Elucidating Lymphocyte Allelic Exclusion Mechanisms and Functions

阐明淋巴细胞等位基因排除机制和功能

• 批准号: 10020899
• 负责人: CRAIG H BASSING
• 金额: $ 44万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-19 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10020899
• 关键词: Alleles; Antigen Receptors; Autoimmune Diabetes; Autoimmunity; B-Lymphocytes; Binding; Biochemical; Biological Assay; Cells; Complex; DNA Double Strand Break; Data; Development; Disease; Ensure; Exclusion; Exhibits; Feedback; Frequencies; Genes; Genetic; Genetic Recombination; Genetic Transcription; Homeostasis; IgK; Immune; Immunology; In Vitro; Inbred NOD Mice; Incidence; Investigation; Knowledge; Lymphocyte; Lymphoma; Mediating; Modeling; Molecular; Mus; Oncogenic; Pathology; Peptide Signal Sequences; Physiological; Predisposition; Process; Receptor Gene; Role; S Phase; Signal Transduction; Specificity; T-Cell Development; T-Lymphocyte; Testing; Time; Transgenic Organisms; V(D)J Recombination; Work; autoreactivity; base; endonuclease; in vivo; mouse model; receptor; receptor expression; response; theories; thymocyte

ABSTRACT Most T and B cells exhibit mono-allelic expression (allelic exclusion) of antigen receptor (AgR) genes. Although allelic exclusion was discovered in 1965, its underlying mechanisms and role(s) remain elusive. The initial and prevailing model for allelic exclusion is that expression of one type (specificity) of AgR inhibits autoimmunity by ensuring negative selection of cells with a self-reactive AgR. Consistent with this view, the expression of a second non-autoreactive AgR enables cells expressing a transgenic autoreactive AgR to evade negative selection. Yet, there remains a knowledge gap regarding effects of bi-allelic expression of endogenous TCRb, IgH, or Igk genes whose stringent allelic exclusion is achieved by mono-allelic initiation and feedback inhibition of V recombination. In addition to the likely benefit of mono-allelic expression, the applicant theorizes that mono-allelic AgR assembly suppresses oncogenic translocations from RAG endonuclease-generated DNA double strand breaks (DSBs). An obstacle to elucidating roles of allelic exclusion is the lack of a proven mechanism for mono-allelic initiation of V recombination. This has precluded experimental approaches to increase bi-allelic assembly and expression of AgR loci without otherwise altering V(D)J recombination, DSB responses, and/or lymphocyte development. Recombination signal sequences (RSSs) mediate V(D)J recombination by directing RAG activity. The applicant shows mice harboring replacement of Vb RSSs with a stronger RSS have substantially increased development of T cells with bi-allelic TCRb expression due to increased bi-allelic Vb recombination. Based on his preliminary data, the applicant hypothesizes that weak Vb and VH RSSs limit V recombination to restrict the incidence of V rearrangements on both alleles before feedback inhibition halts further V recombination. He hypothesizes that this stochastic mechanism of lowering V recombination frequency can be exploited to test roles of mono-allelic assembly and expression of TCRb, and possibly IgH, in immune homeostasis. He proposes two complementary but independent aims to test key aspects of these hypotheses. In Aim 1, the PI will use complementary in vitro biochemical and in vivo molecular assays to determine unequivocally whether inherently weak Vb and VH RSSs limit V recombination to enforce mono-allelic initiation of V recombination. In Aim 2, he will use Vb and VH RSS replacement mice to determine the impact of increasing bi-allelic assembly and expression of TCRb or IgH on negative selection of self-reactive cells, predisposition to autoimmunity, and genesis of oncogenic translocations. This project will show how RSSs control mono-allelic assembly and expression of TCRb, and identify if the same stochastic mechanism enforces IgH allelic exclusion. The data will, for the first time, demonstrate a mechanism for mono-allelic initiation of V recombination. The study has great potential to provide definitive evidence for a fundamental tenet of immunology proposed in 1965, but that remains unproven today. The work also may reveal an additional important benefit of mono-allelic initiation of V rearrangements in suppressing lymphoma.

抽象的 大多数T和B细胞表现出抗原受体（AGR）基因的单相关表达（等位基因排除）。虽然 1965年发现了等位基因排除，其基本机制和角色仍然难以捉摸。最初和 等位基因排除的流行模型是，AGR的一种类型（特异性）的表达抑制自身免疫性 确保用自反应性AGR进行负面选择。与这种观点一致，一秒钟的表达 非AutoreActive AGR使表达转基因自动反应性AGR的细胞能够逃避负面选择。然而， 关于内源性TCRB，IGH或IGK基因的双重平行表达的影响的知识差距仍然存在差距 通过单相关的启动和V重组的反馈抑制，其严格的等位基因排除是实现的。 除了单相表达的可能益处外，申请人还理论上了单相关AGR组装 抑制抹布核酸内切酶产生的DNA双链断裂（DSB）的致癌易位。 阐明等位基因排除作用的障碍是缺乏可靠的单相关启动机制 V重组。这排除了实验方法，以增加双行组装和表达 AGR基因座的of而没有其他改变V（d）J重组，DSB反应和/或淋巴细胞发育。 重组信号序列（RSS）通过指导抹布活性介导V（d）J重组。申请人 显示具有更强RSS的VB RSS替代的小鼠已经大大增加了发育 由于BI-calilleic VB重组的增加而导致具有BI-平行性TCRB表达的T细胞的T细胞。根据他的初步 数据，申请人假设弱VB和VH RSS限制了V重组以限制V的发生率 反馈抑制之前，两个等位基因上的重排停止了V重组。他假设这一点 可以利用这种降低V重组频率的随机机制来测试单相关的角色 免疫稳态中TCRB的组装和表达，甚至可能是IGH。他提出了两个补充 但是独立旨在检验这些假设的关键方面。在AIM 1中，PI将使用互补的体外 生化和体内分子测定，以明确确定固有的弱VB和VH RSSSS是否存在 限制V重组以实施V重组的单相关启动。在AIM 2中，他将使用VB和VH RSS 替换小鼠以确定增加双性组装和TCRB或IGH表达的影响 自我反应性细胞的负选择，自身免疫性的倾向以及致癌易位的起源。 该项目将显示RSS如何控制TCRB的单相关组件和表达，并确定是否相同 随机机制执行IGH等位基因排除。数据将首次演示一种机制 用于V重组的单相关开始。该研究具有很大的潜力，可以为 1965年提出的免疫学的基本原则，但今天仍未得到证实。这项工作也可能揭示 抑制淋巴瘤中V重排的单相关开始的另一个重要优势。