Fig. 6: SLAMF5 fine-tunes the survival of human Bregs.

a, b Healthy human PBMCs were activated for 5 h with PMA, ionomycin, brefeldin A, and monensin or with monensin alone (control) and analyzed by FACS for SLAMF5 expression on B-cell populations: total B cells: CD19⁺; memory B cells: CD19⁺CD24^highCD38^low; naive mature B cells: CD19⁺CD24^lowCD38 ^low; immature B cells: CD19⁺CD24^highCD38^high; and plasma B cells: CD19⁺CD24^lowCD38^high. Regulatory B-cell subpopulations were analyzed with similar markers under the CD19⁺IL-10 gate. Gating is shown in Supplementary Fig. 9a. a Representative histograms, and (b) bar charts of SLAMF5 expression on IL-10⁺ (Breg) and IL-10^neg (non-Breg) populations. (non-Bregs n = 7; Bregs n = 7; memory B cells n = 8; memory Bregs n = 7; naive B cells n = 5; naive Bregs n = 5; transitional B cells n = 7; transitional Breg n = 8; plasma B cell n = 6; plasma Breg n = 5, four independent experiments). c–f Purified human B cells from healthy donors were treated with SLAMF5-blocking or IgG-control antibody for 48 h. For the last 5 h, the cells were activated with PIM and analyzed for (c) cell survival by Zombie dye staining. Bars chart of live Bregs (CD19⁺IL-10⁺Zombie^neg) or non-Breg B cells (CD19⁺IL-10⁻Zombie⁻) out of total CD19⁺. (IgG: Breg n = 5; non-Breg = 5, SLAMF5-blocking: Breg n = 5; non-Breg n = 5, three independent experiments). d After 24 h of treatment, cells were collected for mRNA analysis of c-Maf on total B cells. Results are shown as x-fold of treatment compared to IgG-control. (IgG n = 4; SLAMF5-blocking n = 4, two independent experiments). e, f c-Maf expression in Bregs was analyzed by FACS after 48 h. e Representative histogram, and (f) bar chart of c-Maf expression. Results are shown as x-fold of treatment compared to IgG-control. (IgG = 5; SLAMF5-blocking n = 5, two independent experiments). g, h Blood samples derived from newly diagnosed untreated multiple sclerosis patients and matched healthy controls were processed, and PBMC were activated for 5 h with PMA, ionomycin, monensin, and brefeldin A, and stained for SLAMF5 and IL-10 on Breg populations. Total Bregs: CD19⁺IL-10⁺; immature Bregs: CD19⁺IL-10⁺CD24⁺CD38⁺; B10 Bregs: CD19⁺IL-10⁺, CD24⁺CD27⁺. g Representative histograms showing the gate used to define SLAMF5^high expression and h bar charts of SLAMF5^high expression on Breg populations presented as fold increase over each sex- and age-matched control. Data expressed as mean ± s.e.m. (b) or mean ± s.d. (c, d, f, h). Paired Student’s t test with 95% confidence levels two-tailed (b). Ratio paired Student’s t test with 95% confidence levels two-tailed unpaired Student’s t test with 95% confidence levels two-tailed (c, d, f, h). The gating strategy for a and b is shown in Supplementary Fig. 9a, gating strategy for c is shown in Supplementary Fig. 8a, gating strategy for g and h is shown in Supplementary Fig. 9b.