Figure 1.

PD-1 and Tim-3 expression on CD8⁺ T cells during pregnancy. (a) Frequency of Tim-3 and PD-1 expression on gated CD8⁺ T cells from peripheral blood mononuclear cells (PBMCs) and decidual immune cells (DICs) during human first trimester pregnancy. Freshly isolated PBMCs and DICs were stained with antibodies against CD8, PD-1, and Tim-3 to assess PD-1 and Tim-3 expression on CD8⁺ T cells by flow cytometry. n=30. **P<0.01; ***P<0.001. (b and c) Freshly isolated trophoblasts were seeded at 2 × 10⁵ cells/ml per well in matrigel-coated 24-well plates overnight. The supernatants were aspirated completely, and the cells were washed twice with phosphate-buffered saline (PBS). Equal numbers of DICs or PBMCs were added to each well. Flow-cytometric analysis (right) and quantification (left) of Tim-3 and PD-1 expression on decidual (b) and peripheral (c) CD8⁺ T cells with or without co-culture with trophoblasts (Tros) for 48 h. n=9. *P<0.05; **P<0.01; ***P<0.001. (d) Freshly isolated trophoblasts (Tros), decidual stromal cells (DSCs), and human HTR8/SVneo cells were seeded at 2 × 10⁵ cells/ml per well in 24-well plates. After overnight, the supernatants were aspirated completely, and the cells were washed twice with PBS. Equal numbers of DICs or PBMCs were added to each well. The anti-CD3 and anti-CD28 antibodies were used to activate CD8⁺ T cells in some wells. ***P<0.001, compared with the control. ^#P<0.5, ^##P<0.01, ^###P<0.001, compared with the group co-cultured with trophoblasts. Data represent mean±S.E.M. The flow-cytometry plots are representative of three independent experiments. αHLA-C: anti-HLA-C antibody, αHLA-G: anti-HLA-G antibody, αCD80: anti-CD80 antibody, αCD86: anti-CD86 antibody, αCD3/CD28: anti-CD3 antibody plus anti-CD28 antibody