PROJECT DESCRIPTION/ABSTRACT Programmed death ligand 1 (PD-L1), which promotes immune escape, is overexpressed in triple negative breast cancer (TNBC), an aggressive subtype of breast cancer characterized by poor prognosis. Clinically approved PD-L1 antibodies augment anti-tumor immunity by blocking extracellular PD-1/PD-L1 binding. However, the contribution of intracellular PD-L1 to anti-tumor immunity and therapeutic resistance has remained poorly understood. We have discovered a novel role for intracellular PD-L1 as an RNA binding protein that promotes the stability of target RNAs. This new intracellular PD-L1 function in regulating RNA expression was independent of the established extracellular role of PD-L1 as the ligand for PD-1. The activity of the anti-tumor immune response is governed by a balance between immune effector cells and immune suppressor cells. Regulatory T cells (Tregs) are a CD4+ T cell subpopulation that inhibit effector cell activity, suppress anti-tumor immunity and promote therapeutic resistance. A hallmark of Tregs is the expression of the transcription factor, Foxp3, which binds to the promoters of genes that support Treg activity. Foxp1 is a closely related family member of Foxp3. Emerging data has demonstrated that Foxp1 cooperates with Foxp3 to encourage Foxp3-mediated transcription and Treg function by maintaining Foxp3 occupancy at promoters of target genes. In our preliminary data, we have identified Foxp1 as a key PD-L1 target RNA. We have found that the PD-L1 cytoplasmic domain, but not the PD-L1 extracellular domain, interacts with Foxp1 RNA and promotes Foxp1 expression. In addition, we have discovered that intracellular PD-L1’s promotion of Foxp1 expression is necessary for proper Treg differentiation, Treg function, and TNBC progression. The Akt-mammalian target of rapamycin (mTOR) pathway regulates metabolic reprogramming for proper T cell maturation and function. Our preliminary data suggests that PD-L1 and Foxp1 are required for proper Akt-mTOR pathway activation and metabolism specifically in Tregs, but not effector T cells, suggesting that targeting the PD-L1-Foxp1 pathway may preferentially inhibit Tregs to address therapeutic resistance. Our overarching hypothesis is that intracellular PD-L1 stabilizes Foxp1 RNA to promote Treg immunosuppressive activity and therapeutic resistance. Further, inhibiting intracellular PD-L1 will promote anti-TNBC immunity by blocking Treg activity. This hypothesis will be tested in a series of three aims: Aim 1 will determine the influence of intracellular PD-L1 on Foxp1 mRNA stability and Treg differentiation; Aim 2 will determine the effect of intracellular PD-L1 on Treg function; Aim 3 will compare intracellular PD-L1 and extracellular PD-L1/PD-1 directed TNBC therapy. Clear delineation of the impact of intracellular PD-L1 on cancer therapy will provide important insight for optimizing combination strategies aimed at overcoming immune escape and therapeutic resistanc...