Regulation of Immunoglobulin Class Switch in Aged Mice

描述（由申请方提供）：老年人和实验动物对传染性病原体和疫苗的免疫应答较差。抗体介导的体液免疫应答在老年人中是定性缺陷的，产生亲和力较低的抗体并具有自身反应性。虽然T细胞的问题肯定会导致这些B细胞在衰老中的缺陷，但内在的B细胞变化在这个提议中显示。转换免疫球蛋白（IG）重链类别（同种型）的能力对于IG蛋白的适当效应子功能是关键的，并且人类经历类别转换重组（CSR）能力的遗传缺陷导致免疫缺陷，对许多病原体的易感性增加。这些研究将集中在老年小鼠IG类转换减少的分子和细胞机制。我们假设CSR的减少是激活诱导的胞苷脱氨酶（AID）减少的直接结果，AID反过来又受转录因子E47减少的调节。该提议将比较来自老年与年轻小鼠的脾B细胞的体外E47、AID和类别转换同种型（IgG、IgE、伊加）的存在和功能以及体内抗原特异性应答。 在具体目标1中，我们将扩展初步数据，以确定老年BALB B/c、C57 BL/6和E2 A +/-小鼠中IG类别转换的内在脾B细胞缺陷。纯化的脾B细胞将用有丝分裂原或抗CD 40和各种细胞因子活化，并通过ELISA测量分泌的IG，通过检测生殖系转录物（GLT）和转换后转录物（PST），用荧光细胞术测量细胞表面IG的分子水平。将进行用NP-KLH或NP-Ficoll（胸腺依赖性和胸腺非依赖性抗原）免疫以及将年轻和年老B/T细胞的组合过继转移到SCID小鼠中的体内实验。在具体目标2中，将研究调节年轻和老年小鼠类别转换的分子机制。将表征老化B细胞中的E47、Id 2、Notch、NF κ B和AID表达。实验将包括染色质免疫沉淀（ChIP）与抗E2 A（用于AID基因），以及试图挽救类转换在老龄B细胞与逆转录病毒转导的AID或E47。在具体目标3中，将建立调节E47和AID的分子机制。对蛋白质和mRNA稳定性、翻译后调节和包括MAPK在内的信号转导途径的研究将确定老年小鼠中控制IG类转换的关键分子元件。

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.