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

研究T细胞谱系多样性原因作用的遗传开关 T细胞携带着在其发育过程中选择的不同的抗原受体(TCR)谱系。因为 TCR基本上是通过基因片段的重组以随机的方式产生的，并可能发生反应 对于自身抗原，人们发现了几种机制来限制T细胞的自身反应，包括胸腺阴性 调节性T(Treg)细胞的选择和诱导。在这些机制中，Treg细胞发挥着至关重要的、非 在维持自我耐受和抑制自身免疫性疾病方面的多余作用。先前的研究表明 多克隆常规T细胞(非Treg T细胞)是有效发育Treg细胞所必需的。然而， 这一观察结果的免疫学功能尚未被研究。不断积累的证据也将T 细胞淋巴细胞减少到自身免疫。尽管潜在的机制尚不清楚，但T细胞的多样性 曲目可能在决定免疫耐受性方面发挥关键作用。一如既往地，自身免疫力的提高 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。 鉴于传统T细胞为Treg细胞的发育和免疫抑制提供了必要的线索 功能，我们假设整个T细胞谱系的多样性在促进Treg细胞方面起着因果作用 诱导和作用，从而抑制免疫激活和自身免疫性疾病。对这一点的严格考验 假说和确定这一过程的机制将为研究Treg细胞提供新的见解。 介导的免疫耐受。 然而，要实现这一目标有几个技术障碍。为了解决这个问题，我们建议 用创新的遗传工具模拟TCR多样性。具体地说，我们将利用 CD3e在T细胞分化中的作用，并将loxP-Stop-loxP(LSL)盒插入到CD3e基因的5‘端，导致 在CD3eLSL小鼠中。先前的研究表明，胸腺T细胞不能发育超过双阴性DN3 Cd_3e缺乏小鼠的发育阶段。同样，在没有Cre的情况下，CD3e不会在体内合成 纯合子CD3eLSL/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。去控制 T细胞发育和TCR谱系多样性，我们将把CD3eLSL与我们可用的Rag1Creer敲入鼠杂交 在T细胞分化早期瞬时表达诱导性CRER。我们将用滴定的方法治疗这些小鼠 持续服用他莫昔芬以控制T细胞发育的概率。然后我们将使用例程 方法评估T细胞发育的剂量依赖性诱导，以及Treg和常规的多样性 T细胞库，稳态免疫激活，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细胞谱系的因果作用 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改善我们的 基于Treg的免疫性疾病和癌症治疗的基本知识和方法。

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.