Regulation of B Lymphocyte Defects in Senescent Humans

衰老人类 B 淋巴细胞缺陷的调节

• 批准号: 7742557
• 负责人: BONNIE B. BLOMBERG
• 金额: $ 30.5万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7742557
• 关键词: 3' 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; 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; Time; Transcript; Untranslated Regions; 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; public health relevance; response; senescence; transcription factor; vaccine development

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.

描述（由申请人提供）：我们的实验室之前已证明老年小鼠和最近的老年人类受试者存在内在 B 淋巴细胞缺陷。在老年人中，抗体介导的体液免疫反应不是最理想的，其产生的免疫球蛋白 (Ig) 的亲和力和自身反应性较低，而且正如我们所表明的，激活的 B 细胞转换其 Ig 的能力急剧下降。 Ig 类别（或同种型）的类别转换重组 (CSR) 对于免疫反应的质量和效应功能非常重要； CSR 中存在原发性（遗传性）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、自身免疫和对疫苗接种无效。我们的长期目标是改善老年人的免疫反应。具体目标 1 提出：在老化激活的人类 B 细胞中产生较少 Ig 类别转换的分子机制是什么？子目标是： a) 各种 B 细胞刺激是否会在衰老的人类 B 细胞中产生同样次优的反应？ B 细胞将受到抗 CD40/IL-4、CpG/IL-4 或 BAFF/IL-4 的刺激。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（针对细胞表面 Ig）和 ELISA（针对分泌 Ig）以及 AID 测量 Ig 产生。 b) 哪些分子机制有助于下调衰老 B 细胞中的 AID？我们已在小鼠中显示转录因子在衰老的活化 B 细胞、E47、NF-B 和 Pax5 中下调，将通过 qPCR 进行分析。 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 E47 逆转录病毒能否挽救 AID 和 CSR？ E47（和 AID）的逆转录病毒构建体将用于上调人类 B 细胞系和原代 B 细胞中的 AID（和 CSR）。在具体目标 2 中，我们将确定下调衰老人类 B 细胞中 E47 的分子机制。子目标是： a) 确定衰老的人类 B 细胞中 E47 的减少是否是由于 mRNA 稳定性降低所致，正如我们之前在衰老的鼠 B 细胞中所看到的那样； b) 确定 mRNA 稳定性降低是否是由于蛋白质（例如 tristetraprolin、TTP）与 3' 非翻译区 (UTR) 结合所致； c) 建立TTP调节机制，包括MAPK、ERK和PI3K通路； d) 确定 microRNA 参与 E47 mRNA 稳定性和老化 B 细胞功能。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 细胞反应欠佳的结果？ a) 接种流感疫苗的受试者的年龄连续体的体内免疫反应是什么？这与他们的 B 和 T 细胞表型有何关系？ b) 这些受试者的具体体外 B 细胞免疫反应是什么？公共卫生相关性：老年人对传染源、疫苗和癌症的免疫反应较差，导致发病率和死亡率增加。本申请中的研究将揭示老年受试者体液免疫反应中的细胞和分子缺陷，并改进这些缺陷，以实现老年人更好的免疫反应和疫苗开发。