Regulation of immunoglobulin class switch in senescent humans

描述（由申请方提供）：老年人对传染性病原体、疫苗和癌症的免疫应答较差，这导致发病率和死亡率增加。老年个体具有质量缺陷的抗体介导的体液免疫应答，产生亲和力较低的抗体并具有增加的自身反应性。免疫球蛋白（IG）类别（或同种型）的类别转换重组（CSR）对于免疫应答的质量和效应子功能非常重要;患者中CSR的遗传缺陷导致呼吸道感染、自身免疫和对疫苗接种应答失败等。这些研究将确定我们在年龄刺激的小鼠B淋巴细胞中观察到的IG类转换、AID（活化诱导的胞苷脱氨酶）和E2 A的减少是否在人类中重演，然后将关注导致其减少的分子和细胞机制。我们假设人类CSR的减少是AID减少的直接结果，AID在人类和小鼠中受转录因子E47的调节。本提案将在体外比较来自老年与年轻健康人类志愿者的外周血B细胞中E47、AID和类别转换同种型（IgG、IgE、伊加）的存在和功能。在具体目标1中，我们将确定老年人B细胞中的IG类转换缺陷。将用抗-CD 40和IL-4、BAFF和IL-4或CpG和IL-4/IL-10刺激来自年轻和老年供体的外周血单核细胞（PBMC）的纯化B细胞，并通过荧光细胞术测定细胞表面IG的类别转换表型（IgG、IgE和伊加），通过ELISA测定分泌型IG，通过RT-PCR测定生殖系转录物（GLT）和环状转录物（CT），表示CSR。在具体目标2中，将研究调节年轻和老年人B细胞类别转换的分子机制。将表征在刺激的富集B细胞中E47、AID和NF κ B的表达。在具体目标3中，将建立调节人B细胞中E47的分子机制。将进行mRNA稳定性和包括MAPK在内的信号转导途径的研究，并将有助于确定老年人调节IG类转换的关键分子要求。本申请中的研究将允许在细胞和分子水平上更好地理解老年人受试者的免疫缺陷，并将改善老年人群的免疫应答和疫苗开发。

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.