B cells from patients undergoing CBT show an early and robust reconstitution of both the CD19⁺ B cells and the IL-10⁺ B-cell pools. (A) IL-10 secretion by total CD19⁺ B cells. PBMCs collected from 27 CBT patients and cultured with CD40L for 48 hours were stained for the CD19⁺IL-10⁺ phenotype by intracellular flow cytometric staining. The samples represented the following collection time points: pretransplant (n = 19), 1 month (n = 20), 3 months (n = 22), 6 months (n = 16), 9 months (n = 12), 1 year (n = 12) and 2 years (n = 8) post-CBT. Supernatants were also harvested from cultures of activated patient-derived B cells and assayed for IL-10 secretion by ELISA from samples collected at the following times: pretransplant (n = 12), 1 month (n = 10), 3 months (n = 10), 6 months (n = 10), 9 months (n = 8), 1 year (n = 12), and 2 years (n = 8) post-CBT. Bars indicate mean values and ranges (whiskers). (B) Absolute counts of IL-10⁺CD19⁺ B cells in PBMCs collected from 27 CB recipients, available at the following time points: pretransplant (n = 19), 1 month (n = 20), 3 months (n = 22), 6 months (n = 16), 9 months (n = 12), 1 year (n = 12) and CD19⁺B cells at 2 years (n = 8) post-CBT. (C) Comparison of IL-10 secretion by activated total CD19⁺ B cells in PB (n = 10) and CB (n = 10) from healthy donors and patients at 6 months posttransplant (n = 10). Bars denote median values and ranges (whiskers). *P < .05 by nonparametric ANOVA. (D) CD19⁺ B-cell frequencies and absolute counts per microliter in patients with cGVHD (n = 12) compared with patients without cGVHD (n = 15). *P < .05 and **P < .01 by unpaired t test. (E) CD19⁺IL-10⁺ B-cell frequencies and absolute counts per microliter in patients with cGVHD (n = 12) compared with patients without cGVHD (n = 15). *P < .05 and **P < .01 by unpaired t test. In both panels D and E, samples from patients with GVHD were available at the following time points: pretransplant (n = 9), 1 month (n = 10), 3 months (n = 10), 6 months (n = 7), 9 months (n = 6), 1 year (n = 5), and 2 years (n = 3) post-CBT. Samples from patients without GVHD were available at the following time points: pretransplant (n = 10), 1 month (n = 10), 3 months (n = 12), 6 months (n = 9), 9 months (n = 6), 1 year (n = 7), and 2 years (n = 5) post-CBT. The data points are mean values with ranges (whiskers). (F) Reconstituting IL-10–producing regulatory CD19⁺ B cells from patients possess greater suppressive function on T cells as compared with healthy CB B cells or PB B cells from healthy donors on a cell-per-cell basis. CD19⁺ B cells magnetically isolated from post-CBT patients at different time points post-CBT were cultured at a 1:1 ratio with anti-CD3/anti-CD28-activated CFSE⁺CD4⁺ T cells from healthy individuals for 96 hours, and then harvested and stained for CD4⁺CFSE⁺ proliferating T cells (healthy cord blood CD19⁺ B cells [n = 12]; CD19⁺ B cells at 1 month [n = 3]; CD19⁺ B cells at 3 months [n = 5]; CD19⁺ B cells at 6 months [n = 8]; CD19⁺ B cells at 9 months [n = 7]; CD19⁺ B cells at 1 year [n = 8]; CD19⁺ B cells at 2 years [n = 3]). (G) Patients with cGVHD (n = 6) had higher frequencies and absolute numbers of IFN-γ⁺CD4⁺ T cells compared with those without cGVHD (n = 4). (H) Patients with cGVHD (n = 6) had a reduced ratio of IL-10⁺ B cells to IFN-γ⁺CD4⁺ T cells compared with patients without cGVHD (n = 4). In both panels, bars denote mean values and ranges (whiskers). *P < .05 by unpaired t test. PBMC, peripheral blood mononuclear cell.