The THD-modulated MSCs induced mDCs into tolerogenic DCs in patients with ITP. (A) The inhibitory effect of MSCs on lymphocyte proliferation. CFSE-labeled allogeneic CD4+ T cells (1 × 105 per well) were stimulated with PMA (1 μg/mL) or mDCs (1 × 104 per well) and cocultured in the absence (−) or presence (+) of 1 × 104 per well MSCs (control, n = 9), MSCs (ITP, n = 9), or THD-MSCs (ITP, n = 9). After 3 days, the cells were collected and the division index was analyzed via FACS. Each bar denotes the division index of different conditions. Statistical results represent median with interquartile range. (B-E) The inhibitory effect of MSCs on cytokine secretion. CD4+ T cells (1 × 105 per well), mDCs (1 × 104 per well), and MSCs (1 × 104 per well) were cultured alone or in combination with MSCs (ITP, n = 16) or THD-MSCs (ITP, n = 16) as indicated. The levels of IL-4, IFN-γ, IDO, and IL-12 in the culture supernatant were determined using ELISA after 3 days’ culture. Each dot denotes the level of IL-4, IFN-γ, IDO, and IL-12. Statistical results represent median with interquartile range. (F) The tolerogenic effect of MSC-DCs on T-cell proliferation. After coculture with MSCs (control, n = 9), MSCs (ITP, n = 9), or MSCs (ITP, n = 9) modulated by THD, the MSC-DCs were sorted using anti-HLA-DR immunomagnetic beads. CFSE-labeled allogeneic CD4+ T cells (1 × 105 per well) were cocultured for 3 days with PMA (1 μg/mL) or mDCs (1 × 104 per well) in the presence of 1 × 104 per well MSC (control)–DCs, MSC (ITP)–DCs, or THD-MSC (ITP)–DCs, and analyzed via FACS. All DCs (mDC and MSC-DCs) in each experiment were from the same person. Each bar denotes the division index from different conditions. Statistical results represent median with interquartile range. (G-H) MSC-modulated mDC exhibit a unique pattern of activation markers. The mDCs were cultured in absence or presence of MSCs (control, n = 8), MSCs (ITP, n = 14), or THD-MSCs (ITP, n = 18) in a 1:1 ratio (mDC:MSC). After 3 days, the cells were collected to determine the MFI of CD80 and CD86 on mDCs by flow cytometry. Statistical results represent median with min to max of fluorescence intensity of CD80 and CD86. (I-J) The inhibitory effect of tolerogenic DCs was mediated by IL-10 and TGF-β. mDCs (1 × 104 per well) and MSCs (1 × 104 per well) were cultured alone or in combination for 3 days as indicated. The levels of IL-10 or TGF-β in the culture media were determined using ELISA. Data represent median with interquartile range. Each dot denotes the level of IL-10 or TGF-β. Statistical results represent median with interquartile range from 16 ITP patients. Differences between 2 groups were compared using the Mann-Whitney U test. *P < .05; **P < .01. MFI, mean fluorescence intensity.
