Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76

Abstract

T cell receptor (TCR) activation and signaling precede immunological synapse formation and are sustained for hours after initiation. However, the precise physical sites of the initial and sustained TCR signaling are not definitively known. We report here that T cell activation was initiated and sustained in TCR-containing microclusters generated at the initial contact sites and the periphery of the mature immunological synapse. Microclusters containing TCRs, the tyrosine kinase Zap70 and the adaptor molecule SLP-76 were continuously generated at the periphery. TCR microclusters migrated toward the central supramolecular cluster, whereas Zap70 and SLP-76 dissociated from these microclusters before the microclusters coalesced with the TCR-rich central supramolecular cluster. Tyrosine phosphorylation and calcium influx were induced as microclusters formed at the initial contact sites. Inhibition of signaling prevented recruitment of Zap70 into the microclusters. These results indicated that TCR-rich microclusters initiate and sustain TCR signaling.

Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2

“…PD-1 was shown to accumulate at the immunological synapse in a linear shape depending on the affinity for its ligands (Pentcheva-Hoang et al, 2007). Meanwhile, we and others have revised the immunological synapse concept by identifying the TCR microcluster as a minimal activation unit for T cell activation (Bunnell et al, 2002;Campi et al, 2005;Yokosuka et al, 2005;Saito and Yokosuka, 2006). TCR microclusters contain most of the proximal TCR signaling molecules, for example, Zap70, SLP-76 (SH2 domain-containing leukocyte protein of 76 kD; Yokosuka et al, 2005), Vav1 (Miletic et al, 2006), and PLC1 (Braiman et al, 2006).…”

“…Meanwhile, we and others have revised the immunological synapse concept by identifying the TCR microcluster as a minimal activation unit for T cell activation (Bunnell et al, 2002;Campi et al, 2005;Yokosuka et al, 2005;Saito and Yokosuka, 2006). TCR microclusters contain most of the proximal TCR signaling molecules, for example, Zap70, SLP-76 (SH2 domain-containing leukocyte protein of 76 kD; Yokosuka et al, 2005), Vav1 (Miletic et al, 2006), and PLC1 (Braiman et al, 2006). Previous biochemical studies demonstrated that Zap70, PI3K, and PKC- could be the substrates of PD-1-mediated dephosphorylation in T cells (Sheppard et al, 2004).…”

“…The conventional idea has been that the immunological synapse is composed of a central supramolecular activation cluster (c-SMAC) containing the TCR/CD3-MHCp complex and a peripheral SMAC (p-SMAC) containing the adhesion molecules LFA-1 (leukocyte function-associated antigen 1) and intercellular adhesion molecule 1 (ICAM-1). Based on single cell imaging analysis of T cell signaling, we and others have revised this idea and proposed that T cell activation is induced by a minimal T cell activation unit, the TCR microcluster, which consists of TCRs and their proximal signaling molecules (Bunnell et al, 2002;Campi et al, 2005;Yokosuka et al, 2005;. We further found that T cell costimulation is also regulated based on the formation of microclusters.…”

Antigen-Dependent Integration of Opposing Proximal TCR-Signaling Cascades Determines the Functional Fate of T Lymphocytes

“…7). Upon interaction between a CTL and a target cell, TCR microclusters are formed, containing 10-100 TCRs (63,64). Ag recognition by TCRs induces the phosphorylation of the ITAM of CD3 molecules, particularly the CD3z chains, by Lck kinase, leading to recruitment of ZAP70 and the subsequent activation of LAT and SLP-76.…”

Testing the organization of the immunological synapse

“…The form of this synapse has also been extensively characterized at the level of molecular organization. When TCRs or pMHC complexes are attached to fluorophores, it is found that they first form dynamic and relatively small structures (clusters or 'microclusters') all over the contact area and perhaps predominantly at the outskirts of the synapse [2][3][4]. These ultimately coalesce [3,4] to a central spot and this spot corresponds to a structure first characterized by Kupfer and co-workers in fixed couples and termed the central 'supramolecular activating cluster' or cSMAC [5].…”