Recombinant il 6 ril 6

PBMCs were isolated from peripheral blood using Ficoll density-gradient centrifugation. Naïve CD4⁺T cells were purified from PBMCs according to the manufacturer’s instruction (Miltenyi Biotec, Bergisch Gladbach, Germany). These purified naïve CD4⁺T cells (1 × 10⁶/well) were differentiated into Tfh cells under Tfh cell-polarizing condition (3 μg/ml soluble anti-CD3/28 (eBioscience, San Diego, CA, USA), 50 ng/ml recombinant IL-6 (rIL-6, PeproTech Inc, Rocky Hill, NJ, USA), 50 ng/ml rIL-21 (Abcam, Cambridge, MA, USA), 10 μg/ml anti-IL-4 antibody (eBioscience), 10 μg/ml anti-IFNγ antibody (eBioscience) and 10 μg/ml anti-TGF-β antibody (R&D, Minneapolis, MN, USA)) for 3 days. After initial culture, these differentiating Tfh cells were washed with PBS for 2 times and further expanded alone or cocultured with UC-MSCs (1 × 10⁵/well) in the presence of 3 μg/ml soluble anti-CD3/28 for another 2 days. To detect the factors involved in UC-MSCs-mediated suppression, UC-MSCs were collected after 2 days’ coculture with differentiating Tfh cells and then were fixed by Trizol. Furthermore, 100 μM 1-methyl-DL-tryptophan (1-MT, Sigma), the inhibitor of IDO enzyme activity or 10 μg/ml anti-IL-10 antibody (eBioscience) or 10 μg/ml anti-HLA-G antibody (Biolegend, San Diego, CA, USA) was added to the MSCs-Tfh cells coculture system to block their effects on Tfh cells.