Supplementary Figures and Legends from Stereotactic Radiotherapy Increases Functionally Suppressive Regulatory T Cells in the Tumor Microenvironment

Muroyama, Yuki; Nirschl, Thomas R.; Kochel, Christina M.; Lopez-Bujanda, Zoila; Theodros, Debebe; Mao, Wendy; Carrera-Haro, Maria A.; Ghasemzadeh, Ali; Marciscano, Ariel E.; Velarde, Esteban; Tam, Ada J.; Thoburn, Christopher J.; Uddin, Muniza; Meeker, Alan K.; Anders, Robert A.; Pardoll, Drew M.; Drake, Charles G.

doi:10.1158/2326-6066.22535884.v1

Supplementary Figures and Legends from Stereotactic Radiotherapy Increases Functionally Suppressive Regulatory T Cells in the Tumor Microenvironment

journal contribution

posted on 2023-04-03, 22:40 authored by Yuki Muroyama, Thomas R. Nirschl, Christina M. Kochel, Zoila Lopez-Bujanda, Debebe Theodros, Wendy Mao, Maria A. Carrera-Haro, Ali Ghasemzadeh, Ariel E. Marciscano, Esteban Velarde, Ada J. Tam, Christopher J. Thoburn, Muniza Uddin, Alan K. Meeker, Robert A. Anders, Drew M. Pardoll, Charles G. Drake

Supplementary Figure 1: Radiotherapy suppresses tumor growth. Supplementary Figure 2: Characterization of the TME of B16/F10 tumors post-RT. Supplementary Figure 3: Stereotactic radiation does not change Treg in draining lymph nodes nor in spleens of treated mice. Supplementary Figure 4: Persistently increased Treg Post-RT. Supplementary Figure 5: Stereotactic radiation increases the suppressive markers of Treg in the RENCA tumor model. Supplementary Figure 6: Stereotactic radiation increases the suppressive markers of Treg in the MC38 tumor model. Supplementary Figure 7: Stereotactic radiation does not change the expression of the suppressive markers of Treg in DLNs. Supplementary Figure 8: Stereotactic radiation does not change the expression of the suppressive markers of Treg in spleens. Supplementary Figure 9: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the B16/F10 model. Supplementary Figure 10: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the RENCA model. Supplementary Figure 11: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the MC38 model. Supplementary Figure 12: Expression of 4-1BB on TIL-CD8+ cells. Supplementary Figure 13: The effect of TGF-beta blockade on different T cell subsets. Supplementary Figure 14: TGF-beta expression in the tumor microenvironment. Supplementary Figure 15: The effect of Fingolimod (FTY720) on peripheral blood lymphocytes counts. Supplementary Figure 16: Chemokine/cytokine expression of a selected panel in the tumor microenvironment post-radiation.

Funding

NIH

History

ARTICLE ABSTRACT

Radiotherapy (RT) enhances innate and adaptive antitumor immunity; however, the effects of radiation on suppressive immune cells, such as regulatory T cells (Treg), in the tumor microenvironment (TME) are not fully elucidated. Although previous reports suggest an increased Treg infiltration after radiation, whether these Tregs are functionally suppressive remains undetermined. To test the hypothesis that RT enhances the suppressive function of Treg in the TME, we selectively irradiated implanted tumors using the small animal radiation research platform (SARRP), which models stereotactic radiotherapy in human patients. We then analyzed tumor-infiltrating lymphocytes (TIL) with flow-cytometry and functional assays. Our data showed that RT significantly increased tumor-infiltrating Tregs (TIL-Treg), which had higher expression of CTLA-4, 4-1BB, and Helios compared with Tregs in nonirradiated tumors. This observation held true across several tumor models (B16/F10, RENCA, and MC38). We found that TIL-Tregs from irradiated tumors had equal or improved suppressive capacity compared with nonirradiated tumors. Our data also indicated that after RT, Tregs proliferated more robustly than other T-cell subsets in the TME. In addition, after RT, expansion of Tregs occurred when T-cell migration was inhibited using Fingolimod, suggesting that the increased Treg frequency was likely due to preferential proliferation of intratumoral Treg after radiation. Our data also suggested that Treg expansion after irradiation was independent of TGFβ and IL33 signaling. These data demonstrate that RT increased phenotypically and functionally suppressive Tregs in the TME. Our results suggest that RT might be combined effectively with Treg-targeting agents to maximize antitumor efficacy. Cancer Immunol Res; 5(11); 992–1004. ©2017 AACR.