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

Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Tsurumi-ku, Yokohama 230-0045, Japan.
Immunity (Impact Factor: 21.56). 11/2008; 29(4):589-601. DOI: 10.1016/j.immuni.2008.08.011
Source: PubMed


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.

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    • "CD28 is a co-receptor that is engaged by ligands CD80 and CD86 on antigen presenting cells (APCs) [4] [5]. The activation process is accompanied by the clustering of CD28 in a manner that regulates the localization of protein kinase C theta, a marker for the formation of central supramolecular activation complex (cSMAC) [6] [7] [8]. CD28 is required for productive T-cell responses and survival [5,9–12,60]. "
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    ABSTRACT: The transcription factor NF-κB is needed for the induction of inflammatory responses in T-cells. Whether its activation by the antigen-receptor and CD28 is mediated by the same or different intracellular signaling pathways has been unclear. Here, using T-cells from various knock-out (Cd28−/−, adap−/−) and knock-in (i.e. Cd28 Y-170F) mice in conjunction with transfected Jurkat T-cells, we show that the TCR and CD28 use distinct pathways to activate NF-κB in T-cells. Anti-CD28 ligation alone activated NF-κB in primary and Jurkat T-cells as measured by NF-κB reporter and EMSA assays. Anti-CD28 also activated NF-κB normally in primary T-cells from adap−/− mice, while anti-CD3 stimulation required the adaptor ADAP. Over-expression of ADAP or its binding partner SKAP1 failed to enhance anti-CD28 activation of NF-κB, while ADAP greatly increased anti-CD3 induced NF-κB activity. By contrast, CD28 activation of NF-κB depended on GRB-2 binding to CD28 as seen in CD28 deficient Jurkat T-cells reconstituted with the CD28 YMN-FM mutant, and in primary T-cells from CD28 Y170F mutant knock-in mice. CD28 associated with GRB-2, and GRB-2 siRNA impaired CD28 NF-κB activation. GRB-2 binding partner and guanine nucleotide exchange factor, VAV1, greatly enhanced anti-CD28 driven activation of NF-κB. Further, unlike in the case of anti-CD28, NF-κB activation by anti-CD3 and its cooperation with ADAP was strictly dependent on LAT expression. Overall, we provide evidence that CD28 and the TCR complex regulate NF-κB via different signaling modules of GRB-2/VAV1 and LAT/ADAP pathways respectively.
    Full-text · Article · Oct 2014 · Immunology Letters
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    • "Stimulation of 5C.C7 T cells on bilayers containing pMHC, ICAM-1, and the CD28 ligand B7.1 induced the recruitment of full-length PKCθ into centrally located microclusters (Figure 3A, B). Consistent with previous reports [3], the formation of these microclusters was strictly dependent on the presence of B7.1. Neither θV3 nor εV3, however, formed microclusters in this manner (Fig. 3A, B). "
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    ABSTRACT: The immunological synapse (IS) formed between a T cell and its cognate antigen-presenting cell (APC) enables the directional secretion of cytolytic and inflammatory molecules. Synaptic architecture is established in part by a two-step cascade of novel protein kinase C (nPKC) isozymes. PKCε and PKCη arrive at the IS first, and occupy the entire synaptic membrane. Then, PKCθ accumulates in a smaller zone at the center of the contact. We investigated the molecular basis for this differential recruitment behavior using chimeric nPKC constructs and total internal reflection fluorescence microscopy. Our studies revealed that the V3 linker just N-terminal to the kinase domain plays a crucial role in specifying nPKC localization. Substitution of this linker switched the scope and the kinetics of PKCθ accumulation to that of PKCε and PKCη, and vice versa. Although the V3 was necessary for synaptic compartmentalization, it was not sufficient, as the tandem C1 domains were also required to mediate membrane association. Together, these results suggest a model whereby the V3 linker controls nPKC sub-compartmentalization after initial C1 domain-mediated accumulation at the IS.
    Full-text · Article · Apr 2014 · PLoS ONE
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    • "T cell signaling is initiated and sustained by the formation of TCR microclusters that form at the periphery of the contact site and move to and coalesce within the center of the immunological synapse [7]. TCR and CD28 are rapidly colocalized in these microclusters [8]. Although the precise mechanisms of CD28 costimulation are not fully understood, CD28 functions in part through direct amplification of the TCR signal, for example though activation of PI3K and Lck [6], and through unique contributions, notably the recruitment of PKCθ [9], [10], [11], [12], [13]. "
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    ABSTRACT: T cell activation takes place in the context of a spatial and kinetic reorganization of cell surface proteins and signaling molecules at the contact site with an antigen presenting cell, termed the immunological synapse. Coordination of the activation, recruitment, and signaling from T cell receptor (TCR) in conjunction with adhesion and costimulatory receptors regulates both the initiation and duration of signaling that is required for T cell activation. The costimulatory receptor, CD28, is an essential signaling molecule that determines the quality and quantity of T cell immune responses. Although the functional consequences of CD28 engagement are well described, the molecular mechanisms that regulate CD28 function are largely unknown. Using a micropipet adhesion frequency assay, we show that TCR signaling enhances the direct binding between CD28 and its ligand, CD80. Although CD28 is expressed as a homodimer, soluble recombinant CD28 can only bind ligand monovalently. Our data suggest that the increase in CD28-CD28 binding is mediated through a change in CD28 valency. Molecular dynamic simulations and in vitro mutagenesis indicate that mutations at the base of the CD28 homodimer interface, distal to the ligand-binding site, can induce a change in the orientation of the dimer that allows for bivalent ligand binding. When expressed in T cells, this mutation allows for high avidity CD28-CD80 interactions without TCR signaling. Molecular dynamic simulations also suggest that wild type CD28 can stably adopt a bivalent conformation. These results support a model whereby inside-out signaling from the TCR can enhance CD28 ligand interactions by inducing a change in the CD28 dimer interface to allow for bivalent ligand binding and ultimately the transduction of CD28 costimulatory signals that are required for T cell activation.
    Full-text · Article · Feb 2014 · PLoS ONE
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