Regulation of B Lymphocyte Defects in Senescent Humans

描述（由申请人提供）：我们的实验室以前已经证明了老年小鼠和最近老年人类受试者的内在B淋巴细胞缺陷。抗体介导的肱骨免疫反应在老年个体中是次优的，因为免疫球蛋白（Ig）的产生具有较低的亲和力和自我反应性，并且正如我们所显示的，激活的B细胞切换其Ig的能力急剧下降。Ig类（或同型）的类开关重组（Class switch recombination， CSR）对免疫应答的质量和效应功能至关重要；原发性（遗传）免疫缺陷的CSR患者有严重的免疫并发症，包括呼吸道感染、自身免疫和对疫苗接种无反应。我们的长期目标是改善老年人的免疫反应。特异性目标1问：在衰老激活的人类B细胞中产生较少的Ig类开关的分子机制是什么？次级目标是：a)不同的B细胞刺激是否会在老年B细胞中产生同样的次优反应？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) PCR进行评估。我们将通过qPCR测量环转录本（CT），通过流式细胞术（检测细胞表面Ig）和ELISA（检测分泌Ig）以及AID测量Ig的产生。b)衰老b细胞AID下调的分子机制是什么？我们在小鼠中发现的在衰老激活的B细胞中下调的转录因子，E47， NF-B和Pax5将进行qPCR分析。IRF4，也被认为对企业社会责任很重要，也将被测量。c)体外添加逆转录病毒E47能拯救AID和CSR吗？E47（和AID）的逆转录病毒构建物将用于上调人B细胞系和原代B细胞中的AID（和CSR）。在特异性目标2中，我们将确定衰老人B细胞中下调E47的分子机制。次级目标是；a)确定衰老的人类B细胞中E47的减少是否与我们之前在衰老的小鼠B细胞中看到的mRNA稳定性下降有关；b)确定mRNA稳定性下降是否由于蛋白质（如tristetrprolin， TTP）与3'非翻译区（UTR）结合所致；c)建立包括MAPK、ERK和PI3K通路在内的TTP调控机制；d)确定microRNA参与E47 mRNA稳定性和衰老B细胞功能。特异性目标3将调查：老年人对流感疫苗的抗体反应降低是由于B细胞反应不佳所致吗？a)接种流感疫苗的年龄连续受试者体内免疫反应是什么，这与他们的B细胞和T细胞表型有何关系？b)这些受试者的特异性体外b细胞免疫反应是什么？公共卫生相关性：老年人对传染性病原体、疫苗和癌症的免疫反应较差，导致发病率和死亡率增加。这项应用研究将揭示老年人肱骨免疫反应中的细胞和分子缺陷，并导致这些缺陷的改善，以获得更好的老年人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.