Figure 3: The Notch/RBP-J/FOXP3/RORγt pathway was involved in modulating the Treg/Th17 balance after MSC treatment in vitro.

PBMCs were collected from patients, and the CD4+ lymphocyte subpopulation cells were isolated using antibody-coated immunomagnetic beads. Next, the isolated lymphocyte subpopulation cells were co-cultured with BMSCs from AA patients (A-MSCs) and donor-derived BMSCs (H-MSCs) at ratios of 10:1 for 4 days. (A) The concentrations of cytokines IL-2, INF-γ, TNF-α, and TGF-β in the culture supernatant were measured by ELISA. (B) After co-culturing the lymphocytes with BMSCs from AA patients (A-MSCs) and donor-derived BMMSCs (H-MSCs), the percentage of CD4 + IFNγ+ Th1 cells was analyzed by flow cytometry. (C) The percentage of CD4⁺ IL-4⁺Th2 cells was analyzed by flow cytometry (D) The percentage of CD8⁻CD4⁺IL-17A⁺Th17 cells was analyzed by flow cytometry. (E) The percentage of CD4⁺CD25⁺FOXP3⁺Treg cells was analyzed by flow cytometry. (F) Q-PCR analyses of Notchl, Notch2, Dll1, Jaggedl, RBP-J, RORγT, and Foxp3 expression in lymphocyte subpopulation cells after they were co-cultured with MSCs. (G) Western blot analyses of Notchl, Notch2, Dll1, Jaggedl, RBP-J, RORγT, and Foxp3 expression in lymphocyte subpopulation cells after they were co-cultured with MSCs. GAPDH was used as a loading control. (H) Densitometry plot of the results shown in Fig. 3G. The relative expression levels were normalized to GAPDH. Data represent the mean ± standard error (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.