Figure 1. Enhancement of IL-2 therapy by entinostat is associated with inhibition of Tregs.

Mice with orthotopic inoculation of RENCA cells were treated for 5 days. Blood was drawn from mice on the fifth day and stained for antibodies specific for CD4, CD25, and Foxp3. Tumor weights were measured at the end of two weeks of treatment. A and B, Effect of entinostat on Tregs in tumor bearing mice. A, Effects of vehicle, IL-2, entinostat, or combination treatment on Tregs Foxp3 expression. Cells were stained and subjected to flow cytometry analysis. The dot plots were gated for CD4⁺ cells. The rectangular area encloses the CD4⁺Foxp3⁺ cells, the numbers on the top represent the percentage of Foxp3⁺ cells. The numbers on the bottom in the area represent the mean fluorescence intensity (MFI) of Foxp3 PE staining of CD4⁺Foxp3⁺ cells. B, Quantification of Tregs percentage in CD4 population (left panel) and Foxp3 levels (MFI) in Tregs (right panel) by FACS analysis. Values are means and error bars represent S.D. for 5–7 samples per group. In right panel, p = 0.0011 for IL-2 vs. vehicle; p = 0.000009 for entinostat vs. vehicle. Results are representative of three separate experiments. C, Tumor weight measurements. Columns, mean grams of tumor; Bars, S.D. n = 5–7. p = 0.0209 for entinostat vs. vehicle; p = 0.0077 for combination vs. vehicle; p = 0.0272 for combination vs. entinostat. Results are representative of three separate experiments. D, Entinostat enhanced IFN-γ type immune response induced by IL-2 treatment. Splenocytes (1×10⁶ cells) were harvested and stimulated with PMA (20 ng/ml) and Ionomycin (1 µg/ml) for 5 hours in the presence of Brefeldin A. Cells were then stained for surface markers and intracellular IFN-γ. Histograms show percentage of IFN-γ expressing cells in CD8 population. p = 0.01 for combination vs. IL-2. E, Entinostat reduced tumor infiltration of Tregs. Tumor sections were stained with anti-Foxp3 antibody to show infiltration of Tregs. Histogram shows average numbers of stained Tregs in random 20× resolution bright fields (Tregs number in each field was obtained by blinded count). F, Entinostat treatment induced H3 histone acetylation in splenocytes. BALB/c mice were treated with vehicle (0.5% methocel) or 5 mg/kg/day entinostat by gavage for 5 days. Cells were harvested from spleens and subjected to Western blot analysis for acetylated-H3 histone.