Fig. 3.

Vasoactive intestinal peptide (VIP) specifically increases the frequency of regulatory T cells (T_reg) during the early trophoblast–maternal dialogue. Swan 71 cell line was co-cultured with maternal–peripheral blood mononuclear cells (PBMCs) from recurrent spontaneous abortions (RSA) or fertile women, in the absence or presence of VIP (10⁻⁷ M) or VIP-antagonist (10⁻⁵ M). (a) After 48 h of culture, cells in suspension were recovered and triple-stained for surface CD4, CD25 and intranuclear forkhead box protein P3 (FoxP3) markers and analysed by fluorescence activated cell sorter (FACS). The upper panel shows representative dot-plots and the frequency of the CD4⁺CD25⁺FoxP3⁺ population expressed as a percentage of CD4⁺ cells. (b) Lower panel shows mean ± standard error of the mean of three independent PBMC preparations (n = 3, *P < 0·05, Student's t-test).