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. 2023 May 5;220(8):e20221166. doi: 10.1084/jem.20221166

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© 2023 Yang et al.

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Figure 5. — IT rMVA preferentially depletes OX40^hi Tregs in the injected tumors in a type I IFN signaling dependent manner. (A) Representative flow cytometry plots of OX40 expression on tumor infiltrating CD8⁺, Tconv (CD4⁺Foxp3⁻), and CD4⁺Foxp3⁺ T cells in tumors 15 d after implantation. Mice were treated as described in Fig. 2 A. (B and C) Representative flow cytometry plots and statistical analysis of mean fluorescence intensity (MFI) of OX40 on tumor-infiltrating CD8⁺, Tconv (CD4⁺Foxp3⁻), and CD4⁺Foxp3⁺ T cells. Data are means ± SD in C (n = 6 ∼ 8; ****P < 0.0001, t test). (D) Representative flow cytometry plots of OX40^hi CD4⁺Foxp3⁺ in the injected tumors. Mice were treated as described in Fig.2 A. The experiment was repeated twice. (E) Percentages and absolute number of OX40^hi CD4⁺Foxp3⁺ T cells in the injected tumors. Data are means ± SD (n = 7 or 9; **P < 0.01, ****P < 0.0001, t test). (F) Percentages of CD4⁺Foxp3⁺ T cells in the injected tumors from WT and Ox40^−/− mice. Mice were treated as described in Fig. 3 A. Data are means ± SD (n = 5 or 6; t test). A representative experiment is shown, repeated once. (G) Schematic diagram of IT rMVA in the presence or absence of IT αIFNAR-1 antibody in a unilateral B16-F10 melanoma implantation model. (H) Percentages of CD4⁺Foxp3⁺, CD8⁺Granzyme B⁺, and CD4⁺Granzyme B⁺ T cells in the injected tumors. Data are means ± SD (n = 6; **P < 0.01, ***P < 0.001, ****P < 0.0001, t test). A representative experiment is shown, repeated once. (I) Percentages of CD4⁺Foxp3⁺ T cells in the injected tumors in Ifnar1^fl/fl and Foxp3^creIfnar1^fl/fl mice treated with IT rMVA or PBS. Data are means ± SD (n = 4–5; *P < 0.05, **P < 0.01, t test). A representative experiment is shown, repeated once. (J) Correlation of the percentages of OX40^hi Tregs in the tumors with tumor weight. Mice were implanted with B16-F10 tumors intradermally. Tumors with different sizes were analyzed for OX40 expression on tumor-infiltrating Tregs. (K and L) Representative flow cytometry plots (K) and percentage of T_con (conventional CD4⁺ T cells) proliferation (L) as measured by CellTrace Violet (CTV) dye dilution co-cultured with tumor OX40^hi, OX40^low, Ox40^−/− Tregs, or spleen Tregs. Data are means ± SD in L (***P < 0.001, ****P < 0.0001, t test). A representative experiment is shown, repeated once.