The Combination Timing of Radio-Immunotherapy Determines Immune Microenvironment Remodeling and Abscopal Effect through Eosinophils

Abstract

<h3>Purpose/Objective(s)</h3> Several studies have confirmed the abscopal effect of tumor shrinkage outside the irradiated field induced by local radiotherapy (RT) combined with immunotherapy. The timing of the combination, including concurrent and sequential treatment, may affect the anti-tumor efficacy of radiotherapy and immune checkpoint inhibitors (ICIs). However, the optimal timing of RT combined with immunotherapy to enhance the abscopal effect and the underlying mechanism is still unknown. <h3>Materials/Methods</h3> The animal model of bilateral subcutaneous tumor of colon cancer (MC38) in C57BL/6J mice were randomly divided into four groups: PD-1 therapy (P1) group, radiotherapy (unilateral irradiation, 8Gy × 3F, the same below) concurrent with anti-PD-1 therapy (Con.) group, anti-PD-1 therapy one week after radiotherapy (Seq.1) group and anti-PD-1 therapy two weeks after radiotherapy (Seq.2) group. RNA sequencing was employed to identify differential genes. The changes of the immune microenvironment of tumor-draining lymph nodes (TDLN) and tumors were detected by flow cytometry. The expression of chemokine receptors, ligands, and cytokines was detected by qPCR, western blot, and ELISA. Anti-mouse Siglec-F antibody was used to neutralize the eosinophils in vivo. <h3>Results</h3> The results showed that the survival benefit of sequential PD-1 inhibitor after RT was higher than that of the Con. group and P1 group., the survival benefited most in Seq.1 group, particularly. Tumor growth on both sides was significantly inhibited in Seq.1 group compared with the Con. group, P1 group, and Seq2. group. Furthermore, a combination of RT with anti-PD-1 inhibitor induced changes of immune microenvironment in irradiated tumor, abscopal tumor and TDLN. In the Seq.1 group, the tumor immune microenvironment was reshaped, including increased eosinophils and CD8+T cells infiltration, while decreased Treg, MDSCs, and TAMs frequency. Correspondingly, the expression of crucial chemokine receptors and ligands involved in chemotaxis of eosinophils and CTL CD8+T cells was up-regulated in tumors of mice in the Seq.1 group. After eosinophil neutralization in vivo, the survival of mice in Seq.1 group was significantly shortened. The anti-tumor effect and abscopal effect were significantly weakened, accompanied by deduction of the infiltration of T cells, especially cytotoxicity CD8+T cells in the immune microenvironment. <h3>Conclusion</h3> Our results showed that RT combined with ICIs could induce a synergistic anti-tumor effect. Sequential PD-1 inhibitor one week after RT could enhance the anti-tumor effect most, including the abscopal effect. The mechanism related to eosinophils recruitment established a tumor-suppressive immune microenvironment mainly through enrichment of anticancer T lymphocytes in abscopal tumors. This study provides an insight into clinical precision treatment in a combination of RT and immunotherapy.