The goal of this application is to gain insight into the mechanisms underlying immunoglobulin (Ig) gene somatic hypermutation. Ig hypermutation is central to the affinity maturation of antibodies to exogenous antigens, such as those on infectious pathogens, and autoantibodies to self-antigens in autoimmune diseases, including systemic lupus erythematosus. We hypothesize that the Ig somatic hypermutation process: (i) displays well defined induction requirements, in which triggering of the surface B cell receptor for antigen (BCR) and engagement of the co-stimulatory molecules CD40 and CD80/CD86 by the specific counter-receptors CD154 and CD28/CTA-4 (CD154) on T lymphocytes and other cells, play a central role, (ii) depends on the transcription of the V(D)J DNA segment and involves the BCR-induced down-regulation of the lesion bypass DNA polymerase (pol) eta, which can carry out short-patch DNA repair with a relatively high fidelity, possibly concomitant with an up-regulation of the error-prone pol zeta and, perhaps, the other recently reported error-prone polymerases, including mu, iota and kappa, which would be recruited to repair the DNA nicked by the postulated """"""""mutator factor""""""""; (iii) relies on the presence of the H chain intronic enhancer and its flanking matrix attachment regions; (iv) is specifically mediated by trans-factors induced by signals emanating from the BCR and the co-stimulatory molecules CD80 and/or CD86; and, finally, (v) is similar in induction requirements and modalities of expression in lupus and normal B cells. To test these hypotheses, and to define the molecular and cellular triggers, the mechanisms, and the modalities of expression of Ig hypermutation in human B cells, we will capitalize on the findings and the reagents generated in the previous funding period of this grant. By using a monoclonal model of germinal center B cell differentiation, human IgM+, IgD+ CL-01 lymphocytes, as well as IgM+, IgD+ B cells freshly isolated from healthy subjects and lupus patients, the investigator proposes to (i) define the minimal requirements for and the role of dendritic cells in the induction of somatic hypermutation; (ii) analyze the DNA strand that is being mutated, analyze the BCR-dependent modulation of the expression levels of pol h, pol z, m, i and k, and the concomitant error-prone DNA polymerase activity in hypermutating B cells; (iii) define the cis regulatory elements and the (iv) trans-factors involved in the hypermutation process; and, finally, (v) determine the induction requirements and the modalities of Ig somatic hypermutation in lupus B cells. The proposed experiments are designed to provide important new clues to the understanding of the affinity maturation of the antibody and autoantibody response in health and disease.
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