Compromised immunoregulatory capacity of BM Bregs from patients with ITP. (A) Quantification of CD14⁺TNF-α⁺ monocytes cultured alone or with activated BM B cells that contained Bregs from patients with ITP and HCs, respectively. (B, C) Percentage of TNF-α⁺ monocytes cocultured with BM B cells was significantly lower than that cultured alone, both in patients with ITP (21.24 ± 4.90% vs 36.61 ± 7.82%; P = .001) and in HCs (15.70 ± 5.44% vs 41.52 ± 11.90%; P = .014). (D) BM B cells from patients with ITP exhibited decreased capacity to suppress monocyte TNF-α production compared with HCs (39.95 ± 5.45% vs 61.55 ± 6.89%; P = .029). The percentage of inhibition was calculated as [1 – (percentage of TNF-α⁺ monocytes cocultured with B cells/percentage of TNF-α⁺ monocytes cultured alone)] × 100%. (E) IFN-γ secreted by CD4⁺ T cells cultured alone or with BM B cells from patients with ITP and HCs, respectively. (F, G) BM B cells from the ITP (16.31 ± 4.89% vs 14.46 ± 4.09%; P = .283) and HC (26.22 ± 3.47% vs 15.85 ± 2.31%; P < .001) groups both inhibited IFN-γ secretion by CD4⁺ T cells, but statistical significance was not reached in the ITP group. (H) Inhibition of CD4⁺IFN-γ⁺ T cells by BM B cells from patients with ITP was weaker than that from HCs (median [range], 22.78% [6.84%-35.80%] vs 42.10% [30.16%-48.72%]; P = .009). Percentage of inhibition was calculated as [1 – (percentage of CD3⁺IFN-γ⁺ T cells cocultured with B cells/percentage of CD3⁺IFN-γ⁺ T cells cultured alone)] × 100%. (I) CD4⁺ T cells cultured alone or with BM B cells for 72 hours, and frequencies of CD4⁺CD25⁺Foxp3⁺ Tregs were determined by using flow cytometry. (J, K) BM B cells from patients with ITP and HCs both enhanced the percentage of Tregs in CD4⁺ T cells (patients with ITP, 4.04 ± 1.31% vs 15.09 ± 3.40% [P = .007]; HCs, 1.32 ± 0.38% vs 21.93 ± 1.89% [P < .001]). (L) BM B cells from patients with ITP had weaker ability to promote Treg differentiation compared with HCs (median [range], 3.74 [1.58-14.55] vs 20.70 [8.82-25.82]; P = .009). Promotion of Tregs was calculated as Tregs % cocultured with B cells/Tregs % cultured alone. (M) Platelet counts of active ITP mice transfused with BM Bregs from immunized CD61-KO mice were higher than that of control group mice on days 14, 21, and 28 after splenocyte transfusion. *P < .05; **P < .01; ***P < .001. APC, allophycocyanin; PE, phycoerythrin; SSC, side scatter.