Opposing Two-Fraction Regimens Combining Low and High Doses of Ionizing Radiation Elicit Differential Immune Responses.

Abstract

Radiation therapy exerts direct cytotoxic effects on cancer cells, but also induces immunogenic responses in the tumor microenvironment (TME), eliciting both immune-stimulatory and immune-suppressive dynamics. These effects are influenced by fraction size, fractionation regimen, and timing of radiation therapy-immunotherapy co-administration. In this study, 2 opposing 2-fraction regimens were investigated, in which the same cumulative physical dose was applied to the tumor, but low- and high-dose fractions were administered in opposite order. Using 2 murine tumor models (MC38 and B16F10-Luc) implanted heterotopically, we investigated how dose sequencing affects the immune dynamics interplay in the TME and characterized the TME in response to low (6 Gy) and high (12 Gy) single doses of ionizing radiation and to 2 opposing fractionation regimens (6 + 12 Gy vs 12 + 6 Gy). Furthermore, we assessed the effect of the different radiation therapy regimens on tumor growth and survival and strategically combined the 2-fraction regimens with an immune checkpoint blockade. We demonstrated that the 2 opposing fractionation regimens generated distinct TMEs, depending on the sequence of low- and high-dose fractions. Although the 12 + 6-Gy regimen resulted in a TME enriched with CD8+ T cells with increased effector function, tumors treated with the 6 + 12-Gy regimen exhibited an enhanced proportion of suppressive CD4+ FOXP3+ regulatory T cells, thereby shaping ionizing radiation-induced antitumor immunity. By combining an immune checkpoint blockade with radiation therapy, we effectively counteracted the immune-suppressive effect, predominantly associated with the 6 + 12-Gy regimen. We demonstrated superior tumor control and a strengthened immunologic memory in response to this combinatorial approach and corroborated these findings in a secondary tumor model. The sequence of low and high radiation doses impacts the immunologic response and must be carefully considered for the delivery of heterogeneous fractionation schemes, particularly when combined with immunotherapy.