Expression and secretion of galectin-1 in human CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells. (A) Galectin-1 up-regulation is observed by comparative analysis of 2-DE of total-cell lysates obtained from activated CD4⁺CD25⁻ and CD4⁺CD25⁺ human peripheral blood CD4⁺ T cells. Human CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells were activated with anti-CD3/anti-CD28–coated beads and cell lysates were prepared for 2-DE analysis. Proteins were separated using 18-cm 3-10 nonlinear IPG strips in the first dimension and 12% SDS-PAGE in the second dimension. Shown are the silver-stained gel images. Protein spots were sequenced by electrospray tandem mass spectrometry on the Micromass Q-Tof (high-performance liquid chromatography/tandem mass spectrometry [HPLC-MS/MS]). These results are representative of 3 independent experiments. (B) Kinetic analysis of the expression of galectin-1 in freshly isolated and in vitro–activated human CD4⁺, CD4⁺CD25⁻, and CD4⁺CD25⁺ T-cell populations by Western blot analysis. Human samples were prepared from total-cell lysates obtained from fresh and anti-CD3/anti-CD28–activated human T cells. The membrane was blotted with anti–human galectin-1 mAb (Clone 25C1; Novo Castra). Data are representative of 5 samples analyzed. (C) Detection of galectin-1 in the supernatants of anti-CD3/anti-CD28–activated human CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells and in cocultures of both populations (1:1 and 1:4 ratios) by immunoprecipitation. Cell-free supernatants were harvested from 48-hour cultures and galectin-1 was detected by immunoprecipitation with anti–human galectin-1 mAb. Data are representative of 2 experiments.