Regulation of B Lymphocyte Defects in Senescent Humans

描述（由申请方提供）：我们的实验室先前已在老年小鼠和最近的老年人受试者中证明了固有的B淋巴细胞缺陷。抗体介导的肱骨免疫应答在老年个体中是次优的，产生的免疫球蛋白（IG）具有较低的亲和力和自身反应性，并且如我们所示，活化的B细胞类别转换其IG的能力显著降低。IG类（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; CSR中原发性（遗传性）免疫缺陷的患者具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应答失败。我们的长期目标是改善老年人的免疫反应。具体目标1：在老化激活的人B细胞中产生较少IG类转换的分子机制是什么？子目标是：a）各种B细胞刺激是否在老年人B细胞中产生同样次优的反应？B细胞将用抗CD 40/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测量环状转录物（CT），通过流式细胞术（用于细胞表面IG）和ELISA（用于分泌型IG）测量IG产生以及AID。B）什么样的分子机制有助于老年B细胞中AID的下调？我们已经在小鼠中显示的转录因子在老化的活化B细胞中下调，将对E47、NF-B和Pax 5进行qPCR分析。IRF 4也被认为对CSR很重要，也将被测量。c）体外逆转录病毒添加E47能拯救AID和CSR吗？E47（和AID）的逆转录病毒构建体将用于上调人B细胞系和原代B细胞中的AID（和CSR）。在具体目标2中，我们将确定在老年人B细胞中下调E47的分子机制。子目标是; a）确定老化的人B细胞中E47的减少是否是由于mRNA稳定性的降低，正如我们先前在老化的鼠B细胞中所看到的; B）确定mRNA稳定性的降低是否是由于蛋白质c）建立TTP调节机制，包括MAPK、ERK和PI 3 K途径;和d）确定微小RNA参与E47 mRNA稳定性和老化B细胞功能。具体目标3将研究：老年人对流感疫苗的抗体应答降低是否是次优B细胞应答的结果？a）在给予流感疫苗的受试者的年龄连续体中，体内免疫应答是什么，以及这与他们的B和T细胞表型如何相关？B）这些受试者的体外特异性B细胞免疫应答是什么？公共卫生相关性：老年人对传染性病原体、疫苗和癌症的免疫应答较差，这导致发病率和死亡率增加。本申请中的研究将揭示老年人受试者肱骨免疫应答中的细胞和分子缺陷，并导致这些缺陷的改善，以在老年人群中实现更好的免疫应答和疫苗开发。

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.