Carbon ion irradiation plus CTLA4 blockade elicits therapeutic immune responses in a murine tumor model

Radiotherapy can act as an in situ vaccine thereby activating tumor-specific immune responses that prevent tumor outgrowth in treated patients. While carbon ion radiotherapy has shown superior biophysical properties over conventional photon irradiation, the immunological effects induced have remained largely uncovered. The combination of radiotherapy with immune checkpoint inhibition (radioimmunotherapy) aims at further enhancement of anti-tumor immunity; however, studies on the immune cell composition in irradiated and distant tumors following radioimmunotherapy with carbon ions are scarce. We have established a bilateral tumor model by time shifted transplantation of murine, Her2+ EO771 tumor cells onto the flanks of immune competent mice followed by selective irradiation of the primal tumor, while sparing the consecutive tumor. We demonstrate that CTLA4-but not PD-L1-based radioimmunotherapy induces complete tumor rejection in our model. Intriguingly, local tumor control caused in situ immunization resulting even in eradication of non-irradiated, distant tumors. Moreover, cured mice were protected against EO771 rechallenge indicative of long lasting, tumor-protective immunological memory. Deconvolution of the treatment induced immunological effects by single cell RNA-sequencing (scRNA-seq) and concomitant flow cytometric analyses revealed in irradiated tumors predominating myeloid cells that developed into distinct tumor-associated macrophage clusters with upregulated expression of TNF and IL1 responsive genes, as well as activation of NK cells. Non-irradiated tumors showed higher frequencies of naive T cells in irradiated mice, which were activated when combined with CTLA4 blockade. In conclusion, radioimmunotherapy with carbon ions plus CTLA4 inhibition reshapes the tumor-infiltrating immune cell composition and can induce complete rejection even of non-irradiated tumors. Our data present a rationale to combine radiotherapy approach with CTLA4 blockade to achieve durable anti-tumor immunity. Evaluation of future radioimmunotherapy approaches should thus not only focus on the immunological impacts at the site of irradiation but should also consider systemic immunological effects that might affect outgrowth of non-irradiated tumors.