Tadashi Yokosuka

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

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.

Spatiotemporal Regulation of T Cell Costimulation by TCR-CD28 Microclusters and Protein Kinase C θ Translocation

T cell activation is mediated by microclusters (MCs) containing T cell receptors (TCRs), kinases, and adaptors. Although TCR MCs translocate to form a central supramolecular activation cluster (cSMAC) of the immunological synapse at the interface of a T cell and an antigen-presenting cell, the role of MC translocation in T cell signaling remains unclear. Here, we found that the accumulation of MCs at cSMAC was important for T cell costimulation. Costimulatory receptor CD28 was initially recruited coordinately with TCR to MCs, and its signals were mediated through the assembly with the kinase PKCtheta. The accumulation of MCs at the cSMAC was accompanied by the segregation of CD28 from the TCR, which resulted in the translocation of both CD28 and PKCtheta to a spatially unique subregion of cSMAC. Thus, costimulation is mediated by the generation of a unique costimulatory compartment in the cSMAC via the dynamic regulation of MC translocation.

A motif in the V3 domain of the kinase PKC-[theta] determines its localization in the immunological synapse and functions in T cells via association with CD28

Protein kinase C-θ (PKC-θ) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. We found that the PKC-θ V3 domain is necessary and sufficient for IS localization mediated by Lck-dependent association with CD28. We identified a conserved proline-rich motif in V3 required for CD28 association and IS localization. CD28 association was essential for PKC-θ-mediated downstream signaling and TH2 and TH17, but not TH1, differentiation. Ectopic V3 expression sequestered PKC-θ from the IS and interfered with its functions. These results identify a unique mode of CD28 signaling, establish a molecular basis for the IS localization of PKC-θ, and implicate V3-based “decoys” as therapeutic modalities for T cell-mediated inflammatory diseases. The induction of an immune response depends on effective communication between antigen-specific T cells and antigen presenting cells (APCs). When a T cell expressing a cognate T cell receptor (TCR) encounters an activated APC, both cells actively redistribute their receptors and ligands to the interface, creating a platform for effective signaling, known as the immunological synapse (IS). At steady state, the mature IS is composed of concentric rings with a central core (cSMAC) containing clusters of TCR and costimulatory molecules, and a peripheral ring (pSMAC) of adhesion molecules1. The engagement of these surface molecules triggers signaling cascades resulting in the recruitment of *Correspondence: Amnon Altman, Ph.D., Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, California 92037, Tel.: (858) 752 6808, FAX: (858) 752 6986, amnon@liai.org. AUTHOR CONTRIBUTIONS K.-F.K. and A.A. designed the experiments and wrote the manuscript. K.-F.K. generated and analyzed the data. T.S. and T.Y. provided their expertise in cell imaging, T.S. participated in discussion of the data, and T.Y. generated the PKC-GFP and CD28-CFP fusion vectors. N.I. participated actively in discussions leading to this work and in experimental design. A.J.C.-B. performed various experiments and animal work. COMPETING FINANCIAL INTERESTS The authors have no competing financial interests. NIH Public Access

Spatiotemporal Basis of CTLA-4 Costimulatory Molecule-Mediated Negative Regulation of T Cell Activation

T cell activation is positively and negatively regulated by a pair of costimulatory receptors, CD28 and CTLA-4, respectively. Because these receptors share common ligands, CD80 and CD86, the expression and behavior of CTLA-4 is critical for T cell costimulation regulation. However, in vivo blocking of CD28-mediated costimulation by CTLA-4 and its mechanisms still remain elusive. Here, we demonstrate the dynamic behavior of CTLA-4 in its real-time competition with CD28 at the central-supramolecular activation cluster (cSMAC), resulting in the dislocalization of protein kinase C-θ and CARMA1 scaffolding protein. CTLA-4 translocation to the T cell receptor microclusters and the cSMAC is tightly regulated by its ectodomain size, and its accumulation at the cSMAC is required for its inhibitory function. The CTLA-4-mediated suppression was demonstrated by the in vitro anergy induction in regulatory T cells constitutively expressing CTLA-4. These results show the dynamic mechanism of CTLA-4-mediated T cell suppression at the cSMAC.

The Immunological Synapse, TCR Microclusters, and T Cell Activation

T cell activation begins with the interaction between an antigen-specific T cell and an antigen-presenting cell (APC). This interaction results in the formation of the immunological synapse, which had been considered to be responsible for antigen recognition and T cell activation. Recent advances in imaging analysis have provided new insights into T cell activation. The T cell receptor (TCR) microclusters, TCRs, kinases, and adaptors are generated upon antigen recognition at the interfaces between the T cells and the APCs and serve as a fundamental signaling unit for T cell activation. CD28-mediated costimulation is also found to be regulated by the formation of microclusters. Therefore, the dynamic regulations of TCR and CD28 microcluster formation, migration, and interaction are the key events for the initiation of T cell-mediated immune responses. Comprehensive analyses of the composition and characteristics of the TCR microcluster have identified its dynamic features. This review will outline new discoveries of the microclusters and its related concept in T cell activation.

A Critical Role for the Innate Immune Signaling Molecule IRAK-4 in T Cell Activation

IRAK-4 is a protein kinase that is pivotal in mediating signals for innate immune responses. Here, we report that IRAK-4 signaling is also essential for eliciting adaptive immune responses. Thus, in the absence of IRAK-4, in vivo T cell responses were significantly impaired. Upon T cell receptor stimulation, IRAK-4 is recruited to T cell lipid rafts, where it induces downstream signals, including protein kinase Cθ activation through the association with Zap70. This signaling pathway was found to be required for optimal activation of nuclear factor κB. Our findings suggest that T cells use this critical regulator of innate immunity for the development of acquired immunity, suggesting that IRAK-4 may be involved in direct signal cross talk between the two systems.

Dynamic regulation of T-cell costimulation through TCR–CD28 microclusters

Summary:  T‐cell activation requires contact between T cells and antigen‐presenting cells (APCs) to bring T‐cell receptors (TCRs) and major histocompatibility complex peptide (MHCp) together to the same complex. These complexes rearrange to form a concentric circular structure, the immunological synapse (IS). After the discovery of the IS, dynamic imaging technologies have revealed the details of the IS and provided important insights for T‐cell activation. We have redefined a minimal unit of T‐cell activation, the ‘TCR microcluster’, which recognizes MHCp, triggers an assembly of assorted molecules downstream of the TCR, and induces effective signaling from TCRs. The relationship between TCR signaling and costimulatory signaling was analyzed in terms of the TCR microcluster. CD28, the most valuable costimulatory receptor, forms TCR–CD28 microclusters in cooperation with TCRs, associates with protein kinase C θ, and effectively induces initial T‐cell activation. After mature IS formation, CD28 microclusters accumulate at a particular subregion of the IS, where they continuously assemble with the kinases and not TCRs, and generate sustained T‐cell signaling. We propose here a ‘TCR–CD28 microcluster’ model in which TCR and costimulatory microclusters are spatiotemporally formed at the IS and exhibit fine‐tuning of T‐cell responses by assembling with specific players downstream of the TCR and CD28.

Predominant Role of T Cell Receptor (TCR)-α Chain in Forming Preimmune TCR Repertoire Revealed by Clonal TCR Reconstitution System

The CDR3 regions of T cell receptor (TCR)-α and -β chains play central roles in the recognition of antigen (Ag)-MHC complex. TCR repertoire is created on the basis of Ag recognition specificity by CDR3s. To analyze the potential spectrum of TCR-α and -β to exhibit Ag specificity and generate TCR repertoire, we established hundreds of TCR transfectants bearing a single TCR-α or -β chain derived from a cytotoxic T cell (CTL) clone, RT-1, specific for HIVgp160 peptide, and randomly picked up TCR-β or -α chains. Surprisingly, one-third of such TCR-β containing random CDR3β from naive T cells of normal mice could reconstitute the antigen-reactive TCR coupling with RT-1 TCR-α. A similar dominant function of TCR-α in forming Ag-specific TCR, though low-frequency, was obtained for lymphocytic choriomeningitis virus–specific TCR. Subsequently, we generated TCR-α and/or -β transgenic (Tg) mice specific for HIVgp160 peptide, and analyzed the TCR repertoire of Ag-specific CTLs. Similar to the results from TCR reconstitution, TCR-α Tg generated CTLs with heterogeneous TCR-β, whereas TCR-β Tg-induced CTLs bearing a single TCR-α. These findings of Ag recognition with minimum involvement of CDR3β expand our understanding regarding the flexibility of the spectrum of TCR and suggest a predominant role of TCR-α chain in determining the preimmune repertoire of Ag-specific TCR.

Protein kinase C-η controls CTLA-4–mediated regulatory T cell function

Regulatory T (Treg) cells, which maintain immune homeostasis and self-tolerance, form an immunological synapse (IS) with antigen-presenting cells (APCs). However, signaling events at the Treg cell IS remain unknown. Here we show that the kinase PKC-η associated with CTLA-4 and was recruited to the Treg cell IS. PKC-η–deficient Treg cells displayed defective suppressive activity, including suppression of tumor immunity but not of autoimmune colitis. Phosphoproteomic and biochemical analysis revealed an association between CTLA-4–PKC-η and the GIT2-αPIX-PAK complex, an IS-localized focal adhesion complex. Defective activation of this complex in PKC-η–deficient Treg cells was associated with reduced depletion of CD86 from APCs by Treg cells. These results reveal a CTLA-4–PKC-η signaling axis required for contact-dependent suppression and implicate this pathway as a potential cancer immunotherapy target.

The lymphoid lineage–specific actin-uncapping protein Rltpr is essential for costimulation via CD28 and the development of regulatory T cells

Although T cell activation can result from signaling via T cell antigen receptor (TCR) alone, physiological T cell responses require costimulation via the coreceptor CD28. Through the use of an N-ethyl-N-nitrosourea–mutagenesis screen, we identified a mutation in Rltpr. We found that Rltpr was a lymphoid cell–specific, actin-uncapping protein essential for costimulation via CD28 and the development of regulatory T cells. Engagement of TCR-CD28 at the immunological synapse resulted in the colocalization of CD28 with both wild-type and mutant Rltpr proteins. However, the connection between CD28 and protein kinase C-θ and Carma1, two key effectors of CD28 costimulation, was abrogated in T cells expressing mutant Rltpr, and CD28 costimulation did not occur in those cells. Our findings provide a more complete model of CD28 costimulation in which Rltpr has a key role.