Word count: 7316 One sentence summary:Movement of sub-synaptic vesicles rich in LAT is constrained by protein microclusters at T cell immune synapses, dependent on LAT residues important for its binding to SLP-76 and consistent with a role for vesicular LAT in T cell signal transduction.

T-cell receptors (TCRs) undergo microclustering and supramolecular activation cluster formation at the immunological synapse (IS) during conjugation between T cells and antigen-presenting cells (APCs) (5,21,35). Microclustering can in turn activate GTP-binding proteins, protein kinases, phosphatases, and the phosphorylation of adaptor proteins. CD4-and CD8-p56lck activation leads to immunoreceptor tyrosinebased activation motif phosphorylation on the CD3 and TCR chains, the recruitment of ZAP-70 (zeta-associated proteintyrosine kinase of 70 kDa), and the activation of TEC kinases ITK/RLK (interleukin-2-inducible/resting lymphocyte kinase) (1,(31)(32)(33)41). Adaptor proteins possess binding sites and domains needed for complex-complex formation (31,32,41). Immune cell-specific adaptors include LAT (linker for activation of T cells), GADS (Grb-2-like adaptor downstream of Shc), SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), ADAP (adhesion-and degranulation-promoting adaptor protein; previously known as FYN T-binding protein/SLP-76-associated protein [FYB/SLAP]), and SKAP-55 (Src kinase-associated phosphoprotein of 55 kDa; also known as SCAP1) (3,23,24,26,31,32,(40)(41)(42). Phosphorylation of LAT recruits phospholipase C␥1, Grb-2, and GADS-SLP-76 and induces Ca 2ϩ mobilization and cytokine transcription (32,33,41,44).Binding of leukocyte function-associated antigen 1 (LFA-1; also known as CD11/CD18 or ␣ L ␤ 2 ) to intercellular adhesion molecules 1 and 2 (ICAM-1 and -2) on APCs mediates T-cell-APC conjugation (5,8,11,21,37). Following initial adhesion, TCR/CD3 ligation induces signals (i.e., "inside-out signaling") that further activate integrin adhesion (2,5,8,11,37). Conversion of LFA-1 to intermediate-or higher-affinity forms involves changes in conformation and receptor clustering (11,37). Multiple signaling proteins mediate this process. They include the GTP-binding protein Rap-1, its ligand RapL (regulator of cell adhesion and polarization enriched in lymphoid tissues), RIAM (Rap1-GTP-interacting adaptor molecule), the guanine nucleotide exchange factor Vav-1, and the adaptors ADAP,10,13,15,16,17,18,20,22,27,28,34). The protein-tyrosine kinase ZAP-70 phosphorylates YESP sites in SLP-76, which allows binding to the Src homology 2 (SH2) domain of Vav-1 (29), while the SH2 domain of SLP-76 binds to two YDDV sites in ADAP (30, 39). T-cell lines lacking SLP-76 show impaired superantigen-induced conjugation (44). ADAP is an immune cell-specific adaptor with a unique N-terminal region, a proline-rich region, a canonical and a noncanonical SH3 domain, one Ena/VASP homology 1 (EVH1) binding domain, and two putative nuclear localization motifs (3,4,19,26,31). ADAP is preferentially phosphorylated by the Src kinase p59fynT (4,25) and can cooperate with p59 fynT and SLP-76 in amplifying TCR-induced interleukin-2 (IL-2) transcription (30). The adaptor can up-regulate integrin-mediated adhesion in certain basophilic cell lines (9), while ADAP Ϫ/Ϫ T cells show profound defects in ␤1 and ␤2 integrin clusterin...

CTLA-4 is a co-receptor that modulates the threshold of T cell activation and autoimmunity. We previously showed that CTLA-4 reverses the TCR-mediated stop signal needed for T cell/APC interactions [Schneider et al., Science 2006, 313: 1972. In this study, using a different T cell system, we show that CTLA-4 expression changed the behavior of T8.1 T cells by reducing the contact time between T cell and APC, preventing re-inforced contacts, and reducing the contact area at the immunological synapse. This led to a major reduction in Ca 2+ influx/mobilization and interleukin-2 production. Further, anti-CD3/CTLA-4 increased T cell motility on antibody-coated glass slides, concurrent with an abrogation of ZAP70 microcluster formation. Our findings further support a role for CTLA-4 in limiting the interaction between T cell and APC that is needed for optimal activation.