Figure 1.

Effect of MIP immunotherapy on the frequency and function of tumor infiltrating Tregs and CD8⁺ T cells: (A) Twenty one days post tumor implantation, immune cells were separated from tumor tissue and stained for FoxP3 on live CD3⁺CD4⁺ T cells and analyzed by flow cytometry. The control tumor bearing mice were treated with PBS (vehicle control). The contour plots represent one tumor bearing mouse per group showing the percentage of FoxP3⁺ cells within tumor-infiltrating CD4⁺ T cells. (B) (Left) Specific number of tumor-infiltrating CD4⁺ FoxP3⁺ Tregs; (Right) Number of tumor-infiltrating CD4⁺ FoxP3⁺ Tregs per gram of tumor (n=5 mice/group). (C) Percent frequency of Tregs expressing CTLA4, IL10, TGF-β and PD1 in Control and MIP treated TME (n=4 mice/group). (D) Flow-cytometric analysis of percentage of intratumoral CD8⁺ T cells in control/MIP treated mice. The contour plots represent one tumor bearing mouse per group. (E) Percent frequency of activated CD8⁺ T cells in the TME. (F) Flow cytometric analysis of IFN-γ producing CD8⁺ T cells in the TME of control and MIP treated mice. (G) Percent frequency of potentially proliferating CD8⁺ T cells was determined in the TME by analyzing expression of Ki67 in them. (H) To determine whether MIP immunotherapy suppresses Treg migration into the TME, 24 hours after the third dose of MIP, CFSE-labeled splenocytes of healthy donor mice were adoptively transferred by retro-orbital route into the tumor bearing mice. Tumors were resected 18 hours after adoptive transfer, followed by flow cytometric analysis of intratumoral CFSE⁺ Treg cells and CFSE⁺ total CD4⁺ T cells. Representative contour plots showing the frequency of CFSE⁺ Tregs in one mouse per group. All findings are shown as bar graphs that represent the mean ± SEM of five mice per group in one experiment. Statistical significance was determined by unpaired non-parametric student’s t-test (*:p<0.05; **:p<0.01; ****:p<0.0001; ns, non-significant). Two/three independent repeats were done for all experiments.