Receptor Mediated T and B Cell Activation

Division Of Basic Sciences - Nci

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Abstract

We investigated the function of tumor suppressor p53 in regulating proliferation and function of T lymphocytes. These studies elucidate a critical role of p53 as a negative regulator of T cell proliferation. It is the termination of p53 elevation by TCR signaling that allows proliferative responses to occur, enforcing antigen specificity. Preliminary studies of p53 effect on antigen-inexperienced and memory T cell repertoire have strongly suggested that p53 affects the threshold of TCR signaling required for activation of unprimed T cells by specific antigen, and their subsequent differentiation into memory T cells. p53 thus appears to be an important regulator of antigen-specific T cell activation and in vivo response, proliferation, and differentiation. T cell-dependent germinal center (GC) responses require coordinated interactions of T cells with two distinct antigen-presenting cell populations (APCs), B cells and dendritic cells (DCs), in the presence of B7- and CD40-dependent costimulatory pathways. Conventional models describe the expression of both of these costimulatory molecules on the same APC, both for T cell presentation and for cross-regulation of B7 and CD40 expression. Here, we report that, contrary to the conventional paradigm, cellular requirements for B7 and CD40 expression were distinct for GC TFH , GC B cell, and high affinity antibody responses. B7 expression was required on DCs but not on B cells, while CD40 was required on B cells but not DCs; and there was in fact no requirement for co-expression of B7 and CD40 on the same cell for GC responses. Our findings thus identify a much revised model for costimulatory function in the GC response, with crucial and distinct contributions of B7- and CD40-dependent pathways expressed by distinct APC populations. T cell-dependent germinal center (GC) responses require coordinated interactions of T cells with two distinct antigen-presenting cell populations (APCs)-B cells and dendritic cells (DCs)-in the presence of B7- and CD40-dependent costimulatory pathways. T-APC interactions with both populations are generally considered to depend on similar molecular mechanisms, including the involvement of these two costimulatory receptor-ligand pairs, but direct assessment of the role of each pathway in germinal center-dependent adaptive responses has not been conducted. Here we have utilized models that enable the selective elimination of CD28-B7 and CD40-CD40L signaling during T-DC vs. T-B antigen-driven interactions to probe this issue. In contrast to prevailing views that both pathways are critical for productive T-dependent humoral immunity at both the early (T-DC) and late (T-B) phases of the response, we found that the cellular requirements for B7 and CD40 expression were distinct. B7 expression was required on DCs but not on B cells, while CD40 was required on B cells but not DCs. These data emphasize the emerging evidence that distinct molecular rules apply to CD4+ T cell-myeloid cell and CD4+ T cell-lymphoid cell interactions, with important implications for understanding how to optimize or inhibit these events to promote vaccine responses or limit autoimmunity To further elucidate the role of CD40-CD40L interactions in multiple T cell-developmental and functional events, we have generated unique mouse genetic models. To analyze the role of signaling through CD40 on diverse cell populations, we have produced by CRISPR-Cas9 a set of CD40 cytoplasmic domain mutants that disrupt putative binding sites for TRAFs 2,3, and 6, and have generated initial data indicating differential dependence of these cytoplasmic domains for diverse functions, including B cell germinal center responses, Ig class switching, and affinity maturation; susceptibility to experimental autoimmune encephalomyelitis (EAE); in vivo T cell cytokine responses; and iNKT cell selection. Costimulatory CD40 plays an essential role in autoimmune disease models including EAE, a murine model of human multiple sclerosis (MS). However, the mechanism underlying CD40 function are not well defined in these processes. Conditional knockout of CD40 on either dendritic cells (DCs) or B cells led to profoundly reduced severity of EAE induced by recombinant human MOG (rhMOG). CD40 expression on DCs, but not on B cells, was required for priming of pathogenic T helper (Th) cells in peripheral draining lymph nodes and for appearance of these pathogenic T cells in the CNS. In marked contrast, CD40 on B cells, but not on DCs, was essential for class-switched MOG-specific antibody production. The distinct function of CD40 on B cells and DC was confirmed by the ability of transferred MOG-immune serum to restore sensitivity to EAE in mice lacking CD40 on B cells but not in mice lacking CD40 on DC. Thus, CD40 expressed on B cells and on DC provides distinct and complementary pathways essential for EAE pathogenesis, providing multiple targets for intervention in EAE, and potentially for MS and other autoimmune diseases. Our studies of EAE have identified requirements for CD40 expression by B cells and DC for distinct functions in disease induction. They have further identified requirements for distinct cytoplasmic domains of CD40 in EAE. To determine CD40L function, we are in the process of generating mutants that express only cell surface or only secreted forms of CD40L. Invariant natural killer T (iNKT) cells develop in the thymus, where iNKT cell development depends on TCR recognition of CD1d ligand on CD4/CD8 double positive thymocytes. We previously reported that B7-CD28 co-stimulation is required for thymic iNKT cell development, while underlying cellular and molecular mechanisms are largely not understood. Here we report the unexpected finding that CD28 expression on CD1d expressing antigen presenting T cells is required for thymic iNKT cell development. Mechanistically, antigen-presenting T cells provide costimulation through a novel mechanism, acquiring B7 molecule via CD28-dependent trogocytosis from B7-expressing thymic epithelial cell, DC and B cells and providing critical B7 co-stimulation to developing iNKT cells. Thus, these studies demonstrate a previously unappreciated mechanism of B7 co-stimulation in thymic T cell development by antigen-presenting T cells. We have most recently identified an additional mechanism of iNKT regulation, demonstrating a role for p53 selectively in generation of the iNKT17 subset. We have in addition initiated studies of generation of mucosal-associated invariant (MAIT) cells, another non-conventional T cell population with semi-invariant TCR expression. Initial findings include a functional role of B7 costimulation in thymic MAIT cell generation.