Fig. 5. Proinflammatory potency of transitional B cells in active SLE patients.

a PBMC from 22 HDs, 64 SLE patients and 20 RA patients were stimulated with PMA/Ionomycin in the presence of Brefeldin A for 5 h, and intracellular IL-6 and TNF-α were detected by flow cytometry. Scatter plots show the frequencies of IL-6 and TNF-α production in different B-cell subsets. b, c Purified B cells from healthy donors were cultured with 25% plasma from 4 healthy donors or 11 SLE patients (Neg: IFN-α was undetectable (4 patients); Pos: IFN-α was detectable (7 patients)) for 24 h. PMA/Ionomycin and Brefeldin A were added in the last 5 h. b Representative flow cytometry plots show IL-6 production in Btr and Bn cells in different plasmas. c Bar plots show increased IL-6 secretion in Btr cells with SLE or RA plasma treatment after normalization to healthy donor plasma treatment. d Plasma was pre-treated with medium or B18R for 30 min at 4 °C. Then, purified B cells from healthy donors were cultured with 25% pre-treated plasma. Scatter plots show the frequency of IL-6-producing Btr cells after different pre-treated plasma treatment. Data are presented as means ± SEM (a, c) and are representative of three experiments (b–d). Data were analyzed by the Kruskal–Wallis test with post hoc analysis by Dunn’s multiple comparison test (a) and two-way ANOVA with Bonferroni correction for multiple comparisons (c, d). *P < 0.05, **P < 0.01, ***P < 0.001