Fig. 4. Intratumoral injection of Heat-iMVA leads to immunological changes in the tumor microenvironment.

5 × 10⁵ B16-F10 melanoma cells were implanted intradermally to the right flank of the mice. Seven days post implantation, we injected either Heat-iMVA (an equivalent of 2× 10⁷ pfu) or PBS into the tumors on the right flank. The injection was repeated three days later. Tumors were harvested 3 days post last injection and cell suspensions were generated. The live immune cell infiltrates in the tumors were analyzed by FACS. (A-F) Representative flow cytometry plot of CD3⁺ CD45⁺ T cells (A), CD8⁺ cells expressing Granzyme B⁺ (B) or Ki-67 (C), CD4⁺ cells expressing FoxP3 (D), Granzyme B (E), or Ki-67 (F). (G-L) Percentages of CD45⁺ CD3⁺ (G), CD8⁺ Granzyme B⁺ (H), CD8⁺ Ki-67⁺ (I), CD4⁺ Foxp3⁺ (J), CD4⁺ Granzyme B⁺ (K), and CD4⁺ Ki67⁺ (L) cells within tumors of mice treated with PBS (n=5) or Heat-iMVA (n=5; ***P < 0.001; ****P < 0.0001, t test). A representative experiment is shown, repeated at least twice. (M) The absolute numbers of tumor infiltrating CD3⁺, CD8⁺, CD4⁺ FoxP3⁻, and CD4⁺ FoxP3⁺ cells per gram of injected tumors treated with either Heat-iMVA or PBS (n=5; **P < 0.01; ***P < 0.001, t test). (N) The absolute numbers of CD8⁺ Granzyme B⁺, CD8⁺ Ki67⁺, CD4⁺ Granzyme B⁺, and CD4⁺ Ki67⁺ cells in the injected tumors treated with either Heat-iMVA or PBS (n=5; ***P < 0.001; ****P < 0.0001, t test).