Regulation of immunoglobulin class switch in senescent humans

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DESCRIPTION (provided by applicant): Aged humans have poor immune responses to infectious agents, vaccines, and cancers, which contribute to increased morbidity and mortality. Aged individuals have a qualitatively deficient antibody mediated humoral immune response with the production of antibodies of lower affinity and with increased self reactivity. Class switch recombination (CSR) of the immunoglobulin (Ig) class (or isotype) is very important for the quality and effector functions of the immune response; genetic deficiencies of CSR in patients results in respiratory tract infections, autoimmunity and failure to respond to vaccination, among others. These studies will establish whether the decreases in Ig class switch, AID (activation-induced cytidine deaminase) and E2A we have seen in age-stimulated B lymphocytes in mice is recapitulated in humans, and will then focus on the molecular and cellular mechanisms responsible for their decrease. We hypothesize that there will be a decrease in human CSR as a direct result of a decrease in AID, which is regulated by the transcription factor, E47 in humans as in mice. This proposal will compare peripheral blood B cells from aged vs. young healthy human volunteers for the presence and function of E47, AID and class switched isotypes (IgG, IgE, IgA) in vitro. In Specific Aim 1, we will determine the Ig class switch defects in aged human B cells. Purified B cells from peripheral blood mononuclear cells (PBMC) from young and old donors will be stimulated with anti- CD40 and IL-4, BAFF and IL-4 or CpG and IL-4/IL-10, and class switched phenotypes (IgG, IgE, and IgA) assayed by fluorescent cytometry for cell surface Ig, ELISA for secreted Ig and RT-PCR for germline transcripts (GLT) and circle transcripts (CTs), denoting CSR. In Specific Aim 2, the molecular mechanisms regulating class switch in young and old human B cells will be investigated. E47, AID, and NFKB expression in stimulated enriched B cells will be characterized. In Specific Aim 3, the molecular mechanisms for regulation of E47 in human B cells will be established. Studies of mRNA stability and signal transduction pathways including MAPK will be performed and will help to determine key molecular requirements for the regulation of Ig class switch in aged humans. The studies in this application will allow a better understanding, at the cellular and molecular levels, of the immune deficits in aged human subjects and will lead to improvement of these for immune response and vaccine development in the elderly population.