B lymphocytes participate in immune responses through production of antibodies, antigen presentation to T cells, and cytokine secretion. In ITP, B cells that produce platelet-specific autoantibodies play a major role in the pathogenesis of disease. Recent data from mouse models of autoimmune and inflammatory diseases suggest that B cells have regulatory functions mediated by the production of regulatory cytokines such as IL-10 and/or through inhibitory interactions with effector T cells. In humans, CD19+CD24hiCD38hi B cells, originally identified as immature transitional B cells, were recently shown to possess regulatory capacity mediated in part by IL-10. Given that ITP pathogenesis is in part related to defective T helper functions and since Bregs are important for controlling CD4+ T cell responses, we initiated studies to characterize the CD19+CD24hiCD38hi B cell compartment in ITP patients. Phenotypic analysis in patients with ITP showed statistically significant increases in the frequency of CD19+CD24hiCD38hi B cells (13.2± 1.5% versus 7.7±0.6%, p=0.0015) as well as in the percentage of CD19+CD24hiCD38int mature B cells (72.0± 1.7% versus 62.3±1.6%, p=0.0003). In contrast there was a pronounced decrease in CD19+CD24hiCD38− memory B cells (9.3± 2% versus 26.2±2%, p<0.0001 as confirmed by expression of CD27 memory marker) in patients (n=23) compared to healthy controls (n=23). The suppressive capacity of human CD19+CD24hiCD38hi Bregs involves CD80. We found a statistically significant decrease in the overall frequency of CD80 expressing CD19+ B cells in ITP patients compared to healthy controls (12.40± 1.3% versus 21.8±2.1%, p=0.002), indicating that despite the increase in the Breg frequency, their regulatory capacity may be impaired. To determine the functional activity of the Bregs, PBMCs from 4 patients with ITP were depleted of CD19+CD24hiCD38hi B cells by cell sorting and levels of cytokine production in the CD4+ T cell population following stimulation with plate-bound anti-CD3 for 72h was evaluated. As has been reported previously, we observed a >30% increase in the frequency of CD4+IFN-g+ in CD19+CD24hiCD38hi B cell-depleted compared with non-depleted PBMCs from healthy controls. However, depletion of CD19+CD24hiCD38hi B cells from PBMCs of ITP patients did not alter CD4+IFN-g+ production in 3 /4 patients, indicating a possible defect in the suppressive activity of Bregs in those 3 patients. IL-10 secretion following CD40 engagement in human Bregs is pivotal in mediating their suppressive activity. Given that platelets express CD40L, we tested the ability of Bregs to respond to platelets. We found two-fold lower IL-10 production in CD19+CD24hiCD38hi Bregs (1.9±2.1%, versus 5.0± 1.0%, p<0.05), but not in CD19+CD24hiCD38− B cells (p=0.9) from patients (n=3, different from above) compared to controls (n=5) when PBMCs were stimulated with acid-treated platelets (to remove HLA molecules) from normal controls, indicating a defect in IL-10 production in the Breg population in patients with ITP. Altogether, our in vitro studies of circulating Bregs suggest that these cells may be functionally compromised in some patients with ITP patients as indicated by reduced IL-10 production and indirectly by Breg depletion studies showing inability to dampen effector T cell responses. Given the important role of Bregs in controlling CD4+T responses, the data implicates defective Bregs as an additional mechanism to explain increased T cell responses in patients with ITP. The impaired Breg activity may also explain the variability in response to treatment with anti-CD20 in patients with ITP. It may be that B cell depletion therapy in patients who have defective Breg activity will result in the removal of primarily pathogenic B cells and therefore these patients will have a good response to rituximab treatment. However, in patients with intact Bregs, the same treatment will deplete both pathogenic and regulatory B cells, causing a less effective response. Ongoing studies to test patients' platelets as well as CD40-specific Breg responsiveness are currently underway to further explore the role of Bregs in the pathogenesis of ITP.
Bussel:Portola: Consultancy; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai, Inc.: Membership on an entity's Board of Directors or advisory committees; Cangene: Research Funding; Genzyme: Research Funding.
Asterisk with author names denotes non-ASH members.