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The immune modulatory effects of radiation therapy (RT) on the innate and adaptive arms of the immune system are increasingly recognised to be important. Utilising RT in a manner that supports host anti-cancer immune responses is a paradigm gaining interest, but how best to employ this remains unclear. Here we examine the impact of radiation dose-fractionation on tumor-associated natural killer (NK) cells, which form an important line of host innate immune defense against cancer. From this, we identify ways of better leveraging the therapeutic potential of NK cells with RT and cancer immunotherapy. 

Established AT3-OVA intra-mammary and MC-38 subcutaneous tumors growing in wildtype C57BL/6 mice were locally irradiated with different radiation dose-fractionations to dissect the effects of radiation dose per fraction (DPF) from total radiation dose (biological effective dose, BED). Depleting antibodies were used to assess the impact of immune subsets on tumor growth post irradiation. Effects on the tumor microenvironment were examined with flow cytometry and RNA sequencing.  Such information guided the selection and application of antibody-based checkpoint blockade therapy to promote host innate and adaptive anti-cancer immune defenses.

Radiation dose-fractionations are not equivalent in their capacity to engage CD8+ T cell and NK cell responses. Treatment with the 3x4Gy, 9x4Gy and 3x8Gy regimens elicited effective CD8+ T cell responses that supported the anti-cancer activity of anti-PD-1 therapy. The high DPF regimen of 1x20Gy, despite having a similar BED to 9x4Gy, was unable to engage effective CD8+ T cell responses. In contrast, only higher BED regimens (36-45Gy) evoked NK cell responses that significantly slowed tumor growth. While all radiation regimens tested promoted an acute increase in tumor-associated NK cell numbers, only the higher BED regimen supported a sustained elevation in tumor-associated NK cell numbers with higher activation markers and corresponding enrichment of the NK cell-mediated cytotoxicity gene signature. The anti-cancer activity of these NK cells is highly sensitive to control by a concurrent but brief wave of radiation-induced regulatory T cells.  These early and late phase effects of RT on NK cell responses could be leveraged with checkpoint blockade therapy to achieve prolonged tumor control and, in some instances, complete regression.

NK cells may play a critical role in supporting the therapeutic capacity of RT and immunotherapy combinations, particularly in the context of high BED RT. Understanding the regulation and kinetics of radiation-induced NK cell responses reveals opportunities for optimising RT and immunotherapy strategies.