Regulation of Immunoglobulin Class Switch in Aged Mice

老年人和实验动物对感染剂和疫苗的免疫反应很差。这个 抗体介导的体液免疫反应在老年人中存在质的缺陷，产生 亲和力较低且具有自身反应性的抗体。尽管T细胞的问题肯定会导致这些B细胞 衰老的缺陷，固有的B细胞变化，在这个提案中显示出来。转换免疫球蛋白(Ig)的能力 链类(同型)对于免疫球蛋白的正确效应功能和人类能力的遗传缺陷是至关重要的 经历类交换重组(CSR)导致免疫缺陷，增加对许多疾病的易感性 病原体。这些研究将集中在导致免疫球蛋白水平下降的分子和细胞机制上。 换入衰老的小鼠。我们假设CSR的减少是激活诱导的减少的直接结果 胞苷脱氨酶，AID，反过来由老年小鼠中转录因子E47的减少来调节。这 该计划将比较老年小鼠和年轻小鼠的脾B细胞中E47、AID和CLASS的存在和功能 在体外和体内的抗原特异性反应中，转换的同型(免疫球蛋白G、免疫球蛋白E、免疫球蛋白A)。 在特定的目标1中，我们将扩展初步数据以建立Ig类转换中固有的脾B细胞缺陷。 老龄BALB/c、C57BL/6和E2a/-小鼠。纯化的脾B细胞将被有丝分裂原或抗CD40和 用ELISA法测定分泌型Ig、荧光细胞仪测定细胞表面Ig 通过检测生殖系转录本(GLT)和转换后转录本(PST)的分子水平。活体实验： 胸腺依赖和非胸腺非依赖性抗原的免疫和过继转移 将年轻的和老年的B/T细胞组合成SCID小鼠。在特定目标2中，分子 将对幼龄和老年小鼠的等级转换调节机制进行研究。E47、Id2、Notch、NFicB和AID 老年B细胞的表达将被表征。实验将包括染色质免疫沉淀(CHIP)和 抗E2a(针对AID基因)以及试图通过逆转录病毒转导 救援或E47。在具体目标3中，将建立调控E47和AID的分子机制。研究 蛋白质和信使核糖核酸的稳定性，翻译后调节，以及包括MAPK在内的信号转导通路，将 确定老年小鼠免疫球蛋白类开关调控的关键分子元素。

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, NFicB 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. hi 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.