Altered B-regulatory activity in ITP patients. (A) PBMCs from healthy controls and patients with ITP (see Tables 1,Table 2–3) were stimulated with CpG and following short stimulation with PMA and ionomycin, intracellular expression of IL-10 relative to isotype-matched control antibody in various B-cell subsets was analyzed. Representative dot blot of CD19⁺ cells positive for IL-10 expression is shown and the gating strategy based on isotype control for anti–IL-10 antibody is indicated. (B) Frequency of IL-10–positive CD19 cells in healthy controls versus ITP patients whose platelet counts were below or above 50 × 10⁹/L are shown. As indicated by the P value, patients with less than 50 × 10⁹/L counts have reduced levels of CD19⁺ IL-10 expression. Similar data were obtained for comparison of IL-10 expression in various B cells subsets based on CD24 and CD38 staining as shown in supplemental Figure 3. (C) Purified CD14⁺ monocytes from ITP patients (see Table 1) and healthy controls were cultured either alone or with purified CD19⁺ cells that had been previously stimulated (for 24 hours) with sCD40L plus CpG. After a further stimulation with LPS for 4 hours in presence of brefeldin A, the cells were stained for surface CD14 and cytoplasmic TNF-α expression. Representative dot blots of TNF-α in CD14⁺ monocytes when stimulated CD14⁺ monocytes were cultured alone or with equal numbers of activated CD19 cells for healthy controls and patients are shown. Differences in levels of TNF-α produced by stimulated monocytes between healthy and ITP patients when cultured alone did not reach significance (see supplemental Figure 4, P = .07). (D) The percentage of inhibition of CD14⁺ TNF-α expression in cocultures of monocytes plus activated B cells relative to CD14⁺ TNF-α expressed when monocytes were cultured alone is shown. As indicated, monocyte cytokine inhibition was significantly lower in ITP patients with low platelet counts compared with controls. In patients with higher platelet counts, the inhibitory activity was increased, although it did not reach the levels found in healthy controls (P value not shown in figure, but the difference between healthy controls and patients with increased platelet counts at P = .04 is still significant).