Define regulatory T cell repertoire sustainability

About This Grant

SUMMARY Regulatory T cells (Tregs) actively suppress effector T cells that not only cause autoimmune diseases but also mount anti-tumor immune response. A diverse repertoire of T cell antigen receptors (TCRs) is conferred upon Tregs during their selection and development. We previously demonstrated that Treg repertoire diversity is essential for immune regulatory function. However, Treg repertoire is not static, but rather undergoes dynamic changes, involving continuous new Treg generation, incorporation, and likely turnover of aged Tregs. This raises questions about the nature of Treg repertoire dynamics, sustainability, and immunological roles. Despite the substantial advancements in Treg research, the answers to these questions remain largely speculative due to technical challenges in tracking and controlling specific Treg repertoire without introducing adverse effects. To address this critical knowledge gap, we developed novel mouse genetic tools. Rag1CreER knock-in mice were engineered to perform timestamp tracing of thymic T cells. In the presence of a Rosa reporter, such as RosaLSL-tdTomato (LSL, loxP-Stop-loxP), tamoxifen treatment labeled newly derived thymic T cells, allowing us to investigate the dynamics of thymic Treg generation, egression, and incorporation into the peripheral Treg repertoire in lymphoid and non-lymphoid organs at various stages of life. When integrated with advanced analytical platforms, this will enable us to resolve the developmental heterogeneity of Treg repertoire. We also developed Foxp3LSL mice to seamlessly switch Treg fate in an inducible manner via its master regulator Foxp3. When combined with CreER lines, such as Rag1CreER or available Cd4CreER, these Foxp3LSL mice, upon tamoxifen treatment, would restore normal Foxp3 regulation in precursor cells or Treg cells, depending on the timing of CreER expression. In preliminary experiments, we demonstrated the reliability and feasibility of these methods and unveiled unexpected findings of the activity of Tregs with a finite repertoire. In this study, based on preliminary results, we propose that existing Tregs undergo “exhaustion-like” deterioration over time or upon immune challenges. Consequently, continuous new Treg generation is required to maintain Treg repertoire integrity. To test this hypothesis, we will first rigorously define the exhaustion-like nature of Tregs with a finite repertoire in non-competitive conditions with or without immune challenges. We will then use these innovative genetic tools to determine the causal role of continuous thymic Treg generation. Finally, we will test the hypothesis that excessive stimulation via TCR signaling drives the exhaustion-like activity of Tregs in lineage stability and Foxp3-dependent gene regulation. Overall, we will utilize innovative approaches to define Treg deterioration in the resting state over time or after immune challenges, addressing a long-standing question in the field. Successful completion of our study would lead to a significant conceptual advancement and potential novel strategies to improve Treg-based treatment of cancer and autoimmune diseases.