Our immunocytochemical data confirmed that the greatest majority of CD4+ CD25+ cells were Foxp3+ (Fig. 3b). Furthermore, we performed Foxp3 staining on cytospin preparations of the CD4+ CD25−

fraction as well. Foxp3-positive cells were observed in this fraction in agreement with our flow cytometric data (Fig. 3b). In conclusion, the immunocytochemical stainings of the cytospin preparations confirmed that, indeed, there is a CD4+ CD25− cell population that expresses Foxp3 in human normal early pregnancy decidua. Finding the presence of CD4+ CD25− Foxp3+ cells in decidua, we wanted to clarify whether these cells belonged to the Treg phenotype or whether they were conventional Th cells. It has been shown that small amounts of Foxp3 could be present in conventional signaling pathway effector cells, while naïve Treg- precursor cells express higher and steady state Foxp3.38

Accordingly, we analyzed the relative expression of Foxp3 mRNA in CD4+ CD25− Foxp3+ and CD4+ CD25+ Foxp3+ cell subsets isolated from 10 consecutive decidual and PBMC samples from first trimester normal pregnancies. The results are summarized in Fig. 4. As can be seen, the expression of Foxp3 mRNA in the CD4+ CD25− subpopulation was comparable to that of the CD4+ CD25+ subpopulation while the expression of TGFβ mRNA was very low. In addition to TGFβ1, we evaluated the mRNA expression in these cells for a panel of 14 cytokines: IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, TNFα, IFN-γ, GM-CSF, and TGFβ1, designed to discriminate between Th1, Th2, Th17, aminophylline and the regulatory Th3 and Tr1 cytokine profiles. The results are summarized selleck products in Table I where the cytokine profile of the CD4+ CD25− cells from each individual decidual sample is presented (n = 10). As can be seen, the CD4+ CD25− cells in 4 of 10 samples had a cytokine

profile similar to Th3, although with a low expression of TGFβ1. In fact, the general impression from this analysis was that rather few cytokines were expressed in the CD4+ CD25− samples (Table I). In contrast, the CD4+ CD25− cells in the peripheral blood of pregnant and non-pregnant women showed a very low expression of both Foxp3 and TGFβ mRNA compared with the decidual CD4+ CD25− Foxp3+ and circulating CD4+ CD25+ Foxp3+ Treg cells suggesting that they are another, non-regulatory T-cell subset, e.g. T effector cells (Fig. 4). Summarizing these results, we can conclude that: (i) the majority of the decidual CD4+ CD25− Foxp3+ cell subset, with a stable and comparable Foxp3 mRNA expression and a very low TGFβ mRNA expression, might be Treg cell precursors that have not yet acquired production of the immunosuppressive TGFβ, however, we cannot exclude that some of these cells are CD4+ activated effector cells; and (ii) the naïve CD4+ CD25− Foxp3+ Treg cells were absent in the periphery, suggesting that they are produced in the decidua and might be a reservoir for a local maturation of decidual CD4+ CD25+Foxp3+ Treg cells.