Figure 3.

IL-12-variant treatments modify the tumor microenvironment and improve the therapeutic effect with PD-1 blockade. B6 mice were intraperitoneally inoculated with 5×10⁵ MC38-luc cells and treated with PBS, vvDD, vvDD-IL-12, or vvDD-IL-12-FG at 2×10⁸ PFU/mouse 9 days post-tumor inoculation. Tumor-bearing mice were sacrificed 5 days post-treatment and primary tumors were collected and analyzed using flow cytometry to determine CD4⁺Foxp3⁻ (A) and CD8⁺ T cells (B), IFN-γ⁺CD8⁺ (C), IFN-γ⁺CD4⁺ (D), exhausted CD8⁺ T cell (E–H), G-MDSCs (I), or regulatory T cells (CD4⁺Foxp3⁺) (J), TGF-β⁺Treg (N), TGF-β⁺CD45⁻ (O), using RT-qPCR to determine TGF-β, COX-2, and VEGF (K–M). In a separate experiment, B6 mice were intraperitoneally inoculated with 5×10⁵ MC38-luc cells and treated with vvDD-IL-12-FG or PBS 9 days post-tumor inoculation. Anti-CD8 Ab (250 µg/injection), α-CD4 Ab (150 µg/injection), PK136 (300 µg/injection), α-IFN-γ Ab (200 µg/injection), or α-PD-1 Ab (200 µg/injection), (n≥7) were intraperitoneally injected into mice to deplete CD8⁺ T cells, CD4⁺ T cells, or NK1.1⁺ cells, neutralize circulating IFN-γ, or enhance virotherapy with α-PD-1 Ab (P), and a log-rank (Mantel–Cox) test was used to compare survival rates (Q and R), respectively. *P<0.05; **P<0.01; ***P<0.001; and ****P<0.0001. COX-2, cyclooxygenase-2; IFN-γ, interferon γ; IL-12, interleukin 12; G-MDSC, granulocytic myeloid-derived suppressor cells; NS, not significant; PBS, phosphate-buffered saline; PFU, plaque-forming units; TGF-β, transforming growth factor β; VEGF, vascular endothelial growth factor.