Deficient IL-10–producing Breg compartment in cGVHD patients. Whole PBMCs from AHSCT recipients with no GVHD, active cGVHD, cGVHD in remission, and healthy donors were cultured for 66 hours with CpG-B (3 μg/mL) or CpG-B and recombinant human CD40L (1 μg/mL) followed by restimulation with PMA + ionomycin in the presence of brefeldin A during the last 5 hours of culture, fixed, permeabilized, and intracellular IL-10 was measured in CD19⁺ cells by flow cytometry. CD19⁺IL-10⁺ cells in proportion of total live CD19⁺ cells in AHSCT recipients with no GVHD, patients with active cGVHD, patients with cGVHD in remission, and healthy blood donors (A) after CpG stimulation (left panel) and after CpG + CD40L stimulation (right panel). (B) Representative dot plots showing the proportion of CD19⁺IL-10⁺ cells after CpG in an AHSCT recipient without GVHD, a patient with active cGVHD, a patient with cGVHD in remission, and a healthy donor. (C) Frequencies of CD19⁺IL-10⁺ cells in proportion to live CD19⁺ cells after CpG stimulation in patients with cGVHD (active or in remission, derived from panel A) according to the NIH global severity score (mild vs moderate and severe cGVHD). (D) Longitudinal follow-up of Breg frequencies in cGVHD patients. Patients were included at the time of active cGVHD and prospectively followed during the course of immunosuppressive treatment. An increase in Breg frequencies was observed in patients achieving a partial remission of cGVHD over time, whereas no or little change was observed in patients with stable or worsening symptoms.