Abstract: Breast cancer (BC), the most common cancer globally as of 2021 and accounting for 12% of all new annual cancer cases worldwide, is the most commonly diagnosed cancer among American women. Based on the expression of estrogen receptor alpha (ERα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), breast cancer can be classified into three major subtypes: luminal subtype (ERa/PR+, HER2-), HER2+ subtype (ERa/HR-, HER2+), and triple-negative subtype (TNBC, ERa/HR-, HER2-). All types of BC have metastatic potential. TNBC is the deadliest form. Chemoresistance is a major obstacle to therapeutic efficacy. Once chemoresistance develops, metastatic BC is incurable. Cancer immunotherapy has achieved unprecedented success in treating many types of advanced cancers, including TNBC. However, the response rate of BC patients to cancer immunotherapy is low because of the poor tumor infiltration of tumor-infiltrating lymphocytes (TIL). Developing more effective cancer immunotherapy approaches is critical to treating and curing TNBC patients. Using a T cell receptor (TCR)-alpha deficient Ja281 KO mouse model, we found that transferring thymocytes into Ja281 KO mice could completely inhibit EO771 and Py8119 TNBC growth in these cell- transferred mice. We further found that the cell transfer-induced antitumor immunity was mediated by tissue- resident αβ+CD4-CD8- double-negative T (DN T) cells formed from the transplanted donor population and depended on host NK cells. Deciphering the underlying mechanism will allow us to develop a powerful immunotherapy approach for TNBC treatment. The long-term goal of our research is to develop new immunotherapeutic regimens for cancer treatment. The objective of this project is to decode the mechanism of DN T cell antitumor immunity to TNBC. Our central hypothesis is that DN T cells initiate antitumor immunity and interact with NK cells to control TNBC growth and to shape tumor microenvironment (TME) and that a population of immunoinhibitory T cells modulate DN T cell antitumor function. We will test this hypothesis by pursuing the following three specific aims: Aim 1: Determine how tissue-resident DN T cells are generated and how they mediate antitumor immunity using the Ja281 KO mouse model. Aim 2. Determine the antitumor function of tissue-resident DN T/NK cell axis. Aim 3: Define the immunoregulatory cells that govern tissue-resident DN T cell generation and their antitumor function. The finding that tissue-resident DN T cells can inhibit TNBC growth and eradicate breast cancer is novel. The completion of the proposed research will not only greatly advance our knowledge of DN T cells in antitumor immunity, but also allow us to develop more effective approaches for TNBC immunotherapy.