Bidirectional regulation between 1st trimester HTR8/SVneo trophoblast cells and in vitro differentiated Th17/Treg cells suggest a fetal-maternal regulatory loop in human pregnancy

Abstract

Problem: During normal pregnancy, delicate crosstalk is established between fetus-derived trophoblasts and maternal immune cells to ensure maternal-fetal tolerance and successful placentation. Dysfunction in these interactions has been highly linked to certain pregnancy complications.

Method of study: Naïve CD4⁺ T cells were cultivated with or without 1st trimester derived trophoblast cell line HTR8/SVneo cells in the absence or presence of T helper 17 (Th17) or regulatory (Treg)cell-inducing differentiation conditions. After 5 days of co-culture, HTR8/SVneo cells and CD4⁺ T cells were harvested and analyzed using flow cytometry.

Results: CD4⁺ T cells exposed to HTR8/SVneo cells showed enhanced induction of CD4⁺ Foxp3⁺ Treg cells with strong expression of TGF-β1 and inhibitory molecules (cytotoxic T lymphocyte-associated protein-4 [CTLA-4], T-cell immunoglobulin mucin-3 [Tim-3], and programmed cell death-1 [PD-1]). Though not effecting Th17 differentiation, exposure to HTR8/SVneo cells promoted increased expression of proliferative and apoptotic markers on Th17 cells. Co-culture with Th0 cells, or differentiated Th17 or Treg cells, down-regulated Caspase-3 and MMP-9 (but not MMP-2) expression in HTR8/SVneo cells, while promoting Ki67 expression.

Conclusions: HTR8/SVneo cells regulated maternal CD4⁺ T-cell differentiation, resulting in the expansion of immunosuppressive Treg cells, while CD4⁺ T cells might promote the growth, and control the invasiveness of HTR8/SVneo cells. Thus, a bidirectional regulatory loop might exist between trophoblasts and maternal immune cell subsets, thereby promoting harmonious maternal-fetal crosstalk.

Keywords: HTR8/SVneo cells; Th17 cells; Treg cells; pregnancy; trophoblasts.

Figure 1

HTR8/SVneo cells contributed to the differentiation of Treg cells from maternal naïve CD4⁺T cells. (A) Maternal naïve CD4⁺T cells were differentiated in the presence of IL‐2 (Th0) or IL‐2 + TGF‐β1 + IL‐6 + anti‐IFN‐γ mAb + anti‐IL‐4 mAb (Th17) for 5 days. In some wells, CD4⁺T cells were co‐cultured with HTR8/SVneo cells. IL‐17A expression in T cells was analyzed through flow cytometry. (B and C) Maternal naïve CD4⁺T cells were differentiated in the presence of IL‐2 (Th0) or IL‐2 + TGF‐β1 + anti‐IL‐6 mAb + anti‐IFN‐γ mAb + anti‐IL‐4 mAb (Treg) for 5 days. In some wells, CD4⁺T cells were co‐cultured with HTR8/SVneo cells. Foxp‐3 expression and IL‐10 and TGF‐β1 production in CD4⁺T cells were analyzed through flow cytometry. **P < 0.01, ***P < 0.001, compared to the Th0 group. ##P < 0.01, ###P < 0.001, compared to Treg group. Data are represented as the mean ± SD, n = 18. Flow cytometry plots are representative of three independent experiments

Figure 2

HTR8/SVneo cells regulated the apoptosis and proliferation of differentiated T cells. Maternal naïve CD4⁺T cells were differentiated in the presence of IL‐2 (Th0), IL‐2 + TGF‐β1 + IL‐6 + anti‐IFN‐γ mAb + anti‐IL‐4 mAb (Th17) or IL‐2 + TGF‐β1 + anti‐IL‐6 mAb + anti‐IFN‐γ mAb + anti‐IL‐4 mAb (Treg) for 5 days. In some wells, T cells were co‐cultured with HTR8/SVneo cells. Caspase‐3 (A) and Ki‐67 (B) expression in CD4⁺T cells was analyzed through flow cytometry.*P < 0.05, ***P < 0.001, compared to Th0 group. ###P < 0.001, compared to Th17 group. ^P < 0.05, compared to Treg group. Data are represented as the mean ± SD, n = 18. Flow cytometry plots are representative of three independent experiments

Figure 3

HTR8/SVneo cells promoted the expression of inhibitory receptors in differentiated Treg cells. Maternal naïve CD4⁺T cells were differentiated in the presence of Th0‐, Th17‐, or Treg‐inducing differentiation agents for 5 days. In some wells, CD4⁺T cells were co‐cultured with HTR8/SVneo cells. Cytotoxic T lymphocyte‐associated protein‐4 (CTLA‐4) (A), Tim‐3 (B), and programmed cell death‐1 (PD‐1) (C) expression in CD4⁺T cells was analyzed through flow cytometry. **P < 0.01, ***P < 0.001, compared to Th0 group. #P < 0.05, ###P < 0.001, compared to Treg group. Data are represented as the mean ± SD, n = 18. Flow cytometry plots are representative of three independent experiments

Figure 4

Differentiated CD4⁺T cells modulated the biological functions of HTR8/SVneo cells. HTR8/SVneo cells were co‐cultured with Th0, Th17 and Treg cells (with the indicated agents) for 5 days. Caspase‐3 (A), Ki‐67 (B), MMP‐2 (C) and MMP‐9 (D) expression in HTR8/SVneo cells was analyzed through flow cytometry.***P < 0.001, compared to the Ctrl group. Data are represented as the mean ± SD. Flow cytometry plots are representative of three independent experiments