Immunological synapse and microclusters: the site for recognition and activation of T cells

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An immunological synapse (IS) is formed at the interface between antigen-presenting cells and T cells, and is believed to be the structure responsible for antigen recognition and T-cell activation. However, recent imaging analyses reveal that T-cell receptor microclusters (MCs) formed prior to IS are the site for antigen recognition and T-cell activation. MCs are continuously generated at the periphery of the interface, even after IS formation, and induce sustained activation signals. MC formation is not accompanied by lipid-raft clustering. Central supramolecular activation cluster is considered functional in recycling and degradation of T-cell receptors, directional secretion of cytokines and cytolytic granules, generation of sustained signals, or maintenance of the cell–cell conjugation.

Introduction

Antigen (Ag)-specific immune responses are elicited when T cells that express a specific Ag receptor (the T-cell receptor [TCR]) interact with Ag-presenting cells (APCs) that bear a cognate Ag peptide–MHC (pMHC). This interaction is responsible for the formation of a unique ‘immunological synapse’ (IS). The IS was originally found between T cells and B cells, or between T cells and MHC-containing planar bilayers [1, 2]. It is formed by the accumulation of TCR–pMHC in the central region, termed the central supramolecular activation cluster (c-SMAC), and the accumulation of leukocyte function-associated antigen 1 (LFA-1)–intercellular adhesion molecule-1 (ICAM-1) in outside regions, called the peripheral (p-)SMAC. A variety of molecules have been found to be involved in IS, and their dynamic recruitment and contribution to T-cell activation has been elucidated.

In this review, we will summarize current understanding of the relationship between IS formation and TCR signaling. We will focus on a new view of dynamic regulation of initial and sustained T-cell activation signals from the perspective of newly found microclusters (MCs) and on the contribution of lipid-raft clustering to this process, and will discuss the possible functions of c-SMAC.

Section snippets

Immunological synapse formation

Monks et al. [2] were the first to describe the spatial segregation of several proteins at the contact interface between T cells and B cells that are presenting specific Ag peptides. Using confocal microscopy, they found that the contact interface is composed of a doughnut-like structure in T cells: TCR and protein kinase C (PKC)θ are accumulated in the central region and are surrounded by a ring structure of LFA-1 and talin. Similarly, pMHC and ICAM-1 are accumulated at the contact interface

Regulation of immunological synapse by co-stimulation and cytoskeleton

Co-stimulatory molecules, the most representative ones of which are CD28 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), are crucial for IS formation. CTLA-4 is predominantly present in intracellular vesicles, and translocates to the IS upon TCR activation [12, 13]. Allison et al. [14, 15•] demonstrated that accumulation of CLTA-4 at the IS was dependent upon TCR signal strength. They found that stronger stimuli more effectively recruit CTLA-4 to the IS and inhibit T-cell responses [14

Heterogeneity of synapse formation

The term ‘immunological synapse’ is widely used to indicate the accumulation of TCR and related signaling molecules at the interface between any lymphoid cells (T, B or natural killer cells) [32, 33, 34] and APCs or target cells, and is not necessarily associated with any molecular segregation into c- and p-SMACs. Experimentally, it is necessary to analyze the X–Z axis of the contact interface to show such segregation; most analyses, however, show only the X–Y axis. A number of reports have

Biochemical versus imaging analyses of initial TCR signaling

The biochemical features of the initial events that occur upon TCR activation have been analyzed extensively. Upon Ag recognition, Lck, an Src-family tyrosine kinase, is first activated to phosphorylate immunotyrosine-based activation motif (ITAM) present within the cytoplasmic regions of TCR-associated CD3 chains. Tyrosine kinase ZAP-70 is then recruited to the phosphorylated CD3ζ–ITAMs through its tandem SH2 domains, and is activated to phosphorylate several adaptor proteins that are crucial

TCR cluster formation prior to immunological synapse formation

It has been acknowledged that small TCR clusters are formed prior to IS formation. Krummel et al. [46] were the first to observe small dispersed clusters of CD3ζ and CD4 using green fluorescent protein (GFP)-fusion proteins, which appeared at the same time as initial Ca2+ responses upon cell–cell contact between T-cell clones and APCs. These clusters were unstable and eventually formed c-SMACs. Therefore, the initial T-cell activation appeared to be correlated not with c-SMACs but with

Microcluster formation and initial T-cell activation

The more precise regulation of MC-mediated initial activation upon TCR engagement was recently analyzed using a combination of the supported planar bilayer system and total internal reflection fluorescence (TIRF) microscopy [50••, 51••]. Because TIRF microscopy can analyze fluorescence exclusively on the plasma membrane with high resolution using evanescence, it is ideal for the analysis of IS formation. The supported planar bilayer system contains glycosylphosphatidylinositol (GPI)-anchored

Maintenance of sustained T-cell receptor signals

Although initial T-cell activation signals including tyrosine phosphorylation, Ca2+ influx and PI turnover are induced within a few minutes, continuous stimulation of T cells for at least several hours is required for the final induction of T-cell activation, such as cytokine production and proliferation, before T cells commit to an activation program. Although the exact length of time required for commitment of T cells to activation is still a matter of debate, it is generally accepted that

Lipid-raft clusters and T-cell receptor microclusters

The function and the dynamic movement of lipid rafts for T-cell activation have been the subject of extensive debate. Biochemical analyses clearly showed that many signaling molecules critical for T-cell activation, such as Lck and LAT, are localized in the detergent-insoluble fraction of the plasma membrane, namely, the lipid raft. It has been widely thought that lipid raft plays crucial roles as a platform for signal transduction by recruiting various signaling molecules.

It is suggested that

Function of c-SMAC

The finding that MCs are the site for initial and sustained TCR signaling leads us to re-evaluate the function of c-SMACs. The following is a summary of the current possibilities of their function, which are not necessarily mutually exclusive (Figure 2).

First, c-SMACs are proposed to be the site for degradation of the TCR complex and signaling molecules by which T-cell activation is balanced, on the basis of the finding that CD2-AP-deficient T cells failed to make c-SMACs showed increased

Conclusions

Imaging analyses have now identified TCR-MCs (but not c-SMACs) to be the sites for antigen recognition and TCR-mediated activation on the plasma membrane of T cells upon TCR engagement. MCs are crucial not only for the initial activation but also for sustained signals required for full activation of T cells. TCR-MCs accumulate into the center of the interface between T cells and APC to form c-SMAC, whereas tyrosine kinases and adaptors do not accumulate in c-SMAC and dissociate from TCR-MCs

Update

A recent study has unveiled the function of Wave2 protein complex in calcium entry [66]. Wave2 complex is recruited to the IS for the actin reorganization and cell–cell adhesion upon TCR stimulation, and regulates calcium entry via calcium release-activated calcium channel. However, this mechanism was shown to be independent of phospholipase Cγ1 (PLCγ1) activation. Samelson and co-workers [67] demonstrated that the two SH2 domains and the one SH3 domain of PLCγ1 were necessary for its

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank M Tokunaga, K Sakata-Sogawa, R Varma, G Campi, ML Dustin, S Yamasaki and A Hashimoto-Tane for collaboration and discussion as well as for giving permission to describe data prior to publication, W Kobayashi for technical help, and H Yamaguchi for secretarial assistance.

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