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. 2020 Oct 24;35(11):2454–2466. doi: 10.1093/humrep/deaa223

Figure 4.

Figure 4.

Accumulation of Tim-3+ Treg cells is associated with IL-27 and Gal-9 secretion by trophoblasts. (A) IL-27 and Gal-9 expression in primary trophoblast cells from early pregnancy villi were determined by immunohistochemistry. Original magnification: ×200. (B) Levels of IL-27 and Gal-9 secretion by primary trophoblasts were analyzed by ELISA. (C–F) Primary trophoblast cells were co-cultured with CD4+ T cells from early pregnancy peripheral blood for 72 h, while soluble IL-27 receptor (neutralizing IL-27) (100 ng/ml) or anti-Tim-3 mAb (blocking the Tim-3 signaling pathway) (10 µg/ml) or Gal-9 (activating the Tim-3 signal pathway) (1 µg/ml) were added to the co-culture system. Then, Treg cell differentiation and Tim-3 expression on CD4+ T cells and Treg cells were detected by flow cytometry. Before co-culture, CD4+ T cells were activated with 2 μg/ml plate-bound anti-CD3, 1 μg/ml soluble anti-CD28, and 25 U/ml IL-2 for 2 days. A cell culture treatment schematic is shown in C. Representative and statistical results are shown in D and E. CD4+ T cells group: CD4+ T cells alone; Co-culture group: CD4+ T cells co-cultured with primary trophoblast cells; Co-culture + sIL-27R group: CD4+ T cells co-cultured with primary trophoblasts with sIL-27R; Co-culture + anti-Tim-3 group: CD4+ T cells co-cultured with primary trophoblasts with anti-Tim-3mAb; Co-culture + anti-Tim-3mAb + Gal-9 group: CD4+ T cells co-cultured with primary trophoblast cells with anti-Tim-3mAb and Gal-9. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.