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Figure 4: Activated HMCs induce the differentiation of naïve CD4+ T cells. (a) The mRNA expression of TGF-β, IL-4, IL-6, IL-12A, and IL-23A in HMCs treated with IFN-γ for 48 h was analysed by RT-PCR. (b) The protein expression of TGF-β, IL-4, IL-6, IL-12, and IL-23 in cell culture supernatants from HMCs treated with IFN-γ for 48 h was analysed via ELISA. (c) HMCs were treated with IFN-γ for 48 h, extensively washed, irradiated, and cocultured with naïve CD4+ T cells for 48 h. ELISA was used to determine the expression of the cytokines IFN-γ, IL-4, IL-10, and IL-17 in the culture supernatant. (d) HMCs were treated with IFN-γ for 48 h, extensively washed, irradiated, and cocultured with naïve CD4+ T cells for 48 h. The mRNA expression of IFN-γ, IL-4, IL-10, and IL-17 in CD4+ T cells was analysed via RT-PCR. (e) HMCs were treated with IFN-γ for 48 h, extensively washed, irradiated, and cocultured with naïve CD4+ T cells for 48 h. Two days after stimulation, proliferation was analysed using an EdU assay and detected by FCM. The results are expressed as fold changes in EdU-positive CD4+ T cells. (f) HMCs cultured alone or in the presence of IFN-γ were used to activate naïve CD4+ T cells. The differentiation of activated T cells into Th1 (IFN-γ+), Th2 (IL-4+), Th17 (IL-17+), or Treg (FOXP3+, CD25+) effectors was assessed via intracellular staining and flow cytometry after 48 h. The data in (a) and (b) were analysed by Student’s -test, and the data in (c), (d), and (e) were analysed by one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test. The data are representative of three independent experiments, and the error bars represent the . Con: HMCs treated without IFN-γ. .