New insights into the T cell synapse from single molecule techniques

Dustin, Michael L.; Depoil, David

doi:10.1038/nri3066

Cited by 179 publications

(153 citation statements)

References 119 publications

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“…To test these hypotheses, proximal TCR signaling events were examined using immobilized fusion proteins VISTA-Ig and PD-L1-Ig, both of which suppressed T-cell proliferation and cytokine production in vitro (3,12). LAT is a proximal signaling adaptor that is phosphorylated on TCR stimulation and forms a complex with multiple signaling molecules including SH2 domain containing leukocyte protein of 76kDa (SLP76) and phospholipase C (PLC)-γ1 (24). To determine whether VISTA functions by interfering with the phosphorylation of LAT, a solid phase immunoprecipitation assay was performed, in which the proximal signaling complexes could be recovered as the bound fraction to the plastic surface (25,26).…”

Section: Resultsmentioning

confidence: 99%

Immune-checkpoint proteins VISTA and PD-1 nonredundantly regulate murine T-cell responses

Yuan

Chen

et al. 2015

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

286

249

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V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a negative immune-checkpoint protein that suppresses T-cell responses. To determine whether VISTA synergizes with another immune-checkpoint, programmed death 1 (PD-1), this study characterizes the immune responses in VISTA-deficient, PD-1-deficient (KO) mice and VISTA/PD-1 double KO mice. Chronic inflammation and spontaneous activation of T cells were observed in both single KO mice, demonstrating their nonredundancy. However, the VISTA/PD-1 double KO mice exhibited significantly higher levels of these phenotypes than the single KO mice. When bred onto the 2D2 T-cell receptor transgenic mice, which are predisposed to development of inflammatory autoimmune disease in the CNS, the level of disease penetrance was significantly enhanced in the double KO mice compared with in the single KO mice. Consistently, the magnitude of T-cell response toward foreign antigens was synergistically higher in the VISTA/PD-1 double KO mice. A combinatorial blockade using monoclonal antibodies specific for VISTA and PD-L1 achieved optimal tumor-clearing therapeutic efficacy. In conclusion, our study demonstrates the nonredundant role of VISTA that is distinct from the PD-1/PD-L1 pathway in controlling T-cell activation. These findings provide the rationale to concurrently target VISTA and PD-1 pathways for treating T-cell-regulated diseases such as cancer.

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Section: Resultsmentioning

confidence: 99%

Immune-checkpoint proteins VISTA and PD-1 nonredundantly regulate murine T-cell responses

Yuan

Chen

et al. 2015

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

286

249

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“…Lck-, TCR-, and LAT-regulated traffic is pivotal to TCR signal transduction at the immunological synapse TCR signaling is mediated by formation of transient, heterogeneous clusters of signaling molecules whose structure and subcellular origin remain uncertain (Dustin and Depoil, 2011). Our experimental build-up provided us with the means to disentangle the contributions of regulated signaling molecule exocytosis for TCR signaling.…”

Section: Lck Tcr and Lat Reside In Distinct Exocytic Vesicular Commentioning

confidence: 99%

Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse

Soares¹,

Henriques²,

Sachse³

et al. 2013

J Gen Physiol

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Abbreviations used: dSTORM, direct stochastic optical reconstruction microscopy; MAL, myelin and lymphocyte; Syt7, synaptotagmin-7; TIRF, total internal reflection fluorescence.M.-I. Thoulouze and A. Alcover contributed equally to this paper. Fig. 1, D and H). LAT vesicular compartment co-localized with Rab27a (35%) and Rab37 (30%; Fig. 1, M and N), two Rab molecules known to regulate cytokine and cytotoxic granule exocytosis in CD4 + and CD8 + T cells, respectively (Stinchcombe et al., 2001;Huse et al., 2006). TCR peripheral vesicles colocalized to the fast recycling Rab4b compartment (8%), whereas its core co-localized to Rab3d-(25%) and Rab8b-regulated (30%) exocytic compartments (Huse et al., 2006;Fukuda, 2008; Fig. 1, Q-S). Noteworthy, the secretory v-SNARE protein Ti-VAMP (Chaineau et al., 2009) co-localized with both LAT (30%) and TCR (30%) vesicular compartments (Fig. 1, O and W). Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse LAT and TCR vesicular release is regulated by calcium and synaptotagmin-7The traffic regulators present in LAT and TCR vesicles (Fig. 1) have been reported to be involved in stimulus-induced exocytosis at cytotoxic synapses (Ménager et al., 2007; MarcetPalacios et al., 2008). To pinpoint the molecular mechanism regulating Lck, LAT, and TCR release from vesicular compartments, we assessed the role of calcium, a known regulator of stimulus-dependent exocytosis (Stojilkovic, 2005;Pang and Südhof, 2010). To increase cytosolic calcium levels, we treated CD4 T cells either with thapsigargin or ionomycin. To accurately quantify Lck, LAT, and TCR distribution between intracellular vesicles and the plasma membrane, we stained for surface CD2 and then used it to automatically segment the plasma membrane. The resulting automated mask delimiting the internal and external outlines of the plasma membrane allowed us to discriminate and quantify the subcellular distribution (plasma membrane versus vesicular compartment) of our proteins of interest (materials and methods; unpublished data). We found LAT and TCR distribution to the vesicular compartment to be higher than anticipated by others (Bonello et al., 2004), 75% (Fig. 2, A, B, H, and I). In fact, through plasma membrane segmentation, we were able to distinguish plasma membrane-resident LAT and TCR from their intracellular compartments and subcortical vesicles, within the limits of the confocal microscopy resolution (unpublished data). Lck displayed a higher plasma membrane distribution with 25% in intracellular vesicles (Fig. 2, C and J; and not depicted).Consistent with LAT localization in Rab27a and Rab37 exocytic compartments, increased cytosolic calcium led to a clear release of its vesicular compartment into the plasma membrane (Fig. 2, A, I, and O; and not depicted). In agreement with TCR-segregated distribution into a Rab3d and Rab8b exocytic compartment and also into Rab4b rapid recycling endosomes, cytosolic calcium increase induced an inc...

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“…TCR microclusters have been defined as a third compartment important for sustained signalling. Microcluster formation is F-actin-dependent, but large microclusters formed by high-avidity ligands become F-actin-independent as they progress toward the centre of the immunological synapse (reviewed in [5]). Dustin presented evidence that the endosomal sorting complexes required for transport (ESCRT) protein TSG101 is required for cSMAC formation.…”

Section: Immunoreceptor Dynamicsmentioning

confidence: 99%