Purpose/Objective(s): Multiple preclinical studies and case reports have described synergistic interactions between radiation (RT) and checkpoint blockade immunotherapy (CBI), and the combination has the potential to enhance locoregional as well as systemic control. We previously described a role for RT in enhancing T cell activation and proliferation via antigen cross-presentation in the draining lymph node when combined with anti-PD-1 CBI. Here, we further define this role and specifically analyze the clonal diversity of T cell immune responses induced by RT alone and RT combined with CBI. Materials/Methods: Stereotactic image guided RT was delivered to B16-OVA melanoma flank tumors via the small animal radiation research platform (SARRP). Cohorts of C57B/6 mice received either control IgG Ab or three IP injections of 200ug anti-PD-1 Ab before, during, and after RT. Development of antigen-specific immune responses was analyzed by flow cytometry on LSR II. T cell clonal diversity in the tumor draining lymph node (DLN) and matched tumor infiltrating lymphocytes (TIL) was assayed by TCR sequencing. Results: Radiation therapy enhanced cross presentation of tumor derived antigens in the DLN, which resulted in increased proliferation and activation of tumor specific CD8 T cells in vivo. These effects were abrogated in MHC Class I KO mice indicating that cross-presentation may be required for the immune stimulatory effects of RT. We observed a significant increase in antigen specific TIL when RT was combined with anti-PD-1 CBI, which correlated with tumor regression and improved local control. Preliminary analysis of T cell clonal diversity reported that the mean total number of sequences was 968,092 +/-183,938 in the DLN versus 15 225 +/-16,003 in the TIL. The mean total number of productive sequences was 679,321 +/-127,734 in the DLN versus 9,836 +/-9,386 in the TIL. We observed a statistically significant increase in the clonality of TIL samples compared to the DLN (DLN 0.0323 +/-0.009 vs TIL 0.0946 +/-0.0274; P < .0001). Our preliminary results identified a significant increase in the number of unique productive TCR sequences in the TIL of the group that received RT+CBI compared to untreated controls (controls 127 +/-98 vs RT+CBI 350 +/-15, P < .05). Conclusion: The immune-stimulating effects of RT were significantly increased when combined with anti-PD-1 CBI resulting in improved local tumor control. Mechanistically, we found that RT enhanced antigen cross-presentation in the DLN and increased T-cell activation, proliferation, and infiltration into tumors. Analysis of T-cell clonal diversity identified significant differences between the DLN and TIL samples and between treatment groups. These findings demonstrate the ability of RT to prime endogenous antitumor immune responses and suggest that RT + PD-1 blockade may result in an increased repertoire of antitumor T cells.

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