Summary Radiotherapy (RT) can stimulate anti-cancer T cell responses that target primary and distant tumors. In addition to antigen-mediated stimulation of effector T cells, signals from stimulatory cytokines, notably interleukin-2 (IL-2), are necessary for optimal T cell function and memory. However, timing and IL-2 receptor (IL-2R) bias of such signals are ill-defined. Using image-guided RT in a mouse colon cancer model, we observed that single high-dose (1 x 20 Gy) RT transiently upregulated IL-2Rα (CD25) on effector CD8 + T cells, facilitating the use of CD25-biased IL-2 immunotherapy. Timed administration of CD25-biased IL-2 treatment after RT favored the expansion of tumor-infiltrating CD8 + T cells over regulatory T cells and IL-2Rβ (CD122) high CD8 + T cells, which resulted in comparable anti-tumor effects as with RT plus CD122-biased IL-2 immunotherapy. Moreover, intratumoral CD8 + T cells from animals receiving combined IL-2R-biased IL-2 and RT showed reduced signatures of T cell exhaustion. Finally, these combination treatments affected both primary irradiated and distant non-irradiated tumors, achieving durable responses. We demonstrate that timed and IL-2R subunit-biased IL-2 immunotherapy synergized with single high-dose RT to achieve potent anti-cancer immunity.

IL-2 immunotherapy rescues irradiation-induced T cell exhaustionin vivo