is known about the requirements for T lymphocyte locomotion within LNs. Imaging of murine T lymphocyte migration in explanted LNs using two-photon laser-scanning fluorescence microscopy provides an opportunity to systematically study these requirements. We have developed a closed system for imaging an intact LN with controlled temperature, oxygenation, and perfusion rate. Naive T lymphocyte locomotion in the deep paracortex of the LN required a perfusion rate of >13 microm/s and a partial pressure of O(2) (pO(2)) of >7.4%. Naive T lymphocyte locomotion in the subcapsular region was 38% slower and had higher turning angles and arrest coefficients than naive T lymphocytes in the deep paracortex. T lymphocyte activation decreased the requirement for pO(2), but also decreased the speed of locomotion in the deep paracortex. Although CCR7(-/-) naive T cells displayed a small reduction in locomotion, systemic treatment with pertussis toxin reduced naive T lymphocyte speed by 59%, indicating a contribution of Galpha(i)-mediated signaling, but involvement of other G protein-coupled receptors besides CCR7. Receptor knockouts or pharmacological inhibition in the adenosine, PG/lipoxygenase, lysophosphatidylcholine, and sphingosine-1-phosphate pathways did not individually alter naive T cell migration. These data implicate pO(2), tissue architecture, and G-protein coupled receptor signaling in regulation of naive T lymphocyte migration in explanted LNs
— id: 73300, year: 2007, vol: 178, page: 7747, stat: Journal Article,

Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells
Kaizuka, Yoshihisa; Douglass, Adam D; Varma, Rajat; Dustin, Michael L; Vale, Ronald D
2007 Dec 18;104(51):20296-20301, Proceedings of the National Academy of Sciences of the United States of America
T cells interacting with antigen-presenting cells (APCs) form an 'immunological synapse' (IS), a bull's-eye pattern composed of a central supramolecular activation cluster enriched with T cell receptors (TCRs) surrounded by a ring of adhesion molecules (a peripheral supramolecular activation cluster). The mechanism responsible for segregating TCR and adhesion molecules remains poorly understood. Here, we show that immortalized Jurkat T cells interacting with a planar lipid bilayer (mimicking an APC) will form an IS, thereby providing an accessible model system for studying the cell biological processes underlying IS formation. We found that an actin-dependent process caused TCR and adhesion proteins to cluster at the cell periphery, but these molecules appeared to segregate from one another at the earliest stages of microdomain formation. The TCR and adhesion microdomains attached to actin and were carried centripetally by retrograde flow. However, only the TCR microdomains penetrated into the actin-depleted cell center, whereas the adhesion microdomains appeared to be unstable without an underlying actin cytoskeleton. Our results reveal that TCR and adhesion molecules spatially partition from one another well before the formation of a mature IS and that differential actin interactions help to shape and maintain the final bull's-eye pattern of the IS
— id: 78780, year: 2007, vol: 104, page: 20296, stat: Journal Article,

The lymphocyte function-associated antigen-1 receptor costimulates plasma membrane Ras via phospholipase D2
Mor, Adam; Campi, Gabriele; Du, Guangwei; Zheng, Yang; Foster, David A; Dustin, Michael L; Philips, Mark R
2007 Jun;9(6):713-719, Nature cell biology
Ras activation as a consequence of antigen receptor (T-cell receptor; TCR) engagement on T lymphocytes is required for T-cell development, selection and function. Lymphocyte function-associated antigen-1 (LFA-1) mediates lymphocyte adhesion, stabilization of the immune synapse and bidirectional signalling. Using a fluorescent biosensor we found that TCR activation with or without costimulation of CD28 led to activation of Ras only on the Golgi apparatus, whereas costimulation with LFA-1 induced Ras activation on both the Golgi and the plasma membrane. Ras activation on both compartments required RasGRP1, an exchange factor regulated by calcium and diacylglycerol (DAG), but phospholipase C (PLC) activity was required only for activation on the Golgi. Engagement of LFA-1 increased DAG levels at the plasma membrane by stimulating phospholipase D (PLD). PLD2 and phosphatidic acid phosphatase (PAP) were required for Ras activation on the plasma membrane. Thus, LFA-1 acts through PLD2 to reshape the pattern of Ras activation downstream of the TCR
— id: 73108, year: 2007, vol: 9, page: 713, stat: Journal Article,

Small GTPases and LFA-1 reciprocally modulate adhesion and signaling
Mor, Adam; Dustin, Michael L; Philips, Mark R
2007 Aug;218:114-125, Immunological reviews
Leukocyte-function-associated antigen-1 (LFA-1) is an integrin that is critical for T-cell adhesion and immunologic responses. As a transmembrane receptor and adhesion molecule, LFA-1 signals bidirectionally, whereby information about extracellular ligands is passed outside-in while cellular activation is transmitted inside-out to the adhesive ectodomain. Here, we review the role of small guanosine triphosphatases (GTPases) in LFA-1 signaling. Rap1, a Ras-related GTPase, appears to be central to LFA-1 function. Rap1 is regulated by receptor signaling [e.g. T-cell receptor (TCR), CD28, and cytotoxic T-lymphocyte antigen-4 (CTLA-4)] and by adapter proteins [e.g. adhesion and degranulation-promoting adapter protein (ADAP) and Src kinase-associated phosphoprotein of 55 kDa (SKAP-55)]. Inside-out signaling flows through Rap1 to regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and Rap1-GTP interacting adapter molecule (RIAM) that act in conjunction with the cytoskeleton on the cytosolic domain of LFA-1 to increase adhesion of the ectodomain. Outside-in signaling also relies on small GTPases such as Rho proteins. Vav-1, a guanine nucleotide exchange factor for Rho proteins, is activated as a consequence of LFA-1 engagement. Jun-activating binding protein-1 (JAB-1) and cytohesin-1 have been implicated as possible outside-in signaling intermediates. We have recently shown that Ras is also downstream of LFA-1 engagement: LFA-1 signaling through phospholipase D (PLD) to RasGRP1 was required for Ras activation on the plasma membrane following stimulation of TCR
— id: 73951, year: 2007, vol: 218, page: 114, stat: Journal Article,

In vivo imaging of germinal centres reveals a dynamic open structure
Schwickert, Tanja A; Lindquist, Randall L; Shakhar, Guy; Livshits, Geulah; Skokos, Dimitris; Kosco-Vilbois, Marie H; Dustin, Michael L; Nussenzweig, Michel C
2007 Mar 1;446(7131):83-87, Nature
Germinal centres are specialized structures wherein B lymphocytes undergo clonal expansion, class switch recombination, antibody gene diversification and affinity maturation. Three to four antigen-specific B cells colonize a follicle to establish a germinal centre and become rapidly dividing germinal-centre centroblasts that give rise to dark zones. Centroblasts produce non-proliferating centrocytes that are thought to migrate to the light zone of the germinal centre, which is rich in antigen-trapping follicular dendritic cells and CD4+ T cells. It has been proposed that centrocytes are selected in the light zone on the basis of their ability to bind cognate antigen. However, there have been no studies of germinal-centre dynamics or the migratory behaviour of germinal-centre cells in vivo. Here we report the direct visualization of B cells in lymph node germinal centres by two-photon laser-scanning microscopy in mice. Nearly all antigen-specific B cells participating in a germinal-centre reaction were motile and physically restricted to the germinal centre but migrated bi-directionally between dark and light zones. Notably, follicular B cells were frequent visitors to the germinal-centre compartment, suggesting that all B cells scan antigen trapped in germinal centres. Consistent with this observation, we found that high-affinity antigen-specific B cells can be recruited to an ongoing germinal-centre reaction. We conclude that the open structure of germinal centres enhances competition and ensures that rare high-affinity B cells can participate in antibody responses
— id: 78781, year: 2007, vol: 446, page: 83, stat: Journal Article,

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

Peptide-MHC potency governs dynamic interactions between T cells and dendritic cells in lymph nodes
Skokos, Dimitris; Shakhar, Guy; Varma, Rajat; Waite, Janelle C; Cameron, Thomas O; Lindquist, Randall L; Schwickert, Tanja; Nussenzweig, Michel C; Dustin, Michael L
2007 Aug;8(8):835-844, Nature immunology
T cells survey antigen-presenting dendritic cells (DCs) by migrating through DC networks, arresting and maintaining contact with DCs for several hours after encountering high-potency complexes of peptide and major histocompatibility complex (pMHC), leading to T cell activation. The effects of low-potency pMHC complexes on T cells in vivo, however, are unknown, as is the mechanism controlling T cell arrest. Here we evaluated T cell responses in vivo to high-, medium- and low-potency pMHC complexes and found that regardless of potency, pMHC complexes induced upregulation of CD69, anergy and retention of T cells in lymph nodes. However, only high-potency pMHC complexes expressed by DCs induced calcium-dependent T cell deceleration and calcineurin-dependent anergy. The pMHC complexes of lower potency instead induced T cell anergy by a biochemically distinct process that did not affect T cell dynamics
— id: 73915, year: 2007, vol: 8, page: 835, stat: Journal Article,

Dynamics of host defense: the view at the front lines
Velazquez, Peter; Waite, Janelle C; Dustin, Michael L
2007 Nov;8(11):1153-1157, Nature immunology
— id: 75668, year: 2007, vol: 8, page: 1153, stat: Journal Article,

Analysis of two-dimensional dissociation constant of laterally mobile cell adhesion molecules
Zhu, De-Min; Dustin, Michael L; Cairo, Christopher W; Golan, David E
2007 Feb 1;92(3):1022-1034, Biophysical journal
We formulate a general analysis to determine the two-dimensional dissociation constant (2D Kd), and use this method to study the interaction of CD2-expressing T cells with glass-supported planar bilayers containing fluorescently labeled CD58, a CD2 counter-receptor. Both CD2 and CD58 are laterally mobile in their respective membranes. Adhesion is indicated by accumulation of CD2 and CD58 in the cell-bilayer contact area; adhesion molecule density and contact area size attain equilibrium within 40 min. The standard (Scatchard) analysis of solution-phase binding is not applicable to the case of laterally mobile adhesion molecules due to the dynamic nature of the interaction. We derive a new binding equation, B/F=[(Ntxf)/(KdxScell)]-[(Bxp)/Kd], where B and F are bound and free CD58 density in the contact area, respectively; Nt is CD2 molecule number per cell; f is CD2 fractional mobility; Scell is cell surface area; and p is the ratio of contact area at equilibrium to Scell. We use this analysis to determine that the 2D Kd for CD2-CD58 is 5.4-7.6 molecules/microm2. 2D Kd analysis provides a general and quantitative measure of the mechanisms regulating cell-cell adhesion
— id: 78784, year: 2007, vol: 92, page: 1022, stat: Journal Article,

Quantum dot/peptide-MHC biosensors reveal strong CD8-dependent cooperation between self and viral antigens that augment the T cell response
Anikeeva, Nadia; Lebedeva, Tatiana; Clapp, Aaron R; Goldman, Ellen R; Dustin, Michael L; Mattoussi, Hedi; Sykulev, Yuri
2006 Nov 7;103(45):16846-16851, Proceedings of the National Academy of Sciences of the United States of America
Cytotoxic T lymphocytes (CTL) can respond to a few viral peptide-MHC-I (pMHC-I) complexes among a myriad of virus-unrelated endogenous self pMHC-I complexes displayed on virus-infected cells. To elucidate the molecular recognition events on live CTL, we have utilized a self-assembled biosensor composed of semiconductor nanocrystals, quantum dots, carrying a controlled number of virus-derived (cognate) and other (noncognate) pMHC-I complexes and examined their recognition by antigen-specific T cell receptor (TCR) on anti-virus CD8(+) T cells. The unique architecture of nanoscale quantum dot/pMHC-I conjugates revealed that unexpectedly strong multivalent CD8-MHC-I interactions underlie the cooperative contribution of noncognate pMHC-I to the recognition of cognate pMHC-I by TCR to augment T cell responses. The cooperative, CD8-dependent spread of signal from a few productively engaged TCR to many other TCR can explain the remarkable ability of CTL to respond to virus-infected cells that present few cognate pMHC-I complexes
— id: 78785, year: 2006, vol: 103, page: 16846, stat: Journal Article,

Dissecting lymphocyte stop signal hierarchies with chemokine receptor chimeras
Cameron, TO; Wu, T; Dustin, ML
2006 APR 1 ;176(4):S30-S31, Journal of immunology
— id: 68835, year: 2006, vol: 176, page: S30, stat: Journal Article,

Control of antigen presentation with a photoreleasable agonist peptide
DeMond, Andrew L; Starr, Toby; Dustin, Michael L; Groves, Jay T
2006 Dec 6;128(48):15354-15355, Journal of the American Chemical Society
The immunological synapse is a specialized intercellular junction between a T cell and a target cell that orchestrates the engagement of receptors and ligands in space and time as a means of regulating function. Here we introduce a reagent for controlling the spatial and temporal presentation of natural antigen to T cells. Moth cytochrome c (88-103) peptide (MCC), an agonist to the murine T cell receptor AND when presented in the context of H2 IEk major histocompatibility complex (IEk), was synthesized with the side-chain amine of Lys99 conjugated to a photosensitive protecting group, 6-nitroveratryloxycarbonyl (NVOC). Cells plated on supported bilayers displaying mobile intercellular adhesion molecule-1 (ICAM-1) and NVOC-MCC loaded IEk did not form immunological synapses and exhibited low intracellular calcium levels, similar to cells presented with self-peptide. Irradiation with UV light was sufficient to restore agonist activity in situ
— id: 78783, year: 2006, vol: 128, page: 15354, stat: Journal Article,

Lateral membrane waves constitute a universal dynamic pattern of motile cells
Dobereiner, Hans-Gunther; Dubin-Thaler, Benjamin J; Hofman, Jake M; Xenias, Harry S; Sims, Tasha N; Giannone, Gregory; Dustin, Michael L; Wiggins, Chris H; Sheetz, Michael P
2006 Jul 21;97(3):038102-038102, Physical review letters
We have monitored active movements of the cell circumference on specifically coated substrates for a variety of cells including mouse embryonic fibroblasts and T cells, as well as wing disk cells from fruit flies. Despite having different functions and being from multiple phyla, these cell types share a common spatiotemporal pattern in their normal membrane velocity; we show that protrusion and retraction events are organized in lateral waves along the cell membrane. These wave patterns indicate both spatial and temporal long-range periodic correlations of the actomyosin gel
— id: 68159, year: 2006, vol: 97, page: 038102, stat: Journal Article,

Immunology. When F-actin becomes too much of a good thing
Dustin, Michael L
2006 Aug 11;313(5788):767-768, Science
— id: 67855, year: 2006, vol: 313, page: 767, stat: Journal Article,

Impact of the immunological synapse on T cell signaling
Dustin, Michael L
2006 ;43:175-198, Results & problems in cell differentation
T cell activation requires interactions of T cell antigen receptors and peptides presented by major histocompatibility complex molecules in an adhesive junction between the T cell and antigen-presenting cell (APC). Stable junctions with bull's-eye supramolecular activation clusters have been defined as immunological synapses (IS). These structures maintain T cell-APC interaction and allow directed secretion. T cells can also be activated by asymmetric hemisynapses (HS) that allow migration during signal integration. IS and HS dominate in different stages of T cell priming. Optimal effector functions may also depend upon cyclical use of IS and HS
— id: 78786, year: 2006, vol: 43, page: 175, stat: Journal Article,

T cell-dendritic cell immunological synapses
Dustin, Michael L; Tseng, Su-Yi; Varma, Rajat; Campi, Gabriele
2006 Aug;18(4):512-516, Current opinion in immunology
Dendritic cells (DCs) are myeloid lineage cells that are imprinted by their environment and that mature in response to microbial products. A crucial role of the DC is to impart this context-specific information to T cells as well as to present self and foreign MHC-peptide complexes through formation of an immunological synapse. The structure of the T cell-DC immunological synapse departs from the canonical structure formed with B cells or with supported planar bilayers in that it has multiple foci of T-cell receptor interactions rather than a central focus. Recent studies on model systems provide insight into the mechanisms and biological consequences of the unique T cell-DC synaptic patterns
— id: 68161, year: 2006, vol: 18, page: 512, stat: Journal Article,

T cells like a firm molecular handshake
Dustin, Michael L; Zhu, Cheng
2006 Mar 21;103(12):4335-4336, Proceedings of the National Academy of Sciences of the United States of America
— id: 64164, year: 2006, vol: 103, page: 4335, stat: Journal Article,

Dynamic imaging of the immune system: progress, pitfalls and promise
Germain, Ronald N; Miller, Mark J; Dustin, Michael L; Nussenzweig, Michel C
2006 Jul;6(7):497-507, Nature reviews. Immunology
Both innate and adaptive immunity are dependent on the migratory capacity of myeloid and lymphoid cells. Effector cells of the innate immune system rapidly enter infected tissues, whereas sentinel dendritic cells in these sites mobilize and transit to lymph nodes. In these and other secondary lymphoid tissues, interactions among various cell types promote adaptive humoral and cell-mediated immune responses. Recent advances in light microscopy have allowed direct visualization of these events in living animals and tissue explants, which allows a new appreciation of the dynamics of immune-cell behaviour. In this article, we review the basic techniques and the tools used for in situ imaging, as well as the limitations and potential artefacts of these methods
— id: 68160, year: 2006, vol: 6, page: 497, stat: Journal Article,

Requirements for T lymphocyte migration in explanted lymph nodes
Huang, JH; Cardenas-Navia, LI; Caldwell, CC; Sitkovsky, M; Dewhirst, MW; Dustin, ML
2006 APR 1 ;176(4):S44-S45, Journal of immunology
— id: 68836, year: 2006, vol: 176, page: S44, stat: Journal Article,

Innate response to focal necrotic injury inside the blood-brain barrier
Kim, Jiyun V; Dustin, Michael L
2006 Oct 15;177(8):5269-5277, Journal of immunology
We have studied the initial innate immune response to focal necrotic injury on different sides of the mouse blood-brain barrier by two-photon intravital microscopy. Transgenic mice in which the promoter of the myeloid isoform of lysozyme drives GFP were used to track granulocytes and monocytes. Necrotic injury in the meninges, but not the brain parenchyma, recruited GFP+ cells within minutes that fully surrounded the necrotic site within a day. Recently, it has been suggested that microglial cells and astrocytes cooperate to mount a distinct response to laser injury behind the blood-brain barrier. We followed the microglial response in heterozygous knockin mice in which GFP replaces CX3CR1 coding sequence. Prior to injury, microglial cell bodies were immobile over days, but moved to the laser injury site within 1 day. We followed astrocytes, which have been proposed to cooperate with microglial cells in response to focal injury, using transgenic mice in which glial fibrillary acidic protein promoter drives GFP expression. Before injury fine astrocyte processes permeate the parenchyma. Astrocytes polarized toward the injury in an ATP, connexin hemichannels, and intracellular Ca2+ -dependent process. The astrocytes network established a cytoplasmic Ca2+ gradient that preceded the microglial response. This is consistent with astrocyte-microglial collaboration to mount this innate response that excludes blood leukocytes
— id: 70304, year: 2006, vol: 177, page: 5269, stat: Journal Article,

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

Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo
Tadokoro, Carlos E; Shakhar, Guy; Shen, Shiqian; Ding, Yi; Lino, Andreia C; Maraver, Antonio; Lafaille, Juan J; Dustin, Michael L
2006 Mar 20;203(3):505-511, Journal of experimental medicine
Regulatory T (T reg) cells exert powerful down-modulatory effects on immune responses, but it is not known how they act in vivo. Using intravital two-photon laser scanning microscopy we determined that, in the absence of T reg cells, the locomotion of autoantigen-specific T cells inside lymph nodes is decreased, and the contacts between T cells and antigen-loaded dendritic cells (DCs) are of longer duration. Thus, T reg cells can exert an early effect on immune responses by attenuating the establishment of stable contacts during priming of naive T cells by DCs
— id: 64138, year: 2006, vol: 203, page: 505, stat: Journal Article,

CD28 : CD80 interactions mediate antigen independent T cell adhesion and ring junction formation
Thomas, VK; Dustin, ML
2006 APR 1 ;176(4):S172-S172, Journal of immunology
— id: 68843, year: 2006, vol: 176, page: S172, stat: Journal Article,

T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster
Varma, Rajat; Campi, Gabriele; Yokosuka, Tadashi; Saito, Takashi; Dustin, Michael L
2006 Jul;25(1):117-127, Immunity
T cell receptor (TCR) signaling is initiated and sustained in microclusters; however, it's not known whether signaling also occurs in the TCR-rich central supramolecular activation cluster (cSMAC). We showed that the cSMAC formed by fusion of microclusters contained more CD45 than microclusters and is a site enriched in lysobisphosphatidic acid, a lipid involved in sorting ubiquitinated membrane proteins for degradation. Calcium signaling via TCR was blocked within 2 min by anti-MHCp treatment and 1 min by latrunculin-A treatment. TCR-MHCp interactions in the cSMAC survived these perturbations for 10 min and hence were not sufficient to sustain signaling. TCR microclusters were also resistant to disruption by anti-MHCp and latrunculin-A treatments. We propose that TCR signaling is sustained by stabilized microclusters and is terminated in the cSMAC, a structure from which TCR are sorted for degradation. Our studies reveal a role for F-actin in TCR signaling beyond microcluster formation
— id: 67540, year: 2006, vol: 25, page: 117, stat: Journal Article,

CXCR6 and CXCL16 are expressed by breast cancer cells and may play a dual role in tumor progression
Wang, B; Badura, M; He, C; Cameron, T; Dustin, M; Formenti, SC; Schneider, RJ; Demaria, S
2006 FEB ;100(2):S299-S299, Breast cancer research & treatment
— id: 71013, year: 2006, vol: 100, page: S299, stat: Journal Article,

Mechanisms of cellular avidity regulation in CD2-CD58-mediated T cell adhesion
Zhu, DM; Dustin, ML; Cairo, CW; Thatte, HS; Golan, DE
2006 OCT 22 ;1(10):649-658, ACS chemical biology
The CD2 receptor on T lymphocytes is essential for T cell adhesion and stimulation by antigen presenting cells (APCs). Blockade of CD2 function is immunosuppressive in both model systems and humans, indicating the importance of CD2 for the cellular immune response. Although the affinity of the molecular interaction between CD2 and its counter- receptor, CD58, is relatively low when measured in solution, this interaction mediates tight adhesion within the 2D cell cell interface. To understand the mechanisms responsible for regulating the avidity of the CD2-CD58 interaction, we measured the number, affinity, and lateral mobility of CD2 molecules on resting and activated T cells. Cell activation caused a 1.5-fold increase in the number of CD2 sites on the cell surface, and the 2D affinity of CD2 for CD58 increased by 2.5-fold. The combination of T cell activation and CD2 ligation to CD58 decreased the laterally mobile fraction of the ligated CD2. Together, these changes would substantially enhance CD2 avidity and strengthen T cell-APC adhesion. The change in CD2 mobile fraction suggests that the cell uses cytoskeletal regulators to immobilize the receptor selectively at the site of contact with surfaces expressing CD58. Our observations are consistent with a model in which T cell activation initially induces increased CD2 affinity, cell surface receptor expression, and lateral mobility, allowing the CD2 molecules to diffuse to sites of contact with CD58-bearing APCs. Subsequently, T cell activation causes the CD58-bound CD2 to be recognized and immobilized at sites of cell-cell contact, thereby strengthening T cell-APC adhesion
— id: 70773, year: 2006, vol: 1, page: 649, stat: Journal Article,

Distinct role of lymphocyte function-associated antigen-1 in mediating effective cytolytic activity by cytotoxic T lymphocytes
Anikeeva, Nadia; Somersalo, Kristina; Sims, Tasha N; Thomas, V Kaye; Dustin, Michael L; Sykulev, Yuri
2005 May 3;102(18):6437-6442, Proceedings of the National Academy of Sciences of the United States of America
Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecules (ICAMs) facilitates T cell antigen receptor (TCR)-mediated killing. To dissect TCR and LFA-1 contributions, we evaluated cytolytic activity and granule release by cytotoxic T lymphocytes (CTL) as well as intracellular granule redistribution and morphology of CTL stimulated with natural TCR ligand in the presence or absence of LFA-1 engagement. Although other adhesion mechanisms, e.g., CD2-CD58 interaction, could substitute for LFA-1 to trigger CTL degranulation, productive LFA-1 ligation was indispensable for effective target cell lysis by the released granules. LFA-1-mediated adhesion to glass-supported bilayers containing intercellular adhesion molecule-1 was characterized by a much larger junction area, marked by LFA-1 segregation, and a more compact cell shape compared with those observed for CD2-mediated adhesion to bilayers containing CD58. A larger contact induced by intercellular adhesion molecule 1 determined a unique positioning of granules near the interface. These data provide evidence that LFA-1 delivers a distinct signal essential for directing released cytolytic granules to the surface of antigen-bearing target cells to mediate the effective destruction of these cells by CTL
— id: 68168, year: 2005, vol: 102, page: 6437, stat: Journal Article,

Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling
Campi, Gabriele; Varma, Rajat; Dustin, Michael L
2005 Oct 17;202(8):1031-1036, Journal of experimental medicine
T cell receptor (TCR) microclusters form within seconds of T cell contact with supported planar bilayers containing intercellular adhesion molecule-1 and agonist major histocompatibility complex (MHC)-peptide complexes, and elevation of cytoplasmic Ca2+ is observed within seconds of the first detectable microclusters. At 0-30 s after contact, TCR microclusters are colocalized with activated forms of Lck, ZAP-70, and the linker for activation of T cells. By 2 min, activated kinases are reduced in the older central microclusters, but are abundant in younger peripheral microclusters. By 5 min, TCR in the central supramolecular activation cluster have reduced activated kinases, whereas faint peripheral TCR microclusters efficiently generated activated Lck and ZAP-70. TCR microcluster formation is resistant to inhibition by Src family kinase inhibitor PP2, but is abrogated by actin polymerization inhibitor latrunculin A. We propose that Src kinase-independent formation of TCR microclusters in response to agonist MHC-peptide provides an actin-dependent scaffold for signal amplification
— id: 68164, year: 2005, vol: 202, page: 1031, stat: Journal Article,

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

A dynamic view of the immunological synapse
Dustin, Michael L
2005 Dec;17(6):400-410, Seminars in immunology
T cell activation requires interactions of T cell antigen receptors (TCR) and peptides presented by major histocompatibility complex molecules (MHCp) in an adhesive junction between the T cell and antigen-presenting cell. Stable junctions with bull's eye supramolecular activation clusters (SMACs) have been defined as immunological synapses (IS). These structures maintain T cell-APC interaction and allow directed secretion. T cells can also be activated by asymmetric hemi-synapses (HS) that allow migration during signal integration. IS and HS operate in different stages of T cell priming. Optimal effector functions may also depend upon cyclical use of IS and HS
— id: 62394, year: 2005, vol: 17, page: 400, stat: Journal Article,

Antibody catches T-cell receptor in the act
Dustin, ML
2005 JUL 15 ;106(2):396-396, Blood
— id: 56297, year: 2005, vol: 106, page: 396, stat: Journal Article,

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

A molecular dissection of lymphocyte unresponsiveness induced by sustained calcium signalling
Heissmeyer, Vigo; Macian, Fernando; Varma, Rajat; Im, Sin-Hyeog; Garcia-Cozar, Francisco; Horton, Heidi F; Byrne, Michael C; Feske, Stefan; Venuprasad, K; Gu, Hua; Liu, Yun-Cai; Dustin, Michael L; Rao, Anjana
2005 ;267:165-174, Novartis foundation symposium
In lymphocytes, integration of Ca2+ and other signalling pathways results in productive activation, while unopposed Ca2+ signalling leads to decreased responsiveness to subsequent stimulation (anergy). The Ca(2+)-regulated transcription factor NFAT has an integral role in both aspects of lymphocyte function. NFAT cooperates with the transcription factor AP-1 (Fos/Jun) to up-regulate genes involved in productive activation of lymphocytes. However, in the absence of AP-1, NFAT imposes an opposing genetic programme that leads to lymphocyte anergy. Anergy is implemented at least partly through proteolytic degradation of the key signalling proteins PKCtheta and PLCgamma1. Sustained Ca(2+)-calcineurin signalling increases mRNA and protein levels of the E3 ubiquitin ligases Itch, CblB and Grail and induces expression of Tsg1O1, the ubiquitin-binding component of the ESCRT1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promotes membrane translocation of Itch and the related protein Nedd4, resulting in PKCtheta and PLCgamma1 degradation. T cells from Itch- and CblB-deficient mice are resistant to anergy induction. Anergic T cells show impaired calcium mobilization after TCR triggering and are unable to maintain a mature immunological synapse. Thus Ca(2+)-calcineurin-NFAT signalling links gene transcription to a multi-step programme that leads to impaired signal transduction in anergic T cells
— id: 68165, year: 2005, vol: 267, page: 165, stat: Journal Article,

ICAM-1 co-stimulates target cells to facilitate antigen presentation
Lebedeva, Tatiana; Dustin, Michael L; Sykulev, Yuri
2005 Jun;17(3):251-258, Current opinion in immunology
Adhesion molecules are known to mediate cell-cell interactions, particularly those between T cells and antigen-presenting or target cells. Recent studies identified ICAM-1 as a co-stimulatory ligand that binds to lymphocyte function associated antigen-1 (LFA-1), thereby promoting the activation of T cells. As ICAM-1 is expressed on virtually any cell, it becomes a crucial molecule for the activation of CD8(+) T cells in the absence of co-stimulation provided by CD80 and CD86 molecules. In addition, ICAM-1 might function as cell-surface receptor, capable of initiating intracellular signaling. ICAM-1 is associated with other cell molecules, including MHC-I proteins, and our recent data show that productive engagement of ICAM-1 on target cells leads to recruitment of the MHC-I proteins to the contact area and enhances presentation of cognate peptide MHC-I complexes to cytotoxic T cells
— id: 68167, year: 2005, vol: 17, page: 251, stat: Journal Article,

Altered TCR signaling from geometrically repatterned immunological synapses
Mossman, Kaspar D; Campi, Gabriele; Groves, Jay T; Dustin, Michael L
2005 Nov 18;310(5751):1191-1193, Science
The immunological synapse is a specialized cell-cell junction that is defined by large-scale spatial patterns of receptors and signaling molecules yet remains largely enigmatic in terms of formation and function. We used supported bilayer membranes and nanometer-scale structures fabricated onto the underlying substrate to impose geometric constraints on immunological synapse formation. Analysis of the resulting alternatively patterned synapses revealed a causal relation between the radial position of T cell receptors (TCRs) and signaling activity, with prolonged signaling from TCR microclusters that had been mechanically trapped in the peripheral regions of the synapse. These results are consistent with a model of the synapse in which spatial translocation of TCRs represents a direct mechanism of signal regulation
— id: 68162, year: 2005, vol: 310, page: 1191, stat: Journal Article,

Stable T cell-dendritic cell interactions precede the development of both tolerance and immunity in vivo
Shakhar, Guy; Lindquist, Randall L; Skokos, Dimitris; Dudziak, Diana; Huang, Julie H; Nussenzweig, Michel C; Dustin, Michael L
2005 Jul;6(7):707-714, Nature immunology
The maturation status of dendritic cells (DCs) determines whether they prime or tolerize T cells. We targeted ovalbumin peptide exclusively to DCs in situ using an antibody to DEC-205 and studied the interaction of DCs with naive CD4(+) T cells in tolerizing or priming conditions. We used two-photon microscopy to simultaneously track antigen-specific OT-II T cells, nonspecific T cells and DCs in lymph nodes of living mice. In both tolerance and immunity, OT-II cells arrested on DCs near high endothelial venules beginning shortly after extravasation and regained their baseline speed by 18 h. Thus, early antigen-dependent T cell arrest on DCs is a shared feature of tolerance and priming associated with activation and proliferation
— id: 56023, year: 2005, vol: 6, page: 707, stat: Journal Article,

A model for CD2/CD58-mediated adhesion strengthening
Shao, Jin-Yu; Yu, Yan; Dustin, Michael L
2005 Apr;33(4):483-493, Annals of biomedical engineering
Stable cell adhesion is vital for structural integrity and functional efficacy. Yet how low affinity adhesion molecules such as CD2 and CD58 can produce stable cell adhesion is still not completely understood. In this paper, we present a theoretical model that simulates the accumulation of CD2 and CD58 in the contact area of a Jurkat T lymphoblast and a CD58-containing substrate. The cell is assumed to have a spherical shape initially and it is allowed to spread gradually on a circular substrate. Mobile CD2 and CD58 can diffuse freely on both the cell and substrate. Their binding in the contact area is controlled by first-order kinetics. The contact area grows linearly with the total number of CD2/CD58 bonds. Cellular deformation and cytoskeleton involvement were not considered. This time-dependent moving-boundary problem was solved with the Crank-Nicolson finite difference scheme and the variable space grid method. Our simulated results are in reasonable agreement with the experimental observations. The role of diffusion becomes more and more prominent during the contact area increase, which is not sensitive to the kinetic rate constants tested in this study. However, it is very sensitive to the dissociation equilibrium constant and the concentrations of CD2 and CD58
— id: 68166, year: 2005, vol: 33, page: 483, stat: Journal Article,

CD80 cytoplasmic domain controls localization of CD28, CTLA-4, and protein kinase Ctheta in the immunological synapse
Tseng, Su-Yi; Liu, Mengling; Dustin, Michael L
2005 Dec 15;175(12):7829-7836, Journal of immunology
The binding of costimulatory ligand CD80 to CD28 or CTLA-4 on T cells plays an important role in the regulation of the T cell response. We have examined the role of the cytoplasmic domain of CD80 in murine T cell costimulation and its organization in the immunological synapse (IS). Removal of CD80 cytoplasmic tail decreased its effectiveness in costimulating T cell proliferative response and early IL-2 production in response to agonist MHC-peptide complexes. Immunofluorescent study showed a decreased tailless CD80 accumulation in the IS of naive T cells. The two forms of CD80 accumulated differently at the IS; the tailless CD80 was colocalized with the TCR whereas the full-length CD80 was segregated from the TCR. In addition, we showed that CD80, CD28, and protein kinase Ctheta colocalized in the presence or absence of the CD80 cytoplasmic tail. Thus, the cytoplasmic tail of CD80 regulates its spatial localization at the IS and that of its receptors and T cell signaling molecules such as protein kinase Ctheta, and thereby facilitates full T cell activation
— id: 67433, year: 2005, vol: 175, page: 7829, stat: Journal Article,

Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76
Yokosuka, Tadashi; Sakata-Sogawa, Kumiko; Kobayashi, Wakana; Hiroshima, Michio; Hashimoto-Tane, Akiko; Tokunaga, Makio; Dustin, Michael L; Saito, Takashi
2005 Dec;6(12):1253-1262, Nature immunology
T cell receptor (TCR) activation and signaling precede immunological synapse formation and are sustained for hours after initiation. However, the precise physical sites of the initial and sustained TCR signaling are not definitively known. We report here that T cell activation was initiated and sustained in TCR-containing microclusters generated at the initial contact sites and the periphery of the mature immunological synapse. Microclusters containing TCRs, the tyrosine kinase Zap70 and the adaptor molecule SLP-76 were continuously generated at the periphery. TCR microclusters migrated toward the central supramolecular cluster, whereas Zap70 and SLP-76 dissociated from these microclusters before the microclusters coalesced with the TCR-rich central supramolecular cluster. Tyrosine phosphorylation and calcium influx were induced as microclusters formed at the initial contact sites. Inhibition of signaling prevented recruitment of Zap70 into the microclusters. These results indicated that TCR-rich microclusters initiate and sustain TCR signaling
— id: 68163, year: 2005, vol: 6, page: 1253, stat: Journal Article,

LFA-1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation
Carrasco, Yolanda R; Fleire, Sebastian J; Cameron, Thomas; Dustin, Michael L; Batista, Facundo D
2004 May;20(5):589-599, Immunity
The integrin LFA-1 and its ligand ICAM-1 mediate B cell adhesion, but their role in membrane-bound antigen recognition is still unknown. Here, using planar lipid bilayers and cells expressing ICAM-1 fused to green fluorescence protein, we found that the engagement of B cell receptor (BCR) promotes B cell adhesion by an LFA-1-mediated mechanism. LFA-1 is recruited to form a mature B cell synapse segregating into a ring around the BCR. This distribution is maintained over a wide range of BCR/antigen affinities (10(6) M(-1) to 10(11) M(-1)). Furthermore, the LFA-1 binding to ICAM-1 reduces the level of antigen required to form the synapse and trigger a B cell. Thus, LFA-1/ICAM-1 interaction lowers the threshold for B cell activation by promoting B cell adhesion and synapse formation
— id: 44915, year: 2004, vol: 20, page: 589, stat: Journal Article,

What is the importance of the immunological synapse?
Davis, Daniel M; Dustin, Michael L
2004 Jun;25(6):323-327, Trends in immunology
The immunological synapse (IS) has proved to be a stimulating concept, particularly in provoking discussion on the similarity of intercellular communication controlling disparate biological processes. Recent studies have clarified some of the underlying molecular mechanisms and functions of the IS. For both T cells and natural killer (NK) cells, assembly of the IS can be described in stages with distinct cytoskeletal requirements. Functions of the IS vary with circumstance and include directing secretion and integrating positive and negative signals to determine the extent of response
— id: 44914, year: 2004, vol: 25, page: 323, stat: Journal Article,

A supercode for inflammation
Dustin, Michael
2004 Apr;20(4):361-362, Immunity
Siegelman and colleagues demonstrate unexpected synergism of CD44 and VLA-4 during lymphocyte extravasation. This is the first time that molecules mediating rolling and firm adhesion have been shown to associate biochemically leading to direct functional cooperation
— id: 44916, year: 2004, vol: 20, page: 361, stat: Journal Article,

Stop and go traffic to tune T cell responses
Dustin, Michael L
2004 Sep;21(3):305-314, Immunity
Adaptive immune responses are initiated by interactions of T cells with antigen-presenting cells, but the basic nature of these interactions during an immune response in vivo has been a matter of speculation. While some in vitro systems provide evidence for stable interactions, referred to as immunological synapses, compelling evidence supports T cell activation through serial transient interactions. Deep tissue intravital and organ culture microscopy studies suggest that both modes of interaction are employed, but new issues have emerged. This review will discuss in vitro results that framed the hypotheses that are currently being tested in vivo. I present a model in which TCR stop signals compete with chemokine-mediated go signals to adjust the duration of immunological synapse formation and tune the immune response between tolerance and full activation
— id: 44912, year: 2004, vol: 21, page: 305, stat: Journal Article,

Membranes as messengers in T cell adhesion signaling
Dustin, Michael L; Bivona, Trever G; Philips, Mark R
2004 Apr;5(4):363-372, Nature immunology
Talin and RapL are components of molecular pathways that regulate the avidity of the integrin lymphocyte function-associated antigen 1 (LFA-1) for its ligand, intercellular adhesion molecule 1. In this review, we discuss recent advances in our understanding of LFA-1 affinity regulation and signaling and discuss a scenario for how Talin and Rap1 might act in synergy to achieve regulation of LFA-1 that is tailored to the specific functional requirements of different situations. Speedy delivery of signals may be crucial, and membrane trafficking from endosomes and the Golgi apparatus seem to be essential in delivering the messages from spatially segregated surface receptors
— id: 44917, year: 2004, vol: 5, page: 363, stat: Journal Article,

New ways for lymphocytes to meet
Dustin, ML
2004 NOV 1 ;104(9):2618-2619, Blood
— id: 46911, year: 2004, vol: 104, page: 2618, stat: Journal Article,

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

Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins
Heissmeyer, Vigo; Macian, Fernando; Im, Sin-Hyeog; Varma, Rajat; Feske, Stefan; Venuprasad, K; Gu, Hua; Liu, Yun-Cai; Dustin, Michael L; Rao, Anjana
2004 Mar;5(3):255-265, Nature immunology
Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca(2+)-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-theta and PLC-gamma1. T cells from Itch- and Cbl-b-deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function-associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells
— id: 44918, year: 2004, vol: 5, page: 255, stat: Journal Article,

Visualizing dendritic cell networks in vivo
Lindquist, Randall L; Shakhar, Guy; Dudziak, Diana; Wardemann, Hedda; Eisenreich, Thomas; Dustin, Michael L; Nussenzweig, Michel C
2004 Dec;5(12):1243-1250, Nature immunology
In the steady state, dendritic cells (DCs) in the lymph node induce T cell tolerance to self antigens. Innate signals trigger the maturation of tissue DCs, which migrate into lymph nodes and activate T cells. To examine DCs in vivo, we produced transgenic mice whose DCs expressed enhanced yellow fluorescent protein. Two-photon microscopy of lymph nodes in live mice showed that most of the steady-state DCs were enmeshed in an extensive network and remained in place while actively probing adjacent T cells with their processes. Mature DCs were more motile than steady-state DCs and were rapidly dispersed and integrated into the sessile network, facilitating their interaction with migrating T cells
— id: 68169, year: 2004, vol: 5, page: 1243, stat: Journal Article,

Dissecting the immune synapse using nanopatterned substrates
Mossman, KD; Groves, JT; Dustin, ML
2004 JAN ;86(1):523A-523A, Biophysical journal
— id: 42460, year: 2004, vol: 86, page: 523A, stat: Journal Article,

A polarizing situation
Sims, Tasha N; Dustin, Michael L
2004 Oct;5(10):1012-1013, Nature immunology
— id: 68170, year: 2004, vol: 5, page: 1012, stat: Journal Article,

Cytotoxic T lymphocytes form an antigen-independent ring junction
Somersalo, Kristina; Anikeeva, Nadja; Sims, Tasha N; Thomas, V Kaye; Strong, Roland K; Spies, Thomas; Lebedeva, Tatiana; Sykulev, Yuri; Dustin, Michael L
2004 Jan;113(1):49-57, Journal of clinical investigation
Immunological synapses are organized cell-cell junctions between T lymphocytes and APCs composed of an adhesion ring, the peripheral supramolecular activation cluster (pSMAC), and a central T cell receptor cluster, the central supramolecular activation cluster (cSMAC). In CD8(+) cytotoxic T lymphocytes, the immunological synapse is thought to facilitate specific killing by confining cytotoxic agents to the synaptic cleft. We have investigated the interaction of human CTLs and helper T cells with supported planar bilayers containing ICAM-1. This artificial substrate provides identical ligands to CD4(+) and CD8(+) T cells, allowing a quantitative comparison. We found that cytotoxic T lymphocytes form a ring junction similar to a pSMAC in response to high surface densities of ICAM-1 in the planar bilayer. MICA, a ligand for NKG2D, facilitated the ring junction formation at lower surface densities of ICAM-1. ICAM-1 and MICA are upregulated in tissues by inflammation- and stress-associated signaling, respectively. Activated CD8(+) T cells formed fivefold more ring junctions than did activated CD4(+) T cells. The ring junction contained lymphocyte function associated antigen-1 and talin, but did not trigger polarization and granule translocation to the interface. This result has specific implications for the mechanism of effective CTL hunting for antigen in tissues. Abnormalities in this process may alter CTL reactivity
— id: 42628, year: 2004, vol: 113, page: 49, stat: Journal Article,

T cell receptor antagonism interferes with MHC clustering and integrin patterning during immunological synapse formation
Sumen, Cenk; Dustin, Michael L; Davis, Mark M
2004 Aug 16;166(4):579-590, Journal of cell biology
T cell activation by nonself peptide-major histocompatibility complex (MHC) antigenic complexes can be blocked by particular sequence variants in a process termed T cell receptor antagonism. The inhibition mechanism is not understood, although such variants are encountered in viral infections and may aid immune evasion. Here, we study the effect of antagonist peptides on immunological synapse formation by T cells. This cellular communication process features early integrin engagement and T cell motility arrest, referred to as the 'stop signal.' We find that synapses formed on membranes presenting antagonist-agonist complexes display reduced MHC density, which leads to reduced T cell proliferation that is not overcome by the costimulatory ligands CD48 and B7-1. Most T cells fail to arrest and crawl slowly with a dense ICAM-1 crescent at the leading edge. Similar aberrant patterns of LFA-1/ICAM-1 engagement in live T-B couples correlate with reduced calcium flux and IL-2 secretion. Hence, antagonist peptides selectively disable MHC clustering and the stop signal, whereas LFA-1 valency up-regulation occurs normally
— id: 44913, year: 2004, vol: 166, page: 579, stat: Journal Article,

In silico models for cellular and molecular immunology: successes, promises and challenges
Chakraborty, Arup K; Dustin, Michael L; Shaw, Andrey S
2003 Oct;4(10):933-936, Nature immunology
— id: 44919, year: 2003, vol: 4, page: 933, stat: Journal Article,

Viral spread through protoplasmic kiss
Dustin, Michael
2003 Apr;5(4):271-272, Nature cell biology
— id: 44921, year: 2003, vol: 5, page: 271, stat: Journal Article,

Coordination of T cell activation and migration through formation of the immunological synapse
Dustin, Michael L
2003 Apr;987(4):51-59, Annals of the New York Academy of Sciences
T cell activation is based on interactions of T cell antigen receptors with MHC-peptide complexes in a specialized cell-cell junction between the T cell and antigen-presenting cell-the immunological synapse. The immunological synapse coordinates naive T cell activation and migration by stopping T cell migration with antigen-presenting cells bearing appropriate major histocompatibility complex (MHC) peptide complexes. At the same time, the immunological synapse allows full T cell activation through sustained signaling over a period of several hours. The immunological synapse supports activation in the absence of continued T cell migration, which is required for T cell activation through serial encounters. Src and Syk family kinases are activated early in immunological synapse formation, but this signaling process returns to the basal level after 30 min; at the same time, the interactions between T cell receptors (TCRs) and MHC peptides are stabilized within the immunological synapse. The molecular pattern of the mature synapse in helper T cells is a self-stabilized structure that is correlated with cytokine production and proliferation. I propose that this molecular pattern and its specific biochemical constituents are necessary to amplify signals from the partially desensitized TCR
— id: 37211, year: 2003, vol: 987, page: 51, stat: Journal Article,

In vivo imaging approaches in animal models of rheumatoid arthritis
Dustin, Michael L
2003 ;5(4):165-171, Arthritis research & therapy
The interaction of activated leukocytes with the rheumatoid synovial environment is a key process in arthritis. Understanding this process will play an important role in designing effective treatments. In vivo imaging approaches combined with molecular genetics in animal models provide important tools to address these issues. The present review will focus on approaches to in vivo imaging, with particular attention to approaches that are proving useful for, or have promise for, research on animal models of rheumatoid arthritis. These approaches will probably shed light on the specific local mechanisms involved in chronic inflammation and provide real time monitoring approaches to follow cellular and molecular events related to disease development
— id: 37210, year: 2003, vol: 5, page: 165, stat: Journal Article,

Supported planar bilayers in studies on immune cell adhesion and communication
Groves, Jay T; Dustin, Michael L
2003 Jul 1;278(1-2):19-32, Journal of immunological methods
Supported planar bilayers have been used extensively in immunology to study molecular interactions at interfaces as a model for cell-cell interaction. Examples include Fc receptor-mediated adhesion and signaling and formation of the immunological synapse between T cells and antigen-presenting cells. The advantage of the supported planar bilayer system is control of the bilayer composition and the optical advantages of imaging the cell-bilayer or bilayer-bilayer interface by various types of trans-, epi- and total internal reflection illumination. Supported planar bilayers are simple to form by liposome fusion and recent advances in micro- and nanotechnology greatly extend the power of supported bilayers to address key questions in immunology and cell biology
— id: 37209, year: 2003, vol: 278, page: 19, stat: Journal Article,

The immunological synapse balances T cell receptor signaling and degradation
Lee, Kyeong-Hee; Dinner, Aaron R; Tu, Chun; Campi, Gabriele; Raychaudhuri, Subhadip; Varma, Rajat; Sims, Tasha N; Burack, W Richard; Wu, Hui; Wang, Julia; Kanagawa, Osami; Markiewicz, Mary; Allen, Paul M; Dustin, Michael L; Chakraborty, Arup K; Shaw, Andrey S
2003 Nov 14;302(5648):1218-1222, Science
The immunological synapse is a specialized cell-cell junction between T cell and antigen-presenting cell surfaces. It is characterized by a central cluster of antigen receptors, a ring of integrin family adhesion molecules, and temporal stability over hours. The role of this specific organization in signaling for T cell activation has been controversial. We use in vitro and in silico experiments to determine that the immunological synapse acts as a type of adaptive controller that both boosts T cell receptor triggering and attenuates strong signals
— id: 44920, year: 2003, vol: 302, page: 1218, stat: Journal Article,

Spatial accumulation of costimulatory molecules in the immunological synapse
Tseng, SY; Pardon, D; Dustin, ML
2003 APR 14 ;17(7):C312-C312, FASEB journal
— id: 37136, year: 2003, vol: 17, page: C312, stat: Journal Article,

WIP deficiency reveals a differential role for WIP and the actin cytoskeleton in T and B cell activation
Anton, Ines M; de la Fuente, Miguel A; Sims, Tasha N; Freeman, Sheryl; Ramesh, Narayanaswamy; Hartwig, John H; Dustin, Michael L; Geha, Raif S
2002 Feb;16(2):193-204, Immunity
WIP stabilizes actin filaments and is important for filopodium formation. To define the role of WIP in immunity, we generated WIP-deficient mice. WIP(minus sign/minus sign) mice have normal lymphocyte development, but their T cells fail to proliferate, secrete IL-2, increase their F-actin content, polarize and extend protrusions following T cell receptor ligation, and are deficient in conjugate formation with superantigen-presenting B cells and anti-CD3 bilayers. In contrast, WIP-deficient B lymphocytes have enhanced proliferation and CD69 expression following B cell receptor ligation and mount normal antibody responses to T-independent antigens. Both WIP-deficient T and B cells show a profound defect in their subcortical actin filament networks. These results suggest that WIP is important for immunologic synapse formation and T cell activation
— id: 26978, year: 2002, vol: 16, page: 193, stat: Journal Article,

Stimulation of naive T-cell adhesion and immunological synapse formation by chemokine-dependent and -independent mechanisms
Bromley, Shannon K; Dustin, Michael L
2002 Jul;106(3):289-298, Immunology
Chemokines adsorbed to the cell surface play an important role in the initial interactions of T cells with endothelial cells, and may also have a role in T-cell interactions with dendritic cells. Therefore, we examined the effect of surface-adsorbed chemokines on the interaction of naive murine splenic T cells with supported bilayers containing intercellular adhesion molecule (ICAM)-1, or with bone marrow-derived cultured dendritic cells in the presence and absence of relevant MHC-peptide complexes. Naive T cells formed immunological synapses, defined as a ring of lymphocyte function associated (LFA)-1-ICAM-1 interactions surrounding a central cluster of MHC-peptide complexes, on supported planar bilayers containing ICAM-1 and relevant MHC-peptide complexes. Chemokines stimulated an increase in the percentage of naive cells that adhered to ICAM-1, but did not increase the average number of LFA-1-ICAM-1 interactions in the contact area. In contrast, relevant MHC-peptide complexes resulted in a small increase in the proportion of interacting T cells, but stimulated an 8-fold increase in the number of LFA-1-ICAM-1 interactions in each contact formed. Naive T cells displayed a significant basal adhesion to bone marrow dendritic cells that was further increased when relevant chemokines were adsorbed to the dendritic cell surface. However, basal and antigen-stimulated T-cell adhesion to dendritic cells was not sensitive to pertussis toxin. Thus, there are chemokine-independent mechanisms that initiate adhesion between T cells and dendritic cells
— id: 37221, year: 2002, vol: 106, page: 289, stat: Journal Article,

Cutting edge: quantitative imaging of raft accumulation in the immunological synapse
Burack, W Richard; Lee, Kyeong-Hee; Holdorf, Amy D; Dustin, Michael L; Shaw, Andrey S
2002 Sep 15;169(6):2837-2841, Journal of immunology
Although the accumulation of lipid rafts at the immunological synapse is now well accepted, the degree of the accumulation, the localization within the fine structure of the immunological synapse, and the region from which lipid rafts are recruited have not been defined. In this work we show that lipid rafts preferentially accumulate in the central zone of the immunological synapse, the central supramolecular activation complex (C-SMAC). However, quantitative analyses indicate that the level of recruitment of lipid rafts to the C-SMAC is relatively small and suggests that rearrangement of lipid rafts from the peripheral zone of the synapse into the C-SMAC can account for this accumulation. We also assessed the effects of CD28 deficiency on lipid raft recruitment to the immunological synapse. The accumulation of lipid occurred independently of the CD28/B7 system and was not measurably altered by CD28
— id: 37218, year: 2002, vol: 169, page: 2837, stat: Journal Article,

Membrane domains and the immunological synapse: keeping T cells resting and ready
Dustin, Michael L
2002 Jan;109(2):155-160, Journal of clinical investigation
— id: 26980, year: 2002, vol: 109, page: 155, stat: Journal Article,

Regulation of T cell migration through formation of immunological synapses: the stop signal hypothesis
Dustin, Michael L
2002 ;512(4):191-201, Advances in experimental medicine & biology
— id: 37212, year: 2002, vol: 512, page: 191, stat: Journal Article,

Shmoos, rafts, and uropods- the many facets of cell polarity
Dustin, Michael L
2002 Jul 12;110(1):13-18, Cell
The recent Juan March Foundation meeting on 'Regulation and functional insights in cellular polarity' focused on cellular polarity in yeasts, Dictyostelium, epithelial cells, fibroblasts, and immune cells. The molecular systems covered included membrane rafts, actin and tubulin cytoskeleton, polarized transcription, signaling, and cell-cell adhesion. Across these diverse biological and molecular systems, important general concepts emerged, including new ideas for establishing and maintaining polarity that are likely to be applicable across models and experimental systems
— id: 32453, year: 2002, vol: 110, page: 13, stat: Journal Article,

The immunological synapse
Dustin, Michael L
2002 ;4 Suppl 3(6):S119-S125, Arthritis research
T-cell activation requires interaction of T-cell antigen receptors with proteins of the major histocompatibility complex (antigen). This interaction takes place in a specialized cell-cell junction referred to as an immunological synapse. The immunological synapse contains at least two functional domains: a central cluster of engaged antigen receptors and a surrounding ring of adhesion molecules. The segregation of the T-cell antigen receptor (TCR) and adhesion molecules is based on size, with the TCR interaction spanning 15 nm and the lymphocyte-function-associated antigen-1 (LFA-1) interaction spanning 30-40 nm between the two cells. Therefore, the synapse is not an empty gap, but a space populated by both adhesion and signaling molecules. This chapter considers four aspects of the immunological synapse: the role of migration and stop signals, the role of the cytoskeleton, the role of self-antigenic complexes, and the role of second signals
— id: 37220, year: 2002, vol: 4 Suppl 3, page: S119, stat: Journal Article,

Neural and immunological synaptic relations
Dustin, Michael L; Colman, David R
2002 Oct 25;298(5594):785-789, Science
A synapse is a stable adhesive junction between two cells across which information is relayed by directed secretion. The nervous system and immune system utilize these specialized cell surface contacts to directly convey and transduce highly controlled secretory signals between their constituent cell populations. Each of these synaptic types is built around a microdomain structure comprising central active zones of exocytosis and endocytosis encircled by adhesion domains. Surface molecules that may be incorporated into and around the active zones contribute to modulation of the functional state of the synapse
— id: 37213, year: 2002, vol: 298, page: 785, stat: Journal Article,

Immature CD4(+)CD8(+) thymocytes form a multifocal immunological synapse with sustained tyrosine phosphorylation
Hailman, Eric; Burack, W Richard; Shaw, Andrey S; Dustin, Michael L; Allen, Paul M
2002 Jun;16(6):839-848, Immunity
The immunological synapse formed during mature T cell activation consists of a central cluster of TCR and MHC molecules surrounded by a ring of LFA-1 and ICAM-1. We examined synapse formation in thymocytes undergoing activation in a lipid bilayer system by following the movement of fluorescent MHC and ICAM-1 molecules. Immature CD4(+)CD8(+) thymocytes formed a decentralized synapse with multiple foci of MHC accumulation corresponding to areas of exclusion of ICAM-1. The MHC clusters and ICAM-1 holes were mobile and transient and correlated with active and sustained signaling, as shown by staining with antibodies against phosphotyrosine and activated Lck. Our findings show that signaling in immature thymocytes can result from a novel, multifocal pattern of receptor accumulation
— id: 37219, year: 2002, vol: 16, page: 839, stat: Journal Article,

T cell receptor signaling precedes immunological synapse formation
Lee, Kyeong-Hee; Holdorf, Amy D; Dustin, Michael L; Chan, Andrew C; Allen, Paul M; Shaw, Andrey S
2002 Feb 22;295(5559):1539-1542, Science
The area of contact between a T cell and an antigen-presenting cell (APC) is known as the immunological synapse. Although its exact function is unknown, one model suggests that it allows for T cell receptor (TCR) clustering and for sustained signaling in T cells for many hours. Here we demonstrate that TCR-mediated tyrosine kinase signaling in naive T cells occurred primarily at the periphery of the synapse and was largely abated before mature immunological synapses had formed. These data suggest that many hours of TCR signaling are not required for T cell activation. These observations challenge current ideas about the role of immunological synapses in T cell activation
— id: 26979, year: 2002, vol: 295, page: 1539, stat: Journal Article,

Correlation of a dynamic model for immunological synapse formation with effector functions: two pathways to synapse formation
Lee, Sung-Joo E; Hori, Yuko; Groves, Jay T; Dustin, Michael L; Chakraborty, Arup K
2002 Oct;23(10):492-499, Trends in immunology
During antigen recognition by T cells different receptors and ligands form a pattern in the intercellular junction called the immunological synapse, which might be involved in T-cell activation. Recently, a synapse assembly model has been proposed, which enables the calculation of the propensity for synapse assembly driven by membrane-constrained protein binding interactions. We bring together model predictions of mature synapse assembly with data on the dependence of T-cell responses on T-cell receptor (TCR)-MHC-peptide (pMHC) binding kinetics. Predictions of mature synapse assembly, based on TCR-pMHC binding kinetics, correlate well with observed cytokine responses by T cells bearing the relevant TCR but not with cytotoxic T lymphocyte-mediated killing. We discuss the suggested different role for the synapse in pre- and post-nuclear activation events in T cells. The view of immunological synapse assembly given here emphasizes the importance of both the on and off rates for the TCR-pMHC interaction and in this context recent data on a positive role for analogs of self-peptides in synapse assembly is considered
— id: 37215, year: 2002, vol: 23, page: 492, stat: Journal Article,

The synapse assembly model
Lee, Sung-Joo E; Hori, Yuko; Groves, Jay T; Dustin, Michael L; Chakraborty, Arup K
2002 Oct;23(10):500-502, Trends in immunology
A framework for quantitative analysis of the mechanisms underlying immunological synapse assembly has been recently developed. This model uses partial differential equations to describe the binding interactions of receptors and ligands, with the constraint that they are embedded in apposed deformable membranes linked to a cytoskeletal complex
— id: 37214, year: 2002, vol: 23, page: 500, stat: Journal Article,

Polar redistribution of the sialoglycoprotein CD43: implications for T cell function
Savage, Nigel D L; Kimzey, Stephanie L; Bromley, Shannon K; Johnson, Kenneth G; Dustin, Michael L; Green, Jonathan M
2002 Apr 15;168(8):3740-3746, Journal of immunology
Contact between T cells and APCs results in the orchestrated segregation of molecules at the cell-cell interface and formation of a specialized structure termed the immunological synapse. This model predicts the topological seclusion of large molecules such as CD43 from the site of closest contact between the T cell and APC, allowing for the close apposition of cell membranes and effective TCR engagement. Similarly, during T cell migration segregation of CD43 to the uropod is thought to aid integrin adhesion at the leading edge of the cell by removing steric hindrance. We show in this work that CD43 distribution on T cells is regulated by a membrane proximal ezrin binding site and that failure to displace CD43 from the immunological synapse has no inhibitory effects on primary T cell activation. We also report that CD43 expression at the contact zone between T cells and matrix does not negatively regulate motility but may regulate LFA-1 de-adhesion. These results suggest that the steric barrier model of CD43 is inadequate and that alternative mechanisms account for the negative regulatory properties of CD43
— id: 37222, year: 2002, vol: 168, page: 3740, stat: Journal Article,

The immunological synapse: integrins take the stage
Sims, Tasha N; Dustin, Michael L
2002 Aug;186(10):100-117, Immunological reviews
Adhesive interactions play important roles in coordinating T-cell migration and activation, specifically in the formation of the immunological synapse (IS), a specialized cell-cell junction. Recent demonstrations show several molecules implicated in T-cell signaling, including Vav, ADAP, and Rap-1, have major roles in integrin regulation and place adhesion molecules at center stage in addressing the question: what are the signals involved in the formation of the IS and full T-cell activation? This review focuses on the role of integrins as an essential system for both physical adhesion and signaling in T-cell activation. The role of integrins appears to be quite distinct from classical costimulation and has been largely overlooked due to the ubiquitous use of serum in lymphocyte functional assays. Each major signal transduction pathway has branches leading to the nucleus and others that feed back on cytoskeletal and membrane regulation at the IS
— id: 37216, year: 2002, vol: 186, page: 100, stat: Journal Article,

T-cell activation: a multidimensional signaling network
Tseng, Su-Yi; Dustin, Michael L
2002 Oct;14(5):575-580, Current opinion in cell biology
Naive T cell activation requires the interactions of antigen receptors, adhesion molecules and co-stimulatory molecules. Antigen receptors and adhesion molecules are involved in spatio-temporal movement to form a stable immunological synapse. This stable junction interrupts T cell migration, and provides a platform for temporally regulated co-stimulatory receptor signaling spanning a period of days
— id: 37217, year: 2002, vol: 14, page: 575, stat: Journal Article,

Costimulation and endogenous MHC ligands contribute to T cell recognition
Wulfing, Christoph; Sumen, Cenk; Sjaastad, Michael D; Wu, Lawren C; Dustin, Michael L; Davis, Mark M
2002 Jan;3(1):42-47, Nature immunology
To initiate an immune response, key receptor-ligand pairs must cluster in 'immune synapses' at the T cell-antigen-presenting cell (APC) interface. We visualized the accumulation of a major histocompatibility complex (MHC) class II molecule, I-E(k), at a T cell-B cell interface and found it was dependent on both antigen recognition and costimulation. This suggests that costimulation-driven active transport of T cell surface molecules helps to drive immunological synapse formation. Although only agonist peptide-MHC class II (agonist pMHC class II) complexes can initiate T cell activation, endogenous pMHC class II complexes also appeared to accumulate. To test this directly, we labeled a 'null' pMHC class II complex and found that, although it lacked major TCR contact residues, it could be driven into the synapse in a TCR-dependent manner. Thus, low-affinity ligands can contribute to synapse formation and T cell signaling
— id: 26981, year: 2002, vol: 3, page: 42, stat: Journal Article,

The immunological synapse
Bromley SK; Burack WR; Johnson KG; Somersalo K; Sims TN; Sumen C; Davis MM; Shaw AS; Allen PM; Dustin ML
2001 ;19(1):375-396, Annual review of immunology
The adaptive immune response is initiated by the interaction of T cell antigen receptors with major histocompatibility complex molecule-peptide complexes in the nanometer scale gap between a T cell and an antigen-presenting cell, referred to as an immunological synapse. In this review we focus on the concept of immunological synapse formation as it relates to membrane structure, T cell polarity, signaling pathways, and the antigen-presenting cell. Membrane domains provide an organizational principle for compartmentalization within the immunological synapse. T cell polarization by chemokines increases T cell sensitivity to antigen. The current model is that signaling and formation of the immunological synapse are tightly interwoven in mature T cells. We also extend this model to natural killer cell activation, where the inhibitory NK synapse provides a striking example in which inhibition of signaling leaves the synapse in its nascent, inverted state. The APC may also play an active role in immunological synapse formation, particularly for activation of naive T cells
— id: 20356, year: 2001, vol: 19, page: 375, stat: Journal Article,

The immunological synapse and CD28-CD80 interactions
Bromley SK; Iaboni A; Davis SJ; Whitty A; Green JM; Shaw AS; Weiss A; Dustin ML
2001 Dec;2(12):1159-1166, Nature immunology
According to the two-signal model of T cell activation, costimulatory molecules augment T cell receptor (TCR) signaling, whereas adhesion molecules enhance TCR-MHC-peptide recognition. The structure and binding properties of CD28 imply that it may perform both functions, blurring the distinction between adhesion and costimulatory molecules. Our results show that CD28 on naive T cells does not support adhesion and has little or no capacity for directly enhancing TCR-MHC-peptide interactions. Instead of being dependent on costimulatory signaling, we propose that a key function of the immunological synapse is to generate a cellular microenvironment that favors the interactions of potent secondary signaling molecules, such as CD28
— id: 26982, year: 2001, vol: 2, page: 1159, stat: Journal Article,

Role of adhesion molecules in activation signaling in T lymphocytes
Dustin ML
2001 Jul;21(4):258-263, Journal of clinical immunology
The T cell and antigen-presenting cell communicate to initiate an immune response through formation of an immunological synapse. This specialized cell-cell junction is compartmentalized into adhesion molecule and T cell receptor enriched regions or SMACs. Distinct signals seem to be generated in the T cell receptor and adhesion molecule-dominated regions. This review focuses on how these distinct signaling pathways may be integrated within the T cell to set thresholds for T cell activation, proliferation, and survival
— id: 26516, year: 2001, vol: 21, page: 258, stat: Journal Article,

Environmental control of immunological synapse formation and duration
Dustin ML; Allen PM; Shaw AS
2001 Apr;22(4):192-194, Trends in immunology
The coordination of T-cell migration and antigen recognition is crucial for an effective immune response. We have proposed that this coordination is achieved by formation of an immunological synapse between the T cell and the antigen-presenting cell (APC). Our view contrasts with the serial encounter model also proposed in this issue of Trends in Immunology, which is based on transient T cell-APC interactions when surrounded by collagen. Here, we propose a model that reconciles immunological synapse formation and serial encounters based on environmental control of immunological synapse formation
— id: 20354, year: 2001, vol: 22, page: 192, stat: Journal Article,

Identification of self through two-dimensional chemistry and synapses
Dustin ML; Bromley SK; Davis MM; Zhu C
2001 ;17(22):133-157, Annual review of cell & developmental biology
Cells in the immune and nervous systems communicate through informational synapses. The two-dimensional chemistry underlying the process of synapse formation is beginning to be explored using fluorescence imaging and mechanical techniques. Early analysis of two-dimensional kinetic rates (k(on) and k(off)) and equilibrium constants (K(d)) provides a number of biological insights. First, there are two regimes for adhesion-one disordered with slow k(on) and the other self-ordered with 10(4)-fold faster k(on). Despite huge variation in two-dimensional k(on), the two-dimensional k(off) is like k(off) in solution, and two-dimensional k(off) is more closely related to intrinsic properties of the interaction than the two-dimensional k(on). Thus difference in k(off) can be used to set signaling thresholds. Early signaling complexes are compartmentalized to generate synergistic signaling domains. Immune antigen receptor components have a role in neural synapse editing. This suggests significant parallels in informational synapse formation based on common two-dimensional chemistry and signaling strategies
— id: 26536, year: 2001, vol: 17, page: 133, stat: Journal Article,

Reprograming T cells: the role of extracellular matrix in coordination of T cell activation and migration
Dustin ML; de Fougerolles AR
2001 Jun;13(3):286-290, Current opinion in immunology
The stable immunological synapse between a T cell and antigen-presenting cell coordinates migration and activation. Three-dimensional collagen gels transform this interaction into a series of transient hit-and-run encounters. Here we integrate these alternative modes of interaction in a model for primary T cell activation and effector function in vivo
— id: 21184, year: 2001, vol: 13, page: 286, stat: Journal Article,

The immunological relay race: B cells take antigen by synapse
Dustin ML; Dustin LB
2001 Jun;2(6):480-482, Nature immunology
— id: 20353, year: 2001, vol: 2, page: 480, stat: Journal Article,

The immunological synapse: Signaling networks coordinating T cell migration and antigen recognition
Dustin, ML
2001 NOV ;12(1):394A-394A, Molecular biology of the cell
— id: 55364, year: 2001, vol: 12, page: 394A, stat: Journal Article,

Cytoskeletal polarization and redistribution of cell-surface molecules during T cell antigen recognition
Anton van der Merwe P; Davis SJ; Shaw AS; Dustin ML
2000 Feb;12(1):5-21, Seminars in immunology
T cell antigen recognition is accompanied by cytoskeletal polarization towards the APC and large-scale redistribution of cell surface molecules into 'supramolecular activation clusters' (SMACs), forming an organized contact interface termed the 'immunological synapse' (IS). Molecules are arranged in the IS in a micrometer scale bull's eye pattern with a central accumulation of TCR/peptide-MHC (the cSMAC) surrounded by a peripheral ring of adhesion molecules (the pSMAC). We propose that segregation of cell surface molecules on a much smaller scale initiates TCR triggering, which drives the formation of the IS by active transport processes. IS formation may function as a checkpoint for full T cell activation, integrating information on the presence and quality of TCR ligands and the nature and activation state of the APC
— id: 20361, year: 2000, vol: 12, page: 5, stat: Journal Article,

Cutting edge: hierarchy of chemokine receptor and TCR signals regulating T cell migration and proliferation
Bromley SK; Peterson DA; Gunn MD; Dustin ML
2000 Jul 1;165(1):15-19, Journal of immunology
Chemokines play an important role in establishing the distribution of lymphocyte subpopulations in primary and secondary lymphoid tissues and in the recruitment of leukocytes to sites of inflammation. However, the potential of chemokines to down-regulate immune responses has not been demonstrated. We now show that certain chemokine gradients have the potential to suppress T cell activation by preventing formation of the immunological synapse, the specialized cell-cell junction that forms before a T cell can be fully activated. Our data reveals an immunosuppressive potential of chemokines engaging the CXCR3 and CCR7 receptors, but not the CXCR4, CCR2, CCR4, or CCR5 receptors. These results suggest a novel mechanism for T cell ignorance of agonist MHC-peptide complexes based on dominant chemokine gradients
— id: 20360, year: 2000, vol: 165, page: 15, stat: Journal Article,

Signaling takes shape in the immune system
Dustin ML; Chan AC
2000 Oct 13;103(2):283-294, Cell
— id: 20358, year: 2000, vol: 103, page: 283, stat: Journal Article,

The immunological synapse and the actin cytoskeleton: molecular hardware for T cell signaling
Dustin ML; Cooper JA
2000 Jul;1(1):23-29, Nature immunology
The actin cytoskeleton seems to play two critical roles in the activation of T cells. One of these roles is T cell shape development and movement, including formation of the immunological synapse. The other is the formation of a scaffold for signaling components. This review focuses on the recent convergence of cell biology and immunology studies to explain the role of the actin cytoskeleton in creating the molecular basis for immunological synapse formation and T cell signaling
— id: 20355, year: 2000, vol: 1, page: 23, stat: Journal Article,

A supramolecular basis for CD45 tyrosine phosphatase regulation in sustained T cell activation
Johnson KG; Bromley SK; Dustin ML; Thomas ML
2000 Aug 29;97(18):10138-10143, Proceedings of the National Academy of Sciences of the United States of America
Transmembrane protein tyrosine phosphatases, such as CD45, can act as both positive and negative regulators of cellular signaling. CD45 positively modulates T cell receptor (TCR) signaling by constitutively priming p56lck through the dephosphorylation of the C-terminal negative regulatory phosphotyrosine site. However, CD45 can also exert negative effects on cellular processes, including events triggered by integrin-mediated adhesion. To better understand these opposing actions of tyrosine phosphatases, the subcellular compartmentalization of CD45 was imaged by using laser scanning confocal microscopy during functional TCR signaling of live T lymphocytes. On antigen engagement, CD45 was first excluded from the central region of the interface between the T cell and the antigen-presenting surface where CD45 would inhibit integrin activation. Subsequently, CD45 was recruited back to the center of the contact to an area adjacent to the site of sustained TCR engagement. Thus, CD45 is well positioned within a supramolecular assembly in the vicinity of the engaged TCR, where CD45 would be able to maintain src-kinase activity for the duration of TCR engagement
— id: 20359, year: 2000, vol: 97, page: 10138, stat: Journal Article,

Visualizing T-cell recognition
Davis MM; Wulfing C; Krummel MF; Savage PA; Xu J; Sumen C; Dustin ML; Chien YH
1999 ;64(1):243-251, Cold Spring Harbor symposia on quantitative biology
— id: 20357, year: 1999, vol: 64, page: 243, stat: Journal Article,

Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration
Ding ZM; Babensee JE; Simon SI; Lu H; Perrard JL; Bullard DC; Dai XY; Bromley SK; Dustin ML; Entman ML; Smith CW; Ballantyne CM
1999 Nov 1;163(9):5029-5038, Journal of immunology
To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-alpha was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-alpha in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased
— id: 20362, year: 1999, vol: 163, page: 5029, stat: Journal Article,

Costimulation: building an immunological synapse
Dustin ML; Shaw AS
1999 Jan 29;283(5402):649-650, Science
— id: 20365, year: 1999, vol: 283, page: 649, stat: Journal Article,

The immunological synapse: a molecular machine controlling T cell activation
Grakoui A; Bromley SK; Sumen C; Davis MM; Shaw AS; Allen PM; Dustin ML
1999 Jul 9;285(5425):221-227, Science
The specialized junction between a T lymphocyte and an antigen-presenting cell, the immunological synapse, consists of a central cluster of T cell receptors surrounded by a ring of adhesion molecules. Immunological synapse formation is now shown to be an active and dynamic mechanism that allows T cells to distinguish potential antigenic ligands. Initially, T cell receptor ligands were engaged in an outermost ring of the nascent synapse. Transport of these complexes into the central cluster was dependent on T cell receptor-ligand interaction kinetics. Finally, formation of a stable central cluster at the heart of the synapse was a determinative event for T cell proliferation
— id: 20364, year: 1999, vol: 285, page: 221, stat: Journal Article,

Congenital nephrotic syndrome in mice lacking CD2-associated protein
Shih NY; Li J; Karpitskii V; Nguyen A; Dustin ML; Kanagawa O; Miner JH; Shaw AS
1999 Oct 8;286(5438):312-315, Science
CD2-associated protein (CD2AP) is an 80-kilodalton protein that is critical for stabilizing contacts between T cells and antigen-presenting cells. In CD2AP-deficient mice, immune function was compromised, but the mice died at 6 to 7 weeks of age from renal failure. In the kidney, CD2AP was expressed primarily in glomerular epithelial cells. Knockout mice exhibited defects in epithelial cell foot processes, accompanied by mesangial cell hyperplasia and extracellular matrix deposition. Supporting a role for CD2AP in the specialized cell junction known as the slit diaphragm, CD2AP associated with nephrin, the primary component of the slit diaphragm
— id: 20363, year: 1999, vol: 286, page: 312, stat: Journal Article,

Making a little affinity go a long way: a topological view of LFA-1 regulation
Dustin ML
1998 ;6(2-3):255-262, Cell adhesion & communications
Lymphocytes utilize adhesion to navigate in the body and to transiently interact with a variety of potential antigen presenting cells. Interactions of adhesion molecules are governed by the law of mass action and the less understood rules of apposed biological membranes. Biochemical parameters such as adhesion molecule affinity only tell part of the story. Factors such as lateral mobility, membrane alignment and cytoskeletal interactions are equally important in determining the final outcome. Therefore it is important to determine mechanisms by which the properties of cell membranes and the cytoskeleton reinforce or hinder adhesion molecule interactions. Work from my lab has shown that one mechanism by which lymphocyte adhesion molecules cooperate is to align adhering membranes with nanometer precision. Here, I discuss a model for LFA-1 regulation that is dependent on three independent processes: LFA-1 lateral mobility, ligand induced generation of a small amount of high affinity LFA-1 and local membrane alignment. I propose that coordination of these processes allows rapid interconversion between stable adhesion and detachment
— id: 20366, year: 1998, vol: 6, page: 255, stat: Journal Article,

A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts
Dustin ML; Olszowy MW; Holdorf AD; Li J; Bromley S; Desai N; Widder P; Rosenberger F; van der Merwe PA; Allen PM; Shaw AS
1998 Sep 4;94(5):667-677, Cell
Recognition of antigen by T cells requires the formation of a specialized junction between the T cell and the antigen-presenting cell. This junction is generated by the recruitment and the exclusion of specific proteins from the contact area. The mechanisms that regulate these events are unknown. Here we demonstrate that ligand engagement of the adhesion molecule, CD2, initiates a process of protein segregation, CD2 clustering, and cytoskeletal polarization. Although protein segregation was not dependent on the cytoplasmic domain of CD2, CD2 clustering and cytoskeletal polarization required an interaction of the CD2 cytoplasmic domain with a novel SH3-containing protein. This novel protein, called CD2AP, is likely to facilitate receptor patterning in the contact area by linking specific adhesion receptors to the cytoskeleton
— id: 20367, year: 1998, vol: 94, page: 667, stat: Journal Article,

Survival of FimH-expressing enterobacteria in macrophages relies on glycolipid traffic
Baorto DM; Gao Z; Malaviya R; Dustin ML; van der Merwe A; Lublin DM; Abraham SN
1997 Oct 9;389(6651):636-639, Nature
Strains of Escherichia coli persist within the human gut as normal commensals, but are frequent pathogens and can cause recurrent infection. Here we show that, in contrast to E. coli subjected to opsonic interactions stimulated by the host's immune response, E. coli that bind to the macrophage surface exclusively through the bacterial lectin FimH can survive inside the cell following phagocytosis. This viability is largely due to the attenuation of intracellular free-radical release and of phagosome acidification during FimH-mediated internalization, both of which are triggered by antibody-mediated internalization. This different processing of non-opsonized bacteria is supported by morphological evidence of tight-fitting phagosomes compared with looser, antibody-mediated phagosomes. We propose that non-opsonized FimH-expressing E. coli co-opt internalization of lipid-rich microdomains following binding to the FimH receptor, the glycosylphosphatidylinositol-linked protein CD48, because (1) the sterol-binding agents filipin, nystatin and methyl beta-cyclodextrin specifically block FimH-mediated internalization; (2) CD48 and the protein caveolin both accumulate on macrophage membranes surrounding bacteria; and (3) antibodies against CD48 inhibit FimH-mediated internalization. Our findings bring the traditionally extracellular E. coli into the realm of opportunistic intracellular parasitism and suggest how opportunistic infections with FimH-expressing enterobacteria could occur in a setting deprived of opsonizing antibodies
— id: 20369, year: 1997, vol: 389, page: 636, stat: Journal Article,

Adhesive bond dynamics in contacts between T lymphocytes and glass-supported planar bilayers reconstituted with the immunoglobulin-related adhesion molecule CD58
Dustin ML
1997 Jun 20;272(25):15782-15788, Journal of biological chemistry
The interaction of the T cell glycoprotein CD2 and its ligand CD58 is important for T cell interaction with antigen-presenting and target cells. The binding interaction is of low affinity and has a fast off-rate (>5 s-1) in solution. However, solution measurements may not accurately predict the behavior of molecules in an adhesive contact area. Interaction between T cells that express CD2 and glass-supported planar bilayers containing purified and fluorescently labeled CD58 leads to accumulation of CD58 (fluorescence) in the cell/bilayer contact area. CD58 molecules accumulated within the contact area in excess of the CD58 density in the bilayer outside the contact area can be considered as bound by cell surface CD2. Here, this phenomena and fluorescence photobleaching recovery were utilized to determine whether CD2-CD58 bonds are transient in contact areas. Fluorescent CD58 molecules accumulated in the T cell-bilayer interface were completely bleached. The bleached CD58 molecules accumulated in the contact area were rapidly replaced by fluorescent CD58 that diffused into the contact area from adjacent bilayer regions outside the contact area. Rapid recovery of the accumulated fluorescence directly demonstrates that the CD2-CD58 bonds are dissociating and that the dissociation leads to partner exchange, rather than rebinding of the same CD2-CD58 pairs. This suggests that the solution off-rate provides an accurate description of CD2-CD58 interaction in contact areas. Accumulated fluorescent IgG in contacts between K562 cells expressing low affinity Fc receptors and planar bilayers with fluorescent IgG bound to hapten-derivitized phospholipids displayed slower recovery than CD58 by a factor of 10. This suggests that the Fc receptor-IgG interaction has a longer lifetime than the CD2-CD58 interaction. These findings have implications for the mechanism of signaling by CD2 and the mechanism of cell detachment from large numbers of transient interactions
— id: 20372, year: 1997, vol: 272, page: 15782, stat: Journal Article,

Antigen receptor engagement delivers a stop signal to migrating T lymphocytes
Dustin ML; Bromley SK; Kan Z; Peterson DA; Unanue ER
1997 Apr 15;94(8):3909-3913, Proceedings of the National Academy of Sciences of the United States of America
We investigated the role of the T cell antigen receptor (TcR) in control of T cell migration in an in vitro system. We used T cells from transgenic mice bearing a TcR for the lysozyme peptide 48-62 bound to I-A(k) (3A9). T cells from the 3A9 TcR transgenic mice crawled on purified intercellular adhesion molecule-1 substrates, but strikingly, stopped upon interaction with the physiological ligand, i.e., the mouse I-A(k) with covalently attached hen egg white lysozyme peptide residues 48-62 complex. TcR-triggered stopping was reversible by treatment with adhesion-strengthening phorbol esters. The microtubule organizing center of stopped cells was positioned adjacent to the site of stable cell anchorage. Direct conversion of lymphocyte function associated-1 to the high-affinity conformation with antibodies also stopped T cells in a similar manner to antigen. Thus, physiological TcR engagement triggers a stop signal through lymphocyte function associated-1. We propose that the stop signal is an early and essential event in T cell activation that also will play an important role in control of T cell migration
— id: 20373, year: 1997, vol: 94, page: 3909, stat: Journal Article,

Low affinity interaction of human or rat T cell adhesion molecule CD2 with its ligand aligns adhering membranes to achieve high physiological affinity
Dustin ML; Golan DE; Zhu DM; Miller JM; Meier W; Davies EA; van der Merwe PA
1997 Dec 5;272(49):30889-30898, Journal of biological chemistry
The mechanism by which low affinity adhesion molecules function to produce stable cell-cell adhesion is unknown. In solution, the interaction of human CD2 with its ligand CD58 is of low affinity (500 mM-1) and the interaction of rat CD2 with its ligand CD48 is of still lower affinity (40 mM-1). At the molecular level, however, the two systems are likely to be topologically identical. Fluorescently labeled glycosylphosphatidylinositol-anchored CD48 and CD58 were prepared and incorporated into supported phospholipid bilayers, in which the ligands were capable of free lateral diffusion. Quantitative fluorescence imaging was used to study the binding of cell surface human and rat CD2 molecules to the fluorescent ligands in contact areas between Jurkat cells and the bilayers. These studies provide two major conclusions. First, CD2/ligand interactions cooperate to align membranes with nanometer precision leading to a physiologically effective two-dimensional affinity. This process does not require the intact cytoplasmic tail of CD2. Second, the degree of membrane alignment that can be achieved by topologically similar receptors deteriorates with decreasing affinity. This suggests an affinity limit for the ability of this mode of cooperativity to achieve stable cell-cell adhesion at approximately 10 mM-1
— id: 20368, year: 1997, vol: 272, page: 30889, stat: Journal Article,

Low affinity of cell surface lymphocyte function-associated antigen-1 (LFA-1) generates selectivity for cell-cell interactions
Ganpule G; Knorr R; Miller JM; Carron CP; Dustin ML
1997 Sep 15;159(6):2685-2692, Journal of immunology
We examined binding of soluble intercellular adhesion molecule-1 (ICAM-1) dimers and a range of ICAM-1-coated particles to activated T cells. Lymphocyte function-associated antigen-1 (LFA-1) on the surface of activated T cells did not bind soluble ICAM-1 dimers with high affinity. In contrast, activated T cells adhered avidly to ICAM-1-coated planar surfaces. Between these two extremes, a range of ICAM-1-bearing particles was tested for binding. Activated T cells bound particles of 1-microm diameter or larger, but did not bind particles of 0.5-microm diameter or smaller. This threshold was eliminated, and all forms of ICAM-1 bound to LFA-1 when LFA-1 was converted to a high affinity form with an activating antibody. We show that high affinity LFA-1 is generated only as a consequence of an initial low affinity interaction of LFA-1 with ICAM-1 under physiological conditions. Therefore, the selectivity of cell surface LFA-1 for cell-sized particles bearing ICAM-1 appears to be due to the maintenance of low affinity LFA-1 on the surface of activated T cells. These findings alter the definition of inside-out signaling for LFA-1
— id: 20370, year: 1997, vol: 159, page: 2685, stat: Journal Article,

The lymphocyte function-associated antigen 1 I domain is a transient binding module for intercellular adhesion molecule (ICAM)-1 and ICAM-3 in hydrodynamic flow
Knorr R; Dustin ML
1997 Aug 29;186(5):719-730, Journal of experimental medicine
The I domain of lymphocyte function-associated antigen (LFA)-1 contains an intercellular adhesion molecule (ICAM)-1 and ICAM-3 binding site, but the relationship of this site to regulated adhesion is unknown. To study the adhesive properties of the LFA-1 I domain, we stably expressed a GPI-anchored form of this I domain (I-GPI) on the surface of baby hamster kidney cells. I-GPI cells bound soluble ICAM-1 (sICAM-1) with a low avidity and affinity. Flow cell experiments demonstrated a specific rolling interaction of I-GPI cells on bilayers containing purified full length ICAM-1 or ICAM-3. The LFA-1 activating antibody MEM-83, or its Fab fragment, decreased the rolling velocity of I-GPI cells on ICAM-1-containing membranes. In contrast, the interaction of I-GPI cells with ICAM-3 was blocked by MEM-83. Rolling of I-GPI cells was dependent on the presence of Mg2+. Mn2+ only partially substituted for Mg2+, giving rise to a small fraction of rolling cells and increased rolling velocity. This suggests that the I domain acts as a transient, Mg2+-dependent binding module that cooperates with another Mn2+-stimulated site in LFA-1 to give rise to the stable interaction of intact LFA-1 with ICAM-1
— id: 20371, year: 1997, vol: 186, page: 719, stat: Journal Article,

Making the T cell receptor go the distance: a topological view of T cell activation
Shaw AS; Dustin ML
1997 Apr;6(4):361-369, Immunity
— id: 20374, year: 1997, vol: 6, page: 361, stat: Journal Article,

Visualization of CD2 interaction with LFA-3 and determination of the two-dimensional dissociation constant for adhesion receptors in a contact area
Dustin ML; Ferguson LM; Chan PY; Springer TA; Golan DE
1996 Feb;132(3):465-474, Journal of cell biology
Many adhesion receptors have high three-dimensional dissociation constants (Kd) for counter-receptors compared to the KdS of receptors for soluble extracellular ligands such as cytokines and hormones. Interaction of the T lymphocyte adhesion receptor CD2 with its counter-receptor, LFA-3, has a high solution-phase Kd (16 microM at 37 degrees C), yet the CD2/LFA-3 interaction serves as an effective adhesion mechanism. We have studied the interaction of CD2 with LFA-3 in the contact area between Jurkat T lymphoblasts and planar phospholipid bilayers containing purified, fluorescently labeled LFA-3. Redistribution and lateral mobility of LFA-3 were measured in contact areas as functions of the initial LFA-3 surface density and of time after contact of the cells with the bilayers. LFA-3 accumulated at sites of contact with a half-time of approximately 15 min, consistent with the previously determined kinetics of adhesion strengthening. The two-dimensional Kd for the CD2/LFA-3 interaction was 21 molecules/microns 2, which is lower than the surface densities of CD2 on T cells and LFA-3 on most target or stimulator cells. Thus, formation of CD2/LFA-3 complexes should be highly favored in physiological interactions. Comparison of the two-dimensional (membrane-bound) and three-dimensional (solution-phase) KdS suggest that cell-cell contact favors CD2/LFA-3 interaction to a greater extent than that predicted by the three-dimensional Kd and the intermembrane distance at the site of contact. LFA-3 molecules in the contact site were capable of lateral diffusion in the plane of the phospholipid bilayer and did not appear to be irreversibly trapped in the contact area, consistent with a rapid off-rate. These data provide insights into the function of low affinity interactions in adhesion
— id: 20378, year: 1996, vol: 132, page: 465, stat: Journal Article,

A mannose 6-phosphate-containing N-linked glycopeptide derived from lysosomal acid lipase is bound to MHC class II in B lymphoblastoid cell lines
Dustin ML; McCourt DW; Kornfeld S
1996 Mar 1;156(5):1841-1847, Journal of immunology
Binding of peptides to MHC class II Ag generates ligands for TCR of Th lymphocytes. We have identified a novel class of peptides bound to MHC class II: mannose 6- phosphate (Man-6-P) containing glycopeptides from lysosomal enzymes. These species were identified in the process of characterizing mannose 6-phosphate/insulin-like growth factor II (M-6-P/IGF-II) receptor binding to the surface of B lymphoblasts. Surface iodination and Man-6-P/IGF-II receptor affinity chromatography implicated MHC class II as a carrier of Man-6-P-modified oligosaccharides. These oligosaccharides were found to be primarily associated with the bound peptide. Peptides eluted from the Man-6-P/IGF-II receptor-binding fraction of immunoaffinity-purified MHC class II from the Swei cell line contained a sequence derived from the propiece of lysosomal acid lipase. Partial sequences were also obtained for peptides from other HLA-DR alleles but none of these were attributable to known proteins. This study defines a novel approach for isolating rare glycan-modified peptides from MHC class II and demonstrates that very large secondary modifications are tolerated in peptides bound to MHC class II
— id: 20377, year: 1996, vol: 156, page: 1841, stat: Journal Article,

TCR-mediated adhesion of T cell hybridomas to planar bilayers containing purified MHC class II/peptide complexes and receptor shedding during detachment
Dustin ML; Miller JM; Ranganath S; Vignali DA; Viner NJ; Nelson CA; Unanue ER
1996 Sep 1;157(5):2014-2021, Journal of immunology
T cell recognition of foreign Ag/MHC class II complexes is sensitive down to approximately 100 complexes per cell or approximately 0.2 complexes/micron2. To better understand the physical basis of the recognition stage of Ag presentation, we examined adhesion of the lysozyme- specific T cell hybridoma, 3A9, to artificial bilayers containing covalent MHC class II/peptide complexes or adhesion molecules. Adhesion of 3A9 cells required a superphysiologic density of the MHC class II/peptide complex and was partly dependent on CD4; cells adhered but did not crawl. No adhesion was observed to bilayers containing MHC class II molecules without the lysozyme peptide. Activated 3A9 cells adhered and crawled on bilayers containing ICAM-1. The physical strength of contacts was tested with fluid shear. 3A9 cells adherent to bilayers containing MHC class II/peptide complexes shed their contact, which remained on the substrate and contained TCR. In contrast, 3A9 cells peeled from the ICAM-1 bilayer, and held firmly on LFA-1 bilayers; in a manner dependent on filamentous actin. When ICAM-1 and the MHC/peptide complexes were combined, the 3A9 cells adhered tightly and spread, but did not crawl, on the bilayers and TCR clustered at the center of the contact area. Physiologically, the TCR is unlikely to directly initiate adhesion. TCR clusters formed with the assistance of adhesion mechanisms may have to be shed to allow de-adhesion, and this may contribute to TCR down-regulation
— id: 20375, year: 1996, vol: 157, page: 2014, stat: Journal Article,

Adhesion-activating phorbol ester increases the mobility of leukocyte integrin LFA-1 in cultured lymphocytes
Kucik DF; Dustin ML; Miller JM; Brown EJ
1996 May 1;97(9):2139-2144, Journal of clinical investigation
Lymphocytes activate adhesion to intracellular adhesion mlecule 1 (ICAM-1) via leukocyte function associated antigen 1 (LFA-1), their major beta 2 integrin, in response to PMA (phorbol 12-myristate 13-acetate) without an increase in the number of receptors expressed. The molecular details of the mechanism are unknown. To determine the effect of PMA activation on LFA-1 movement within the plasma membrane, we used the single particle tracking technique to measure the diffusion rate of LFA-1 molecules on EBV-transformed B cells before and after PMA activation. Diffusion of LFA-1 on unactivated cells was restricted compared to CR1 (CD35), another transmembrane protein of equivalent size. PMA caused a 10-fold increase in the diffusion rate of LFA-1 without any effect on CD35. The increased LFA-1 motion induced by PMA was random, not directed, indicating that it was due to a release of constraints rather than the application of forces. The diffusion rates of LFA-1 are consistent with cytoskeletal attachment before and free diffusion after PMA. Cytochalasin D led to an equivalent increase in mobility and, at low doses, stimulated adhesion, implying that the nonadhesive state of LFA-1 is actively maintained by the lymphocyte cytoskeleton
— id: 20376, year: 1996, vol: 97, page: 2139, stat: Journal Article,

A novel mutagenesis strategy identifies distantly spaced amino acid sequences that are required for the phosphorylation of both the oligosaccharides of procathepsin D by N-acetylglucosamine 1-phosphotransferase
Dustin ML; Baranski TJ; Sampath D; Kornfeld S
1995 Jan 6;270(1):170-179, Journal of biological chemistry
A novel combinatorial mutagenesis strategy (shuffle mutagenesis) was developed to identify sequences in the propiece and amino lobe of cathepsin D which direct oligosaccharide phosphorylation by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. Propiece restriction fragments and oligonucleotide cassettes corresponding to 13 regions of the cathepsin D and glycopepsinogen amino lobes were randomly shuffled together to generate a large library of chimeric molecules. The library was inserted into an expression vector encoding the carboxyl lobe of cathepsin D with a carboxyl-terminal myc epitope and a CD8 transmembrane extension. Transfected COS1 cells expressing the membrane-anchored forms of the cathepsin D/glycopepsinogen chimeras at the cell surface were selected with solid phase mannose 6-phosphate receptor or an antibody to the myc epitope. Plasmids were rescued in Escherichia coli and sequenced by hybridization to the original oligonucleotide cassettes. Two regions of the cathepsin D amino lobe (segments 7 and 12) were found to contribute to proper folding, surface expression, and selective phosphorylation of the carboxyl lobe oligosaccharide. Two different cathepsin D regions (the propiece and segment 5) cooperated with a previously identified recognition element in the carboxyl lobe to allow efficient phosphorylation of both the amino and carboxyl lobe oligosaccharides. Three general models for extending the catalytic reach of N-acetylglucosamine 1-phosphotransferase to widely spaced oligosaccharides are presented
— id: 20381, year: 1995, vol: 270, page: 170, stat: Journal Article,

Characterization of intercellular adhesion molecule-1 ectodomain (sICAM-1) as an inhibitor of lymphocyte function-associated molecule-1 interaction with ICAM-1
Meyer DM; Dustin ML; Carron CP
1995 Oct 1;155(7):3578-3584, Journal of immunology
Intercellular adhesion molecule-1 (ICAM-1) is a member of the Ig superfamily, contains five Ig-like domains comprising the extracellular portion of the molecule, and interacts with lymphocyte function-associated molecule-1 (LFA-1), a member of the beta 2-integrin family. LFA-1/ICAM-1 interaction is important in a variety of cellular events including Ag-specific T cell activation and leukocyte transendothelial migration. Recently, a soluble circulating form of ICAM-1 has been detected in human serum that appears to result from the proteolytic cleavage of membrane ICAM-1. Native and recombinant soluble forms of ICAM-1 have been reported to inhibit LFA-1/ICAM-mediated adhesion in vitro, and it is conceivable that circulating forms of soluble ICAM-1 are regulators of LFA-1/ICAM-1-mediated cell-cell interaction in vivo. We have investigated the properties of the ICAM-1 ectodomain (sICAM453) as an inhibitor of LFA-1 interaction with ICAM-1 in cell- and molecule-based systems. The results show clearly that recombinant sICAM453 can inhibit LFA-1/ICAM-1 interaction. Soluble ICAM-1 inhibited LFA-1-mediated cell adhesion to immobilized sICAM453 and homotypic T-cell aggregation with IC50 in the 20 to 40 microM range. Definitive evidence that sICAM-1 can inhibit LFA-1 interaction with ICAM-1 was obtained by showing that the sICAM-1 inhibited the interaction between LFA-1 protein micelles and ICAM-1 immobilized on plastic. These results clearly show that sICAM453 can bind to LFA-1 and competitively inhibit ICAM-1/LFA-1-mediated cell-cell interaction, albeit at concentrations much greater than found in plasma. As a consequence, it is unlikely that sICAM-1 would antagonize ICAM-1/LFA-1-mediated cellular events in vivo
— id: 20380, year: 1995, vol: 155, page: 3578, stat: Journal Article,

Intercellular adhesion molecule-1 dimerization and its consequences for adhesion mediated by lymphocyte function associated-1
Miller J; Knorr R; Ferrone M; Houdei R; Carron CP; Dustin ML
1995 Nov 1;182(5):1231-1241, Journal of experimental medicine
Intercellular adhesion molecule-1 (ICAM-1, CD54) is a ligand for the integrins lymphocyte function associated-1 (LFA-1, CD11a/CD18) and complement receptor-3 (Mac-1, CD11b/CD18) making it an important participant in many immune and inflammatory processes. Modified recombinant soluble ICAM-1 formed dimers. This result indicated that the ectodomain of ICAM-1 contains homophilic interaction sites. Soluble ICAM-1 dimers bind to solid-phase purified LFA-1 with high avidity (dissociation constant [Kd] = 8 nM) in contrast to soluble ICAM-1 monomers whose binding was not measurable. Cell surface ICAM-1 was found to be dimeric based on two distinct criteria. First, a monoclonal antibody specific for monomeric soluble ICAM-1, CA7, binds normal ICAM-1 poorly at the cell surface; this antibody, however, binds strongly to two mutant forms of ICAM-1 when expressed at the cell surface, thus identifying elements required for dimer formation. Second, chemical cross-linking of cell surface ICAM-1 on transfected cells and tumor necrosis factor-activated endothelial cells results in conversion of a portion of ICAM-1 to a covalent dimer. Cell surface ICAM-1 dimers are more potent ligands for LFA-1-dependent adhesion than ICAM-1 monomers. While many extracellular matrix-associated ligands of integrins are multimeric, this is the first evidence of specific, functionally important homodimerization of a cell surface integrin ligand
— id: 20379, year: 1995, vol: 182, page: 1231, stat: Journal Article,

Regulation of locomotion and cell-cell contact area by the LFA-1 and ICAM-1 adhesion receptors
Dustin ML; Carpen O; Springer TA
1992 May 1;148(9):2654-2663, Journal of immunology
We demonstrate complementary differences in the behavior of B lymphoblastoid cells adhering to LFA-1 or its counter-receptor ICAM-1. The interaction of B lymphoblastoid cells with glass-supported planar bilayers bearing LFA-1 or ICAM-1 was observed by time-lapse video microscopy, and the distribution of adhesion receptors on cells interacting with the planar bilayers was studied by immunofluorescence microscopy. B lymphoblasts formed a large contact area and crawled rapidly (up to 25 microns/min) on planar bilayers bearing ICAM-1. In contrast, these cells attached to planar bilayers bearing LFA-1 through a fixed point about which the cells actively pivoted, using a single stalk-like projection. Phorbol ester-stimulated lymphoblasts, which adhere more strongly to ICAM-1-bearing substrates than unstimulated lymphoblasts, were still capable of locomotion on ICAM-1. Phorbol ester stimulation of B lymphoblasts on planar bilayers bearing LFA-1 promoted a rapid conversion from 'stalk' attachment to symmetrical spreading of the cell on the substrate. Cellular LFA-1 remained uniformly distributed on the cell surface during interaction with bilayers bearing purified ICAM-1 as determined by immunofluorescence. In contrast, ICAM-1 was concentrated in the stalk-like structure through which the unstimulated B lymphoblasts adhered to LFA-1 in planar bilayers, but ICAM-1 immunofluorescence became more uniformly distributed over the cell surface within minutes of phorbol ester addition. Neither LFA-1 or ICAM-1 colocalized with the prominent staining of filamentous actin in the ruffling membrane regions. Interaction through cell surface LFA-1 and ICAM-1, 2, or 3 promotes different cellular morphologies and behaviors, the correlation of which with previously observed patterns of lymphocyte interaction with different cell types is discussed
— id: 20383, year: 1992, vol: 148, page: 2654, stat: Journal Article,

Micromanipulation of adhesion of phorbol 12-myristate-13-acetate-stimulated T lymphocytes to planar membranes containing intercellular adhesion molecule-1
Tozeren A; Mackie LH; Lawrence MB; Chan PY; Dustin ML; Springer TA
1992 Jul;63(1):247-258, Biophysical journal
This paper presents an analytical and experimental methodology to determine the physical strength of cell adhesion to a planar membrane containing one set of adhesion molecules. In particular, the T lymphocyte adhesion due to the interaction of the lymphocyte function associated molecule 1 on the surface of the cell, with its counter-receptor, intercellular adhesion molecule-1 (ICAM-1), on the planar membrane, was investigated. A micromanipulation method and mathematical analysis of cell deformation were used to determine (a) the area of conjugation between the cell and the substrate and (b) the energy that must be supplied to detach a unit area of the cell membrane from its substrate. T lymphocytes stimulated with phorbol 12-myristate-13-acetate (PMA) conjugated strongly with the planar membrane containing purified ICAM-1. The T lymphocytes attached to the planar membrane deviated occasionally from their round configuration by extending pseudopods but without changing the size of the contact area. These adherent cells were dramatically deformed and then detached when pulled away from the planar membrane by a micropipette. Detachment occurred by a gradual decrease in the radius of the contact area. The physical strength of adhesion between a PMA-stimulated T lymphocyte and a planar membrane containing 1,000 ICAM-1 molecules/micron 2 was comparable to the strength of adhesion between a cytotoxic T cell and its target cell. The comparison of the adhesive energy density, measured at constant cell shape, with the model predictions suggests that the physical strength of cell adhesion may increase significantly when the adhesion bonds in the contact area are immobilized by the actin cytoskeleton
— id: 20382, year: 1992, vol: 63, page: 247, stat: Journal Article,

Micromanipulation of adhesion of a Jurkat cell to a planar bilayer membrane containing lymphocyte function-associated antigen 3 molecules
Tozeren A; Sung KL; Sung LA; Dustin ML; Chan PY; Springer TA; Chien S
1992 Feb;116(4):997-1006, Journal of cell biology
Cell adhesion plays a fundamental role in the organization of cells in differentiated organs, cell motility, and immune response. A novel micromanipulation method is employed to quantify the direct contribution of surface adhesion receptors to the physical strength of cell adhesion. In this technique, a cell is brought into contact with a glass-supported planar membrane reconstituted with a known concentration of a given type of adhesion molecules. After a period of incubation (5-10 min), the cell is detached from the planar bilayer by pulling away the pipette holding the cell in the direction perpendicular to the glass-supported planar bilayer. In particular, we investigated the adhesion between a Jurkat cell expressing CD2 and a glass-supported planar bilayer containing either the glycosyl-phosphatidylinositol (GPI) or the transmembrane (TM) isoform of the counter-receptor lymphocyte function-associated antigen 3 (LFA-3) at a concentration of 1,000 molecules/microns 2. In response to the pipette force the Jurkat cells that adhered to the planar bilayer containing the GPI isoform of LFA-3 underwent extensive elongation. When the contact radius was reduced by approximately 50%, the cell then detached quickly from its substrate. The aspiration pressure required to detach a Jurkat cell from its substrate was comparable to that required to detach a cytotoxic T cell from its target cell. Jurkat cells that had been separated from the substrate again adhered strongly to the planar bilayer when brought to proximity by micromanipulation. In experiments using the planar bilayer containing the TM isoform of LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape
— id: 20384, year: 1992, vol: 116, page: 997, stat: Journal Article,

Motility and ultrastructure of large granular lymphocytes on lipid bilayers reconstituted with adhesion receptors LFA-1, ICAM-1, and two isoforms of LFA-3
Carpen O; Dustin ML; Springer TA; Swafford JA; Beckett LA; Caulfield JP
1991 Nov;115(3):861-871, Journal of cell biology
Large granular lymphocytes, mediators of NK activity, bind to other cells using both the LFA (lymphocyte function-associated)-1-ICAM and the CD2-LFA-3 adhesion pathways. Here we have studied the motility and ultrastructure of large granule lymphocyte (LGL) on lipid bilayers containing purified LFA-1, ICAM-1, and the transmembrane and glycophosphatidylinositol isoforms of LFA-3. LGLs moved at 8 microns/min on ICAM-1 but poorly (less than 1 microns/min) on its receptor pair LFA-1. TM-LFA-3 promoted locomotion at a rate close to ICAM-1, whereas the cells were less motile on GPI-LFA-3. The difference in the rates of locomotion on the two isoforms of LFA-3 is presumably attributable to their difference in anchoring and lateral mobility in the bilayer. In spite of the variation in motility the ultrastructure of the adhering cells was similar on all four ligands. LGLs contacted the membrane variably, i.e., cells adhering only in a few small areas or in larger areas were detected on each ligand. The relative percentage of the plasma membrane facing the lipid bilayer was greatest on ICAM-1 and least on the transmembrane isoform of LFA-3, demonstrating no correlation with motility. The ratio of adjacent plasma membrane to lipid bilayer was virtually constant for all four ligands. Activation of the LGLs with a combination of CD2 mAb T11(2) and T11(3) (T11(2/3) mAb) reduced the movement on ICAM-1 and virtually immobilized the cells on the other bilayers. In the presence of T11(2/3) mAb, the area of cell membrane attaching to bilayers containing ICAM-1 and GPI-LFA-3 was decreased and the percentage of plasma membrane facing other cells was increased. No preferential orientation of the Golgi apparatus or degranulation was detected in the absence or presence of T11(2/3) mAb, but a significantly lower percentage of LGLs on ICAM-1 contained a profile of the Golgi apparatus after exposure to T11(2/3) mAb. The results demonstrate that the motility of LGLs depends on the type of receptor in the opposing bilayer, the receptor mobility in the bilayer, and the activation of the cells. The ultrastructure of LGLs binding to any of the adhesion molecules does not have the characteristics of LGLs in cytolytic contact with target cells, suggesting that the mediation of an attack on a target requires more complex stimulus than any one of the single adhesion proteins tested here
— id: 20385, year: 1991, vol: 115, page: 861, stat: Journal Article,

Influence of receptor lateral mobility on adhesion strengthening between membranes containing LFA-3 and CD2
Chan PY; Lawrence MB; Dustin ML; Ferguson LM; Golan DE; Springer TA
1991 Oct;115(1):245-255, Journal of cell biology
We have used an in vitro model system of glass-supported planar membranes to study the effects of lateral mobility of membrane-bound receptors on cell adhesion. Egg phosphatidylcholine (PC) bilayers were reconstituted with two anchorage isoforms of the adhesion molecule lymphocyte function-associated antigen 3 (LFA-3). The diffusion coefficient of glycosyl phosphatidylinositol (GPI)-anchored LFA-3 approached that of phospholipids in the bilayers, whereas the transmembrane (TM)-anchored isoform of LFA-3 was immobile. Both static and laminar flow assays were used to quantify the strength of adherence to the lipid bilayers of the T lymphoma cell line Jurkat that expresses the counter-receptor CD2. Cell adhesion was dependent on LFA-3 density and was more efficient on membranes containing the GPI isoform than the TM isoform. Kinetic measurements demonstrated an influence of contact time on the strength of adhesion to the GPI isoform at lower site densities (25-50 sites/microns2), showing that the mobility of LFA-3 is important in adhesion strengthening. At higher site densities (1,500 sites/microns2) and longer contact times (20 min), Jurkat cell binding to the TM and GPI isoforms of LFA-3 showed equivalent adhesion strengths, although adhesion strength of the GPI isoform developed twofold more rapidly than the TM isoform. Reduction of CD2 mobility on Jurkat cells at 5 degrees C greatly decreased the rate of adhesion strengthening with the TM isoform of LFA-3, resulting in a 30-fold difference between the two LFA-3 isoforms. Our results demonstrate that the ability of a membrane receptor and its membrane-bound counter-receptor to diffuse laterally enhances cell adhesion both by allowing accumulation of ligands in the cell contact area and by increasing the rate of receptor-ligand bond formation
— id: 20386, year: 1991, vol: 115, page: 245, stat: Journal Article,

Role of lymphocyte adhesion receptors in transient interactions and cell locomotion
Dustin ML; Springer TA
1991 ;9(1):27-66, Annual review of immunology
Lymphocytes adhere to other cells and extracellular matrix in the process of immunological recognition and lymphocyte recirculation. This review focuses on regulation of lymphocyte adhesion and the use of adhesion mechanisms by lymphocytes to obtain information about their immediate environment. The CD2 and LFA-1 adhesion receptors appear to have distinct roles in the regulation of adhesion and modulation of T lymphocyte activation. Adhesion mediated by interaction of CD2 with LFA-3 is dramatically altered by surface charge and adhesion receptor density in such a way that this pathway is latent in resting T lymphocytes but becomes active over a period of hours following T-cell activation. CD2 ligation can mediate or enhance T-cell activation, suggesting that signals from CD2/LFA-3 adhesive interactions are integrated with signals from the T-cell antigen receptor during immunological recognition. A model for the role of LFA-3 lateral diffusion in adhesion is presented, based on the lateral diffusion of different LFA-3 forms in glass supported planar membranes. Interaction of LFA-1 with ICAMs is also regulated by cell activation but in a different way than in interaction of CD2 with LFA-3. LFA-1 avidity for ICAMs is transiently increased by T-cell activation over a period of minutes. Cycles of avidity change are also observed for other T lymphocyte integrins which bind to extracellular matrix components. We propose that integrin avidity cycles may have an important role in the interconnected phenomena of locomotion, initial cell-cell adhesion, and cell-cell deadhesion. Recent observations on recirculation of T lymphocyte subpopulations are discussed in the context of general lessons learned from study of the CD2/LFA-3 and LFA-1/ICAM adhesion mechanisms
— id: 20387, year: 1991, vol: 9, page: 27, stat: Journal Article,

Two-way signalling through the LFA-1 lymphocyte adhesion receptor
Dustin ML
1990 Sep;12(9):421-427, Bioessays
T lymphocyte recognition of foreign antigens and migration throughout the body require the regulated adhesion of lymphocytes to diverse types of cells and to the extracellular matrix. The lymphocyte adhesion 'receptor' LFA-1, a member of the integrin family, interacts with ICAM-1 and other counter-receptors to mediate adhesion. The LFA-1/ICAM-1 interaction is regulated by signals transmitted from the cytoplasm to the extracellular space. Conversely, LFA-1 transmits signals from the extracellular space to the cytoplasm to regulate T lymphocyte activation. The observed properties of LFA-1 and related adhesion 'receptors' are incorporated into a general model for adhesion during immune surveillance and recognition of foreign antigens
— id: 20388, year: 1990, vol: 12, page: 421, stat: Journal Article,

On the species specificity of the interaction of LFA-1 with intercellular adhesion molecules
Johnston SC; Dustin ML; Hibbs ML; Springer TA
1990 Aug 15;145(4):1181-1187, Journal of immunology
Species restrictions in immune cell interactions have been demonstrated both in Ag-specific responses of T lymphocytes and the phenomenon of natural attachment. To determine the possible contribution of adhesion receptors to these restrictions, we have studied binding between the murine and human homologues of LFA-1 (CD11a/CD18) and ICAM employing purified human LFA-1 and ICAM-1 (CD54) bound to solid substrates. Murine cell lines bind to purified human LFA-1 through ICAM-1 and at least one other counter-receptor. This provides evidence for multiple counter-receptors for LFA-1 in the mouse as well as in the human. In contrast to binding of murine ICAM-1 to human LFA-1, murine LFA-1 does not bind to human ICAM-1. The species specificity maps to the LFA-1 alpha subunit, because mouse x human hybrid cells expressing the human alpha subunit associated with a mouse beta subunit bind to human ICAM-1, whereas those with a human beta subunit associated with a murine alpha subunit do not. Increased adhesiveness for ICAM-1 stimulated by phorbol esters could be demonstrated for hybrid LFA-1 molecules with human alpha and murine beta subunits
— id: 20389, year: 1990, vol: 145, page: 1181, stat: Journal Article,

The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus
Staunton DE; Dustin ML; Erickson HP; Springer TA
1990 Apr 20;61(2):243-254, Cell
Intercellular adhesion molecule 1 (ICAM-1, CD54) binds to the integrin LFA-1 (CD11a/CD18), promoting cell adhesion in immune and inflammatory reactions. ICAM-1 is also subverted as a receptor by the major group of rhinoviruses. Electron micrographs show that ICAM-1 is a bent rod, 18.7 nm long, suggesting a model in which the five immunoglobulin-like domains are oriented head to tail at a small angle to the rod axis. ICAM-1 sequences important to binding LFA-1, rhinovirus, and four monoclonal antibodies were identified through the characterization of chimeric ICAM-1 molecules and mutants. The amino-terminal two immunoglobulin-like domains of ICAM-1 appear to interact conformationally. Domain 1 of ICAM-1 contains the primary site of contact for both LFA-1 and rhinovirus; the presence of domains 3-5 markedly affects the accessibility of the binding site for rhinovirus and less so for LFA-1. The binding sites appear to be distinct but overlapping; rhinovirus binding also differs from LFA-1 binding in its lack of divalent cation dependence. Our analysis suggests that rhinoviruses mimic LFA-1 in binding to the most membrane-distal, and thus most accessible, site of ICAM-1
— id: 20390, year: 1990, vol: 61, page: 243, stat: Journal Article,

Structure and regulation of the leukocyte adhesion receptor LFA-1 and its counterreceptors, ICAM-1 and ICAM-2
Dustin ML; Garcia-Aguilar J; Hibbs ML; Larson RS; Stacker SA; Staunton DE; Wardlaw AJ; Springer TA
1989 ;54 Pt 2(2):753-765, Cold Spring Harbor symposia on quantitative biology
— id: 20395, year: 1989, vol: 54 Pt 2, page: 753, stat: Journal Article,

Correlation of CD2 binding and functional properties of multimeric and monomeric lymphocyte function-associated antigen 3
Dustin ML; Olive D; Springer TA
1989 Feb 1;169(2):503-517, Journal of experimental medicine
LFA-3 was purified with an intact (mLFA-3) or an enzymatically removed membrane-anchoring domain (sLFA-3). Gel filtration and sucrose gradient sedimentation showed sLFA-3 to be a single highly glycosylated polypeptide chain in solution, while mLFA-3 formed micelles of 8 LFA-3 monomers. 125I-mLFA-3 bound to Jurkat T leukemic cell surface CD2 with much higher avidity than sLFA-3. mLFA-3 binding had characteristics of a multivalent interaction with cell surface CD2 and had an avidity of 1.5 nM for Jurkat cells and 12 nM for resting T cells. Two CD2 mAbs tested did not block mLFA-3 binding: 9-1 and CD2.1. These mAbs were tested in combination with LFA-3 for their ability to activate T cells. The combination of mLFA-3 and CD2.1 mAbs induced a rapid increase in cytosolic free Ca2+ in Jurkat cells which was proportional to mLFA-3 occupation of CD2 sites. sLFA-3 showed no activity in the Ca2+ flux assay. The combination of mLFA-3 and the CD2.1 mAbs significantly stimulated proliferation of PBMC. The combination of mLFA-3 and 9-1 mAbs was weakly or not mitogenic for the same cells. The combination of CD2.1 and sLFA-3 at concentrations up to 480 nM was not consistently mitogenic. Therefore, monomeric LFA-3/CD2 interaction appears to have a relatively low affinity, while multimeric LFA-3 binds with high avidity. T cell activation by binding of LFA-3 to CD2 appears to require occupation of 10(4) to 10(5) CD2 sites, which is likely to occur during adhesion, but is rare in receptor systems with soluble ligands
— id: 20394, year: 1989, vol: 169, page: 503, stat: Journal Article,

T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1
Dustin ML; Springer TA
1989 Oct 19;341(6243):619-624, Nature
Effective interaction between T cells and their targets requires that recognition of specific antigen be coordinated with increased cell-cell adhesion. We show that antigen-receptor cross-linking increases the strength of the adhesion mechanism between lymphocyte function-associated molecule-1 (LFA-1) and intercellular adhesion molecules (ICAMs), with intracellular signals transmitted from the T-cell antigen receptor to the LFA-1 adhesion molecule. The increase in avidity is rapid and transient, providing a dynamic mechanism for antigen-specific regulation of lymphocyte adhesion and de-adhesion
— id: 20391, year: 1989, vol: 341, page: 619, stat: Journal Article,

The induction of intercellular adhesion molecule 1 (ICAM-1) expression on human fetal astrocytes by interferon-gamma, tumor necrosis factor alpha, lymphotoxin, and interleukin-1: relevance to intracerebral antigen presentation
Frohman EM; Frohman TC; Dustin ML; Vayuvegula B; Choi B; Gupta A; van den Noort S; Gupta S
1989 Jul;23(2):117-124, Journal of neuroimmunology
Antigen presentation reactions are dependent upon the expression of the class II major histocompatibility antigens (MHC), the T-cell receptor, and the presented antigen. Recent studies demonstrate that such processes also require the presence of adhesion molecules such as lymphocyte functional antigen 1 (LFA-1) and its cell surface ligand, intercellular adhesion molecule 1 (ICAM-1). It has been suggested that the brain astrocyte can function as a facultative antigen presenting cell (APC). This hypothesis is based upon the ability to induce the expression of the class II MHC antigens on astrocytes, and on their ability to present myelin basic protein to encephalitogenic T-cells in vitro. The best in vivo data showing that astrocytes serve as intracerebral APCs is the finding that astrocytes in multiple sclerosis plaques are DR+ (class II MHC in human). However, it still remains to be resolved whether the in vivo expression of the MHC antigens in disease states is instrumental to antigen presentation mechanisms or whether these cell surface glycoproteins are expressed secondary to brain immune responses. If astrocytes function as immunocompetent APCs within the brain, it would seem that they would also be able to express molecules important for intercellular adhesion. Here, we present the first data that indicates that human astrocytes are capable of expressing ICAM-1 in response to cytokines that either induce or upregulate the expression of DR. In essence, cytokines derived from different cell types seem to exert similar pleiotropic effects on the modulation of MHC and ICAM-1 expression on astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 20392, year: 1989, vol: 23, page: 117, stat: Journal Article,

The leukocyte integrins
Kishimoto TK; Larson RS; Corbi AL; Dustin ML; Staunton DE; Springer TA
1989 ;46(2):149-182, Advances in immunology
— id: 20396, year: 1989, vol: 46, page: 149, stat: Journal Article,

Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1
Staunton DE; Dustin ML; Springer TA
1989 May 4;339(6219):61-64, Nature
The leukocyte adhesion molecule LFA-1 mediates a wide range of lymphocyte, monocyte, natural killer cell, and granulocyte interactions with other cells in immunity and inflammation. LFA-1 (CD11a/CD18) is a receptor for intercellular adhesion molecule 1 (ICAM-1, CD54), a surface molecule which is constitutively expressed on some tissues and induced on other in inflammation. Induction of ICAM-1 on epithelial cells, endothelial cells and fibroblasts mediates LFA-1-dependent adhesion of lymphocytes. Several lines of evidence have suggested the existence of a second LFA-1 ligand: homotypic adhesion of one cell line was inhibited by a monoclonal antibody to LFA-1, but not by one to ICAM-1; there exists an LFA-1-dependent, ICAM-1-independent pathway of adhesion to endothelial cells; and also, there are some types of target cells in which LFA-1-dependent T-lymphocyte adhesion and lysis are independent of ICAM-1. We have cloned this second ligand, designated ICAM-2, using a novel method for identifying ligands of adhesion molecules. ICAM-2 is an integral membrane protein with two immunoglobulin-like domains, whereas ICAM-1 has five. Remarkably, ICAM-2 is much more closely related to the two most N-terminal domains of ICAM-1 (34% identity) than either ICAM-1 or ICAM-2 is to other members of the immunoglobulin superfamily, demonstrating the existence of a subfamily of immunoglobulin-like ligands that bind the same integrin receptor
— id: 20393, year: 1989, vol: 339, page: 61, stat: Journal Article,

Purified lymphocyte function-associated antigen-3 (LFA-3) activates human thymocytes via the CD2 pathway
Denning SM; Dustin ML; Springer TA; Singer KH; Haynes BF
1988 Nov 1;141(9):2980-2985, Journal of immunology
Defining the cellular and molecular mechanisms of interaction of developing thymocytes with nonlymphoid cells of the thymic microenvironment is critical for understanding normal thymus function. We have previously shown that the CD2/LFA-3 adhesion pathway is important in the interaction of thymocytes with a variety of LFA-3+ nonlymphoid thymic microenvironment cell types. Moreover, T cell activation via the CD2 (alternative, Ag independent) pathway is considered an important mechanism for intrathymic T cell proliferation. To study the relevance of CD2/LFA-3 interactions to human thymocyte activation, we have used purified LFA-3 Ag in several in vitro assays of thymocyte proliferation. Whereas LFA-3 Ag alone did not induce thymocyte proliferation, LFA-3 Ag in combination with the anti-CD2 antibody, CD2.1, and rIL-2 induced marked thymocyte proliferation. Additionally, the anti-CD28 antibody, Kolt2, could substitute for rIL-2, resulting in thymocyte activation induced by LFA-3 Ag in combination with antibodies CD2.1 and Kolt2. In both triggering systems, LFA-3 induced thymocyte activation was dependent upon the concentration of LFA-3 Ag. LFA-3 Ag-dependent thymocyte activation was directed primarily toward CD1-, mature thymocytes. Finally, intact SRBC that express the sheep homolog of LFA-3, T11TS, in combination with antibody CD2.1 and rIL-2 could also induce thymocyte activation. These data suggest that interaction of LFA-3 molecules with thymocyte CD2 molecules may provide a component of the stimulus for normal intrathymic thymocyte activation leading to thymocyte proliferation
— id: 20397, year: 1988, vol: 141, page: 2980, stat: Journal Article,

Adhesion of T lymphoblasts to epidermal keratinocytes is regulated by interferon gamma and is mediated by intercellular adhesion molecule 1 (ICAM-1)
Dustin ML; Singer KH; Tuck DT; Springer TA
1988 Apr 1;167(4):1323-1340, Journal of experimental medicine
The cell surface expression and function of the LFA-1 ligand, intercellular adhesion molecule 1 (ICAM-1), on epidermal keratinocytes (EK) was studied. ICAM-1 expression on the surface of cultured EK was either absent or weak, but was induced by treating EK with rIFN-gamma or TNF for 4-48 h. IFN-gamma and TNF were synergistic. IFN-gamma treatment increased T lymphoblast adhesion from less than 2% to 20-40%, with a concentration dependence similar to that seen for ICAM-1 induction. All of the adhesion to EK was inhibited by LFA-1 and ICAM-1 mAbs, but not by HLA-DR, CD2, or LFA-3 mAbs. There was no difference in the level of T lymphoblast adhesion to IFN-gamma-treated allogeneic or autologous EK. ICAM-1 purified from the HeLa epithelioid cell line and reconstituted into planar membranes also supported efficient adhesion of T lymphoblasts that was blocked by LFA-1 mAb bound to the T lymphoblasts or ICAM-1 mAb bound to the planar membranes. T lymphoblasts adherent to EK or ICAM-1 planar membranes were isolated by panning, and surface markers were analyzed by immunofluorescence flow cytometry. The adherent T cells were a phenotypically skewed subpopulation. They were enriched for CD8+ cells and expressed 1.5-2.5-fold higher LFA-1 and CD2 compared with the unseparated population
— id: 20400, year: 1988, vol: 167, page: 1323, stat: Journal Article,

Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells
Dustin ML; Springer TA
1988 Jul;107(1):321-331, Journal of cell biology
Intercellular adhesion molecule-1 (ICAM-1) on the surface of cultured umbilical vein and saphenous vein endothelial cells was upregulated between 2.5- and 40-fold by rIL-1, rTNF, LPS and rIFN gamma corresponding to up to 5 X 10(6) sites/cell. Endothelial cell ICAM-1 was a single band of 90 kD in SDS-PAGE. Purified endothelial cell ICAM-1 reconstituted into liposomes and bound to plastic was an excellent substrate for both JY B lymphoblastoid cell and T lymphoblast adhesion. Adhesion to endothelial cell ICAM-1 in planar membranes was blocked completely by monoclonal antibodies to lymphocyte function associated antigen-1 (LFA-1) or ICAM-1. Adhesion to artificial membranes was most sensitive to ICAM-1 density within the physiological range found on resting and stimulated endothelial cells. Adhesion of JY B lymphoblastoid cells, normal and genetically LFA-1 deficient T lymphoblasts and resting peripheral blood lymphocytes to endothelial cell monolayers was also assayed. In summary, LFA-1 dependent (60-90% of total adhesion) and LFA-1-independent basal adhesion was observed and the use of both adhesion pathways by different interacting cell pairs was increased by monokine or lipopolysaccharide stimulation of endothelial cells. The LFA-1-dependent adhesion could be further subdivided into an LFA-1/ICAM-1-dependent component which was increased by cytokines and a basal LFA-1-dependent, ICAM-1-independent component which did not appear to be affected by cytokines. We conclude that ICAM-1 is a regulated ligand for lymphocyte-endothelial cell adhesion, but at least two other major adhesion pathways exist
— id: 20398, year: 1988, vol: 107, page: 321, stat: Journal Article,

Supergene families meet in the immune system
Dustin ML; Staunton DE; Springer TA
1988 Jul-Aug;9(7-8):213-215, Immunology today
— id: 20399, year: 1988, vol: 9, page: 213, stat: Journal Article,

ICAM-1 a ligand for LFA-1-dependent adhesion of B, T and myeloid cells
Makgoba MW; Sanders ME; Ginther Luce GE; Dustin ML; Springer TA; Clark EA; Mannoni P; Shaw S
1988 Jan 7;331(6151):86-88, Nature
Cell-cell adhesion is essential for many immunological functions. The LFA-1 molecule, a member of a superfamily of adhesion molecules, participates in adhesion which is critical to the function of each of the three major subsets of leukocytes: lymphocytes, monocytes and granulocytes. Putative LFA-1 ligands have been identified functionally in different laboratories using three different monoclonal antibodies that inhibit LFA-1-mediated leukocyte adhesion in particular model systems; however, there may be more than one LFA-1 ligand. We have directly compared the three relevant monoclonal antibodies, and show that each binds to the same molecule, intercellular-adhesion molecule-1 (ICAM-1). Most important, B, T and myeloid cells adhere specifically to purified ICAM-1-coated surfaces; such adhesion has distinctive requirements for Mg2+ and Ca2+. This constitutes biochemical evidence that ICAM-1 functions as a ligand for LFA-1-dependent adhesion by a variety of leukocytes
— id: 20403, year: 1988, vol: 331, page: 86, stat: Journal Article,

Functional evidence that intercellular adhesion molecule-1 (ICAM-1) is a ligand for LFA-1-dependent adhesion in T cell-mediated cytotoxicity
Makgoba MW; Sanders ME; Ginther Luce GE; Gugel EA; Dustin ML; Springer TA; Shaw S
1988 Apr;18(4):637-640, European journal of immunology
Although intercellular adhesion molecule-1 (ICAM-1) has been implicated as a ligand in some LFA-1-dependent adhesion, its importance to T cell function has not been established. The present studies investigate the importance of ICAM-1 for human cytotoxic T lymphocytes (CTL), both in their formation of antigen-independent conjugates (AIC) and in their lysis of targets. Analysis of monoclonal antibody (mAb) inhibition of AIC formation indicate that ICAM-1 mAb 1 blocks (a) AIC formation with some but not all targets; (b) the LFA-1 pathway but not the CD2/LFA-3 pathway of adhesion; (c) by binding to the target cell, not the T cell. In studies of cell-mediated lysis (CML) ICAM-1 mAb inhibited lysis of some targets, such as U-937, that use ICAM-1 predominantly in AIC formation; CML on some other targets is not inhibited by ICAM-1 mAb. These data indicate that ICAM-1 is a ligand for AIC formation, antigen-specific CTL recognition and cytolysis of particular target cells. The data also indicate that ICAM-1 is not used in LFA-1-dependent CTL interactions with all kinds of target cells, suggesting the existence of alternative ligands for LFA-1
— id: 20401, year: 1988, vol: 18, page: 637, stat: Journal Article,

Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families
Staunton DE; Marlin SD; Stratowa C; Dustin ML; Springer TA
1988 Mar 25;52(6):925-933, Cell
Intercellular adhesion molecule 1 (ICAM-1) is a 90 kd inducible surface glycoprotein that promotes adhesion in immunological and inflammatory reactions. ICAM-1 is a ligand of lymphocyte function-associated antigen-1 (LFA-1), an alpha beta complex that is a member of the integrin family of cell-cell and cell-matrix receptors. ICAM-1 is encoded by an inducible 3.3 kb mRNA. The amino acid sequence specifies an integral membrane protein with an extracellular domain of 453 residues containing five immunoglobulin-like domains. Highest homology is found with neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG), which also contain five Ig-like domains. NCAM and MAG are nervous system adhesion molecules, but unlike ICAM-1, NCAM is homophilic. The ICAM-1 and LFA-1 interaction is heterophilic and unusual in that it is between members of the immunoglobulin and intergrin families. Unlike other integrin ligands, ICAM-1 does not contain an RGD sequence
— id: 20402, year: 1988, vol: 52, page: 925, stat: Journal Article,

Purified lymphocyte function-associated antigen 3 binds to CD2 and mediates T lymphocyte adhesion
Dustin ML; Sanders ME; Shaw S; Springer TA
1987 Mar 1;165(3):677-692, Journal of experimental medicine
CD2 is a T lymphocyte glycoprotein that functions in adhesion of T lymphocytes and also as a putative receptor for activation signals. Functional data suggest that LFA-3, a widely distributed cell surface glycoprotein, may be the biological ligand of CD2. We have purified LFA-3 from human erythrocytes and characterized the purified protein functionally. LFA-3 bound specifically to CD2+ cells, and this binding was inhibited by CD2 mAb. Conversely, purified LFA-3 inhibited binding of CD2 mAb to cells, and the concentration required for this effect suggests that LFA-3 half-saturated CD2 at 1-5 nM LFA-3. Purified LFA-3 inhibited rosetting of human and sheep erythrocytes with CD2+ T lymphoma cells and T lymphocytes, and mediated aggregation of a CD2+ T lymphoma cell line. Purified LFA-3 reconstituted into planar membranes mediated efficient CD2-dependent adhesion of T lymphoblasts. These data demonstrate that LFA-3 is a ligand for CD2 and that LFA-3 can mediate T lymphocyte adhesion
— id: 20410, year: 1987, vol: 165, page: 677, stat: Journal Article,

Anchoring mechanisms for LFA-3 cell adhesion glycoprotein at membrane surface
Dustin ML; Selvaraj P; Mattaliano RJ; Springer TA
1987 Oct 29-Nov 4;329(6142):846-848, Nature
The manner in which a membrane protein is anchored to the lipid bilayer may have a profound influence on its function. Most cell surface membrane proteins are anchored by a membrane-spanning segment(s) of the polypeptide chain, but another type of anchor has been described for several proteins: a phosphatidyl inositol glycan moiety, attached to the protein C terminus. This type of linkage has been identified on membrane proteins involved in adhesion and transmembrane signalling and could be important in the execution of these functions. We report here that an immunologically important adhesion glycoprotein, lymphocyte function-associated antigen 3 (LFA-3), can be anchored to the membrane by both types of mechanism. These two distinct cell-surface forms of LFA-3 are derived from different biosynthetic precursors. The existence of a phosphatidyl-inositol-linked and a transmembrane anchored form of LFA-3 has important implications for adhesion and transmembrane signalling by LFA-3
— id: 20407, year: 1987, vol: 329, page: 846, stat: Journal Article,

Rosetting of activated human T lymphocytes with autologous erythrocytes. Definition of the receptor and ligand molecules as CD2 and lymphocyte function-associated antigen 3 (LFA-3)
Plunkett ML; Sanders ME; Selvaraj P; Dustin ML; Springer TA
1987 Mar 1;165(3):664-676, Journal of experimental medicine
CD2, also known as LFA-2, T11, and the E rosette receptor, is a T lymphocyte surface protein functionally important in adhesion to target cells and T cell triggering. LFA-3 is a widely distributed cell surface protein that functions in adhesion on target cells. We find that LFA-3 is expressed on human E, and that CD2 is a receptor for LFA-3 that mediates T cell adhesion to human E. Pretreatment of T lymphocytes with CD2 mAb or of E with LFA-3 mAb inhibits rosetting. Purified CD2 molecules bind to human E and inhibit rosetting. 125I-CD2 binding to E is inhibited by LFA-3 mAb; reciprocally, binding of LFA-3 mAb to human E is inhibited by pretreatment with purified CD2. Higher concentrations of CD2 aggregate human E; aggregation is inhibited by mAb to LFA-3
— id: 20411, year: 1987, vol: 165, page: 664, stat: Journal Article,

Rosetting of human T lymphocytes with sheep and human erythrocytes. Comparison of human and sheep ligand binding using purified E receptor
Selvaraj P; Dustin ML; Mitnacht R; Hunig T; Springer TA; Plunkett ML
1987 Oct 15;139(8):2690-2695, Journal of immunology
Previous studies have shown that the purified T lymphocyte glycoprotein, cluster differentiation 2 (CD2) (also known as T11, lymphocyte function-associated antigen (LFA)-2, and the erythrocyte (E) rosette receptor) interacts with the LFA-3 molecule on human E. We have examined the interaction of the purified CD2 molecule with the T11 target structure (T11TS) molecule on sheep E, and compared the two interactions. Purified, 125I-labeled CD2 bound to sheep E and the binding was inhibited by anti-T11TS monoclonal antibody (mAb). Reciprocally, the binding of T11TS mAb to sheep E was inhibited by pretreatment of sheep E with purified CD2. High concentrations of purified CD2 aggregated sheep E, possibly by inserting into the membrane, and the aggregation was inhibited by T11TS mAb. The affinity and number of binding sites for purified CD2 on sheep and human E was found to be similar, with Ka of 9 X 10(7)/M and 6 X 10(7)/M and 9800 and 8300 CD2 binding sites/E, respectively. Thus, the human T lymphocyte CD2 molecule is a receptor that cross-reacts between LFA-3 on human E and T11TS on sheep E, suggesting that LFA-3 and T11TS are functionally homologous ligands. As measured by saturation mAb binding, there are 8100 and 3900 ligand molecules/sheep and human E, respectively. Human and sheep E have surface areas of 145 and 54 micron 2, respectively. The 3.2- to 5.6-fold higher ligand density on sheep E appears to account for the ability of sheep but not human E to rosette with certain types of human T lymphocytes
— id: 20405, year: 1987, vol: 139, page: 2690, stat: Journal Article,

Deficiency of lymphocyte function-associated antigen 3 (LFA-3) in paroxysmal nocturnal hemoglobinuria. Functional correlates and evidence for a phosphatidylinositol membrane anchor
Selvaraj P; Dustin ML; Silber R; Low MG; Springer TA
1987 Oct 1;166(4):1011-1025, Journal of experimental medicine
Lymphocyte function-associated antigen 3 (LFA-3) is a widely distributed cell surface glycoprotein that binds to the T lymphocyte CD2 surface glycoprotein. This interaction mediates CTL-target cell conjugate formation and adhesion of thymocytes to thymic epithelial cells. CD2 is also the E rosette receptor of T lymphocytes and mediates rosetting with autologous E by binding to LFA-3. We describe deficient expression of LFA-3 on E from paroxysmal nocturnal hemoglobinuria (PNH) patients. PNH is an acquired defect affecting phosphatidylinositol-anchored membrane proteins, of which decay-accelerating factor (DAF) is most important in the clinical symptoms of PNH. LFA-3-negative, weakly positive, and positive populations were found among PNH E. There was a good correlation with DAF deficiency. PNH E exhibited decreased binding of 125I-CD2 and rosetting with a human T lymphoma cell line. PNH E readily incorporated purified LFA-3, restoring LFA-3 expression and the CD2 binding and rosetting activity to normal levels. The expression of DAF was not restored after the incorporation of purified LFA-3 into PNH E, showing that LFA-3 and DAF are different molecules. Phosphatidylinositol-specific phospholipase C (PIPLC) treatment of a B lymphoma cell line released 35% of the cell surface LFA-3 and 62% of DAF. LFA-3 on E was resistant to PIPLC. However, when LFA-3 purified from human E was reconstituted in sheep E or human E and subjected to PIPLC treatment, 40-50% of LFA-3 was released from the cell membrane. The results show that LFA-3 is attached to the cell membrane by a phosphatidylinositol glycolipid moiety, and confirm previous findings (37-41) that LFA-3 is a cell adhesion molecule that mediates adhesion by interacting with CD2 antigen
— id: 20409, year: 1987, vol: 166, page: 1011, stat: Journal Article,

The lymphocyte function-associated LFA-1, CD2, and LFA-3 molecules: cell adhesion receptors of the immune system
Springer TA; Dustin ML; Kishimoto TK; Marlin SD
1987 ;5(3):223-252, Annual review of immunology
— id: 20412, year: 1987, vol: 5, page: 223, stat: Journal Article,

Serologic cross-reactivity of T11 target structure and lymphocyte function-associated antigen 3. Evidence for structural homology of the sheep and human ligands of CD2
Tiefenthaler G; Dustin ML; Springer TA; Hunig T
1987 Oct 15;139(8):2696-2701, Journal of immunology
T11 target structure (T11TS) and lymphocyte function-associated antigen (LFA) 3 are the cell-surface glycoproteins on sheep and human erythrocytes (E) binding to cluster differentiation 2 (the E-receptor) on T cells in E rosette formation. Here we show that this functional cross-reactivity is most likely due to a structural homology of these molecules. A rabbit antiserum to sheep T11TS is shown to cross-react with LFA-3 in several independent assays: (a) rabbit anti-T11TS antiserum blocks the formation of E rosettes by human T cells with both autologous and xenogeneic (sheep) E by binding to the respective E; (b) the antiserum blocks the binding of anti-LFA-3 monoclonal antibody to human E; and (c) it reacts with purified LFA-3 in Western blotting. Together, these findings demonstrate that T11TS on sheep E and LFA-3 on human E are serologically related, providing further support for the notion that T11TS and LFA-3 are the sheep and human forms of the same cell interaction molecule
— id: 20406, year: 1987, vol: 139, page: 2696, stat: Journal Article,

Purified lymphocyte function-associated antigen-3 and T11 target structure are active in CD2-mediated T cell stimulation
Tiefenthaler G; Hunig T; Dustin ML; Springer TA; Meuer SC
1987 Dec;17(12):1847-1850, European journal of immunology
In this study we have used cells expressing LFA-3 or T11TS, the human and sheep forms of the ligand of CD2, as well as the purified LFA-3 and T11TS molecules themselves to study their effects on T cell activation via the CD2-mediated 'alternative pathway'. Sheep red blood cells, which bind to CD2 via T11TS in E-rosette formation, and human autologous monocytes, which express the LFA-3 molecule, both induce proliferation of resting T cells in the presence of per se submitogenic concentrations of anti-T11(2) plus anti-T11(3) monoclonal antibodies (mAb). This effect is blocked by mAb to LFA-3, T11TS and CD2 known to inhibit CD2-ligand interaction. In addition, purified LFA-3 and T11TS, when added at ng amounts to cultures containing submitogenic concentrations of anti-T11(2 + 3) mAb, are also strongly mitogenic for resting human T cells. Thus, both LFA-3 and T11TS are potent co-stimulators of the alternative pathway of T cell activation but by themselves do not provide a mitogenic signal. This finding is discussed with regard to a physiological role of CD2-LFA-3 interaction in T cell activation
— id: 20404, year: 1987, vol: 17, page: 1847, stat: Journal Article,

Primary structure of lymphocyte function-associated antigen 3 (LFA-3). The ligand of the T lymphocyte CD2 glycoprotein
Wallner BP; Frey AZ; Tizard R; Mattaliano RJ; Hession C; Sanders ME; Dustin ML; Springer TA
1987 Oct 1;166(4):923-932, Journal of experimental medicine
We have isolated the cDNA for human lymphocyte function-associated antigen 3 (LFA-3), the ligand of the T lymphocyte CD2 molecule. The identity of the clones was established by comparison of the deduced amino acid sequence to the LFA-3 NH2-terminal and tryptic peptide sequences. The cDNA defines a mature protein of 222 amino acids that structurally resembles typical membrane-anchored proteins. An extracellular domain with six N-linked glycosylation sites is followed by a hydrophobic putative transmembrane region and a short cytoplasmic domain. The mature glycoprotein is estimated to be 44-68% carbohydrate. Southern blots of human genomic DNA indicate that only one gene codes for human LFA-3. Northern blot analysis demonstrates that the LFA-3 mRNA of 1.3 kb is widely distributed in human tissues and cell lines
— id: 20408, year: 1987, vol: 166, page: 923, stat: Journal Article,

Induction by IL 1 and interferon-gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1)
Dustin ML; Rothlein R; Bhan AK; Dinarello CA; Springer TA
1986 Jul 1;137(1):245-254, Journal of immunology
ICAM-1 is a cell surface glycoprotein originally defined by a monoclonal antibody (MAb) that inhibits phorbol ester-stimulated leukocyte aggregation. Staining of frozen sections and immunofluorescence flow cytometry showed intercellular adhesion molecule-1 (ICAM-1) is expressed on non-hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, certain other epithelial cells, and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal center dendritic cells in tonsils, lymph nodes, and Peyer's patches. ICAM-1 staining on vascular endothelial cells is most intense in T cell areas in lymph nodes and tonsils showing reactive hyperplasia. ICAM-1 is expressed in low amounts on peripheral blood leukocytes. Phorbol ester-stimulated differentiation of myelomonocytic cell lines greatly increases ICAM-1 expression. ICAM-1 expression on dermal fibroblasts is increased threefold to fivefold by either interleukin 1 (IL 1) or interferon-gamma at 10 U/ml over a period of 4 or 10 hr, respectively. The induction is dependent on protein and mRNA synthesis and is reversible. ICAM-1 displays Mr heterogeneity in different cell types with a Mr of 97,000 on fibroblasts, 114,000 on the myelomonocytic cell line U937, and 90,000 on the B lymphoblastoid cell JY. ICAM-1 biosynthesis involves a Mr approximately 73,000 intracellular precursor. The non-N-glycosylated form resulting from tunicamycin treatment has a Mr of 55,000. ICAM-1 isolated from phorbol myristic acetate (PMA) stimulated U937 and from fibroblasts yields an identical major product of Mr = 60,000 after chemical deglycosylation. ICAM-1 MAb interferes with the adhesion of phytohemagglutinin blasts, and the adhesion of the cell line SKW3 to human dermal fibroblast cell layers. Pretreatment of fibroblasts but not lymphocytes with ICAM-1 MAb, and of lymphocytes but not fibroblasts with lymphocyte function-associated antigen 1 MAb inhibits adhesion. Intercellular adhesion is increased by prior exposure of fibroblasts to IL 1, and correlates with induction of ICAM-1
— id: 20414, year: 1986, vol: 137, page: 245, stat: Journal Article,

A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1
Rothlein R; Dustin ML; Marlin SD; Springer TA
1986 Aug 15;137(4):1270-1274, Journal of immunology
Homotypic adhesion by phorbol ester-stimulated lymphocytes requires LFA-1 and Mg+2 and does not involve like-like interactions between LFA-1 molecules on adjacent cells. The latter finding suggested that a second molecule, distinct from LFA-1, also participates in LFA-1-dependent adhesion. The identification of such a molecule was the object of this investigation. After immunization with LFA-1-deficient EBV-transformed lymphoblastoid cells, a MAb was obtained that inhibits phorbol ester-stimulated aggregation of LFA-1+ EBV lines. This MAb defines a novel cell surface molecule, which is designated intercellular adhesion molecule 1 (ICAM-1). ICAM-1 is distinct from LFA-1 in both cell distribution and structure. In SDS-PAGE, ICAM-1 isolated from JY cells is a single chain of Mr = 90,000. As shown by MAb inhibition, ICAM-1 participates in phorbol ester-stimulated adhesion reactions of B lymphocyte and myeloid cell lines and T lymphocyte blasts. However, aggregation of one T lymphocyte cell line (SKW-3) was inhibited by LFA-1 but not ICAM-1 MAb. It is proposed that ICAM-1 may be a ligand in many, but not all, LFA-1-dependent adhesion reactions
— id: 20413, year: 1986, vol: 137, page: 1270, stat: Journal Article,

Effects of insulin receptor down-regulation on hexose transport in human erythrocytes
Dustin ML; Jacobson GR; Peterson SW
1984 Nov 25;259(22):13660-13663, Journal of biological chemistry
D-Glucose and D-galactose influx and efflux rates in human erythrocytes were studied using infinite-cis and zero-trans assay methods. It was found that insulin decreased the infinite-cis Km for both D-glucose and D-galactose influx by 44 and 56%, respectively, while the Vmax was unchanged. The Km for D-glucose efflux in the presence of insulin decreased by 47% when compared to controls, and the change in Vmax was statistically insignificant. If insulin receptors were first down regulated, and then influx and efflux assays were performed, decreases in the infinite-cis and zero-trans Km values were also observed in the absence of exogenous insulin. These affinity changes were not due to persistent surface insulin receptor occupation by the insulin which was used to induce down-regulation. These affinity changes were comparable to those observed in non-down-regulated cells in the presence of insulin
— id: 20415, year: 1984, vol: 259, page: 13660, stat: Journal Article,