Combinations of Radiotherapy with Vaccination and Immune Checkpoint Inhibition Differently Affect Primary and Abscopal Tumor Growth and the Tumor Microenvironment

SIMPLE SUMMARY: Radiotherapy (RT) with higher single doses is capable of creating a beneficial immunological tumor microenvironment for additional immune therapies to trigger systemic anti-tumor immune responses. Here, we aimed to boost RT-induced immune responses with inactivated whole tumor cell vaccines generated with high hydrostatic pressure (HHP) and to break immunosuppression by inhibition of suppressive immune checkpoint molecules, such as PD-1. Our results highlight that HHP vaccines act systemically to retard tumor growth, but only of previously irradiated tumors. In contrast, abscopal anti-tumor immune responses, namely those to non-irradiated tumors, are triggered by a combination of RT of not too many fractions plus anti-PD-1, being characterized by the induction of distinct immune alterations in the primary and abscopal tumor, respectively. This emphasizes that future multimodal radioimmunotherapy approaches need to be carefully optimized regarding RT fractionation and the interaction of different immunotherapies with each other. ABSTRACT: Radiotherapy (RT) is known to have immune-modulatory properties. We hypothesized that RT and inactivated whole tumor cell vaccines generated with high hydrostatic pressure (HHP) synergize to retard the tumor growth which can be additionally improved with anti-PD-1 treatment. In abscopal tumor models, we injected mice with B16-F10 melanoma or TS/A mammary tumors. To evaluate the efficiency of RT in combination with HHP vaccines, we locally irradiated only one tumor with 2 × 8 Gy or 3 × 8 Gy. HHP vaccines further retarded the growth of locally irradiated (2 × 8 Gy) tumors. However, HHP vaccination combined with RT failed to induce abscopal anti-tumor immune responses, namely those to non-irradiated tumors, and even partly abrogated those which were induced with RT plus anti-PD-1. In the latter group, the abscopal effects were accompanied by an elevated infiltration of CD8+ T cells, monocytes/macrophages, and dendritic cells. 3 × 8 Gy failed to induce abscopal effects in association with increased expression of immunosuppressive checkpoint molecules compared to 2 × 8 Gy. We conclude that HHP vaccines induce anti-tumor effects, but only if the tumor microenvironment was previously modulated by hypofractionated RT with not too many fractions, but failed to improve RT plus anti-PD-induced abscopal responses that are characterized by distinct immune alterations.