PROJECT SUMMARY Prognosis is poor for patients diagnosed with locally advanced melanoma. Survival outcomes are improved with checkpoint blockade in the neoadjuvant, adjuvant and unresectable setting. Dual checkpoint blockade further enhances outcomes, but often at the cost of significant clinical toxicity. Despite this progress, for the more than 40% of patients who do not respond, outcomes remain dismal. There is a clear need to develop innovative treatment approaches with higher response rates and less toxicity. Radiation (RT) has immuno- stimulatory activity capable of inducing an anti-tumor CD8+ T cell response within and outside of the irradiated field - an abscopal effect. In preclinical and clinical studies, RT’s immuno-stimulatory activity synergizes with checkpoint blockade (αPD-1) to significantly improve tumor control with limited toxicity. Despite the encouraging clinical potential of RT + αPD-1, the abscopal effect still only occurs in a minority of patients. To determine innovative methods to enhance the abscopal effect, we evaluated tumor-directed RT’s impact on different CD8+ T-cell subsets in a murine model. The Ahmed lab has shown an exhausted PD-1+ T cell subset, stem-like CD8+ TCF-1+ T cells, are essential for robust αPD-1 responses. Dendritic cell (DC) provided peptide- MHC and co-stimulation along with αPD-1 stimulate stem-like T cell expansion and differentiation into terminal effector cells capable of tumor killing. I have shown this stem-like subset is primarily localized to the tumor- draining lymph node (TDLN). Strikingly, tumor-directed RT alone stimulates maturation of DCs and proliferation of this subset in the TDLN. This proliferative response following RT, independent of αPD-1, suggests a novel RT induced mechanism for T-cell reinvigoration which may be exclusively DC dependent. Notably, for locally advanced or high-risk melanoma patients, immunostimulatory therapy (RT and/or αPD-1) are typically given following tumor and TDLN removal. This current approach reduces the immuno-stimulatory potential of either treatment compared to their administration with an intact TDLN. Importantly, combination pre-operative αPD-1 and RT have not been investigated in locally advanced or high-risk melanoma. Based on these preliminary results, our central hypothesis is that the stem-like CD8+ T cells in the TDLN are important mediators of the Tumor- RT stimulated anti-tumor abscopal effect. To test this hypothesis, we will: (1) determine the role of the stem-like CD8+ T cells in the abscopal effect; (2) determine whether dendritic cell mediated stimulation is critical for RT induced stem-like T cell expansion in the TDLN; (3) determine whether neoadjuvant Tumor-RT with αPD-1 is associated with increased stem-like CD8+ T cell infiltration in locally advanced melanoma in a pilot human study. Results of this proposal may identify a novel mechanism for overcoming T-cell exhaustion which has less immunologic toxicity with increased anti-t...