Fig. 2.

ttIL-12 enhanced infiltration of CD8⁺ T cells into tumors, decreased infiltration of Tregs and MDSCs, and increased tumor levels of IFNγ. (a) Treatment scheme. BALB/c mice were inoculated with 4 T1 breast cancer cells by intra-mammary fat pad injection. CH3 mice were inoculated with LM8 sarcoma cells by intratibial injection. Mice from both groups were treated with empty control plasmid DNA (pDNA; pCtrl), wild-type IL-12 pDNA (pwtIL-12), or tumor-targeted IL-12 pDNA (pttIL-12; n = 5~8 mice per treatment group). Primary tumors were removed surgically as indicated. b Frozen sections from 4 T1 primary tumors were stained with CD8 antibody (upper panels) or FOXP3 antibody (lower panels). FOXP3 is a marker of T regulatory cells (Tregs). Representative sections are shown. Scale bars, 100 μm. Absolute numbers of CD8⁺ cells and FOXP3⁺ cells were determined by microscopy. c Calculated CD8⁺/Treg ratios. d Representative flow cytometry plots of percentages of CD11b⁺Gr1⁺ cells (myeloid-derived suppressor cells [MDSCs]); absolute numbers of MDSCs per 10⁵ total tumor cells were calculated from the flow cytometry data. e Levels of the IL-12 and IFNγ proteins in primary 4 T1 tumors were measured by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001