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 T 细胞携带在其发育过程中选择的多种抗原受体 (TCR) 库。因为 TCR 本质上是通过基因片段重组以随机方式生成的，并可能发生反应 针对自身抗原，发现了多种机制来限制 T 细胞的自身反应性，包括胸腺阴性 选择和调节性 T (Treg) 细胞诱导。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发挥着多余的作用。先前的研究表明 高效 Treg 细胞发育需要多克隆常规 T 细胞（非 Treg T 细胞）。然而， 这一观察结果的免疫功能尚未得到研究。不断积累的证据也将T联系起来 细胞淋巴细胞减少至自身免疫。尽管其潜在机制尚不清楚，但 T 细胞的多样性 曲目可能在决定免疫耐受方面发挥关键作用。一致地，自身免疫力升高 在衰老过程中观察到，由于胸腺退化和 T 细胞衰老，T 细胞多样性降低。 鉴于传统 T 细胞为 Treg 细胞发育和免疫抑制提供了重要线索 功能，我们假设整个 T 细胞库的多样性在促进 Treg 细胞中发挥因果作用 诱导和功能，从而抑制免疫激活和自身免疫性疾病。对此进行了严格的测试 假设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，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 使用创新的遗传工具模拟 TCR 多样性。具体来说，我们将充分利用 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，从而产生 在 Cd3eLSL 小鼠中。先前的研究表明，胸腺 T 细胞无法发育至双阴性 DN3 之外 Cd3e 缺陷小鼠的阶段。类似地，如果没有 Cre，则不会合成有功能的 CD3e。 纯合 Cd3eLSL/LSL 小鼠。在我们的初步实验中，我们确认这些小鼠体内没有 T 细胞。控制 T 细胞发育和 TCR 库多样性，我们将把 Cd3eLSL 与我们现有的 Rag1CreER 敲入小鼠杂交 在早期 T 细胞分化过程中瞬时表达诱导型 CreER。我们将用滴定法治疗这些小鼠 持续使用一定量的他莫昔芬来控制 T 细胞发育的可能性。然后我们将使用常规 评估 T 细胞发育的剂量依赖性诱导、Treg 和常规 T 细胞多样性的方法 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改善我们的 基于 Treg 的免疫疾病和癌症治疗的基本认识和方法。

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.