The Effect of Radiotherapy on the Susceptibility of Cancer Cells to Natural Killer Cell Cytotoxicity

Abstract

Natural killer (NK) cells contribute to immune surveillance against cancer, but the impact of common treatments, such as radiotherapy, on their interaction with cancer cells is poorly understood. Here the response of human peripheral blood NK cells against cancer cell lines treated with X-ray irradiation was investigated. Irradiation of cancer cells was found to significantly reduce NK cell cytotoxicity induced either constitutively or via antibody-dependent cellular cytotoxicity against cell lines from multiple cancers. Resistance appeared 72 hours post-irradiation and persisted for two weeks. Resistance was found to be linked to cell cycle arrest, since pharmacological inhibitors of the cell cycle induced similar resistance as radiotherapy. Although radiotherapy induced resistance to NK cell cytotoxicity, NK cell degranulation, conjugation, and detachment were found to be unaffected. These results were confirmed by live imaging of NK cells and cancer cells, which revealed that the dynamics of the NK cell-cancer cell interactions were not affected, but irradiated cancer cells were less likely to be killed. Instead, radiotherapy was found to induce an intrinsic resistance against perforin. Irradiated cancer cells were highly resistant to perforin-induced calcium flux and lytic death, but their susceptibility to death receptor mediated cytotoxicity was enhanced. Surprisingly, radiotherapy did not affect the binding of perforin or membrane repair in response to pore formation. Instead, functional pore formation, which was observed through the uptake of small dyes directly through the pore, was impaired. Radiation-induced time-dependent up-regulation of phosphatidylserine on the surface of cancer cells correlated with resistance to perforin. This link between phosphatidylserine and perforin inhibition was further demonstrated by the finding that increasing concentrations of phosphatidylserine on synthetic liposomes inhibited lysis by perforin. Finally, irradiation of cancer cells was also shown to inhibit cytotoxicity by CD19-specific CAR T cells. Together, these data establish a novel treatment-induced resistance to lymphocyte cytotoxicity, which may be important for the design of radiotherapy-immunotherapy protocols.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jamie Honeychurch (Supervisor) & Daniel Davis (Supervisor)