Regulation of Immunoglobulin Class Switch in Aged Mice

描述（由申请人提供）：老年人和实验动物对传染源和疫苗的免疫反应较差。在老年人中，抗体介导的体液免疫反应在质量上存在缺陷，产生的抗体亲和力较低且具有自身反应性。尽管 T 细胞的问题肯定会导致衰老过程中的 B 细胞缺陷，但该提案显示了 B 细胞的内在变化。转换免疫球蛋白 (Ig) 重链类别（同种型）的能力对于 Ig 蛋白的正常效应功能至关重要，而人类进行类别转换重组 (CSR) 能力的遗传缺陷会导致免疫缺陷，并增加对许多病原体的易感性。这些研究将重点关注老年小鼠 Ig 类别转换减少的分子和细胞机制。我们假设 CSR 的降低是激活诱导的胞苷脱氨酶 AID 减少的直接结果，而 AID 反过来又受到老年小鼠转录因子 E47 减少的调节。该提案将比较老年小鼠和年轻小鼠的脾 B 细胞在体外以及体内抗原特异性反应中 E47、AID 和类别转换同种型（IgG、IgE、IgA）的存在和功能。 在具体目标 1 中，我们将扩展初步数据以确定老年 BALB/c、C57BL/6 和 E2A /- 小鼠 Ig 类别转换中的内在脾 B 细胞缺陷。纯化的脾 B 细胞将用有丝分裂原或抗 CD40 和各种细胞因子激活，并通过 ELISA 测量分泌的 Ig，通过检测种系转录物 (GLT) 和转换后转录物 (PST) 进行细胞表面 Ig 的荧光细胞分析。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 细胞组合过继转移到 SCID 小鼠体内的体内实验。在具体目标2中，将研究调节年轻和年老小鼠类别转换的分子机制。将表征衰老 B 细胞中的 E47、Id2、Notch、NFkappaB 和 AID 表达。实验将包括使用抗 E2A（针对 AID 基因）的染色质免疫沉淀 (ChIP)，以及尝试通过逆转录病毒转导 AID 或 E47 来挽救衰老 B 细胞的类别转换。具体目标3将建立E47和AID调节的分子机制。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 类别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.