PROJECT SUMMARY Breast cancer is estimated to be the most diagnosed cancer in American women and account for 15% of female cancer-related deaths in 2023. Triple-negative breast cancer (TNBC) is an aggressive subtype lacking effective and durable treatment strategies due to the absence of targetable cell surface receptors, including ER, PR, and HER2. Consequently, TNBC patients face the worst prognosis among breast cancer subtypes. Adaptive immune activity in the tumor microenvironment is a well-known prognostic indicator within TNBC patients and is correlated with treatment benefit. However, tumors frequently evolve to avoid immune recognition, which negatively impacts patient prognosis. Clinical data from paired primary and metastatic breast tumors demonstrate that TNBC metastases commonly downregulate expression of genes involved in major histocompatibility complex Class I (MHC-I) antigen presentation, a mechanism by which CD8+ cytotoxic T cells can recognize and kill tumor cells displaying aberrant neoantigens. Therefore, understanding and addressing tumor immune evasion mediated by loss of MHC-I has the potential to improve TNBC patient outcomes. Our central hypothesis is that breast tumor MHC-I loss results in a unique immune-evasive phenotype that can be overcome through novel treatment modalities. This proposed research will functionally and molecularly characterize MHC-I-low breast tumors and evaluate customized combination immunotherapy approaches to enhance anti-tumor adaptive immunity. Experiments in Aim 1 will determine how MHC-I loss affects tumor progression and immune cell infiltration, through both in vivo immunocompetent mouse models and computational analysis of human clinical breast cancer datasets. In Aim 2, I will assess whether a CD40 agonist or demethylating agent will increase the efficacy of immunotherapy against MHC-I-low tumors. Altogether, this work will comprehensively inform how MHC-I-mediated immune evasion affects multiple facets of breast tumor biology and which therapeutic strategies should be explored in the clinic to improve patient outcomes.