Figure 1.

VIP increases the frequency of CD4⁺CD25⁺FoxP3 cells with suppressor ability in PBMC–Swan‐71 cell cocultures. Maternal PBMCs from fertile women were cocultured in the absence or presence of Swan‐71 at 70% of confluence with or without VIP (100 nM). After 48 h, PBMCs were recovered and (A) the frequency of CD4⁺CD25⁺FoxP3⁺ was evaluated by FACS analysis. Results are expressed as the mean percentage of CD4⁺CD25⁺FoxP3⁺ cells ± sem of at least 5 independent experiments using PBMC samples from different fertile women. *P < 0.05, Mann‐Whitney U test. (Right) Representative dot plots and the frequency of CD25⁺FoxP3⁺ cells (inside the electronically gated CD4⁺) after culture or not with trophoblast cells in the absence or presence of VIP. (B) In some experiments, before culture with trophoblast cells in the presence of VIP, freshly isolated PBMCs were first labeled with CFSE, and proliferation was investigated after 0, 24, and 48 h of coculture by FACS analysis. Result shown is representative of 3 similar runs. (C) After coculture, PBMCs in suspension were recovered, and the frequency of CD4⁺CD25⁺CD127⁻, CD4⁺CD25⁺CTLA‐4⁺, and CD4⁺CD25⁺CD39⁺ was evaluated by FACS analysis. The dot plots show the percentage of CD25⁺CD127⁻ cells (inside the electronically gated CD4⁺ cells). The histograms show the percentage of CTLA‐4 or CD39 cells (inside the electronically gated CD4⁺CD25⁺ cells). (D) Maternal PBMCs were cultured with or without Swan‐71 cells in the absence or presence of VIP (100 nM) for 48 h. Then, PBMCs in suspension were recovered and transferred to a culture with mismatched allogeneic healthy donor PBMCs (1 × 10⁵ cells/well). These last PBMCs had previously been treated with mitomycin C to inhibit DNA synthesis by crosslinking DNA at guanine and adenine residues to obtain a unidirectional proliferation. After 5 d, [³H]TdR was added for 18 h, and uptake was determined using a β‐scintillation counter. Results are expressed as mean cpm ± sem of at least 3 independent experiments run in triplicate. *P < 0.05, Mann Whitney U test. (E) At 48 h of coculture, PBMCs recovered were harvested and analyzed by Western blot for FoxP3, T‐bet, and RUNX1 expression. Representative immunoreactive bands and semiquantification, expressed as relative to β‐actin in A.U. ± sem are shown from 4 independent experiments. *P < 0.05, Mann Whitney U test.