Regulation of B Lymphocyte Defects in Senescent Humans

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DESCRIPTION (provided by applicant): Our laboratory has previously demonstrated intrinsic B lymphocyte defects in aged mice and more recently elderly human subjects. The antibody-mediated humeral immune response is suboptimal in aged individuals with the generation of immunoglobulin (Ig) of lower affinity and self-reactivity and, as we have shown, a dramatic decrease in the ability of activated B cells to class switch their Ig. Class switch recombination (CSR) of the Ig class (or isotype) is very important for the quality and effector functions of the immune response; patients with a primary (genetic) immune deficiency in CSR have severe immune complications including respiratory tract infections, autoimmunity and failure to respond to vaccination. Our long-term objective is to improve the immune response in elderly humans. Specific Aim 1 asks: What are the molecular mechanisms which generate less Ig class switch in aged-activated human B cells? Sub aims are: a) Do various B cell stimuli generate equally suboptimal responses in aged human B cells? B cells will be stimulated either with anti-CD40/IL-4, CpG/IL-4, or BAFF/IL-4. Possible AID down regulation will be assessed by quantitative (q) PCR. We will measure circle transcripts (CT) by qPCR, Ig production by flow cytometry (for cell surface Ig) and ELISA (for secreted Ig) as well as AID. b) What molecular mechanisms contribute to the down regulation of AID in aged B cells? Transcription factors we have shown in mice to be down regulated in aged activated B cells, E47, NF-B, and Pax5 will be analyzed qPCR. IRF4, also known to be important for CSR, will also be measured. c) Can in vitro retroviral addition of E47 rescue AID and CSR? Retroviral constructs for E47 (and AID) will be used to up regulate AID (and CSR) in human B cell lines and primary B cells. In Specific Aim 2, we will determine the molecular mechanisms which down-regulate E47 in aged human B cells. Sub aims are to; a) establish whether the decrease in E47 in aging human B cells is due to decreased mRNA stability as we have seen previously in aged murine B cells; b) determine whether the decreased mRNA stability is due to proteins (e.g. tristetraprolin, TTP) binding to the 3' untranslated region (UTR); c) establish mechanisms of TTP regulation including MAPK, ERK and PI3K pathways; and d) determine microRNA involvement in E47 mRNA stability and aged B cell functions. Specific Aim 3 will investigate: Is a decreased antibody response to the influenza vaccine in elderly individuals a result of a suboptimal B cell response? a) What is the in vivo immune response in an age continuum of subjects given the influenza vaccine, and how is this associated with their B and T cell phenotypes? b) What is the specific in vitro B cell immune response in these subjects? PUBLIC HEALTH RELEVANCE: Aged humans have poor immune responses to infectious agents, vaccines, and cancers which contribute to increased morbidity and mortality. The studies in this application will reveal cellular and molecular deficits within the humeral immune response in aged human subjects and lead to improvement of these for better immune response and vaccine development in the elderly population.