ARTICLES Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76 Tadashi Yokosuka1, Kumiko Sakata-Sogawa2, Wakana Kobayashi1, Michio Hiroshima2, Akiko Hashimoto-Tane1, Makio Tokunaga2–4, Michael L Dustin5 & Takashi Saito1 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 http://www.nature.com/natureimmunology 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. Immune responses are triggered by the activation of T cells after However, the initial activation of T cells, including tyrosine phos- recognition of a specific antigen, which is mediated by the interaction phorylation, calcium mobilization and phosphoinositide metabolism, between antigen-presenting cells (APCs), such as dendritic cells, and occurs much earlier than immunological synapse formation10–13. antigen-specific T cells. Such interactions takes place through the Indeed, phosphorylation of the Src tyrosine kinases Lck and Zap70 10 2005 Nature Publishing Group Group 2005 Nature Publishing formation of a specific structure at the interface: the ‘immunological occurs earlier than mature immunological synapse formation . © synapse’1–3. Within 10–15 min of cell-cell contact, dynamic rearrange- Analysis of CD2AP-deficient mice has similarly indicated that ment occurs, accompanied by protein organization of both cell surface T cell activation is induced in the absence of mature immuno- and membrane-associated intracellular molecules. Immunological logical synapses14. Furthermore, partial agonist peptides can trigger synapse formation is dependent on T cell receptor (TCR)–mediated T cells in the absence of mature immunological synapses11,15.Thus, signaling that induces the cellular organization of cytoskeleton and immunological synapse formation may not be required for initial receptors and has effector functions4,5. For example, TCR stimulation T cell activation. induces specific segregation of surface molecules: the TCR-CD3 com- In contrast, the signaling pathways induced after TCR stimulation plex accumulates at the center of the interface as the central supra- have been analyzed extensively by biochemical and genetic molecular activation cluster (c-SMAC) together with other molecules, approaches. Antigen recognition by TCRs induces the phosphoryla- such as CD4-CD8, CD2 and CD28, whereas adhesion molecules such tion of the immunoreceptor tyrosine-based activation motif of as LFA-1 accumulate in a surrounding area called the peripheral CD3 molecules, particularly the CD3z chains, by Lck, which leads supramolecular activation cluster (p-SMAC). In addition, large mole- to recruitment of Zap70 to the phosphorylated immunoreceptor cules such as CD43 and CD45 are excluded from the immunological tyrosine-based activation motifs. Zap70 is activated and then phos- synapse and instead are located in the distal supramolecular activation phorylates several adaptor molecules, including two critical adaptors, cluster (d-SMAC)6–9. From these structural features of the immuno- LAT and SLP-76. The membrane adaptor molecule LAT, localized in logical synapse, particularly the accumulation of TCRs in the c-SMAC, lipid rafts, recruits SLP-76 through association with the adaptor it was thought that the c-SMAC mediates the antigen recognition and protein Gads. Phosphorylated LAT and SLP-76 then recruit many activation of T cells, whereas the p-SMAC serves to support cell ‘downstream’ signaling molecules, leading to the activation of adhesion and maintain the immunological synapse. various effector functions. 1Laboratory for Cell Signaling and 2Single-Molecule Immunoimaging, RIKEN Center for Allergy and Immunology, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. 3Structural Biology Center, National Institute of Genetics, and 4Department of Genetics, The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan. 5Program in Molecular Pathogenesis, Skirball Institute of Biomolecular Medicine and Department of Pathology, New York University School of Medicine, New York, New York 10021, USA. Correspondence should be addressed to T.S. ([email protected]). Received 11 July; accepted 6 October; published online 6 November 2005; doi:10.1038/ni1272 NATURE IMMUNOLOGY VOLUME 6 NUMBER 12 DECEMBER 2005 1253 ARTICLES a 10 s 20 s 30 s 40 s 50 s 60 s 150 s 300 s b CD3ζ Zap70 SLP-76 ζ CD3 0–75 s 0–75 s SLP-76 Zap70 CD3ζ Zap70 SLP-76 c 0 0 0 35–70 s 0–35 s 75–150 s 1 1 1 75–150 s 2 2 2 3 3 3 Time (min) 70–105 s 4 4 4 150–225 s 150–225 s After-contraction Contraction Expansion 5 5 5 Figure 1 Generation of microclusters of CD3z, Zap70 and SLP-76 throughout the cell-bilayer contact area in a spatio-temporal way. (a) AND-Tg T cells expressing EGFP–CD3z, EGFP–Zap70 or EGFP–SLP-76, plated on a planar bilayer containing unlabeled I-Ek and ICAM-1 (prepulsed with PCC(88–104)). Images were obtained at a video rate (30 frames/s) using TIRFM (times, above images). (b) Representative CD3z, Zap70 and SLP-76 microclusters tracked after cell-bilayer contact; paths of movement are presented separately in three phases: expansion (top), contraction (middle) and after contraction with http://www.nature.com/natureimmunology c-SMAC (bottom). Real-time images are available in Supplementary Video 1 (EGFP–CD3z), Supplementary Video 2 (EGFP–Zap70) and Supplementary Video 3 (EGFP–SLP-76) online. (c) Inward movement and accumulation or disappearance of CD3z, Zap70, or SLP-76 microclusters, presented as horizontal elements in kymographs. Scale bars, 5 mm. The dynamic recruitment of signaling molecules during the initial microclusters was necessary for inducing activation. After immuno- activation of T cells has been analyzed using Jurkat cells expressing logical synapse formation, signal-inducing microclusters were gener- various enhanced green fluorescent protein (EGFP) fusion proteins ated only at the periphery of the immunological synapse, which and stimulation with immobilized antibody to CD3 (anti-CD3)16. suggested that sustained activation signals were produced from the Those studies have demonstrated that small clusters containing TCR, microclusters at the periphery of the immunological synapse and not kinase and adaptor molecules are induced immediately after stimula- from the c-SMAC. tion of the TCR. However, stimulation with immobilized anti-CD3 limits analysis to the initial phase of activation, and thus the entire RESULTS 2005 Nature Publishing Group Group 2005 Nature Publishing process up to the formation of the immunological synapse in more Generation of TCR microclusters at the initial contact sites © physiological conditions remains unknown. The immunological Because we intended to analyze the microstructural basis of immu- synapse structure is necessary for the maintenance of sustained nological synapse formation, we used a combined system of a planar signals required for full T cell activation17. It is well known that bilayer that incorporated glycophosphatidylinositol (GPI)–anchored sustained signal transduction, including mitogen-activated protein intercellular adhesion molecule 1 (ICAM-1) and major histocompat- kinase activation, calcium mobilization and activation of Akt (protein ibility complex (MHC) class II (I-Ek) loaded with a pigeon cyto- kinase B), is required for T cell activation and induction of cytokine chrome c (PCC) peptide of amino acids 88–104 (PCC(88–104))18 and secretion and effector function. Immunological synapse structure is objective-type TIRFM to detect the dynamic recruitment of clustering maintained for hours17–20, and disruption of the immunological molecules after antigen stimulation of T cells. We obtained PCC(88– synapse immediately stops sustained signaling, which suggests that 104)-specific T cells from AND TCR-transgenic (AND-Tg) mice that the immunological synapse is required for the maintenance of had been activated in vitro and had been previously transduced by sustained signaling13,21–27. However, the actual physical location for EGFP-tagged CD3z, Zap70 and SLP-76 retroviruses and placed the the induction of such sustained signaling remains unclear. cells on a planar bilayer. Immunological synapse formation, visualized To analyze precisely and in real time the dynamic process of as TCR-enriched c-SMACs and LFA-1-enriched p-SMACs, as immunological synapse formation, particularly its relationship with described before1,2, occurred 5–10 min after cell-bilayer contact. the formation of the TCR signaling complex to induce activation When we analyzed c-SMACs in T cells expressing a fusion protein signals, we used a combined system of a planar bilayer for normal of