Differential Clustering of CD4 and CD3ζ During T Cell Recognition

Krummel, Matthew F.; Sjaastad, Michael D.; Wülfing, Christoph; Davis, Mark M.

doi:10.1126/science.289.5483.1349

Cited by 352 publications

(341 citation statements)

References 27 publications

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“…3A and data not shown). Preliminary evidence suggests that the difference between these observations lies in timing, with an accumulation of CD8 occurring at shorter time-points (5-10 min) following conjugate formation followed by exclusion of CD8 at later time-points; a similar phenomenon has been described for the CD4 coreceptor in T cells [21]. However, in both cases we saw no differences in CD8 distribution between Vav1 +/+ and Vav1 -/-thymocytes.…”

Section: Vav1 Is Not Required For the Assembly Of An Issupporting

confidence: 66%

Vav1 transduces TCR signals required for LFA‐1 function and cell polarization at the immunological synapse

et al. 2003

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Activation of T lineage cells through the TCR by peptide-MHC complexes on APC is critically dependent on rearrangement of the actin cytoskeleton. Vav1 is a guanine nucleotide exchange factor for members of the Rho/Rac family of GTPases which is activated following TCR stimulation, suggesting that it may transduce TCR signals to the activation of some or all actin-controlled processes. We show that Vav1-deficient double-positive thymocytes are less efficient at forming conjugates with APC presenting agonist peptide than wild-type cells are. Furthermore we demonstrate that Vav1 is required for TCR-induced activation of the integrin LFA-1, which is likely to explain the defect in conjugate formation. However, once Vav1-deficient cells form a conjugate, the assembly of proteins into an immunological synapse at the conjugate interface is normal. In contrast, thymocyte polarization is defective in the absence of Vav1, as judged by the relocalization of the microtubule-organizing center. These data demonstrate that Vav1 transduces signals to only a subset of cytoskeletondependent events at the immunological synapse.

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Section: Vav1 Is Not Required For the Assembly Of An Issupporting

confidence: 66%

Vav1 transduces TCR signals required for LFA‐1 function and cell polarization at the immunological synapse

et al. 2003

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“…For example, the structural features of the immunological synapse have been pieced together from in vitro studies -mainly on fixed T cells using specific antibody staining and on the interactions of T cells with planar lipid films. In a few cases, the redistribution of molecules has been observed in living T cells using GFP-tagged proteins 17,[39][40][41] . Close contacts between T cells and APCs, and the clustering of TCRs near to the site of antigen presentation have been observed also in fixed tissue samples from the lymph nodes of TCR-transgenic mice 27,42 , but this provides only a static picture of a dynamic event.…”

Section: The Need For Live-tissue Imagingmentioning

confidence: 99%

Two-photon tissue imaging: seeing the immune system in a fresh light

et al. 2002

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Many lymphocyte functions, such as antigen recognition, take place deep in densely populated lymphoid organs. Because direct in vivo observation was not possible, the dynamics of immune-cell interactions have been inferred or extrapolated from in vitro studies. Two-photon fluorescence excitation uses extremely brief (<1 picosecond) and intense pulses of light to 'see' directly into living tissues, to a greater depth and with less phototoxicity than conventional imaging methods. Twophoton microscopy, in combination with newly developed indicator molecules, promises to extend single-cell approaches to the in vivo setting and to reveal in detail the cellular collaborations that underlie the immune response.Scientific questions drive the development of technology, and new technologies, in turn, influence the type of questions that we pose in an iterative process of discovery. Applied to immunology, this has prompted the innovation and application of powerful techniques for analysing the microscopic world of the immune system. Two methodological lines of investigation have dominated the field: in vivo experiments that examine the behaviour of populations of cells in living animals during an immune response; and in vitro experiments that use individual cells in artificial environments. An immune response is the sum of many complex and dynamic individual cellular behaviours that are shaped by many environmental factors. In vivo experiments maintain this natural environment, but they cannot resolve the behaviours of individual cells. By contrast, in vitro experiments provide information at the subcellular and molecular levels, but they cannot replicate adequately the full repertoire of environmental factors. There is a pressing need for techniques that allow real-time observation of single cells and molecules in intact tissues. Recent developments in imaging technology now make this possible. In this review, we provide a guide to the application of biophotonic techniques to the field of immunology and, in particular, to the use of two-photon laser microscopy. We discuss recent results obtained using this approach and pinpoint some key questions that might be resolved by tissue imaging. The biophotonics tool kitImmunologists have long been adept at raiding other disciplines to acquire new research tools. Fluorescence techniques -for example, flow cytometry, video imaging and more exotic imaging modalities, such as FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) and two-photon microscopy -combined with new probes to track Ca 2+ signalling, Correspondence to M.D.C. mcahalan@uci.edu. NIH Public Access Imaging T-cell dynamics in vitroThe single-cell approach has intrinsic value, because asynchronous behaviours are not indicated by population measurements. The need for live-tissue imagingIn vivo approaches have elucidated the basic properties of immune cells and lymphoid tissues, whereas in vitro cell-culture systems have provided a high-definition analysis of the cellsurface receptors and intracellular signalling pat...

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“…For retroviral enhancement of CD4 expression on Th2 cells, the pIB-CD4-EGFP(C4) vector (a kind gift of M. Davis, HHMI Stanford University School of Medicine [37]) was transfected into Phoenix-Eco packaging cells. Viral supernatant collected from these transduced cells was added to previously generated 5C.C7 Th2 cells that were re-stimulated with splenic antigenpresenting cells and anti-CD3e (clone 145-2C11; 1 lg/mL) in 24-well plates for 24 h. Cells were spun down at 2200 rpm for 90 min at 32 C in the presence of polybrene (8 lg/mL).…”

Section: Retroviral Gene Transductionmentioning

confidence: 99%

Decreased CD4 expression by polarized T helper 2 cells contributes to suboptimal TCR-induced phosphorylation and reduced Ca2+ signaling

et al. 2005

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Polarized Th1 and Th2 cells expressing the same TCR produce distinct biochemical responses to ligand engagement. Compared to Th1 cells, Th2 cells show altered substrate tyrosine phosphorylation and a diminished or transient Ca 2+ response. Here we demonstrate that agonist stimulation of Th1 cells leads to the predominant appearance of fully phosphorylated (p23) TCR f, substantial phosphorylation of zetaassociated protein 70 (ZAP-70), and strong elevation of intracellular Ca 2+ , whereas agonist stimulation of Th2 cells expressing an identical TCR results in an elevated p21:p23 TCR f ratio, little or no detectable ZAP-70 phosphorylation, and a more limited elevation in intracellular Ca 2+ . Th2 cells consistently had twofold lower surface CD4 expression as compared to Th1 cells with the same TCR. When CD4 levels in Th2 cells were raised to Th1 levels using retroviral gene transfer, the transduced cells showed greater generation of p23 phospho-f, measurable phosphorylation of ZAP-70, and increased Ca 2+ responses. These findings suggest that the apparent qualitative differences in TCR signaling characterizing Th1 versus Th2 cells are largely the result of modest quantitative variation in CD4 expression, with decreased CD4 expression playing a significant role in attenuating the proximal signaling responsiveness of Th2 cells to TCR ligands.Supporting information for this article is available at http://www.wileyvch.de/contents/jc_2040/2005/26064_s.pdf IntroductionWhile it is an oversimplification of in vivo physiology, the Th1-Th2 model is a convenient and generally applicable paradigm for naive CD4 + T cell differentiation [1,2]. The set of effector cytokines produced by an antigenactivated CD4 + T cell is largely determined by the particular combination of T cell receptor (TCR), cytokine, and costimulatory signals received by the naive T cell. In particular, T cells expressing the same The altered downstream phosphorylation seen in Th2 cells resembles that reported by this laboratory for Th1 cells in which CD4-MHC class II interactions are disrupted [14]. Therefore, we undertook a re-examination of the status of the CD4 molecule in polarized Th2 cells. Here we report that such cells consistently show a lower surface level of CD4 expression than Th1 cells derived from the same precursor pool. This lower CD4 expression could be linked to the altered signaling pattern of the Th2 cells through experiments showing a switch to a more Th1-like pattern of tyrosine phosphorylation and Ca 2+ signaling following enhancement of CD4 expression using a retroviral vector. These data provide evidence that a modest quantitative change in expression of a surface receptor can have qualitative effects on signaling in differentiated T cells and that decreased CD4 expression plays a significant role in controlling the antigen responsiveness of polarized Th2 cells. Results Differential TCR down-modulation in response to agonist by Th1 vs. Th2 cells expressing identical TCRTo examine in a controlled fashion the origin of the differences...

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Differential Clustering of CD4 and CD3ζ During T Cell Recognition

Cited by 352 publications

References 27 publications

Vav1 transduces TCR signals required for LFA‐1 function and cell polarization at the immunological synapse

Vav1 transduces TCR signals required for LFA‐1 function and cell polarization at the immunological synapse

Two-photon tissue imaging: seeing the immune system in a fresh light

Decreased CD4 expression by polarized T helper 2 cells contributes to suboptimal TCR-induced phosphorylation and reduced Ca2+ signaling

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