Regulation of Immunoglobulin Class Switch in Aged Mice

老年人和实验动物对传染性病原体和疫苗的免疫反应较差。的 抗体介导的体液免疫应答在老年个体中是定性缺陷的， 低亲和力和自身反应性的抗体。虽然T细胞的问题肯定会导致这些B细胞 衰老的缺陷，内在的B细胞的变化显示在这个提议中。转换免疫球蛋白（IG）重链的能力 链类（同种型）对于IG蛋白的适当效应子功能和人类免疫功能的遗传缺陷是关键的。 进行类别转换重组（CSR）导致免疫缺陷，增加了对许多疾病的易感性。 病原体这些研究将集中在负责IG类减少的分子和细胞机制 在老年小鼠中转换。我们假设CSR的降低是激活诱导的细胞凋亡减少的直接结果。 胞苷脱氨酶，AID，这反过来又受到老年小鼠转录因子E47减少的调节。这 一项提案将比较老年与年轻小鼠脾B细胞中E47、AID和类E47的存在和功能。 转换同种型（IgG，IgE，IgA）在体外以及在抗原特异性反应在体内。 在具体目标1中，我们将扩展初步数据，以建立内在脾B细胞缺陷的IG类转换， 老年BALB/c、C57BL/6和E2A +/-小鼠。纯化的脾B细胞将用有丝分裂原或抗CD40活化， 通过ELISA测量分泌型IG的各种细胞因子和类别转换，通过荧光细胞术测量细胞表面IG 通过检测生殖系转录物（GLT）和转换后转录物（PST）在分子上进行。体内实验， 用NP-KLH或NP-Ficoll（胸腺依赖性和胸腺非依赖性抗原）免疫和过继转移 将年轻和年老的B/T细胞组合植入SCID小鼠。在具体目标2中， 将研究调节年轻和老年小鼠中类别转换的机制。E47、Id2、Notch、NFicB和AID 将表征老化B细胞中的表达。实验将包括染色质免疫沉淀（ChIP）， 抗E2 A（针对AID基因）以及尝试用逆转录病毒转导的方法挽救衰老B细胞中的类别转换。 AID或E47。在具体目标3中，将建立用于调节E47和AID的分子机制。研究 蛋白质和mRNA的稳定性，翻译后调节和信号转导途径，包括MAPK，将 确定控制老年小鼠IG类转换的关键分子元件。

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.