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: 10466824
• 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/10466824
• 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.

摘要 大多数T细胞和B细胞表现出抗原受体（AgR）基因的单等位基因表达（等位基因排斥）。虽然 等位基因排阻是在1965年发现的，其潜在的机制和作用仍然是难以捉摸的。初始和 等位基因排斥的流行模型是一种类型（特异性）的AgR的表达通过以下方式抑制自身免疫： 确保具有自身反应性AgR的细胞的阴性选择。与这一观点相一致，第二种表达方式 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， 关于内源性TCRb、IgH或Igk基因的双等位基因表达的影响，仍然存在知识缺口 其严格的等位基因排斥是通过单等位基因起始和V重组的反馈抑制实现的。 除了单等位基因表达的可能益处之外，申请人还推论单等位基因AgR组装 抑制由RAG核酸内切酶产生的DNA双链断裂（DSB）引起的致癌易位。 阐明等位基因排斥作用的一个障碍是缺乏一个已证实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 而不改变V（D）J重组、DSB应答和/或淋巴细胞发育。 翻译信号序列（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的小鼠显著增加了发育 由于增加的双等位基因Vb重组，具有双等位基因TCRb表达的T细胞。根据他初步的 数据，申请人假设弱Vb和VH RSS限制V重组以限制V的发生率。 在反馈抑制停止进一步的V重组之前，两个等位基因上的重排。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， 在免疫稳态中TCRb和可能的IgH的组装和表达。他提出了两个互补的 但独立的目的是测试这些假设的关键方面。在目标1中，PI将在体外使用补充 生物化学和体内分子测定，以明确确定是否固有的弱Vb和VH RSS 限制V重组以加强V重组单等位基因起始。在目标2中，他将使用Vb和VH RSS 以确定增加双等位基因组装和TCR b或IgH表达对小鼠免疫功能的影响。 自身反应细胞的负选择、自身免疫的易感性和致癌易位的发生。 本项目将展示RSS如何控制TCRb的单等位基因组装和表达，并确定是否相同。 随机机制强制IgH等位基因排除。这些数据将首次证明 用于V重组的单等位基因起始。这项研究有很大的潜力为一个 免疫学的基本原理于1965年提出，但至今仍未得到证实。这项工作还可能揭示 单等位基因启动V重排在抑制淋巴瘤中的另一个重要益处。