《Table 2 Nine culture conditions for Treg and Th17 cell differentiation from nave CD4+T cells in NP

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《The altered PD-1/PD-L1 pathway delivers the ‘one-two punch’ effects to promote the Treg/Th17 imbalance in pre-eclampsia》

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Abbreviations:NP，normal pregnancy；PE，pre-eclampsia.

Naive CD4+T and Treg cells were isolated from PBMCs of women with NP and of women with PE by multi-step magnetic sorting using a human Naive CD4+T Cell Isolation Kit II and a human CD4+CD25+Regulatory T Cell Isolation Kit（Miltenyi Biotec，Auburn，CA，USA），respectively，with a Midi&Mini MACS instrument（Miltenyi Biotec）in accordance with the manufacturer’s instructions.Sorted naive CD4+T cells（1×106/well in 24-well plates）were then cultured at 37°C in a moist atmosphere containing 5%CO2in complete RPMI 1640medium supplemented with 10%heat-inactivated fetal calf serum，penicillin（100 U/ml），streptomycin（100μg/ml），2 mM L-glutamine，10 mM HEPES，0.1 mM non-essential amino acids and 1 mM sodium pyruvate（all from Sigma，Oakville，ON，Canada）.The purity of the sorted cells was generally495%.All the cells were cultured under Th0-（anti-CD3mAbs （5μg/ml）/anti-CD28 mAbs（5μg/ml））（eBioscience），Th17-（anti-CD3 mAbs （5μg/ml）/anti-CD28 mAbs（5μg/ml）/TGF-β（5 ng/ml）/IL-1β（5 ng/ml）/IL-6（20 ng/ml）/antiIFN-γmAbs（2μg/ml）/anti-IL-4 mAbs（2μg/ml））（PeproTech，Rocky Hill，NJ，USA）and Treg-prone（anti-CD3 mAbs （5μg/ml）/anti-CD28 mAbs（5μg/ml）/TGF-β（5 ng/ml）/anti-IFN-γmAbs（2μg/ml）/anti-IL-4 mAbs（2μg/ml）） conditions with or without PD-L1 Fc（10μg/ml）（R&D，Minneapolis，MN，USA） or anti-PD-L1 m Abs（10 ng/ml）（eBioscience） for 3 days.For each study case，nine groups were established as shown in the following scheme（Table 2）.Sorted Treg cells（1×105/well in96-well plates）from women with NP were then cultured at 37°C in a moist atmosphere containing 5%CO2in complete RPMI 1640 medium under rh IL-2（0.5 U/ml）（PeproTech） /anti-CD3 mAbs（5μg/ml）/anti-CD28 mAbs（5μg/ml）condition with or without anti-PD-L1 mAbs（10 ng/ml），or under pro-inflammatory conditions（rh IL-2 （0.5 U/ml）/anti-CD3m Abs（5μg/ml）/anti-CD28 mAbs（5μg/ml）/IL-1β（10 ng/ml）/IL-6（20 ng/ml）/IL-23（20 ng/ml））（PeproTech）with or without PD-L1 Fc for 5 days.