Regulation of B Lymphocyte Defects in Senescent Humans

衰老人类 B 淋巴细胞缺陷的调节

基本信息

批准号：
8894635
负责人：
BONNIE B. BLOMBERG
金额：
$ 12.45万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8894635
关键词：
3&apos Untranslated Regions Affinity Age Aging Antibodies Antibody Formation Antigens Autoimmunity B-Lymphocytes Binding Biological Markers Cancer Vaccines Cell Line Cell physiology Cell surface Data Defect Down-Regulation Elderly Enzyme-Linked Immunosorbent Assay Failure Flow Cytometry Generations Genetic Hemagglutination Human IRF4 gene IgE Immune Immune response Immune system Immunoglobulin A Immunoglobulin Class Switching Immunoglobulin G Immunoglobulin Switch Recombination Immunoglobulins In Vitro Individual Infectious Agent Interleukin-4 Laboratories Lead Lymphocyte Function MAP Kinase Gene MAPK14 gene Magnetism Measures Mediating MicroRNAs Molecular Morbidity - disease rate Mus Pathway interactions Patients Phenotype Phosphoric Monoester Hydrolases Phosphorylation Population Production Protein Phosphatase 2A Regulatory Subunit PR53 Proteins Regulation Respiratory Tract Infections Serum Stimulus Surface Immunoglobulins System T-Lymphocyte TCF3 gene TIS11 protein TNFRSF5 gene Time Transcript Vaccination Vaccines activation-induced cytidine deaminase age related aged anti-influenza helix-loop-helix protein E47 human subject improved in vivo influenza virus vaccine mRNA Stability mRNA Transcript Degradation mortality response senescence transcription factor vaccine development

项目摘要

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?

描述（由申请人提供）：我们的实验室先前已经证明了老年小鼠和最近的老年人受试者的内在B淋巴细胞缺陷。抗体介导的肱骨免疫反应在较低亲和力和自我反应性的衰老个体中是次优的，正如我们已经表明的那样，激活B细胞类别切换其Ig的能力急剧下降。 IG类（或同种型）的类开关重组（CSR）对于免疫反应的质量和效应子功能非常重要。企业社会责任中原发性（遗传）免疫缺陷的患者患有严重的免疫并发症，包括呼吸道感染，自身免疫性和对疫苗接种反应。我们的长期目标是改善老年人的免疫反应。特定目标1问：在年龄激活的人类B细胞中产生IG类切换较少的分子机制是什么？子目的是：a）各种B细胞刺激是否会在老年人B细胞中产生同样的次优应答？ B细胞将用抗CD40/IL-4，CPG/IL-4或BAFF/IL-4刺激B细胞。可能的辅助调节将通过定量（Q）PCR评估。我们将通过qPCR测量圆圈转录（CT），流式细胞仪（用于细胞表面IG）和ELISA（用于分泌的Ig）以及AID的Ig产生。 b）哪些分子机制有助于对老化B细胞的辅助下降？我们在老年活化的B细胞E47，NF-B和PAX5中显示的小鼠已显示的转录因子将被分析qPCR。 IRF4（也对CSR很重要）也将被测量。 c）可以在体外逆转录病毒添加E47救援辅助和CSR吗？ E47（和AID）的逆转录病毒构建体将用于增强人类B细胞系和原代B细胞中的AID（和CSR）。在特定的目标2中，我们将确定在老年人B细胞中下调E47的分子机制。子目标是a）确定像我们先前在老化的鼠B细胞中看到的那样，衰老的人类B细胞中E47的降低是否是由于mRNA稳定性的降低。 b）确定降低的mRNA稳定性是否是由于蛋白（例如三翼蛋白，TTP）与3'未翻译区（UTR）结合的蛋白质； c）建立包括MAPK，ERK和PI3K途径在内的TTP调节机制； D）确定MicroRNA参与E47 mRNA稳定性和老化的B细胞功能。具体目的3将研究：在老年人中对流感疫苗的抗体反应降低是次优B细胞反应的结果吗？ a）鉴于流感疫苗的受试者年龄连续体的体内免疫反应是什么，这与它们的B和T细胞表型有何关系？ b）这些受试者的体外B细胞免疫反应是什么？