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: 10546456
• 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/10546456
• 关键词: Affect; Aging; Alleles; Antigen Receptors; Antigens; 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; Play; Probability; Process; Protocols documentation; Reaction; Receptor-CD3 Complex, Antigen, T-Cell; Regulatory T-Lymphocyte; Reporting; Retroviridae; Role; Self Tolerance; Side; Specific qualifier value; Structure; T cell differentiation; T-Cell Development; T-Lymphocyte; T-cell diversity; T-cell receptor repertoire; Tamoxifen; Testing; Thymectomy; Thymus Gland; Titrations; Tumor Immunity; adaptive immunity; anti-tumor immune response; autoreactive T cell; experimental study; immune activation; immune function; improved; innovation; insight; mouse genetics; novel; pharmacologic; 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细胞库多样性因果作用的遗传开关 T细胞携带在其发育过程中选择的不同抗原受体（TCR）库。因为 TCR基本上是通过基因片段的重组以随机方式产生的，并且可能与基因片段的重组反应。 对于自身抗原，发现了几种机制来限制T细胞的自身反应性，包括胸腺阴性T细胞， 选择和调节性T（Treg）细胞诱导。在这些机制中，Treg细胞起着关键的、非- 在维持自身耐受性和抑制自身免疫性疾病中的多余作用。以往的研究表明 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，在这方面， 该观察结果的免疫功能尚未研究。积累证据也将T 细胞淋巴细胞减少到自身免疫。虽然其潜在机制尚不清楚，但T细胞的多样性 库可能在决定免疫耐受中起关键作用。一致地，升高的自身免疫性是 在衰老过程中观察到，由于胸腺退化和T细胞衰老，T细胞多样性减少。 鉴于常规T细胞为Treg细胞发育和免疫抑制提供了必要的线索， 功能，我们假设整个T细胞库的多样性在促进Treg细胞的增殖中起着因果作用。 诱导和功能，从而抑制免疫激活和自身免疫性疾病。严格的测试 这一假说并确定控制这一过程的机制将为Treg细胞提供新的见解， 介导的免疫耐受。 然而，要实现这一目标存在一些技术障碍。为了解决这个问题，我们建议 用创新的遗传工具来模拟TCR多样性。具体而言，我们将利用 在CD 3e基因的5'端插入loxP-Stop-loxp（LSL）盒， 在Cd 3eLSL小鼠中。先前的研究表明，胸腺T细胞不能发育超过双阴性DN 3 在Cd 3e缺陷小鼠中的阶段。类似地，在不存在Cre的情况下，在细胞中将不合成功能性CD 3e。 纯合子Cd 3eLSL/LSL小鼠。在我们的初步实验中，我们证实这些小鼠中没有T细胞。控制 T细胞发育和TCR库多样性，我们将Cd 3eLSL与我们可用的Rag 1CreER敲入小鼠杂交 其在早期T细胞分化期间瞬时表达诱导型CreER。我们将用滴定的 他莫昔芬的量，以控制T细胞发育的可能性。然后我们将使用例程 评估T细胞发育的剂量依赖性诱导、Treg的多样性和常规免疫调节的方法。 T细胞库、稳态免疫激活和抗肿瘤免疫。 总之，我们将开发一种创新的遗传工具来确定T细胞库的因果作用 自身免疫性疾病和癌症的多样性。使用这种遗传工具进行的研究将提高我们的 基本理解和基于Treg的免疫性疾病和癌症治疗方法。