CD56(bright)CD25+ NK cells are preferentially recruited to the maternal/fetal interface in early human pregnancy

Abstract

Decidual natural killer (dNK) cells are believed to be critical for maintaining maternal/fetal tolerance and regulating placental vascular remodeling based upon their abundance and unique phenotype during early pregnancy. However, the mechanism for how the dNK cells play such important roles in successful pregnancy remains undefined. Here, we identified a subtype of dNK cells characterized as having a CD3(-)CD56(bright)CD25(+) phenotype. We found that CD56(bright)CD25(+) NK cells preferentially localize to the maternal/fetal interface during early human pregnancy. CD25(+) dNK cells account for approximately 75% of CD25-expressing decidual immune cells (DICs). However, less than 5% of CD25-positive peripheral blood mononuclear cells are CD25(+) NK cells. Furthermore, CD25(+) and CD25(-) dNK cells exhibit distinct phenotypes: CD25(+) dNK cells display a more activated phenotype and greater cytokine-secreting capacity. Interestingly, coculture of peripheral NK (pNK) cells with primary trophoblasts upregulates the percentage of CD25-expressing pNK cells, resulting in increased expression of activation markers and cytokine production by pNK cells. In addition, we demonstrated that the CXCL12/CXCR4 axis is crucial for the recruitment of CD25(+) dNK cells and contributes to the accumulation of CD3(-)CD56(bright)CD25(+) dNK cells at the maternal/fetal interface. Thus, our data reveal that the crosstalk between trophoblasts and pNK cells leads to the accumulation of CD3(-)CD56(bright)CD25(+) dNK cells, which exert a regulating effect at the maternal/fetal interface.

Figure 1

CD3⁻CD56^brightCD25⁺ dNK cells are preferentially recruited to the maternal/fetal interface in early human pregnancy. (a, b) Comparison of the percentage of CD25⁺ NK cells in pNK and dNK cells in early human pregnancy. CD56⁺ NK cells were gated on prior to analysis of CD25 expression by FCM. The percentage of CD25⁺ NK cells was determined. (c, d) Comparison of the cell subsets constituting CD25⁺, CD25⁻ or total immune cells from PBMC and DICs in early human pregnancy. Data represent the mean±standard error from ten independent experiments with ten deciduas. A representative image is shown. Data were analyzed using a paired t-test (b). ***P<0.001. DIC, decidual immune cell; dNK, decidual natural killer; PBMC, peripheral blood mononuclear cell; pNK, peripheral natural killer.

Figure 2

Differences between CD25⁺ and CD25⁻ dNK cells. (a) Flow cytometry analysis of activating and inhibitory receptors on CD3⁻CD56^bright CD25⁺ and CD3⁻CD56^brightCD25⁻ dNK cells. (b–d) Flow cytometry analysis of intracellular cytokine percentage and MFI of IFN-γ, IL-8 and TGF-β on CD3⁻CD56^brightCD25⁻ and CD3⁻CD56^brightCD25⁻ dNK cells. (e) Flow cytometry analysis of perforin and Granzyme B expression in CD3⁻CD56^bright CD25⁺ and CD3⁻CD56^brightCD25⁻ dNK cells. Data represent the mean±standard error from 10 independent experiments with different deciduas. *P<0.05, **P<0.01; ***P<0.001. dNK, decidual natural killer; MFI, mean fluorescence intensity.

Figure 3

Trophoblasts attract CXCR4⁺CD25⁺ dNK cells to the maternal-fetal interface via secretion of CXCL12. (a) CD3⁻CD56^brightCD25⁺ dNK cells exhibit higher CXCR4 expression at the maternal/fetal interface (n=10). (b) Analysis of the number of migrated dNK cells following treatment with rhCXCL12 in various concentrations or TCM. (c) Analysis of the percentage of CD25⁺ dNK that migrated toward various concentrations of rhCXCL12 and TCM (n=4). Data represent the mean±standard error from ten or four independent experiments with different deciduas. *P<0.05, **P<0.01, compared with control; ^#P<0.05, compared with TCM treatment. dNK, decidual natural killer; TCM, trophoblast culture media.

Figure 4

Trophoblasts instruct the phenotypic shift from a peripheral CD25⁺ NK (CD25⁺ pNK) toward a decidual CD25⁺ NK (CD25⁺ dNK)-like phenotype. (a) Trophoblasts increase the percentage of CD25-expressing NK cells (n=6). (b) Trophoblasts mediate the activation of CD3⁻CD56⁺CD25⁺ pNK cells. (n=3) (c) Trophoblasts increase the production TGF-β and IL-8 by pNK cells (n=3). Data represent the mean±standard error from six or three independent experiments with different deciduas. *P<0.05, **P<0.01; ***P<0.001. dNK, decidual natural killer; pNK, peripheral natural killer.