High-dose Dexamethasone Restores The Regulatory Functions Of CD5~+B Cells From Patients With Immune Thrombocytopenia

[Background] Immune thrombocytopenia (ITP) is an autoimmune-mediated bleeding disorder characterized by the presence of autoreactive antibodies which accelerate the destruction of platelets and/or impair thrombocytogenesis. Given that B lymphocytes produce antibodies, B cells have long been considered to play critical pathogenic roles in ITP. However, a growing body of evidence has revealed that B regulatory cells (Bregs) exist both in mice and in humans and suppress immune response mainly via production of interleukin 10 (IL-10). The expression of CD5 is a common feature, or a hallmark, of Bregs.CD5+ 8 cells are able to produce a large amount of IL-10. However, little is known about the characteristics of CD5+ B cells in ITP, a well-defined Thl polarized autoimmune disorder, and the effects of clinical treatments on CD5+ B cells. As one of the initial treatments in managing ITP, high-dose dexamethasone (HD-DXM) affects various types of immune cells in patients with ITP, such as reducing platelet destruction by macrophages, correcting Thl/Th2 imbalance, arresting the maturation of dendritic cells and reducing synthesis of pro-inflammatory cytokines. However, effects of HD-DXM on CD5+ B cells are still to be unraveled.[Objectives] To investigate the alteration of numbers of CD5+ B cells and their ability of producing IL-10 as well as immune regulatory functions in patients with ITP, and the effects of pulsed HD-DXM therapy on CD5+B cells. Then we attempted to identify the molecular mechanisms underlying the functional and numerical deficencies in CD5+ B cells in patients with ITP.[Methods] (1) Venous whole blood samples (2mL) from eligible newly diagnosed ITP patients (before and after HD-DXM treatment) and healthy controls were collected. Peripheral blood mononuclear cells (PBMCs) were seperated by using Ficoll-Hypaque gradient centrifugation. PBMCs were stained with PE-CD5/FITC-CD19 for analysis of B cell subpopulations. After 24 hours culture with phorbol myristate acetate (PMA), ionomycin and Brefeldin A (BFA), cells were permeabilized and stained with APC-IL-10 antibody to investigate intracellular IL-10 expression. Real time polymerase chain reaction (RT-PCR) was employed to detect the expression of IL-10 mRNA. Supernatant IL-10 concentration was measured by enzyme-linked immuno sorbent assay (ELISA).(2) CD5+ B cells, CD5-B cells and CD4+T cells were magenetically purified out of 20 mL venous blood samples from eligible newly diagnosed ITP patients (before and after HD-DXM treatment) and healthy controls. CD4+T cells were labelled with carboxyfluoresceindiacetate (CFSE) before incubated with CD5+ CD19+ B cells or cells at gradient B/T ratios in CD3 mAb and CD28 mAb pre-coated 96-well platelets for 3-6 days. The proliferation of CD4+T cells was investigated by flow cytometry (FCM). Cells were stained with PI/Annexin-V for analysis of apoptosis. The concentration of IL-4 and IFN-y in cell culture supernatant was analyzed by ELISA. The expression of T-bet and GATA-3 mRNA was also detected by using RT-PCR.(3) CD5+B cells were magenetically purified out of 20 mL venous blood samples from newly diagnosed ITP patients and healthy controls. PCR Arrays were used to analyze the aberrant mRNA expression of moleculars in TLR signaling pathways in CD5+B cells from ITP patients.[Results] (1) The number of CD5+ B cells was elevated in patients with ITP. Expression of IL-10 mRNA, percentage of IL-10+ cells and mean fluorescence intensity (MFI) of intracellular IL-10 in CD5+ B cells from untreated patients were significantly higher than that in controls. In contrast, ITP patients showed lower IL-10 concentration in supernatants than controls. After HD-DXM therapy, the number of CD5+ B cells decreased to normal level, while intracellular IL-10 expression in CD5+ B cells was further enhanced and IL-10 concentration in supernatants was also increased.(2) Purified CD5+ B cells from healthy subjects were capable of suppressing the proliferation of CD4+ T cells in a dose-dependent manner when co-cultured for 72 hours. Meanwhile, the apoptosis of CD4+ T cells was promoted by CD5+ B cells as the percentage of apoptotic CD4+ T cells was increased when co-cultured with CD5+ B cells at 1:1 B/T ratio. In contrast, CD5" B cells did not suppress the proliferation of CD4+ T cells. No effect of CD5- B cells on the apoptosis of CD4+ T cells was observed. In ITP patients, CD5+ B cells showed compromised regulatory functions. The proliferation of CD4+ T cells was not altered by CD5+ B cells until the B/T ratio increased to 3:1. The ability of CD5+ B cells to promote the apoptosis of CD4+ T cells was also impaired in ITP patients. After HD-DXM therapy, the ability of CD5+B cells of repressing T cell proliferation and promoting the apoptosis of CD4+T cells was restored in ITP patients.In healthy subjects, the IFN-y secretion by CD4+ T cells was decreased by CD5+ B cells at a minimal B/T ratio of 1:5. The secretion of IL-4, a representative cytokine of Th2 subset, was not influenced by CD5+ B cells. CD5+ B cells from ITP patients showed no capacity for suppressing the IFN-γ secretion until the ratio of B/T increased as high as to 1:1 and, even under the condition of 5:1 B/T ratio, CD4+ T cells from ITP patients retained about 30% of the ability of IFN-γ secretion. After HD-DXM therapy, the ability of CD5+B cells of suppressing IFN-γ secretion was partially restored.(3) There were 20 moleculars in TLR signaling pathways were differentially expressed between ITP group and healthy controls. Twelve were over-expressed in ITP patients:CLEC4E, FOS, IL1B, IL6, IL8, IRAKI, JUN, PTGS2, SIGIRR, TICAM2, TLR2 and TNFo Eight were low-expressed:CSF2, CSF3, IFNB1, IRF3, TLR10, TLR3, TLR7 and TLR9.[Conclusions] Increased number of CD5+ B cells in which IL-10 is accumulated with decreased IL-10 concentration in supernatants suggests that the ability of CD5+ B cells to secret IL-10 is impaired in ITP patients, which might lead to the diminished regulatory functions including repressing proliferation of T cells and Thl differentiation. Both the aberrant number and impaired ability of immune regulation of CD5+ B cells could be corrected, fully or partially, by successful HD-DXM treatment. The underlying mechanisms of the abnormal CD5+ B cells in ITP might involve several moleculars in TLR signaling pathways.