Suppressed expression of galectin-10 in CD25⁺ Treg cells alters their anergic phenotype, cytokine production, and suppressive activity. Freshly isolated CD25⁺ Treg cells were nucleofected with 1 μM siRNA targeted to galectin-10 (Gal-10) or control siRNA (scrambled control SC). At 48 hours after nucleofection, CFSE labeled and unlabeled T cells were stimulated with anti-CD3 mAb (0.5 μg/mL) and irradiated PBMCs. (A) Proliferation and cytokine release of the CFSE labeled T-cell populations in single culture. CFSE profile: FACS analysis of CFSE⁺ T cells determined proliferation on day 3 of stimulation. Cytokine release: cytokine release for the indicated cytokines was determined in supernatants derived from CFSE stimulation cultures on day 3, using human Cytometric Bead Array. (B) CFSE profile and cytokine release of CD4⁺ T cells in coculture with CD25⁺ Treg cells (untreated/siRNA-treated) in a ratio of 1:2. Cytokine release was determined as described. Results are representative of 4 independent experiments. (C) IL-2 mRNA expression in nucleofected CD25⁺ Treg cells and CD4⁺CD25⁻ T cells. Freshly isolated CD25⁺ Treg cells were nucleofected with 1 μM siRNA targeted to galectin-10 (Gal-10) or control siRNA (scrambled control SC). At 48 hours after nucleofection, T cells were stimulated with anti-CD3 mAb (1 μg/mL) and anti-CD28 mAb (2 μg/mL) and after an additional 4 hours, RNA was prepared and used for qRT-PCR analysis. The expression level of IL-2 was normalized to the expression of EF1-α. Results are representative of 3 independent experiments; measurements were performed in triplicates (mean ± SD from each triplicate are shown; asterisks indicate P values as defined in “Materials and methods, Statistical analysis”).