Galectin-10 specific siRNA abolishes the anergic state and suppressive activity of human CD25⁺ Tregs. (A) Freshly isolated CD25⁺ Treg cells were nucleofected with 0.5 or 1 μM of siRNA targeted to galectin-10 (Gal-10) or with control siRNA (scrambled control SC). At 24 hours after nucleofection, RNA was prepared and used for qRT-PCR analysis. The expression level was normalized to expression of EF1-α. The qRT-PCR data are representative of 4 independent experiments; measurements were performed in triplicates (mean ± SD from each triplicate shown; asterisks indicate P values as defined in “Materials and methods, Statistical analysis”). (B) At 48 hours after nucleofection, galectin-10 protein expression was examined by galectin-10–specific Western blot analysis. (C) At 48 hours after nucleofection, T cells were stimulated with anti-CD3 mAb (0.5 μg/mL) and irradiated PBMCs. CD25⁺ Treg cells in coculture with CD4⁺CD25⁻ T cells were irradiated. Proliferation was determined by ³H-Tdr incorporation. Representative results of 4 independent experiments are shown; measurements were performed in triplicates (mean ± SD) from each triplicate are shown; asterisks indicate P values as defined in “Materials and methods, Statistical analysis”).