Increased output of IL-10–producing Bregs after AHSCT. (A) Gating strategy of 1 representative patient showing the frequency of CD24^hiCD38⁻, CD24^intCD38^int, CD24⁻CD38^hi memory, and CD24^hiCD38^hi Bregs immunophenotyped by flow cytometry at baseline (0 months, pretransplant) and following time points. Quantification of the B-cell subpopulations frequency (B) and absolute values (C) are shown in panel A. The boundaries of the boxes indicate the 25th and 75th percentiles; the lines within the boxes indicate the median, and the whiskers mark the 10th and the 90th percentiles. Plots show mean ± SE. *P < .05; **P < .01 comparing posttransplant values with baseline (Wilcoxon’s). Mean ± SE changes on (D) Breg/CD19⁺CD27⁺IgD⁺ Unswitched memory and on (E) Breg/CD19⁺CD27⁺IgD⁻ Switched memory ratios. *P < .05 comparing posttransplant values to baseline (Wilcoxon’s). N = 17 transplanted patients at baseline, n = 14 at 6 and 12 months, and n = 9 at 18, 24, and >24 months. (F) Correlation between Bregs and sjKREC values (Spearman’s). (G) Whole PBMCs from AHSCT patients at baseline, 6 months, and 12 months posttransplant were cultured for 18 hours with CpG or CpG and rhCD40L followed by restimulation with phorbol myristate acetate + ionomycin + BFA (PIB) in the last 6 hours of culture, fixed, permeabilized, and intracellular IL-10 assessed in CD19⁺ B cells by flow cytometry. The position of all gates was determined using isotype-matched control mAb staining. Negative controls consisted of PBMCs cultured in the presence of CpG control and BFA. These data are representative of those obtained in 6 independent experiments, with numbers representing the frequency of IL-10–producing B10 cells. Quantifications (mean ± SE) are expressed in parentheses. *P < .05; **P < .01 comparing posttransplant values with baseline (Wilcoxon’s). (H) Correlation between CD19⁺CD24^hiCD38^hi Bregs and the C-reactive protein levels (Spearman’s). (I) Responder patients after AHSCT presented higher Breg percentage at 12 months after transplant than nonresponder patients. *P < .05 comparing groups with each other (Mann-Whitney U test). (J) Correlation between sjKREC and sjTREC (Spearman’s).