Regulation of Immunoglobulin Class Switch in Aged Mice

老年小鼠免疫球蛋白类别转换的调节

基本信息

批准号：
7176151
负责人：
BONNIE B. BLOMBERG
金额：
$ 28.73万
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：
2005
资助国家：
美国
起止时间：
2005-02-01 至 2010-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7176151
关键词：
1-Phosphatidylinositol 3-Kinase 4-hydroxy-3-nitrophenylacetyl-keyhole limpet hemocyanin Acetylation Adoptive Transfer Affinity Age Aging Animals Antibodies Antibody Formation Antigens B-Lymphocytes Binding Cell Aging Cell surface Class Cytometry DNA Binding Data Defect Detection E protein EMSA Enzyme-Linked Immunosorbent Assay Ficoll Genes Genetic Transcription Germ Lines Human IgE Immune response Immunization Immunoglobulin A Immunoglobulin Class Switching Immunoglobulin G Immunoglobulin Switch Recombination Immunoglobulins Immunologic Deficiency Syndromes In Vitro Inbred BALB C Mice Indium Individual Infectious Agent Lasers MAP Kinase Gene Measures Mediating Messenger RNA Mitogens Molecular Mus Mutation Phosphorylation Post-Translational Protein Processing Post-Translational Regulation Predisposition Proteins RNA Stability Regulation Retroviridae SCID Mice Serum Signal Transduction Pathway Structure of germinal center of lymph node Surface Immunoglobulins T-Independent Antigens T-Lymphocyte TCF3 gene Thymus Gland Transcript Translations Vaccines activation-induced cytidine deaminase aged cell age chromatin immunoprecipitation cytokine helix-loop-helix protein E47 in vivo mRNA Stability notch protein pathogen protein degradation protein expression research study response retroviral transduction senescence transcription factor

项目摘要

DESCRIPTION (provided by applicant): Aged humans and experimental animals have a poor immune response to infectious agents and vaccines. The antibody-mediated humoral immune response is qualitatively deficient in aged individuals with the production of antibodies of lower affinity and with self reactivity. Although problems with T cells certainly contribute to these B cell deficits in aging, intrinsic B cell changes are shown in this proposal. The ability to switch immunoglobulin (Ig) heavy chain class (isotype) is critical for proper effector functions of the Ig protein and genetic defects in the ability of humans to undergo class switch recombination (CSR) result in immunodeficiency with increased susceptibility to many pathogens. These studies will focus on the molecular and cellular mechanisms responsible for the decrease in Ig class switch in aged mice. We hypothesize that the decrease in CSR is a direct result of a decrease in the activation-induced cytidine deaminase, AID, which in turn is regulated by a decrease in the transcription factor, E47, in aged mice. This proposal will compare splenic B cells from aged vs. young mice for the presence and function of E47, AID and class switched isotypes (IgG, IgE, IgA) in vitro as well as in antigen-specific responses in vivo. In Specific Aim 1 we will extend preliminary data to establish the intrinsic splenic B cell defect in Ig class switch in aged BALB/c, C57BL/6 and E2A+/- mice. Purified splenic B cells will be activated with mitogens or anti-CD40 and various cytokines and class switch measured by ELISA for secreted Ig, fluorescent cytometry for cell surface Ig molecularly by detection of germ line transcripts (GLT) and post-switch transcripts (PST). In vivo experiments with immunization with NP-KLH or NP-Ficoll (thymus-dependent and thymus-independent antigens) and adoptive transfer of combinations of young and old B/T cells into SCID mice will be done. In Specific Aim 2, the molecular mechanisms regulating class switch in young and old mice will be investigated. E47, Id2, Notch, NFkappaB and AID expression in aged B cells will be characterized. Experiments will include chromatin immunoprecipitation (ChIP) with anti-E2A (for the AID gene) as well as attempts to rescue class switch in aged B cells with retroviral transduction of AID or E47. In Specific Aim 3, the molecular mechanisms for regulation of E47 and AID will be established. Studies of protein and mRNA stability, post translational regulation, and signal transduction pathways including MAPK, will determine key molecular elements in the control of Ig class switch in aged mice.

描述（由申请人提供）：老年人和实验动物对感染剂和疫苗的免疫反应较差。抗体介导的体液免疫反应在具有较低亲和力和自反应性的抗体的老年人中在质量上缺乏。尽管T细胞的问题肯定会导致衰老中的这些B细胞缺陷，但本提案中显示了内在的B细胞变化。切换免疫球蛋白（IG）重链类（同种型）的能力对于人类经历类别开关重组（CSR）能力的适当效应子功能至关重要，从而导致免疫缺陷，并增加对许多病原体的易感性。这些研究将集中于导致老年小鼠Ig类转换减少的分子和细胞机制。我们假设CSR的减少是激活诱导的胞苷脱氨酶AID的直接结果，而cytidine脱氨酶AID又受老年小鼠转录因子E47的降低来调节。该建议将在体外以及体外的抗原特异性反应中比较E47，AID和类切换的同种型（IgG，IgE，IgA）的存在和功能的脾B细胞的存在和功能。在特定目标1中，我们将扩展初步数据，以在老年BALB/C，C57BL/6和E2A +/-小鼠中建立IG类开关中的固有性脾B细胞缺陷。纯化的脾脏B细胞将用丝分裂原或抗CD40和各种细胞因子和通过ELISA测量的分泌Ig测量的类转换激活，通过检测生殖线转录本（GLT）和移动后转录物（PST），用于细胞表面Ig分子的荧光细胞仪。用NP-KLH或NP-FICOLL（胸腺依赖性和胸腺独立抗原）进行免疫实验的体内实验，将完成年轻和老年B/T细胞的组合传递到SCID小鼠中。在特定的目标2中，将研究调节年轻小鼠和老鼠的类别转换的分子机制。 E47，ID2，Notch，NFKappab和老年B细胞中的辅助表达将被表征。实验将包括具有抗E2A（用于辅助基因）的染色质免疫沉淀（CHIP），以及试图通过逆转录病毒转导AID或E47来挽救老年B细胞中的类别转换。在特定的目标3中，将建立用于调节E47和AID的分子机制。蛋白质和mRNA稳定性，转化后调节以及包括MAPK在内的信号转导途径的研究将确定老年小鼠Ig类开关控制中的关键分子元素。