Regulation of Immunoglobulin Class Switch in Aged Mice

描述（由申请人提供）：老年人类和实验动物对传染性病原体和疫苗的免疫反应较差。抗体介导的体液免疫反应在老年人中存在质量缺陷，产生的抗体亲和力较低，具有自身反应性。尽管T细胞的问题肯定会导致B细胞在衰老过程中的缺陷，但本研究显示了B细胞的内在变化。切换免疫球蛋白（Ig）重链类（同型）的能力对于Ig蛋白的适当效应功能至关重要，而人类进行类开关重组（CSR）能力的遗传缺陷导致免疫缺陷，并增加对许多病原体的易感性。这些研究将集中在老龄小鼠Ig类转换减少的分子和细胞机制上。我们假设CSR的减少是由激活诱导的胞苷脱氨酶（AID）减少的直接结果，而AID反过来又受衰老小鼠中转录因子（E47）减少的调节。本研究将比较老年小鼠和年轻小鼠脾B细胞在体外以及体内抗原特异性反应中E47、AID和类转换同型（IgG、IgE、IgA）的存在和功能。

DESCRIPTION (provided by applicant): Aged humans and experimental animals have a poor immune response to infectious agents and vaccines. The antibody-mediated humoral immune response is qualitatively deficient in aged individuals with the production of antibodies of lower affinity and with self reactivity. Although problems with T cells certainly contribute to these B cell deficits in aging, intrinsic B cell changes are shown in this proposal. The ability to switch immunoglobulin (Ig) heavy chain class (isotype) is critical for proper effector functions of the Ig protein and genetic defects in the ability of humans to undergo class switch recombination (CSR) result in immunodeficiency with increased susceptibility to many pathogens. These studies will focus on the molecular and cellular mechanisms responsible for the decrease in Ig class switch in aged mice. We hypothesize that the decrease in CSR is a direct result of a decrease in the activation-induced cytidine deaminase, AID, which in turn is regulated by a decrease in the transcription factor, E47, in aged mice. This proposal will compare splenic B cells from aged vs. young mice for the presence and function of E47, AID and class switched isotypes (IgG, IgE, IgA) in vitro as well as in antigen-specific responses in vivo. In Specific Aim 1 we will extend preliminary data to establish the intrinsic splenic B cell defect in Ig class switch in aged BALB/c, C57BL/6 and E2A+/- mice. Purified splenic B cells will be activated with mitogens or anti-CD40 and various cytokines and class switch measured by ELISA for secreted Ig, fluorescent cytometry for cell surface Ig molecularly by detection of germ line transcripts (GLT) and post-switch transcripts (PST). In vivo experiments with immunization with NP-KLH or NP-Ficoll (thymus-dependent and thymus-independent antigens) and adoptive transfer of combinations of young and old B/T cells into SCID mice will be done. In Specific Aim 2, the molecular mechanisms regulating class switch in young and old mice will be investigated. E47, Id2, Notch, NFkappaB and AID expression in aged B cells will be characterized. Experiments will include chromatin immunoprecipitation (ChIP) with anti-E2A (for the AID gene) as well as attempts to rescue class switch in aged B cells with retroviral transduction of AID or E47. In Specific Aim 3, the molecular mechanisms for regulation of E47 and AID will be established. Studies of protein and mRNA stability, post translational regulation, and signal transduction pathways including MAPK, will determine key molecular elements in the control of Ig class switch in aged mice.