A Large T Cell Invagination with CD2 Enrichment Resets Receptor Engagement in the Immunological Synapse

Singleton, Kentner L.; Parvaze, Nadia; Dama, Kavyya R.; Chen, Kenneth; Jennings, Paula; Purtic, Bozidar; Sjaastad, Michael D.; Gilpin, Christopher J.; Davis, Mark M.; Wülfing, Christoph

doi:10.4049/jimmunol.177.7.4402

Cited by 33 publications

(38 citation statements)

References 56 publications

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“…S6). Interestingly, Wülfing and colleagues, nicely showed by electron microscopy how a large invagination in the T cell is formed around a fragment of the APC in a TCR affinity-dependent manner (Singleton et al, 2006). The molecular mechanisms that allow the phagocytic cup to close around the APC membrane fragment are at present unknown.…”

Section: Discussionmentioning

confidence: 99%

“…T cells acquire MHC class I and class II glycoproteins from APCs, together with co-stimulatory molecules and membrane patches, by a mechanism referred to as trogocytosis (Ahmed et al, 2008; Davis, 2007; Joly and Hudrisier, 2003; Wetzel and Parker, 2006). MHC transfer to the T cell is TCR triggering-dependent, and it is potentiated by the T cell co-stimulatory molecule CD28 and by CD2 (Hwang et al, 2000; Singleton et al, 2006). The transfer of pMHC to T cells has been demonstrated both in vitro and in vivo (Huang et al, 1999), although the mechanisms, and physiological meaning of such transfer are not completely understood.…”

Section: Introductionmentioning

confidence: 99%

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T Cell Receptor Internalization from the Immunological Synapse Is Mediated by TC21 and RhoG GTPase-Dependent Phagocytosis

et al. 2011

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Summary The immunological synapse (IS) serves a dual role for sustained T cell receptor (TCR) signaling and for TCR downregulation. TC21 (Rras2) is a RRas subfamily GTPase that constitutively associates with the TCR and is implicated in tonic TCR signaling by activating phosphatidylinositol 3-kinase. In this study, we demonstrate that TC21 both co-translocates with the TCR to the IS and is necessary for TCR internalization from the IS through a mechanism dependent on RhoG, a small GTPase previously been associated with phagocytosis. Indeed, we found that the TCR triggers T cells to phagocytose 1-6 μm beads through a TC21- and RhoG-dependent pathway. We further show that TC21 and RhoG are necessary for the TCR-promoted uptake of major histocompatibility complex (MHC) from antigen presenting cells. Therefore, TC21- and RhoG-dependence underlie the existence of a common phagocytic mechanism that drives TCR internalization from the IS together with its peptide-MHC ligand.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

T Cell Receptor Internalization from the Immunological Synapse Is Mediated by TC21 and RhoG GTPase-Dependent Phagocytosis

et al. 2011

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“…Here electron microscopic studies are particularly valuable for their very high resolution, especially with the power of 3D tomography and highpressure freezing, which preserves cell structure more faithfully. Although a number of high-resolution electron microscopy studies of T cell-APC interactions have been reported (3,(12)(13)(14)(15), these involve studies of cytotoxic T or natural killer cells and their targets for only the first few minutes of CD4 + T-cell recognition (16). In earlier work using fluorescence microscopy, Kupfer et al…”

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confidence: 99%

CD4⁺T-cell synapses involve multiple distinct stages

Ueda

Morphew

McIntosh

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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112

104

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One very striking feature of T-cell recognition is the formation of an immunological synapse between a T cell and a cell that it is recognizing. Formation of this complex structure correlates with cytotoxicity in the case of killer (largely CD8 + ) T-cell activity, or robust cytokine release and proliferation in the case of the much longer lived synapses formed by helper (CD4 + ) T cells. Here we have used electron microscopy and 3D tomography to characterize the synapses of antigen-specific CD4 + T cells recognizing B cells and dendritic cells at different time points. We show that there are at least four distinct stages in synapse formation, proceeding over several hours, including an initial stage involving invasive T-cell pseudopodia that penetrate deeply into the antigen-presenting cell, almost to the nuclear envelope. This must involve considerable force and may serve to widen the search for potential ligands on the surface of the cell being recognized. We also show that centrioles and the Golgi complex are always located immediately beneath the synapse and that centrioles are significantly shifted toward the late contact zone with either B lymphocytes or bone marrow-derived dendritic cells such as antigenpresenting cells, and that there are dynamic, stage-dependent changes in the organization of microtubules beneath the synapse. These data reinforce and extend previous data on cytotoxic T cells that one of the principal functions of the immunological synapse is to facilitate cytokine secretion into the synaptic cleft, as well as provide important insights into the overall dynamics of this phenomenon. microtubule organizing center | centrosome A prominent feature of many T-lymphocyte interactions with other cells on which it recognizes a particular antigen is the formation of an immunological synapse (IS). The formation of this structure correlates with robust signaling, lineage commitment, and fate determination of T cells (1-6) and the directed secretion of cytokines and/or cytotoxic molecules. There is a wholesale reorganization of the T cell's cytoskeleton, surface molecules, and organelles, and the loss of polarity regulators or guidance cues that negatively affect T-cell activation (7,8). Whereas a great deal has been learned about the dynamics of cell-surface and signaling molecules within this interface (9, 10), much less is known about changes within the cell, especially in the long-lived helper T cell, namely in antigen-presenting cell (APC) interactions, which can last for 6 or more h and need continuous T-cell receptor (TCR) engagement (11). Here electron microscopic studies are particularly valuable for their very high resolution, especially with the power of 3D tomography and highpressure freezing, which preserves cell structure more faithfully. Although a number of high-resolution electron microscopy studies of T cell-APC interactions have been reported (3,(12)(13)(14)(15), these involve studies of cytotoxic T or natural killer cells and their targets for only the first few minutes of CD4 + ...

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“…As immortalization of cells in the generation of cell lines likely changes the regulation of cellular polarization, we used primary, in vitro primed T cells. As a T cell model, we used 5C.C7 TCR transgenic T cells, where polarization has been extensively studied before Tskvitaria-Fuller et al, 2003;Purtic et al, 2005;Singelton et al, 2006;Tskvitaria-Fuller et al, 2006). The 5C.C7 TCR recognizes the moth cytochrome C 92-103 peptide (MCC) presented by I-E k (Seder et al, 1992).…”

Section: Protein Transduction As An Approach To Manipulate Cdc42 Actimentioning

confidence: 99%

“…We analyzed Cdc42 activity within the first and the second minute after tight cell couple formation. Within the first minute distinct and dramatic morphologically changes occur, T cell actin spreads towards the edges of the interface (Tskvitaria-Fuller et al, 2003), the uropod retracts (Montoya et al, 2002), and a deep invagination forms at the center of the interface (Singelton et al, 2006). In the subsequent about four minutes, proximal T cell signaling peaks.…”

Section: Single Cell Analysis Of Cdc42 Activitymentioning

confidence: 99%

Protein transduction as a means of effective manipulation of Cdc42 activity in primary T cells

Tskvitaria-Fuller

Mistry

Sun

et al. 2007

Journal of Immunological Methods

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The Rho family GTPase Cdc42 is a critical regulator of cellular polarization from yeast to man. An analysis of its function in T cell activation is therefore of interest. This analysis poses two substantial challenges, similar to the analysis of many other critical T cell signaling intermediates. First, Cdc42 is required for development and cell survival, necessitating short-term manipulation of its activity. Second, Cdc42 is likely involved in multiple signaling pathways, requiring approaches to distinguish multiple roles. To address these challenges, we first determined and quantified spatio-temporal patterns of Cdc42 activity using live cell video fluorescence microscopy. This generates hypotheses at which times and locations Cdc42 might play possibly distinct roles. Second and as the focus of this manuscript, we employed protein transduction to manipulate Cdc42 activity for the generation of causality. Protein transduction allows such manipulation to be short-term, quantitative, and with multiple reagents. Here, we characterize uptake, retention, and subcellular distribution of protein transduction reagents. We describe how a more quantitative single cell analysis of Cdc42 activity provides superior distinction between experimental conditions. And we show how we have used dose responses of the protein transduction reagents to minimize side effects while retaining efficacy. We suggest that our strategy is an important complement to more established techniques to study protein function in primary T cells, in particular in the investigation of signaling intermediates that are essential for cell survival and regulate multiple aspects of T cell activation.

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A Large T Cell Invagination with CD2 Enrichment Resets Receptor Engagement in the Immunological Synapse

Cited by 33 publications

References 56 publications

T Cell Receptor Internalization from the Immunological Synapse Is Mediated by TC21 and RhoG GTPase-Dependent Phagocytosis

T Cell Receptor Internalization from the Immunological Synapse Is Mediated by TC21 and RhoG GTPase-Dependent Phagocytosis

CD4⁺T-cell synapses involve multiple distinct stages

Protein transduction as a means of effective manipulation of Cdc42 activity in primary T cells

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A Large T Cell Invagination with CD2 Enrichment Resets Receptor Engagement in the Immunological Synapse

Cited by 33 publications

References 56 publications

T Cell Receptor Internalization from the Immunological Synapse Is Mediated by TC21 and RhoG GTPase-Dependent Phagocytosis

T Cell Receptor Internalization from the Immunological Synapse Is Mediated by TC21 and RhoG GTPase-Dependent Phagocytosis

CD4+T-cell synapses involve multiple distinct stages

Protein transduction as a means of effective manipulation of Cdc42 activity in primary T cells

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Product

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CD4⁺T-cell synapses involve multiple distinct stages