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

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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.