Regulation of immunoglobulin class switch in senescent humans

描述（由申请人提供）：老年人对传染性病原体、疫苗和癌症的免疫反应较差，这导致发病率和死亡率增加。老年人的抗体介导的体液免疫反应具有质量缺陷，产生亲和力较低的抗体，并增加自身反应性。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至关重要；患者的CSR基因缺陷会导致呼吸道感染、自身免疫和对疫苗接种没有反应等。这些研究将确定我们在小鼠年龄刺激的B淋巴细胞中看到的Ig类开关、AID（激活诱导胞苷脱氨酶）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。本研究将比较老年和年轻健康志愿者外周血B细胞中E47、AID和类转换同型（IgG、IgE、IgA）的存在和功能。在Specific Aim 1中，我们将确定衰老的人B细胞中的Ig类开关缺陷。将用抗CD40和IL-4、BAFF和IL-4或CpG和IL-4/IL-10刺激来自年轻和老年供者的外周血单核细胞（PBMC）纯化的B细胞，并通过荧光细胞术检测细胞表面Ig、ELISA检测分泌Ig和RT-PCR检测种系转录物（GLT）和环转录物（ct），检测类转换表型（IgG、IgE和IgA），表示CSR。在特异性目标2中，将研究调节年轻和老年人类B细胞的类转换的分子机制。E47， AID和NFKB在刺激富集的B细胞中的表达将被表征。在Specific Aim 3中，我们将建立E47在人B细胞中的调控分子机制。对mRNA稳定性和包括MAPK在内的信号转导途径的研究将进行，并将有助于确定老年人Ig类开关调节的关键分子需求。本应用程序中的研究将使我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.