Regulation of Immunoglobulin Class Switch in Aged Mice

描述(申请人提供)：老年人和实验动物对感染性物质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的缺陷，产生的抗体亲和力较低，且具有自我反应性。虽然T细胞的问题肯定会导致这些B细胞在衰老过程中的缺陷，但在这一建议中显示了固有的B细胞变化。转换免疫球蛋白(Ig)重链类(同型)的能力对于Ig蛋白的正常效应功能至关重要，而人类进行类转换重组(CSR)能力的遗传缺陷会导致免疫缺陷，从而增加对许多病原体的易感性。这些研究将集中在导致老年小鼠Ig类开关减少的分子和细胞机制上。我们假设CSR的下降是激活诱导的胞苷脱氨酶AID下降的直接结果，AID反过来又受老年小鼠转录因子E47的下降调节。这项建议将在体外比较老年小鼠和年轻小鼠的脾B细胞E47、AID和类别转换异型(Ig G、Ig E、Ig A)的存在和功能，以及体内抗原特异性反应。 在特定目的1中，我们将扩展初步数据以建立老年BALB/c、C57BL/6和E2A/-小鼠Ig类开关中固有的脾B细胞缺陷。纯化的脾B细胞将被有丝分裂原或抗CD40及各种细胞因子和类开关激活，分泌型免疫球蛋白用ELISA法检测，细胞表面免疫球蛋白用荧光细胞术检测生殖系转录本(GLT)和转换后转录本(PST)的分子水平。将进行NP-KLH或NP-Ficoll(胸腺依赖和胸腺非依赖抗原)免疫以及将年轻和老年B/T细胞组合过继转移到SCID小鼠体内的实验。在特定的目标2中，我们将研究幼年和老年小鼠类转换的分子机制。E47、Id2、Notch、NFkappaB和AID在老年B细胞中的表达将被表征。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AID或E47来挽救老年B细胞的类转换。在具体目标3中，将建立调控E47和AID的分子机制。对蛋白质和mRNA稳定性、翻译后调节以及包括MAPK在内的信号转导途径的研究，将确定控制老年小鼠Ig类开关的关键分子元件。

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.