PROJECT SUMMARY (See instructions): Double-negative T cells (DNTs), characterized by the absence of CD4 and CDB expression but the presence of aBeta T cell receptors (TCRs), play a pivotal role in regulating immune homeostasis within the ovary and uterus. However, how the origin and function of DNTs in the ovary and reproductive tract remains poorly understood, representing a critical gap in our knowledge of ovarian immune regulation. My recent investigations have demonstrated that these DNTs arise from peripheral CD8+ T cells that downregulate the CD8 coreceptor, adopting a unique transcriptional profile and acquiring suppressive functions. In models of autoimmune ovarian failure (AOF), DNTs are markedly reduced, correlating with ovarian dysfunction and infertility, while adoptive transfer of DNTs restores ovarian function and fertility by restraining pathogenic CD8+ T cell responses. Intriguingly, emerging observations indicate that DNTs also accumulate within ovarian tumors, suggesting a complex role in reproductive health by simultaneously modulating autoimmunity and potentially promoting local immune tolerance that could be co-opted by tumors. The central hypothesis of this project is that the reprogramming of peripheral CD8+ T cells into DNTs constitutes a critical immune checkpoint in the ovary, which can be harnessed to treat AOF and strategically modulated to enhance anti-tumor immunity. Specifically, this project will: (1) elucidate the molecular and transcriptional mechanisms driving the conversion of CD8+ T cells into regulatory DNTs within the ovarian microenvironment; (2) determine how DNTs suppress autoreactive T cells to maintain fertility in AOF models; (3) investigate how ovarian tumors exploit this pathway to establish local immune tolerance; (4) develop targeted strategies to enhance ONT-mediated tolerance in autoimmune conditions; and (5) explore engineering DNTs to restore CD8-associated cytotoxic programs as a novel approach