Background Resection followed by radio-chemotherapy remains the mainstay of standard-of-care for patients with malignant brain tumors. Immunotherapies such as vaccinations, checkpoint inhibition or cellular therapies are promising approaches to complement or replace current therapies. Pre-clinical and clinical results indicate that immunotherapies benefit from a combination with radiotherapy by inducing immunogenic cell death and subsequent immune cell activation. However, the optimal dosage and fractionation to induce the optimal immune cell activation for a successful combination have still to be determined. Here we dissect the cellular and molecular immune cell dynamics after different dosages of irradiation in order to find the optimum for future studies testing radio- and immunotherapy combinations for brain tumors.

Methods The syngeneic and orthotopically transplanted glioma model GL261 was irradiated with different dosages of 2Gy (low), 5Gy (medium), and 8Gy (high) and CD45+ immune cells were sorted and analyzed with single-cell RNA sequencing, 3 and 7 days after irradiation. The spatial distribution of cells was investigated by immunocytochemistry as well as clearing of brains with subsequent 3D light sheet microscopy.

Results Single-cell transcriptomics of immune cells after different dosages revealed distinct dose- and time-dependent effects on specific subsets of T cells, NK cells, macrophages, microglia and DCs. Especially single doses of 5 and 8Gy resulted in reduced T cell exhaustion and higher T and NK cell activation. Affected macrophage, microglia and DC subsets showed an increased pro-inflammatory state with a high expression of chemokines such as CCL4, CCL12 and CXCL10. Overall, after higher doses, the TME was reshaped and the highest degree of immune cell activation and stimulation was observed and sustained until day 7. All affected cell subsets demonstrated a strong type I IFN response signature both on day 3 and 7 after irradiation. Immunocytochemistry imaging and 3D light sheet microscopy showed a depletion of T cells directly after irradiation but a high re-infiltration of T cells and perivascular accumulation starting at 48h after irradiation.

Conclusions Higher irradiation doses result in immune cell stimulation, but the impact of different dosages in combination with immunotherapies are subject to current investigation. However, the dominating type I IFN signatures observed in all dosages indicates a crucial role of irradiation-induced type I IFN in shaping the TME after radiotherapy. The observation, that T cells are depleted from the TME after irradiation but infiltrating cells sustain their activated phenotype for at least 7 days will instruct regimes combining immunotherapy with immune stimulating radiotherapy.

Ethics Approval All animal experiments were performed in compliance with the laboratory animal research guidelines and were approved by governmental authorities (animal protocols: G95–16, G170–21, regional administrative authority, Regierungspräsidium Karlsruhe, Germany).