Regulation of Immunoglobulin Class Switch in Aged Mice

老年小鼠免疫球蛋白类别转换的调节

• 批准号: 8220821
• 负责人: BONNIE B. BLOMBERG
• 金额: $ 38.01万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8220821
• 关键词: 1-Phosphatidylinositol 3-Kinase; 4-hydroxy-3-nitrophenylacetyl-keyhole limpet hemocyanin; Adoptive Transfer; Affinity; Aging; Animals; Antibodies; Antibody Formation; Antigens; B-Lymphocytes; Binding; Cell Aging; Cell surface; Cytometry; DNA Binding; Data; Defect; Detection; E protein; EMSA; Enzyme-Linked Immunosorbent Assay; Ficoll; Genes; Genetic Transcription; Germ Lines; Heavy-Chain Immunoglobulins; 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; Protein Acetylation; Proteins; RNA Stability; Regulation; SCID Mice; Serum; Signal Transduction Pathway; Structure of germinal center of lymph node; Surface Immunoglobulins; T-Independent Antigens; T-Lymphocyte; TCF3 gene; TNFRSF5 gene; Thymus Gland; Transcript; Translations; Vaccines; Virus; 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

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, NFicB 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. hi 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.

老年人和实验动物对感染剂和疫苗的免疫反应较差。这 抗体介导的体液免疫反应在生产的老年人中质性不足 较低亲和力和自反应性的抗体。尽管T细胞的问题肯定会导致这些B细胞 该提案中显示了衰老，内在B细胞变化的缺陷。切换免疫球蛋白（IG）重量的能力 链类（同种型）对于人类的能力中Ig蛋白的适当效应子功能和遗传缺陷至关重要 进行类开关重组（CSR）导致免疫缺陷，对许多人的敏感性提高 病原体。这些研究将集中于导致Ig类减少的分子和细胞机制 切换老年小鼠。我们假设CSR的减少是激活诱导的下降的直接结果 胞苷脱氨酶AID，进而受老年小鼠转录因子E47的降低来调节。这 提案将比较年龄与年轻小鼠的脾脏B细胞的存在和功能 在体内的体外以及抗原特异性反应中，开关的同种型（IgG，IgE，IgA）。 在特定目标1中，我们将扩展初步数据以在IG类开关中建立固有的脾B细胞缺陷 老化BALB/C，C57BL/6和E2A +/-小鼠。纯化的脾脏B细胞将用有丝分裂原或抗CD40激活 ELISA测量的各种细胞因子和类开关用于分泌的IG，细胞表面Ig的荧光细胞术 通过检测生殖系转录本（GLT）和开关转录本（PST）的分子。体内实验 用NP-KLH或NP-FICOLL（胸腺依赖性和胸腺独立抗原）和过继转移的免疫接种 将完成将年轻和老年b/t细胞组合成SCID小鼠的组合。在特定的目标2中，分子 调节年轻小鼠和老鼠的班级转换的机制将进行研究。 E47，ID2，Notch，NFICB和AID 在老化的B细胞中的表达将被表征。实验将包括具有染色质免疫沉淀（CHIP） 抗E2A（用于辅助基因），以及试图通过逆转录病毒转导的老年B细胞中的类转换 援助或E47。 HI特异性目标3，将建立用于调节E47和AID的分子机制。研究 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，将 确定老年小鼠Ig类开关的控制中的关键分子元素。