Stereotactic radiotherapy increases functionally suppressive regulatory T cells in the tumor microenvironment
Yuki Muroyama¹, Thomas R. Nirschl¹, Christina M. Kochel¹, Esteban Velarde³, Ada J. Tam¹, Christopher J. Thoburn1, Muniza Uddin4, Alan K. Meeker4, Robert A. Anders4, Drew M. Pardoll¹, Charles G. Drake1,2*
1 Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine
2 Current address: Division of Hematology and Oncology, Columbia University Medical Center
3 Department of Radiation Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine
4 Department of Pathology, Johns Hopkins University School of Medicine
* Corresponding author.
Radiotherapy enhances innate and adaptive anti-tumor immunity; however, the effects of radiation on suppressive immune cells, such as regulatory T cells (Treg), in the tumor microenvironment has not been fully elucidated. Although previous reports suggest a post-radiation increase in Treg infiltration, whether these Treg are fully suppressive has not been determined. To test the hypothesis that radiotherapy increases the suppressive function of Treg in tumor microenvironment, we used the Small Animal Radiation Research Platform (SARRP), which models stereotactic radiotherapy in human patients, to selectively irradiate implanted tumors in mice, followed by flow-cytometry based analysis of tumor-infiltrating lymphocytes (TILs) populations. Our data showed that radiation significantly increased tumor-infiltrating Treg, and that these cells have higher expression of suppressive markers (CTLA-4, 41BB, and Helios) as compared to un-irradiated tumors. This observation held across several tumor models (B16/F10, RENCA, and MC38). Notably, the in vitro suppression assay showed radiated tumor-infiltrating Tregs are fully suppressive. Our data also suggest that Tregs proliferate post-radiation, and this expansion was more robust compared to other T cell subsets (CD3, CD4, and CD8) in the tumor microenvironment. In addition, the post-radiation Treg expansion occurred even when T cell migration was inhibited using Fingolimod - suggesting that Treg expansion is likely due to preferential proliferation post-radiation. Our data also suggested that the post-irradiation Treg expansion is independent on TGF-. Collectively, these data suggest that radiation therapy increases phenotypically and functionally suppressive Tregs in tumor microenvironment, and that treatment regimens that combine radiotherapy with Treg-targeting agents might be required to maximize anti-tumor efficacy.