PerspectiveImmunology

T Cell Activation at the Immunological Synapse: Vesicles Emerge for LATer Signaling

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Science Signaling  11 May 2010:
Vol. 3, Issue 121, pp. pe16
DOI: 10.1126/scisignal.3121pe16

Abstract

Signaling at the immunological synapse (IS) between a T cell and an antigen-presenting cell is initiated by the proximal tyrosine kinases Lck and ζ chain–associated protein kinase of 70 kD (ZAP-70) after engagement of the T cell receptor (TCR) by an antigen peptide–loaded major histocompatibility complex. Activation of these kinases leads to the formation of small protein aggregates (microclusters) within the IS, which contain kinases and adaptor proteins important for signal propagation. Src homology 2 (SH2) domain–containing leukocyte phosphoprotein of 76 kD (SLP-76), which is cytosolic, and LAT, which is membrane-associated, are key adaptor proteins phosphorylated by TCR-associated ZAP-70, and they form microclusters at the IS. Microclusters of either protein move during T cell activation; however, how they come in contact with each other to propagate signals remains unclear. A study addressed this question by monitoring the origin and kinetics of SLP-76 or LAT microclusters. Whereas the majority of LAT formed microclusters at the IS, a fraction of LAT resided on mobile, intracellular vesicles beneath the IS. Although SLP-76 microclusters formed just below the plasma membrane at the IS and moved centripetally, LAT-containing subsynaptic vesicles moved more rapidly, but slowed when in contact with SLP-76 microclusters. This interaction of vesicular LAT with SLP-76 microclusters coincided with the phosphorylation of LAT on key tyrosines that mediate its interaction with the SLP-76–associated adaptor protein GADS. Together, these data provide new insight into signaling at the IS and suggest that the endosomal network likely participates in T cell signaling.

An emerging paradigm suggests that the endosomal matrix does not merely regulate the degradation and recycling of receptors, but is also a site at which receptor signaling is initiated, propagated, or terminated (1). T cell activation is initiated at the contact site [known as the immunological synapse (IS)] between a T cell and an antigen-presenting cell (APC) where the T cell antigen receptor (TCR) recognizes foreign- or self-derived peptide–major histocompatibility complexes (pMHCs) that are presented by the APC (2). Ligation of the TCR results in the clustering of TCRs into small patches at the IS that are known as microclusters, as well as the activation of the proximal tyrosine kinases Lck and ζ chain–associated protein kinase of 70 kD (ZAP-70). These kinases in turn phosphorylate numerous signaling adaptors, which then also accumulate into distinct microclusters within seconds of the engagement of the TCR (3). The cytosolic adaptor protein Src homology 2 (SH2) domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) [which forms a complex with GADS (the Grb2-related adaptor downstream of Shc)] and the membrane-associated adaptor protein LAT both form distinct microclusters at the IS and are targets of ZAP-70 (4). Real-time imaging techniques demonstrated that these signaling microclusters are initially juxtaposed to TCR microclusters but that they then move independently of the TCR (4), even undergoing endocytosis in a manner that is dependent on the E3 ubiquitin ligase c-Cbl (5). Whereas it is appreciated that multiple receptor systems use “signaling endosomes,” whether such tubulovesicular structures participate in T cell signaling remains unclear, despite the observation that the endosomal network polarizes toward the IS during T cell activation (6, 7). Davis and colleagues used multicolor, live-cell microscopy to visualize TCR-initiated microcluster organization and dynamics (8). To their surprise, they observed that a large fraction of LAT was present in subsynaptic vesicles that moved in a restricted fashion among immobile microclusters of ZAP-70 and that were dynamically juxtaposed with microclusters of SLP-76 in a tyrosine phosphorylation–dependent manner during T cell activation. These data provide the first glimpse into a putative role for LAT-containing subsynaptic vesicles in regulating T cell signaling at the IS.

Multiple reports of experiments that used high-resolution imaging techniques, including total internal-reflection fluorescence (TIRF) microscopy, demonstrated the formation of signaling microclusters at the IS that undergo dynamic movement and endocytosis (6, 9). LAT, which undergoes palmitoylation and localizes to lipid rafts, organizes signaling microclusters at the IS. The localization of LAT to the plasma membrane is essential for its recruitment to the IS and its subsequent ZAP-70-dependent phosphorylation at key tyrosine residues (in particular, Tyr171 and Tyr191), which mediate the interaction of LAT with the SLP-76–GADS complex through the SH2 domain of GADS, and ultimately, T cell activation (10). In addition to localizing to lipid rafts, LAT also resides on intracellular vesicles in resting and stimulated T cells (11, 12). These vesicles polarize toward the IS during its formation, but the importance of the vesicular pool of LAT is unclear.

To better understand the spatial and temporal dynamics of the LAT and SLP-76–GADS complexes within the IS, Purbhoo et al. expressed fluorescently tagged proteins in transfected Jurkat cells (a human CD4+ T cell leukemia line) and visualized the dynamics and organization of microclusters during T cell activation. Despite the known physical recruitment of the SLP-76–GADS complex by tyrosine-phosphorylated LAT, the authors found that SLP-76–GADS complexes and LAT complexes were segregated into separate punctae at the IS, in which the more dynamic LAT microclusters moved among the more slowly moving SLP-76 microclusters (8). Interestingly, these distinct microclusters made contact with each other for extended periods of time during T cell activation. Moreover, similar to the movement of LAT, overnight labeling of T cells with the membrane dye 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) to label intracellular vesicles and organelles revealed that not only did intracellular vesicles localize to the IS and move dynamically, but they also made contacts with SLP-76 microclusters, dwelling for extended periods of time before moving off. Indeed, the authors used a combination of confocal and TIRF microscopy to demonstrate that, whereas the majority of SLP-76 microclusters were just below the surface of the IS, microclusters of LAT were present at the surface of the IS and on DiI-stained, membrane-proximal vesicles, as well as on vesicles that contained Rab7, a marker of late endosomes, or Rab8a, a marker of vesicles emerging from the Golgi. However, it was unclear whether either of these two vesicular compartments generated the LAT-containing vesicles that associated with the SLP-76 microclusters, or whether a distinct LAT-containing vesicle was involved (Fig. 1). Thus, an important next step in delineating the nature and mechanisms that regulate the generation and movement of LAT-containing vesicles will require the investigation of Rab proteins and their effectors as they coordinate multiple aspects of the endosomal network, including the formation and transport of vesicles (13).

Fig. 1

Vesicular LAT moves and interacts with surface-bound microclusters of SLP-76. Representation of an immunological synapse (IS) formed between an APC and a T cell; the T cell side of the IS contains TCR–ZAP-70, SLP-76–GADS, and LAT microclusters. LAT is an integral membrane protein that is also palmitoylated and trafficks through the Golgi on its way to the plasma membrane. In addition, intracellular vesicles that contain LAT reside in the cytoplasm of resting and activated T cells. During cellular activation, LAT microclusters form at the IS and are down-regulated from the surface by the E3 ubiquitin ligase c-Cbl. The work of Davis and colleagues suggests that vesicular LAT, which may be derived from the Golgi, the pool of vesicular LAT, or from LAT that is internalized moves and comes into contact with SLP-76–GADS microclusters localized at the IS. During this interaction, vesicular LAT is phosphorylated on critical tyrosine residues. P, phosphorylation; Ub, ubiquitin.

Phosphorylation of Tyr171 and Tyr191 is critical for the interaction between LAT and the SLP-76–GADS complex, and this might be one mechanism that contributes to the increased dwelling time that was observed between vesicular LAT and microclusters of SLP-76. Davis and colleagues showed that the expression of wild-type LAT in a LAT-deficient Jurkat cell line reconstituted the formation of microclusters of SLP-76, as well as the movement and interactions between vesicular LAT and microclusters of SLP-76 (8). In contrast, transfected LAT-deficient cells that expressed the Y171F, Y191F double-mutant LAT (in which tyrosine is replaced by phenylalanine at these numbered residues) exhibited reduced numbers of SLP-76 microclusters, which did not exhibit centripetal movement. Furthermore, the mutant LAT protein did not dwell on the SLP-76 microclusters that formed, which suggests that the direct interaction of LAT with the SLP-76–GADS complex was mediated by tyrosine phosphorylation of LAT. Consistent with this, Purbhoo et al. showed that the vesicular LAT that formed complexes with SLP-76 microclusters was phosphorylated, whereas vesicular LAT that was not in contact with SLP-76 was less likely to be phosphorylated (8). Of interest, stationary TCR–ZAP-70 microclusters that originated on cover slips coated with an antibody against the TCR complex corralled vesicular LAT, which enabled LAT to rapidly migrate in between the TCR–ZAP-70 microclusters. This suggests that the architecture of the IS might influence the movement of these LAT-containing vesicles. It remains to be determined how the LAT that accumulates with the SLP-76 microclusters becomes phosphorylated if it is not phosphorylated to a detectable degree before its interaction with SLP-76. Consistent with an important role for TCR-mediated signaling in the movement of vesicular LAT, engagement of the inhibitory receptor ILT2 abrogated the formation of ZAP-70 and SLP-76 microclusters and the movement of vesicular LAT.

Davis and colleagues have uncovered a previously unappreciated role for vesicular LAT during T cell activation. Although the authors provide compelling evidence that LAT-containing vesicles move and associate with the more membrane-proximal and slower-moving SLP-76 microclusters, where LAT undergoes increased tyrosine phosphorylation, it will be interesting to know whether the SLP-76 microclusters show similar increases in the extent of tyrosine phosphorylation when they are in contact with vesicular LAT, as well as to determine the molecular composition of the SLP-76 microclusters that engage vesicular LAT. For example, do these microclusters also contain other known SLP-76–interacting proteins such as Vav1, Itk or Nck before or after their interaction with vesicular LAT? Moreover, these data suggest that the organization of the IS likely contributes to the sequestration of signaling molecules into distinct compartments to promote functional interactions. Indeed, other high-resolution imaging studies of the T cell plasma membrane demonstrated that the TCR and LAT organize on distinct “protein islands” that concatenate, but remain isolated, upon ligation of the TCR (14). Thus, the capacity of LAT signaling appears to be sequestered not only at the plasma membrane, but also within the subsynaptic vesicles identified by Davis and colleagues (8).

Furthermore, and as indicated above, an outstanding question relates to the origin of vesicular LAT and the mechanisms that contribute to its movement. Although it is generally appreciated that the actin and microtubule cytoskeletons participate in the movement and sequestration of signaling microclusters, it is unclear what drives the movement of the vesicular LAT structures (9). In addition, because the rate of movement of LAT microclusters changes when in contact with SLP-76 clusters and when phosphorylated, it may be possible that TCR ligands might affect signaling by altering the kinetics of the movement of LAT microclusters. A better mechanistic understanding of this process will help to define the roles of these structures in propagating or sustaining T cell signaling.

Ideas about the origins of vesicular LAT are beginning to emerge. Because LAT resides on intracellular vesicles that contain transferrin before activation of the T cell, it remains likely that some LAT is continuously being recycled and might represent a vesicular pool that is rapidly polarized and moved into the membrane during the dynamic cytoskeletal reorganization that occurs during formation of the IS (Fig. 1). Another possibility is that newly synthesized LAT from the Golgi serves to supply the pool of vesicular LAT. Last, LAT internalized from the plasma membrane during T cell activation undergoes distinct internalization and, likely, uses trafficking pathways, some of which may supply the vesicular pool of LAT. Clearly, delineating the origin, molecular components, and mechanisms that contribute to the movement of the vesicular pool of LAT will be an important next step in understanding this unique, dynamic signaling platform and its impact on T cell activation.

Acknowledgments

Funding: This work was supported by NIH grant AI065474. D.D.B. is a Leukemia and Lymphoma Society Scholar.

References and Notes

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