Fig. 7

Effects of butyrate on Treg differentiation and function were dependent on autophagy. (A, B) Magnetically sorted naive CD4⁺ T cells (2 × 10⁵ cells) from the peripheral blood of patients with AChR MG were cultured for 3 days under Treg-polarizing conditions with or without 200 μM butyrate or 20 μM chloroquine (CQ). The frequencies of CD4⁺CD25⁺FOXP3⁺ Tregs were determined by flow cytometry (F = 203.2, p = 0.0003 for difference between Butyrate and Butyrate + CQ with ANOVA). (C) The resultant measurement of glycoPER and (D) the basal ratio of mitoOCR to glycoPER in these cells (F = 30.57, p = 0.0134 for difference between Butyrate and Butyrate + CQ with ANOVA). (E, F) Magnetically sorted CD4⁺CD25⁻ Tresps (2 × 10⁵ cells) from patients with AChR MG were labeled with CFSE and cultured for 5 days with or without 200 μM butyrate or 20 μM CQ in an equal number of CD4⁺CD25⁺CD127_low Tregs. The suppressive function of Tregs was measured by calculating the proliferation of CFSE⁺ cells (F = 75.07, p = 0.0026 for difference between Butyrate and Butyrate + CQ with ANOVA). (G, H) Magnetically sorted CD4⁺CD25⁺CD127_low Tregs (2 × 10⁵ cells) from patients with AChR MG were cultured for 5 days with or without 200 μM butyrate or 20 μM CQ. The frequencies of CTLA-4⁺ Tregs were detected by flow cytometry (F = 490.8, p = 0.0007 for difference between Butyrate and Butyrate + CQ with ANOVA). 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