Expression and Significance of Regulatory B Cells in Patients with Immune Thrombocytopenia

Objective: To detect the expression and significance of regulatory B cells in patients with immune thrombocytopenia. Methods: 73 ITP patients were divided into glucocorticoids treatment group (n = 42) and recombinant human thrombopoietin (rhTPO) treatment group (n = 31). According to the thera-peutic effect, it was divided into effective group and ineffective group. The expression of CD19+ CD24hiCD38hi Breg in peripheral blood was detected by flow cytometry before and after treatment. The expression levels of transforming growth factor (TGF)-β1, interleukin (IL-10) and interferon (IFN)-γ were detected by ELISA before and after treatment. 30 volunteers were se-lected as the control group. Results: The expression of CD19+ CD24hiCD38hi Breg and cytokines IL-10 and TGF-β1 in 73 ITP patients before treatment was lower than that in the control group, while the expression of IFN-γ was higher than that in the control group (p < 0. 05). The expression levels of CD19+ CD24hiCD38hi Breg, IL-10 and TGF-β1 in the effective group were significantly higher than before treatment, while the expression of IFN-γ was significantly lower than before treatment (p < 0. 05). The expression of CD19+ CD24hiCD38hi Breg, IFN-γ, IL-10 and TGF-β1 in the invalid group had no significant change compared with before treatment. Conclusion: Abnormal expression of CD19+ CD24hiCD38hi Breg and related cytokines is involved in the pathogenesis of ITP.


production. Previous studies have shown that the main pathogenesis of ITP is
PLT destruction mediated by PLT-specific autoantibodies, and the common types are anti-platelet membrane glycoprotein (GP) IIb/IIIa and anti-GPIB antibodies [1] [2]. Regulatory B cells (Bregs) identified in mouse and in human have been shown to downregulate inflammation associated with numerous pathological processes, including autoimmune diseases, transplant rejection, anti-tumor responses, and infections [3] [4] [5]. Studies on regulatory B cells' involvement in the pathogenesis of ITP are rare. In this study, flow cytometry was used to detect the expression levels of regulatory B cells and related immune cytokines in peripheral blood of ITP before and after treatment, so as to explore their possible role in the pathogenesis of ITP.

Patients
A total of 73 ITP patients and 30 age-and gender-matched healthy donors were enrolled in this study. The diagnosis of ITP was based on the recently reported criteria [6]. There were 27 males and 46 females, aged from 16 to 82 years, with a median age of 47 years, 25 patients over 40 years old, and 41 patients with PLT count ≤ 10 × 10 9 /L, all of which met the ITP diagnostic criteria. The clinical data of the patients are shown in Table 1. In the control group, there were 30 patients, including 12 males and 18 females, aged from 22 to 68 years, with a median age of 38 years. Our research was approved by the hospital based ethics committee, and written informed consent was obtained from all participants.

Evaluation Criteria of Curative Effect
Refer to relevant diagnostic criteria [6]. 1) Completely response: After treatment, PLT ≥ 100 × 10 9 /L and no bleeding symptoms. 2) Effective: PLT > 30 × 10 9 /L after treatment and increased by at least 2 times compared with basic PLT count and no bleeding symptoms. 3) Ineffective: PLT ≤ 30 × 10 9 /L after treatment or PLT count increased less than 2 times of the base value or bleeding symptoms.

Treatment
The GCs group patients: Dexamethasone 40 mg/d was given intravenously for 4 days. The rhTPO group patients: rhTPO 15,000 units were injected subcutaneously once a day for 14 days.

FAS
To detect the percentage of Breg cells, heparinized PB (100 ul) was incubated with antibodies FITC-CD38, PE-CD24, PECy7-CD19 and the appropriate isotype controls (Biolegend, San Diego, CA, USA) for 20 min at room temperature.
Then, 2 ml of FACS lysing solution (BD Biosciences, San Jose, CA, USA) was added to the samples and they were incubated for 10 min at room temperature, washed twice in phosphate-buffered saline (PBS). Analysis of all samples was conducted using a Beckman Navios.

Enzyme-Linked Immunosorbent Assay (ELISA) for Cytokines
The concentrations of c-interferon (IFN-γ), transforming growth factor-b (TGF-β1) and IL-10 in the cell culture supernatants were measured using an ELISA kit (ABclonal Technology, Wuhan, P. R. China) according to the manufacturer's instructions.

Statistical Analysis
Statistical analysis was performed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA).
Unless indicated, data were expressed as the mean standard error of the mean (SEM). The percentage of Breg cells in PB among ITP patients, ITP patients and controls were analysed by one-way analysis of variance. p < 005 was considered significant.

Decreased Percentage of CD19+ CD24hiCD38hi Breg in Patients with ITP
To detect the abnormal frequency of Breg cells in ITP, we analysed the percen-

Expression of IFN-γ, TGF-β1 and IL10 in Patients with ITP
The expression of cytokines IL-10 and TGF-β1 was lower than that of the control group, and the expression of IFN-γ was higher than that of the normal control group, with statistically significant differences between the two groups. Furthermore, the expression of IL-10, TGF-β1 in patients who are responses to treatment were increased compared with before treatment. These differences between before and after treatment were uniform statistical significance. While, the expression of INF-γ of response to treatment was lower than that of no response ITP, these differences were not statistical significance (Figure 2). The expression of cytokines IL-10 was lower than that of the control group. The expression of IL-10 in patients who are responses to treatment were increased compared with before treatment. (C) The expression of IFN-γ of patients was higher than that of the normal control group, the expression of INF-γ of response to treatment was lower than that of no response ITP, these differences were not statistical significance.

Discussion
The pathogenesis of ITP has not been fully clarified, and existing studies have shown that platelet autoantibodies secreted by B lymphocytes are the main cause of ITP. Platelets bound to autoantibodies are eventually cleared by the body's mononuclear phagocyte system, resulting in peripheral thrombocytopenia7. To elucidate the regulation mechanism of platelet autoantibody production is of great significance to clarify the pathogenesis and treatment of ITP. In this paper, the expression of Breg cells in ITP patients was detected by FCM to explore its possible mechanism in ITP. TGF-β is a cytokine in the body that regulates the body's inflammatory response.
It regulates the proliferation, differentiation and activation of lymphocytes, macrophages and other immune-related cells through autocrine and paracrine forms [11]. TGF-β can induce secretion of Treg cells, and Treg cells play an immunosuppressive role by secreting TGF-β and IL-10. The results of this study showed that the levels of TGF-β in peripheral blood of ITP patients decreased compared with normal controls.

Conclusion
In summary, Breg can inhibit the activity of reactive T cells and inhibit the pro-