Condition of human Th17 cell differentiation induced in vitro

In this study, a series of related indicators were investigated via flow cytometry, enzyme-linked immuno-sorbent assay(ELISA)and quantitative real-time PCR(Q-PCR)technology to assess the in vitro differentiation of human Th17 cells. Human peripheral blood mononuclear cells(PBMCs)were purified from fresh human blood using gradient centrifugation and the Th17 cells were then induced with different cytokines(IL-1β, IL-6, TGF-β and IL-23)at different induction times(1, 2, 3, 4 d)to compare the effects on Th17 cell differentiation under these conditions. Data showed that IL-1β, IL-6, TGF-β or IL-23 alone play a promoting role in Th17 cell differentiation and combination of IL-1β, IL-6, TGF-β and IL-23 could induce efficient human Th17 cell differentiation in vitro to achieve the best. Further optimization of the induction time found that the Th17 cell differentiation efficiency gradually increased with the extension of the time; however, when culturing for 3 d, it reached the peak number and then decreased in spite of the time increase. Finally the optimal condition of in vitro polarization of human Th17 cells was established, in which the purified PBMCs were cultured with anti-CD3 and anti-CD28 as the basal conditions, and co-cultured with IL-1β, IL-6, TGF-β and IL-23 for 3 d to effectively induce the differentiation of Th17 cells. The inducing efficiency is significantly higher than that in normal control. At the optimal condition, Th17 cell differentiation frequency(CD4⁺IL-17A⁺)was found to increase to nearly 10% through flow cytometry analysis and the secretion level of IL-17A in cell supernatants was also detected to reach 3 ng/mL using ELISA methods. In addition, gene expression of IL-17A was determined by quantitative real-time PCR using pre-designed primers by the comparative method of relative quantitation(ΔΔCt)and β-actin gene was used as an internal control for sample normalization. The results showed that the expression of IL-17A mRNA could be increased about 15 times co-culturing with IL-1β, IL-6, TGF-β and IL-23 for 3 d. The protocol of efficient human Th17 cell differentiation presented in this paper is simple, rapid and easy to be repeated. This study provides an effective detection platform for the research of Th17 cell function and development of related drugs targeting Th17 cells for autoimmune disease treatment.