Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by peripheral blood platelet count<100x10^9/L and increased risk of bleeding, but its exact pathogenesis remains unclear. Previous researches mostly emphasized on periphral blood, in order to elucidate the pathogenesis and provide new strategies for treatment, a series of studies about bone marrow B cells were carried out in our investigation.
5ml bone marrow blood samples from 11 newly diagnosed ITP patients and 7 allo-HSCT healthy donors were collected into heparin anticoagulant tubes , bone marrow mononuclear cells (BMMCs) were separated with Ficoll density gradient-centrifugation in two hours. Different subsets of B lymphocytes were determined by multicolor flow cytometry including naive B cells, total memory B cells , plasma cells and regulatory B cells (Bregs) ,as well as some chemokine receptors of B cells (CXCR5, BAFFR, BCMA, TACI) . RNA were extracted from BMMCs using Trizol reagent, transcription factors related to development and differentiation of B cells (PRDM1, Pax5, IRF-4, XBP1) were detected by real-time PCR.
The percentage of B cells (CD19+) in bone marrow lymphocytes in ITP patients was significantly lower than that in healthy donors (6.16±0.74% vs. 17.28±2.43%, P < 0.0001). The proportion of naive B (CD19+CD27-) in B cells was also lower compared with normal controls (59.11±7.60% vs. 81.58±4.00%, P=0.041), and the proportion of memory B (CD19+CD27+) was higher(40.17±7.67% vs. 18.01±3.89%, P=0.045),but there was no significant difference in plasma cells (CD19-CD138+). Besides,there was a decrease of Breg (CD19+CD24highCD38high) in ITP patients compared with healthy donors (20.33±5.05% vs. 57.98±9.76% , P = 0.008), and its percentage of total lymphocytes was also significantly lower unsurprisingly (1.38±0.38% vs. 12.23±2.88%, P < 0.001). The level of BAFFR in mature B cells was elevated in ITP patients (80.72±4.53% vs. 45.81±8.49%, P = 0.002). However, no significant difference was observed in other three chemokine receptors. All four Transcription factors related to B cell development and differentiation was not found to be significantly different between ITP patients and healthy controls.
Our results showed that memory B cells which represent the active form were increased, and Bregs which mediate immune tolerance were much more decreased in ITP patients. As BAFFR is the only specific receptor of B cell activating factor(BAFF), its elevated expression suggested the BAFF-BAFFR system enhanced chemotactic function of B cells in ITP patients. All those results indicated that the bone marrow B cells in patients with ITP were in a state of immune overstimulation, this may potentially constitute a novel therapeutic target.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.