Cutting Edge: Epigenetic Regulation of Foxp3 Defines a Stable Population of CD4⁺ Regulatory T Cells in Tumors from Mice and Humans

Intratumoral Tregs from several mouse tumor models exhibit Foxp3 stability. (A) CpG methylation analysis of Foxp3, CTLA-4, TNFRSF18, and Ikzf4 from Tconvs and Tregs isolated from the indicated tissue of CT26 tumor-bearing and control mice (pooled, n = 5 mice). (B) Foxp3 TSDR methylation analysis of Tconvs and Tregs from LLC, 4T1, MC-38, MCA-induced tumors (pooled, n = 5 mice). Color coding represents percentage methylation. (C) Tconvs (Foxp3^EGFP−) or nTregs (Foxp3^EGFP+) were cultured under iTreg (+TGF-β) or Th0 conditions (−TGF-β) for 3 d. Th0, iTregs, and nTregs were incubated in fresh culture media for an additional 2 d (day 5), then analyzed for Foxp3 expression (profiles from n = 5 spleens). The percentage of cells expressing Foxp3 is relative to freshly isolated Tconvs (upper right of histogram). (D) Ex vivo differentiation schematic for cells cultured with TGF-β (iTreg) or without TGF-β (iTreg-to-Tconv, “Reversion”). (E and F) Representative histograms showing the percent of T cells expressing Foxp3 after 5 days of ex vivo culture. Tconvs and Tregs were isolated from spleens and tumors of 4T1 (E) or LLC (F) tumor-bearing mice and subjected to culture conditions outlined in (D). Samples were pooled from n = 5 mice for each model. Tumors were collected when volumes reached 500 mm³. All data are representative of two or more individual experiments.