Advanced metastatic melanoma (AMM) refractory to current therapies remains difficult to treat. Adoptive cell transfer (ACT) of TILs was approved as a second line therapy for AMM patients refractory to immune checkpoint blockade (ICB). Despite positive responses observed in a subset of patients, many are resistant, highlighting a need to identify strategies to improve TIL-ACT efficacy. We have shown a low precursor frequency (PF) of self-antigen (SAg)-specific CD8+ T cells in ACT products enhances the quality of the antitumor response, whereas SAg-specific CD4+ T cells are susceptible to exhaustion at similar frequencies. These data suggest clonal abundance plays a critical role in determining T cell functional state. ICB reinvigorates exhausted T cells, however, whether ICB can reprogram exhaustion arising from dysregulated PF or subset composition is unknown. We hypothesize TIL therapy efficacy is determined by Ag-specific PF and T cell subset composition of the infusion product, and combining TIL-ACT with ICB reprograms exhausted T cells to sustain the hostile TME. We aim to determine the optimal dose and PF of tumor-specific T cells in the TIL product required for therapeutic efficacy and evaluate rational ICB-based. Our preliminarily findings demonstrate that increasing the number of Ag-specific CD4+ in ACT product enhances early tumor control in B16-bearing mice. Next, we expand ex vivo murine TILs which retain effector function in vitro and exhibit increased expression of inhibitory checkpoints (e.g. PD-1, CTLA-4). In the future we will titrate TIL-ACT doses and assess immune checkpoint expression post-infusion to design the best combination regimen in TIL-ACT.