The importance of the PD-1/PD-L1 pathway at the maternal-fetal interface

Abstract

Background: Our goal with this study was to investigate the contribution of PD-1/PD-L1 immune-checkpoint pathway to maternal immunotolerance mechanisms.

Methods: Thirteen healthy pregnant women and 10 non-pregnant controls were involved in this project. PBMCs and DICs were isolated from peripheral blood and from decidual tissues. After the characterization of different immune cell subsets, we used fluorochrome-conjugated monoclonal antibodies to measure the expression level of PD-1, PD-L1, NKG2D, and CD107a molecules by flow cytometry.

Results: We measured significant alternations in the proportion of decidual immune cell subsets compared to the periphery. Elevated PD-1 expression by decidual CD8+ T, CD4+ T, and NKT-like cells were also detected accompanied by the increased PD-L1 expression by decidual CD4+ T, Treg, NKT-like and CD56 + NK cell subsets compared to peripheral blood. The cytotoxic potential was significantly higher in PD-1- decidual immune cells compared to the periphery, however we measured a significantly lower cytotoxicity in the decidual PD-1+ CD8+ T cells compared with the peripheral subsets. An activation receptor NKG2D expression was decreased by the PD-1+ CD8+ T subsets in the first trimester compared to non-pregnant condition but the expression level of the decidual counterparts was significantly elevated compared to the periphery. The cytotoxic potential of decidual PD1/NKG2D double positive CD8+ T cells was significantly decreased compared to the peripheral subsets.

Conclusions: Based on our results we assume that PD-1/PD-L1 pathway might have a novel role in the maintaining of the local immunological environment. Accompanied by NKG2D activating receptor this checkpoint interaction could regulate decidual CD8 Tc cell subsets and may contribute maternal immunotolerance.

Keywords: Maternal immunotolerance; Maternal-fetal interface; PD-1; PD-L1; Pregnancy.

Fig. 1

Flow cytometry gating strategy for peripheral and decidual immune cell subpopulations a, Lymphocytes from peripheral blood were gated on FSC-A versus SSC-A. Cell surface antibodies were used to identify, CD8+ T, CD4+ T, Treg cells, CD56 + NK, and NKT-like cell subpopulations. b Immune cells from decidual tissues were gated using side-scatter area (SSC-A) and CD45 gate. Decidual lymphocytes were selected from CD45+ cells on the basis of forward-scatter area (FSC-A) and SSC-A. Cell surface antibodies were used to identify CD8+ T, CD4+ T, Treg cells, CD56 + NK, and NKT-like cell subpopulations

Fig. 2

PD-1 expression by different immune cell populations in 1st-trimester healthy pregnant and in non-pregnant women. Box plot of the median, the 25th and, 75th percentiles, range, and individual data values for the expression of the PD-1 receptor by CD8+ T (a), CD4+ T (b), and NKT-like (c) cells in peripheral blood and decidual tissue in healthy pregnant and in non-pregnant women. The middle line within the box represent medians of 10, 13 and 7 determinations, respectively, the middle dot within the box represents the mean, the boxes indicate the interquartile ranges and the whiskers show the most extreme observations. Differences were considered statistically significant for p-values ≤ 0.05. ***p < 0,01

Fig. 3

PD-L1 expression by different immune cell populations in 1st-trimester healthy pregnant and in non-pregnant women. Box plot of the median, the 25th and, 75th percentiles, range, and individual data values for the expression of the PD-L1 receptor by, CD4+ T (a), Treg (b), NKT-like (c), CD56 + NK (d), and CD56^dimNK (e) cells in peripheral blood and decidual tissue in healthy pregnant and in non-pregnant women. The middle line within the box represent medians of 10, 13 and 7 determinations, respectively, the middle dot within the box represents the mean, the boxes indicate the interquartile ranges and the whiskers show the most extreme observations. Differences were considered statistically significant for p-values ≤ 0.05. ***p < 0,01; **p < 0,03

Fig. 4

Cytotoxicity of PD-1 positive/negative immune cell populations in 1st-trimester healthy pregnant and in non-pregnant women. Box plot of the median, the 25th and, 75th percentiles, range, and individual data values for the expression of the CD107a molecule by PD-1+/PD-1 negative immune cells (a and b) and PD-1+/PD-1 negative CD8+ T cells (c and d) in peripheral blood and decidual tissue in healthy pregnant and in non-pregnant women. The middle line within the box represent medians of 10, 13 and 7 determinations, respectively, the middle dot within the box represents the mean, the boxes indicate the interquartile ranges and the whiskers show the most extreme observations. Differences were considered statistically significant for p-values ≤ 0.05. ***p < 0,01; **p < 0,03

Fig. 5

NKG2D expression and cytotoxicity by PD-1+/CD8+ T cells in 1st-trimester healthy pregnant and in non-pregnant women. Box plot of the median, the 25th and, 75th percentiles, range, and individual data values for the expression of the NKG2D receptor by CD8+ T (a) cells and PD-1+ CD8+ T cells (b) and their cytotoxic potential (c) in peripheral blood and decidual tissue in healthy pregnant and in non-pregnant women. The middle line within the box represent medians of 10, 13 and 7 determinations, respectively, the middle dot within the box represents the mean, the boxes indicate the interquartile ranges and the whiskers show the most extreme observations. Differences were considered statistically significant for p-values ≤ 0.05. ***p < 0,01; **p < 0,03; *p < 0,05