Figure 6.

ST2L⁺Tregs suppress CD4⁺CD25⁻ T-cell proliferation and IFN-γ production single-cell suspensions of tumor tissue from tumor-bearing mice were prepared. Gating schemes for analysis of the percentages of ST2L⁺CD4⁺CD25⁺ cells (R5, gated from R1 and R2) or ST2L⁻CD4⁺CD25⁺ cells (R4, gated from R1 and R2) after purification by flow sorting (A) or spleen CD4⁺CD25⁻ cells (R6, gated from R1) after purification by immunomagnetic sorting. Cells were stained with CD4-FITC, ST2L-APC, and CD25-PE-cyanine 7 and then intracellularly stained with PE-conjugated antibodies against Foxp3 for FACS analysis. Freshly sorted tumoral ST2L⁺CD4⁺CD25⁺ cells or ST2L⁻CD4⁺CD25⁺ cells were used in suppression assays, and the proliferation of spleen CD4⁺CD25⁻ cells (B) or the IFN-γ secretion (C) was assessed with and without ST2L⁺CD4⁺CD25⁺ or ST2L⁻CD4⁺CD25⁺ cells at 1:2 ratio. The experiments were Performed twice, with similar results (**P < .01, Student t test). Treg indicates regulatory T; IFN-γ, interferon-γ; PE, phycoerythrin; FACS, fluorescence-activated cell sorting.