The Role of Treg and Tpex Interactions in Tumor-Draining Lymph Node Immunosuppression and Immunotherapy Response - PROJECT ABSTRACT Cytotoxic CD8 T cells are a necessary component of the anti-cancer immune response. When CD8 T cells encounter chronic antigen stimulation, they become dysfunctional and can no longer effectively exude their cytotoxic function1-3. Our lab and others have identified a CD8 stem-like progenitor, called Tpex, that retains its ability to kill cancer cells4. It was found that upon immune checkpoint blockade (ICB) therapy, these Tpex cells differentiate, egress from the tumor-draining lymph node (tdLN), and migrate into the tumor microenvironment to kill the tumor via cell-mediated cytotoxicity4. When the tumor-draining lymph node has been metastasized (metLN), this Tpex response is abrogated, and there is not a productive anti-tumor response with ICB4. Our lab found that in the metLN, Tpex were present but were preferentially surrounded by suppressive Tregs and DCs4. Additionally, Tregs are known to play an immunosuppression role in the TME and have a negative impact on the effectiveness of many current immunotherapies5-8. How Tregs are affecting the environment in the metLN and how Treg immunosuppression impacts other immune cell types, including Tpex, is not yet known. This proposal will test the hypothesis that LN metastasis amplifies Treg function both directly and indirectly to create an immunosuppressive niche in the metLN, limiting the function of Tpex cells and impairing a proper CD8 T cell response to ICB. Aim 1 of this proposal will identify the mechanism by which Tregs are creating an immunosuppressive environment in the tumor-draining lymph node. Aim 2 will define how Tregs are impacting the Tpex functionality. Aim 3 will determine how the Treg-Tpex cell axis affects ICB response and efficacy in the tdLN. This research approach will use a variety of methods, including an ex vivo organoid model, high-dimensional flow cytometry, multiplexed ion beam imaging (MIBI), single-cell RNA sequencing, and in vitro co-culture assays. These proposed studies will be among the first to study immune cell interactions and Tpex cells using patient-derived lymph node organoids. These results could elucidate novel mechanisms of immunosuppression in the tdLN and indicate how those mechanisms impact current cancer immunotherapy. This would result in an improved understanding of Treg and Tpex function in the tdLN and how ICB impacts them in the tdLN. The use of human-derived organoids as the primary model allows this research to be greatly translationally relevant and inform our understanding of cancer immunotherapy response, specifically in patients. Furthermore, this work will better inform novel immunotherapy approaches for the treatment of patients with metastatic cancer, especially in the fields of biologics and adoptive T cells, where preventing CD8 T cell exhaustion is a major goal. This research project and fellowship training will be conducted at a top-funded research institution, the University of California, San Francisco (UCSF), in the laboratory of Dr. Matthew Spitzer. Dr. Spitzer is an investigator with expertise in systems immunology, cancer immunology, and CD8 T cell biology as well as spatial and single-cell sequencing methods. This mentor and institution will provide a high-quality training environment with ample resources for the completion of this research and development of professional skills required for a career in academic research.
