Thymus-dependent humoral immune responses are an essential component in host defense to disease. The initiating event in the development of a specific humoral immune response to an invading pathogen is the interaction between antigen-specific helper T cell (Th) and the antigen- presenting B cell. Subsequent to B cell antigen processing and presentation, Th are activated to express a membrane protein, gp39. This molecule is the ligand for CD40, a receptor on the surface of all mature B cells whose triggering results in profound B cell growth. Therefore, the interactions between gp39 and CD40 play an essential role in the initiation of TD humoral immune responses. The mechanisms by which antigen, tolerogen, lymphokines and co-stimulatory molecules regulate the expression of gp39 on activated Th and the mechanisms through which gp39 exerts its impact on humoral immunity are the focus of this proposal. It appears that for Th to express helper function, they must express a high density of gp39. Using ex vivo CD4+ cells, the role of activation, co-stimulatory molecules and lymphokines in the high density expression of gp39 will be determine. In addition, the high density expression of gp39 on discrete subsets (virgin, memory) of DC4+ cells will be determined. To more fully understand the mechanisms by which antigen regulates the expression of gp39, the requirements for antigen-driven expression of gp39 on Th clones and DC4+ T cells from TCR transgenic mice will be studied. Finally, a model system of polyclonal tolerance induction will be used to determine if tolerogens restrict the expression of gp39. Having developed a fundamental understanding of the parameters that govern gp39 expression, the effect of gp39-CD40 interactions on B cell activation will be studied in vitro. This component of the proposal will exploit the use of mabs specific to murine CD40 as well as recombinant forms of soluble and membrane-bound gp39. Using this battery of reagents that trigger through CD40, in combination with lymphokines, the requirements for inducing maximal B cell cycle entry, progression and differentiation will be determined. Specific attention will be focussed on the signals required to induce B cell growth, the expression of germline Ig transcripts and Ig isotype switching. Administration of anti-gp39 ablates primary and secondary immune responses in vivo. Experiments are proposed that will determine if gp39- CD40 interactions are critical to the generation and maintenance of B cell memory. Assuming that gp39-CD40 interactions mediate B memory cell generation, a novel in vitro system of B cell memory differentiation will be developed. Given the propensity of CD40 signalling to trigger the long-term growth of B cells, it is anticipated that under appropriate conditions, gp39 and lymphokines will induce the differentiation of resting B cells to memory B cells in vitro. Preliminary studies indicate that anti-gp39 can arrest the progression of collagen-induced arthritis in mice. Studies are proposed to investigate if the progression of autoimmune diseases also rely on gp39- CD40 interactions. Experimental allergic encephalomyelitis and the spontaneous autoimmune of NZB mice will be investigated to determine if disease progression is arrested by therapy with anti-gp39. Finally, give the profound B cell stimulatory signals which result from signalling via CD40, recombinant DNA technology will be used to construct chimeric proteins of gp39 that exert adjuvants effects of humoral immunity when co-administered with antigen or engineered as chimeric constructs containing antigenic epitopes. In summary, a new ligand-receptor pair, gp39-CD40, has been identified as mediating a critical event in the initiation of humoral immune responses. These studies will provide the fundamental knowledge which will allow the manipulation of gp39-CD40 interactions for the benefit of host protection against disease.
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