Project Summary Extensive studies have been conducted to define the development, conversion, stability, and regulatory mechanisms of CD4+Foxp3+ regulatory T cells (Tregs) in homeostasis and a variety of disease models. It is well-known that Tregs are recruited, converted, and expanded in the tumor microenvironment and act as one of the major immunosuppressive mechanisms dampening spontaneous tumor-associated antigen (TAA)- specific T cell immunity and immunotherapy and active vaccination induced anti-tumor immunity. However, how Tregs behave in the metabolically abnormal tumor microenvironment remains unknown. The Warburg effect is an important metabolic feature in many types of cancer. Given that nutrients including glucose are poorly replenished in the tumor, it is assumed that T cell glycolytic metabolism has been altered due to the Warburg effect in the tumor microenvironment. In support of this, poor glycolysis can alter effector memory T cell function in the tumor microenvironment. In addition, the oxygen-sensing prolyl-hydroxylase proteins, necrotic cells released potassium ions, and abnormal zinc metabolism can impair effector T cell function in the tumor microenvironment. These studies underscore the significance of metabolic regulation of memory T cells in the tumor. Tregs exhibit a memory and effector phenotype in the human tumor microenvironment. It is unknown whether Tregs are subject to glycolytic regulation in the tumor. Furthermore, oxidative stress is an additional metabolic feature in the tumor microenvironment. Myeloid dendritic cells (DCs) are phenotypically and functionally altered by oxidative stress in the tumor microenvironment. However, it is unknown whether oxidative stress alters Treg phenotype and function in the tumor and affects cancer immunotherapy. To address these questions, we have examined the phenotypic and functional nature of Tregs in human ovarian cancer and several types of mouse cancer. We have found that Tregs are highly apoptotic in the tumor microenvironment. Interestingly, apoptotic Tregs are superior suppressors via a distinct mechanism. Furthermore, oxidative stress, rather than glycolysis, is a metabolic mechanism controlling tumor Treg functional behavior and tempering therapeutic efficacy of immune checkpoint therapy. This project is to conduct comprehensive molecular, functional, translational, and clinical research on the nature of Tregs and their metabolic pathway in the cancer microenvironment. We will provide rich opportunities to take our understanding of Treg biology in the tumor to a new level of basic and practical application. Our specific aims are: Aim 1 is to test our hypothesis that oxidative stress controls Treg apoptosis in the tumor microenvironment. Aim 2 is to determine the molecular mechanisms controlling the energy circuit of Tregs and the interaction between Tregs and APCs in tumor.