Background: The use of immunotherapy strategies for the treatment of advanced cancer is rapidly increasing. Most immunotherapies rely on induction of CD8+ tumor-specific cytotoxic T cells that are capable of directly killing cancer cells. Tumors, however, utilize a variety of mechanisms that can suppress anti-tumor immunity. CD4+ regulatory T cells can directly inhibit cytotoxic T cell activity and these cells can be recruited, or induced, by cancer cells allowing escape from immune attack. The use of ionizing radiation as a treatment for cancer has been shown to enhance anti-tumor immunity by several mechanisms including immunogenic tumor cell death and phenotypic modulation of tumor cells. Less is known about the impact of radiation directly on suppressive regulatory T cells. In this study we investigate the direct effect of radiation on human T REG viability, phenotype, and suppressive activity. Results: Both natural and TGF-β1-induced CD4+ T REG cells exhibited increased resistance to radiation (10 Gy) as compared to CD4+ conventional T cells. Treatment, however, decreased Foxp3 expression in natural and induced T REG cells and the reduction was more robust in induced T REGS. Radiation also modulated the expression of signature iT REG molecules, inducing increased expression of LAG-3 and decreased expression of CD25 and CTLA-4. Despite the disconcordant modulation of suppressive molecules, irradiated iT REGS exhibited a reduced capacity to suppress the proliferation of CD8+ T cells. Conclusions: Our findings demonstrate that while human T REG cells are more resistant to radiation-induced death, treatment causes downregulation of Foxp3 expression, as well as modulation in the expression of T REG signature molecules associated with suppressive activity. Functionally, irradiated TGF-β1-induced T REGS were less effective at inhibiting CD8+ T cell proliferation. These data suggest that doses of radiotherapy in the hypofractionated range could be utilized to effectively target and reduce T REG activity, particularly when used in combination with cancer immunotherapies.

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