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Principal Investigator: RICHARD J. HODES
Organization: DIVISION OF BASIC SCIENCES - NCI
Fiscal Year: 2021
Award: $706,073
Funding agency: National Cancer Institute
We investigated the function of tumor suppressor p53 in regulating proliferation and function of T lymphocytes. We have made the unexpected observation that antigen-specific proliferative responses of naive and memory CD4 T cells require the down-modulation of tumor suppressor p53. In the absence of TCR signal, IL-2 induces a sustained increase in p53 protein, which prevents proliferative responses despite strong signaling through the IL-2 receptor. In contrast, TCR signaling results in early termination of p53 protein expression by decreasing p53 mRNA as well as by strong transcriptional induction of the p53-regulating protein Mdm2. Down-modulation of p53 in response to antigen stimulation is in fact critical for antigen-specific T cell proliferation; and preventing p53 degradation by inhibiting Mdm2 results in sustained p53 protein levels and prevents antigen-specific T cell proliferation. 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, acquiringe B7 molecule via CD28-dependent trogocytosis from B7-expressing thymic epithelial cell, DC and B cells and providinge critical B7 co-stimulation to developing iNKT cells. Thus, the present study demonstrates a previously unappreciated mechanism of B7 co-stimulation in thymic T cell development by antigen-presenting T cells.
Terms: <Ab response><Affect><Affinity><Antibody Affinity><Antibody Formation><Antibody Production><Antibody Response><Antigen-Presenting Cells><Antigens><Antioncogene Protein p53><Antisera><Appearance><Autoimmune Diseases><Autoimmune Status><Autoimmunity><B blood cells><B cell><B cells><B-Cell Activation><B-Cells><B-Lymphocytes><B-cell><Binding Sites><Bp50><CD154><CD28><CD28 gene><CD4 Cells><CD4 Positive T Lymphocytes><CD4 T cells><CD4 helper T cell><CD4 lymphocyte><CD4+ T-Lymphocyte><CD4-Positive Lymphocytes><CD40><CD40L><CD40LG><CD8><CD8B><CD8B1><CD8B1 gene><CDW40><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cell Communication><Cell Communication and Signaling><Cell Interaction><Cell Signaling><Cell surface><Cell-Mediated Lympholytic Cells><Cell-to-Cell Interaction><Cells><Cellular Tumor Antigen P53><Class Switching><Class Switchings><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Co-Stimulator><Combining Site><Costimulator><Critical Paths><Critical Pathways><Cytolytic T-Cell><Cytoplasmic Domain><Cytoplasmic Tail><Cytotoxic T Cell><Cytotoxic T-Lymphocytes><Data><Dendritic Cells><Dependence><Development><Disease><Disease model><Disorder><Disseminated Sclerosis><EAE><Epidermal Thymocyte Activating Factor><Event><Experimental Allergic Encephalitis><Experimental Allergic Encephalomyelitis><Experimental Autoimmune Encephalitis><Experimental Autoimmune Encephalomyelitis><Gene Transcription><General Population><General Public><Generations><Genetic Models><Genetic Transcription><Germinal Center><HDM2><Helper Cells><Helper T-Cells><Helper T-Lymphocytes><Helper-Inducer T-Cells><Helper-Inducer T-Lymphocyte><High Affinity Interleukin-2 Receptor><Human><Humoral Immunities><IL-2><IL-2 Receptors><IL2 Protein><IL2 Receptors><Immune Sera><Immunoglobulin Class Switching><Immunoglobulin Class Switchings><Inducer Cells><Inducer T-Lymphocytes><Interleukin 2><Interleukin 2 Precursor><Interleukin 2 Receptor><Interleukin II><Interleukin-2><Interleukine 2><Interleukine 2 Precursor><Interleukine II><Intervention><Intervention Strategies><Intracellular Communication and Signaling><Isotype Switching><Isotype Switchings><LYT3><Ligands><Lymphocyte Mitogenic Factor><Lymphoid Cell><MDM2><MDM2 gene><MDMX protein><MGC9013><Mdm-2 protein><Mediating><Messenger RNA><Mice><Mice Mammals><Mitogenic Factor><Modeling><Modern Man><Molecular><Mouse Strains><Multiple Sclerosis><Murine><Mus><Myeloid Cells><Oncoprotein MDM2><Oncoprotein p53><P53><Pathogenesis><Pathogenicity><Pathway interactions><Peripheral><Phase><Phosphoprotein P53><Phosphoprotein pp53><Play><Population><Predisposition><Process><Protein TP53><RNA Expression><Reactive Site><Receptor Protein><Recombinants><Regulation><Reporting><Role><Severities><Signal Transduction><Signal Transduction Systems><Signaling><Specificity><Structure of germinal center of lymph node><Susceptibility><T cell growth factor><T memory cell><T-Cell Activation><T-Cell Development><T-Cell Growth Factor><T-Cell Growth Factor Receptors><T-Cell Ontogeny><T-Cell Proliferation><T-Cell Stimulating Factor><T-Cells><T-Lymphocyte><T-Lymphocyte Development><T4 Cells><T4 Lymphocytes><T44><TCGF Receptors><TNFRSF5><TNFRSF5 gene><TNFSF5><TNFSF5 gene><TP53><TP53 gene><TRAP Gene><TRP53><Thymic epithelial cell><Thymocyte Stimulating Factor><Thymus><Thymus Gland><Thymus Proper><Thymus Reticuloendothelial System><Transcription><Tumor Necrosis Factor Receptor Superfamily Member 5 Gene><Tumor Protein p53><Tumor Protein p53 Gene><Veiled Cells><accessory cell><activated B cells><antibody biosynthesis><antibody-based immunity><antigen antibody affinity><antigen-specific T cells><autoimmune condition><autoimmune disorder><autoimmune encephalomyelitis><biological signal transduction><conditional knock-out><conditional knockout><cytokine><developmental><disorder model><draining lymph node><experiment><experimental research><experimental study><human model><immune serum><immunogen><immunoglobulin biosynthesis><in vivo><insight><insular sclerosis><interventional strategy><killer T cell><mRNA><mdm-2 oncogene protein><mdm2 protein><memory CD4 T cell><memory CD4 T lymphocyte><memory T lymphocyte><model of human><mouse genetics><mouse model><murine model><mutant><novel><p50><p53 Antigen><p53 Genes><p53 Tumor Suppressor><p53-Binding Protein MDM2><pathway><prevent><preventing><protein expression><protein p53><receptor><regional lymph node><response><social role><thymocyte><thymus derived lymphocyte><vaccine response>