A genetic switch to study the causal roles of T cell repertoire diversity

研究 T 细胞库多样性因果作用的基因开关

• 批准号: 10452985
• 负责人: Yongqiang Feng
• 金额: $ 9.1万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-05 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10452985
• 关键词: Affect; Aging; Alleles; Antigen Receptors; Autoantigens; Autoimmune Diseases; Autoimmunity; B-Lymphocytes; Biochemical; Birth; Bone Marrow Cells; CD3E gene; CRISPR/Cas technology; Cell Aging; Cell Lineage; Cell physiology; Cells; Cues; Development; Dose; Gene Rearrangement; Genes; Genetic; Genetic Recombination; Goals; Immune System Diseases; Immune Tolerance; Immunologics; Immunosuppression; Immunotherapy; Kinetics; Knock-in Mouse; Link; Lymphopenia; Malignant Neoplasms; Mediating; Methods; Modeling; Mus; Oils; Outcome; Pharmacology; Play; Probability; Process; Protocols documentation; Receptor-CD3 Complex, Antigen, T-Cell; Regulatory T-Lymphocyte; Reporting; Retroviridae; Role; Self Tolerance; Side; Structure; T cell differentiation; T-Cell Development; T-Lymphocyte; T-cell diversity; T-cell receptor repertoire; Tamoxifen; Testing; Thymectomy; Thymus Gland; Tumor Immunity; adaptive immunity; anti-tumor immune response; autoreactive T cell; experimental study; immune activation; immune function; improved; innovation; insight; mouse genetics; novel; reconstitution; side effect; tool

A genetic switch to study the causal roles of T cell repertoire diversity T cells carry a diverse antigen receptor (TCR) repertoire selected during their development. Because TCRs are generated essentially in a random way via the recombination of gene segments and potentially react to self-antigens, several mechanisms are found to restrict the autoreactivity of T cells including thymic negative selection and regulatory T (Treg) cell induction. Among these mechanisms, Treg cells play a crucial, non- redundant role in sustaining self-tolerance and suppressing autoimmune diseases. Previous studies suggest that polyclonal conventional T cells (non-Treg T cells) are required for efficient Treg cell development. However, the immunological function of this observation has not been investigated. Accumulating evidence also links T cell lymphopenia to autoimmunity. Although the underlying mechanisms remain unclear, the diversity of T cell repertoire may play a critical role in dictating immune tolerance. Consistently, elevated autoimmunity was observed during aging when T cell diversity is reduced because of thymic involution and T cell senescence. Given that conventional T cells provide essential cues for Treg cell development and immune suppressive function, we hypothesize that the diversity of the entire T cell repertoire plays causal roles in promoting Treg cell induction and function, thus suppressing immune activation and autoimmune diseases. Rigorous test of this hypothesis and determining the mechanisms governing this process would provide novel insights into Treg cell- mediated immune tolerance. However, there are several technical barriers to achieve this goal. To solve this issue, we propose to model TCR diversity with innovative genetic tools. Specifically, we will take advantage of the essential role of CD3e in T cell differentiation and insert a loxP-Stop-loxp (LSL) cassette into the 5’ end of Cd3e gene resulting in Cd3eLSL mice. Previous study showed that thymic T cells fail to develop beyond the double negative DN3 stage in Cd3e-deficient mice. Similarly, in the absence of Cre, no functional CD3e will be synthesized in homozygous Cd3eLSL/LSL mice. In our preliminary experiment, we confirmed no T cells in these mice. To control T cell development and TCR repertoire diversity, we will cross Cd3eLSL to our available Rag1CreER knock-in mice that transiently express inducible CreER during early T cell differentiation. We will treat these mice with titrated amounts of tamoxifen continuously to control the probability of T cell development. We will then use routine methods to assess the dose-dependent induction of T cell development, the diversity of Treg and conventional T cell repertoires, immune activation in the steady state, and anti-tumor immunity. In summary, we will develop an innovative genetic tool to determine the causal roles of T cell repertoire diversity in autoimmune diseases and cancer. The study performed with this genetic tool will improve our fundamental understanding and the methods for Treg-based treatment of immunological diseases and cancer.

研究T细胞库多样性的因果关系的遗传转换 T细胞在发育过程中选择了发散的抗原受体（TCR）库。因为 TCR基本上是通过基因片段的重组而随机生成的，并可能反应 对于自我抗原，发现几种机制限制了T细胞的自动反应性，包括胸腺阴性 选择和调节t（Treg）细胞诱导。在这些机制中，Treg细胞起着至关重要的，非 - 在维持自我耐受和抑制自身免疫性疾病方面的冗余作用。先前的研究表明 多克隆常规T细胞（非Treg T细胞）是有效的Treg细胞发育所必需的。然而， 该观察的免疫功能尚未研究。累积证据也联系在一起 细胞淋巴细胞减少到自身免疫性。尽管基本机制尚不清楚，但T细胞的多样性 曲目在决定免疫耐受性方面可能起关键作用。一贯，自身免疫升高是 由于胸腺的相关性和T细胞的感应，在老化期间观察到在老化时观察到。 鉴于常规T细胞为Treg细胞发育和免疫抑制提供了必不可少的提示 功能，我们假设整个T细胞库的多样性在促进Treg细胞中扮演因果关系 诱导和功能，从而抑制免疫激活和自身免疫性疾病。严格测试 假设和确定管理此过程的机制将为Treg细胞提供新的见解 介导的免疫耐受性。 但是，实现这一目标有几个技术障碍。为了解决这个问题，我们建议 使用创新的遗传工具模型TCR多样性。具体来说，我们将利用 T细胞分化中的CD3E并将LOXP-Stop-loxp（LSL）盒插入CD3E基因的5'端，结果 在CD3ELSL小鼠中。先前的研究表明，胸腺T细胞无法发育超出双负DN3 CD3E缺陷小鼠的阶段。同样，在不存在CRE的情况下，将不合成功能性CD3E 纯合CD3ELSL/LSL小鼠。在我们的初步实验中，我们证实了这些小鼠没有T细胞。控制 T细胞开发和TCR曲目多样性，我们将越过CD3ELSL到我们可用的rag1creer敲入小鼠 在早期T细胞分化过程中，该瞬时表达可诱导的CRER。我们将用滴定治疗这些老鼠 他莫昔芬的量连续控制T细胞发育的概率。然后我们将使用例程 评估T细胞发育剂量依赖性诱导的方法，Treg的多样性和常规的多样性 T细胞库，稳态的免疫激活和抗肿瘤免疫希金会。 总而言之，我们将开发一种创新的遗传工具来确定T细胞库的因果关系 自身免疫性疾病和癌症的多样性。使用此遗传工具进行的研究将改善我们的 基本的理解以及基于Treg的免疫疾病和癌症治疗的方法。