sCD40L increased Tregs and CD4⁺CD25^int cells in vitro. (A) FACS analysis of the frequency of the CD4⁺CD25^high population in CD3⁺ T cells after adding sCD40L. PBMCs from 5 healthy donors (HD) were incubated in medium with or without 2 μg/mL of sCD40L. Both sCD40L⁻ and sCD40L⁺ cultures contained 25 U/mL of IL-2. Four days later, cells were analyzed by multicolor FACS for CD3, CD4, CD25, and intracellular Foxp3. (B) The cells were analyzed by gating on the CD4⁺CD25^high population. (C) CD4⁺CD25^high cells were mainly Foxp3⁺. Intracellular staining of Foxp3 was performed, and a comparison of Foxp3 expression between CD4⁺CD25^high and CD4⁺CD25⁻ cells is shown for 1 sample that is representative of 5 PBMCs that were tested individually. The data were repeated 3 times. Statistical analysis was performed using a paired t test.