As first primary antibodies against CD45RO, Neuropilin-1, LAG-3, CTLA-4, Venetoclax and CD62L were

used for 30 min incubation followed by washing and incubation with secondary goat anti-mouse IgG FITC-conjugated Ab. Then, the cells were blocked with 10% mouse serum and goat anti-mouse Fab. After a permeabilization step, the second primary mAb against Foxp3 was applied for 30 min, and after washing, the cells were incubated with biotinylated goat anti-mouse Fab Ab, followed by Streptavidin-PE. Finally, the slides were washed and mounted in Shandon medium. Total RNA was isolated from MACS purified CD4+ Treg cells decidual and peripheral blood paired samples (n = 10) as well as from PBMC from non-pregnant women (n = 10) learn more using acid guanidinium thiocyanate-phenol-chloroform method.12 The isolated total RNA samples were subjected to real-time quantitative RT-PCR (Perkin Elmer Gene Amp/RNA PCR kit; Applied Biosystems, Carlsbad, CA, USA) for analysis of the level

of mRNA expression of Foxp3 and a panel of the following 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. The specific primers and probes are described elsewhere.12 The following Foxp3 primers and probes were used: forward primer 5′-GCATGTTTGCCTTCTTCAGAAAC; reverse primer 5′-TGTAGGGTTGGAACACCTGCTG; and probe 5′-AGCGAGAAGGGGGCTGTGTGT. For quantification of gene expression between paired peripheral and decidual samples, the MACS purified decidual and peripheral CD4+ CD25+ and CD4+ CD25− cells were prepared Ribose-5-phosphate isomerase from equal starting numbers of PBMC and DMC. As a positive control of the RT-PCR reactions, we used PMA-Ionomycin stimulated PBMC.12 All sample analyses were normalized to an internal control using S18 rRNA. All results were expressed as mean ± SD. One-way anova and Newman–Keuls post hoc test were used to compare non-paired groups, and Wilcoxon signed rank test was performed for matched pairs using statsoft version 6 (StatSoft, Inc., Tulsa, OK, USA). Values of P < 0.05 were considered significant. To assess the in situ distribution of Treg cells at the materno-fetal

interface, we performed double immunoperoxidase staining with monoclonal antibodies against CD4 and Foxp3. To detect the Foxp3 protein expression, we used 236A/E7 mAb, known to label functional suppressor/Treg cells.37 Both CD4+ and Foxp3+ single positive- as well as double positive CD4+ Foxp3+ cells were found in decidua (Fig. 1a–c). As can be seen in representative photomicrographs illustrated in Fig. 1a–c, CD4+ Foxp3+ cells were constitutively present in human decidua. This is the first demonstration in situ of CD4 and Foxp3 stained cells in decidua. As can be seen, they are very small, displaying the morphology of small lymphocytes with large nucleus and very scarce cytoplasm. They could be found dispersed between decidual stromal cells or in the vicinity of blood vessels (Fig. 1a).