Regulation of B Lymphocyte Defects in Senescent Humans

描述(由申请人提供)：我们的实验室以前在老年小鼠和最近的老年人类受试者中发现了固有的B淋巴细胞缺陷。抗体介导的体液免疫反应在产生亲和力和自我反应性较低的免疫球蛋白(Ig)的老年人中是次优的，正如我们已经表明的那样，激活的B细胞转换其Ig的能力急剧下降。Ig类(或同型)的类切换重组(CSR)对免疫应答的质量和效应功能非常重要；CSR中存在原发(遗传)免疫缺陷的患者会出现严重的免疫并发症，包括呼吸道感染、自身免疫和疫苗接种失败。我们的长期目标是改善老年人的免疫反应。具体目标1问：在老化激活的人B细胞中产生较少Ig类开关的分子机制是什么？次目标是：A)不同的B细胞刺激在老年人类B细胞中是否产生同样次优的反应？B细胞将被抗CD40/IL-4、CpG/IL-4或BAFF/IL-4刺激。可能的AID下调将通过定量(Q)聚合酶链式反应进行评估。用定量聚合酶链式反应(QPCR)检测循环转录本(CT)，用流式细胞仪(FCM)检测细胞表面免疫球蛋白(Ig)，用酶联免疫吸附试验(EL ISA)检测分泌型免疫球蛋白(Ig)，同时检测AID。B)衰老B细胞AID下调的分子机制是什么？我们在小鼠中显示的转录因子在衰老激活的B细胞中下调，E47，NF-B和Pax5将被分析qPCR。IRF4，也被认为对企业社会责任很重要，也将被测量。C)体外加入E47逆转录病毒能挽救艾滋病和CSR吗？针对E47(和AID)的逆转录病毒载体将用于上调人类B细胞系和原代B细胞中的AID(和CSR)。在特定的目标2中，我们将确定下调老年人类B细胞E47的分子机制。亚目标是：a)确定衰老的人B细胞中E47的降低是否是由于我们以前在衰老的小鼠B细胞中看到的mRNA稳定性降低所致；b)确定mRNA稳定性降低是否是由于与3‘非翻译区(UTR)结合的蛋白质(例如Tristetraprolin，TTP)所致；c)建立TTP调控机制，包括MAPK、ERK和PI3K通路；以及d)确定microRNA参与E47 mRNA的稳定性和老年B细胞的功能。具体目标3将调查：老年人对流感疫苗的抗体反应降低是否是B细胞反应不佳的结果？A)接种流感疫苗的受试者在年龄上的体内免疫反应是什么，这与他们的B和T细胞表型有何关联？B)这些受试者在体外的B细胞免疫反应是什么？公共卫生相关性：老年人对感染剂、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。这项应用中的研究将揭示老年人体液免疫反应中的细胞和分子缺陷，并导致这些缺陷的改善，以便在老年人中更好的免疫反应和疫苗开发。

DESCRIPTION (provided by applicant): Our laboratory has previously demonstrated intrinsic B lymphocyte defects in aged mice and more recently elderly human subjects. The antibody-mediated humeral immune response is suboptimal in aged individuals with the generation of immunoglobulin (Ig) of lower affinity and self-reactivity and, as we have shown, a dramatic decrease in the ability of activated B cells to class switch their Ig. Class switch recombination (CSR) of the Ig class (or isotype) is very important for the quality and effector functions of the immune response; patients with a primary (genetic) immune deficiency in CSR have severe immune complications including respiratory tract infections, autoimmunity and failure to respond to vaccination. Our long-term objective is to improve the immune response in elderly humans. Specific Aim 1 asks: What are the molecular mechanisms which generate less Ig class switch in aged-activated human B cells? Sub aims are: a) Do various B cell stimuli generate equally suboptimal responses in aged human B cells? B cells will be stimulated either with anti-CD40/IL-4, CpG/IL-4, or BAFF/IL-4. Possible AID down regulation will be assessed by quantitative (q) PCR. We will measure circle transcripts (CT) by qPCR, Ig production by flow cytometry (for cell surface Ig) and ELISA (for secreted Ig) as well as AID. b) What molecular mechanisms contribute to the down regulation of AID in aged B cells? Transcription factors we have shown in mice to be down regulated in aged activated B cells, E47, NF-B, and Pax5 will be analyzed qPCR. IRF4, also known to be important for CSR, will also be measured. c) Can in vitro retroviral addition of E47 rescue AID and CSR? Retroviral constructs for E47 (and AID) will be used to up regulate AID (and CSR) in human B cell lines and primary B cells. In Specific Aim 2, we will determine the molecular mechanisms which down-regulate E47 in aged human B cells. Sub aims are to; a) establish whether the decrease in E47 in aging human B cells is due to decreased mRNA stability as we have seen previously in aged murine B cells; b) determine whether the decreased mRNA stability is due to proteins (e.g. tristetraprolin, TTP) binding to the 3' untranslated region (UTR); c) establish mechanisms of TTP regulation including MAPK, ERK and PI3K pathways; and d) determine microRNA involvement in E47 mRNA stability and aged B cell functions. Specific Aim 3 will investigate: Is a decreased antibody response to the influenza vaccine in elderly individuals a result of a suboptimal B cell response? a) What is the in vivo immune response in an age continuum of subjects given the influenza vaccine, and how is this associated with their B and T cell phenotypes? b) What is the specific in vitro B cell immune response in these subjects? PUBLIC HEALTH RELEVANCE: Aged humans have poor immune responses to infectious agents, vaccines, and cancers which contribute to increased morbidity and mortality. The studies in this application will reveal cellular and molecular deficits within the humeral immune response in aged human subjects and lead to improvement of these for better immune response and vaccine development in the elderly population.