Regulation of immunoglobulin class switch in senescent humans

描述(由申请人提供)：老年人对感染性物质、疫苗和癌症的免疫反应较差，这些因素导致发病率和死亡率增加。老年人存在抗体介导的体液免疫反应的定性缺陷，产生的抗体亲和力较低，自身反应性增强。免疫球蛋白(Ig)类(或同型)的类开关重组(CSR)对免疫应答的质量和效应功能非常重要，患者CSR的遗传缺陷会导致呼吸道感染、自身免疫和疫苗接种失败等。这些研究将确定我们在年龄刺激的小鼠B淋巴细胞中看到的Ig类开关、AID(激活诱导型胞苷脱氨酶)和E2A的减少是否在人类中重现，然后将重点放在导致它们减少的分子和细胞机制上。我们假设人类CSR的减少将是AID减少的直接结果，AID在人类和小鼠中都是由转录因子E47调节的。这项建议将在体外比较老年和年轻健康志愿者的外周血B细胞E47、AID和类别转换同型(Ig G、Ig E、Ig A)的存在和功能。在特定的目标1中，我们将确定老年人B细胞中的Ig类开关缺陷。用抗CD40和IL-4、BAFF和IL-4或CpG和IL-4/IL-10刺激青年和老年供者外周血单个核细胞(PBMC)，用荧光细胞仪检测细胞表面Ig、EL ISA检测分泌Ig，用RT-PCR检测生殖系转录本(GLT)和循环转录本(CTs)，检测类转换表型(Ig、Ig E和Ig A)。在具体目标2中，我们将研究年轻人和老年人B细胞中调节类转换的分子机制。E47、AID和NFKB在刺激的浓缩B细胞中的表达将被表征。在具体目标3中，将建立人B细胞中E47调控的分子机制。将进行包括MAPK在内的mRNA稳定性和信号转导途径的研究，这将有助于确定调节老年人Ig类开关的关键分子需求。这项应用中的研究将使人们在细胞和分子水平上更好地了解老年人的免疫缺陷，并将有助于改善老年人的免疫反应和疫苗开发。

DESCRIPTION (provided by applicant): Aged humans have poor immune responses to infectious agents, vaccines, and cancers, which contribute to increased morbidity and mortality. Aged individuals have a qualitatively deficient antibody mediated humoral immune response with the production of antibodies of lower affinity and with increased self reactivity. Class switch recombination (CSR) of the immunoglobulin (Ig) class (or isotype) is very important for the quality and effector functions of the immune response; genetic deficiencies of CSR in patients results in respiratory tract infections, autoimmunity and failure to respond to vaccination, among others. These studies will establish whether the decreases in Ig class switch, AID (activation-induced cytidine deaminase) and E2A we have seen in age-stimulated B lymphocytes in mice is recapitulated in humans, and will then focus on the molecular and cellular mechanisms responsible for their decrease. We hypothesize that there will be a decrease in human CSR as a direct result of a decrease in AID, which is regulated by the transcription factor, E47 in humans as in mice. This proposal will compare peripheral blood B cells from aged vs. young healthy human volunteers for the presence and function of E47, AID and class switched isotypes (IgG, IgE, IgA) in vitro. In Specific Aim 1, we will determine the Ig class switch defects in aged human B cells. Purified B cells from peripheral blood mononuclear cells (PBMC) from young and old donors will be stimulated with anti- CD40 and IL-4, BAFF and IL-4 or CpG and IL-4/IL-10, and class switched phenotypes (IgG, IgE, and IgA) assayed by fluorescent cytometry for cell surface Ig, ELISA for secreted Ig and RT-PCR for germline transcripts (GLT) and circle transcripts (CTs), denoting CSR. In Specific Aim 2, the molecular mechanisms regulating class switch in young and old human B cells will be investigated. E47, AID, and NFKB expression in stimulated enriched B cells will be characterized. In Specific Aim 3, the molecular mechanisms for regulation of E47 in human B cells will be established. Studies of mRNA stability and signal transduction pathways including MAPK will be performed and will help to determine key molecular requirements for the regulation of Ig class switch in aged humans. The studies in this application will allow a better understanding, at the cellular and molecular levels, of the immune deficits in aged human subjects and will lead to improvement of these for immune response and vaccine development in the elderly population.