Tumor Cell-Intrinsic PD-L1 Effects on Radiation-Induced Locoregional Antitumor Immunity

Abstract

Targeting PD-L1 is a beneficial strategy to reinvigorate antitumor immunity, however variable response and resistance are challenging and suggest the need for multimodality approaches. Tumor cell-intrinsic PD-L1 signals also regulate non-canonical pathogenic pathways that may impact treatment resistance. Ionizing radiation (IR) can induce antitumor immunity and has demonstrated therapeutic synergy with immunotherapy in some cases, however tumor-driven immunologic mechanisms affecting clinical outcomes remain incompletely understood. In this study, we investigated the impact of tumor cell-intrinsic PD-L1 signals on IR-induced locoregional immune response and tumor control. We used orthotopic B16-F10 melanoma (WT-B16) and 4T1 triple negative breast cancer (WT-4T1) murine tumor models, as well as PD-L1 disabled variants (KO) generated by CRISPR/Cas9, implanted bilaterally. IR (10 Gy) was targeted at one tumor alone to evaluate both direct and indirect IR effects based on tumor PD-L1 status. We evaluated response by tumor volume (TV) measurements, flow cytometry of tumor-infiltrating lymphocytes (TILs) and tumor draining lymph nodes (TDLNs) in both irradiated and unirradiated compartments, and granzyme B (GZB) PET imaging to assess functional in vivo changes. Chemokine-based multiplex assays were used to assess cell lines receiving IR (4Gy) and ex vivo tumor lysates and serum. IR-induced local tumor control was not significantly affected based on tumor PD-L1 status, however deactivation of tumor cell PD-L1 enhanced IR-induced regional tumor control. Unirradiated WT tumors in mice harboring irradiated KO but not irradiated WT tumors demonstrated a significant mean reduction in TV with instances of complete distant tumor regression. PET imaging demonstrated a nearly 2-fold higher concentration of GZB in KO versus WT tumors, in line with known locally immunosuppressive effects of tumor PD-L1. Remarkably, GZB levels were >1.5-fold higher in unirradiated WT tumors in mice harboring an irradiated KO versus WT tumor, which correlated with a 50% increase in PD-1+CD8+ T cells. Higher levels of CD62+CD44- naïve CD4+ (4-fold) and CD8+ (2-fold) memory T cells were seen in TDLNs of irradiated KO versus WT tumors. Cytokine levels positively correlated with immune recruitment and activation status, as CXCL10, CCL2 and CCL5 were significantly upregulated in PD-L1 KO versus WT tumors cells. Results from this study demonstrate cell-intrinsic PD-L1 inhibits IR-induced locoregional immune activation and frequency of regional tumor control, with clinical implications including therapeutic targeting of tumor cell-intrinsic PD-L1 signals to enhance IR-induced immunogenicity, utility of IR based on tumor PD-L1 status particularly in the metastatic setting, and immunotherapy combinations. Future studies investigating mechanisms of resistance to IR-induced immune activation to enhance responsiveness are warranted.