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) 基因的单等位基因表达（等位基因排除）。虽然 等位基因排斥于 1965 年被发现，但其潜在机制和作用仍然难以捉摸。初始和 等位基因排除的流行模型是，一种类型（特异性）AgR 的表达通过以下方式抑制自身免疫： 确保具有自反应 AgR 的细胞的阴性选择。与此观点一致的是，第二个表达式 非自身反应性 AgR 使表达转基因自身反应性 AgR 的细胞能够逃避阴性选择。然而， 关于内源 TCRb、IgH 或 Igk 基因的双等位基因表达的影响仍然存在知识差距 其严格的等位基因排除是通过 V 重组的单等位基因起始和反馈抑制来实现的。 除了单等位基因表达的可能益处之外，申请人还推测单等位基因 AgR 组装 抑制 RAG 核酸内切酶产生的 DNA 双链断裂 (DSB) 导致的致癌易位。 阐明等位基因排除作用的一个障碍是缺乏经过验证的单等位基因起始机制 V重组。这排除了增加双等位基因组装和表达的实验方法 AgR 位点的变化而不改变 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 RAG 活性介导 V(D)J 重组。申请人 显示用更强的 RSS 替换 Vb RSS 的小鼠显着加快了发育 由于双等位基因 Vb 重组增加而具有双等位基因 TCRb 表达的 T 细胞。根据他的初步 根据数据，申请人假设弱 Vb 和 VH RSS 限制 V 重组，从而限制 V 的发生率 在反馈抑制阻止进一步的 V 重组之前，两个等位基因均发生重排。他假设 这种降低 V 重组频率的随机机制可用于测试单等位基因的作用 TCRb 以及可能的 IgH 在免疫稳态中的组装和表达。他提出了两种互补的 但独立的目的是检验这些假设的关键方面。在目标 1 中，PI 将使用互补的体外 生化和体内分子测定，明确确定 Vb 和 VH RSS 是否本质上较弱 限制 V 重组以强制 V 重组的单等位基因起始。在目标 2 中，他将使用 Vb 和 VH RSS 替换小鼠以确定增加双等位基因组装和 TCRb 或 IgH 表达对 自身反应细胞的阴性选择、自身免疫倾向以及致癌易位的发生。 该项目将展示 RSS 如何控制 TCRb 的单等位基因组装和表达，并鉴定是否相同 随机机制强制 IgH 等位基因排除。这些数据将首次展示一种机制 用于 V 重组的单等位基因起始。该研究具有巨大的潜力，可以为以下事实提供明确的证据： 免疫学的基本原则于 1965 年提出，但至今仍未得到证实。该作品还可能揭示 V 重排的单等位基因起始在抑制淋巴瘤方面的另一个重要益处。