Figure 5. 1,25(OH)₂VD₃ promotes FOXP3 expression in human CD25⁻CD4⁺ T cells in the presence of anti-CD3/CD28 Abs and IL-2, and these vitamin D-treated cells (VD-iTreg cells) suppress target T cell proliferation dependently of cell contact and FOXP3.

Human CD25⁻CD4⁺ T cells were purified from peripheral blood of healthy individuals. (A–B) Cells were treated for 5 days with 1,25(OH)₂VD₃ (10 nM or indicated), anti-CD3/CD28 Abs and IL-2 to generate 1,25(OH)₂VD₃-induced Treg (VD-iTreg) cells. Intracellular FOXP3 expression was determined by flow cytometry. (C) VD-iTreg cells were generated as in (A), rested for 2 days and then co-cultured for 5 days with CFSE-stained autologous CD25⁻CD4⁺ T cells (target) at different ratios in a transwell culture system separating the two cell populations or a regular culture plate in the presence of anti-CD3/CD28 Abs. (D–E) Cells were stimulated for 3 days with 1,25(OH)₂VD₃ (10 nM)_, anti-CD3/CD28 Abs and IL-2. Cells were then transfected with control or FOXP3-specific siRNA (Invitrogen, Stealth Select RN, HSS 121458) using electroporation (Amaxa Biosystems). (D) Cells were fixed, permeabilized and stained with Abs to FOXP3 or isotype controls and analyzed on a flow cytometer. (E) CD25⁻CD4⁺ T cells that were transfected with control or FOXP3-specific siRNA and treated with 1,25(OH)₂VD₃, anti-CD3/CD28 Abs and IL-2 were incubated for 5 days with CFSE-stained CD25⁻CD4⁺ T cells (target cells) in the presence of anti-CD3/CD28 Abs. Cells were analyzed on a flow cytometer. Representative data from 7 (A) or 3 (B–E) independent experiments (one experiment per donor).