Fig. 4
Butyrate enhances surface CTLA-4 expression of Tregs in patients with AChR MG. (A, B) The frequencies of surface CTLA-4+ in CD4+FOXP3+ Tregs obtained from the peripheral blood of patients with AChR MG and HCs were determined by flow cytometry (t = 3.525, p = 0.0078 with unpaired t-test). (C, D) Magnetically sorted CD4+CD25+CD127low Tregs (2 × 105 cells) from patients with AChR MG were cultured for 3 days with or without 200 μM butyrate. The frequencies of CTLA-4+FOXP3+ cells were determined by flow cytometry (t = 5.357, p = 0.0017 with unpaired t-test). (E) Immunofluorescent images of CTLA-4 on Tregs (scale bar = 20 μm). Magnetically sorted CD4+ T cells (2 × 105 cells), obtained from the peripheral blood of patients with AChR MG, were cultured for 3 days under Treg-polarizing conditions with or without CTLA-4-Ig in the presence of 200 μM butyrate. (F, G) The percentages of FOXP3+CTLA-4+ cells (F = 51.93, p = 0.0031 for difference between Butyrate and Butyrate + CTLA-4 Ig with ANOVA) and FOXP3−CTLA-4+ cells (F = 94.22, p < 0.001 for difference between Butyrate and Butyrate + CTLA-4 Ig with ANOVA) were determined by flow cytometry. (H) The secretion of IFN-γ (F = 16.85, p = 0.0011 for difference between Butyrate and Butyrate + CTLA-4 Ig with ANOVA), IL-17 A (F = 20.96, p = 0.0004 for difference between Butyrate and Butyrate + CTLA-4 Ig with ANOVA) and TGF-β (F = 12.99, p = 0.0043 for difference between Butyrate and Butyrate + CTLA-4 Ig with ANOVA) in the culture supernatant were quantified using ELISA kits. Each dot represents an individual sample. Data are shown as the mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant