Discussion Of Regulatory B Cells And Primary Sjogren's Syndrome Pathogenesis

Background. Primary Sjogren’s syndrome (pSS) is a chronic, multisystem-involved autoimmune disease in which abnormally activated and differentiated B cells act as one of the most important characteristics. Regulatory B cells (Breg) are newly defined B cell subgroups with immunosuppressive functions. To date, key roles for Breg cells have been identified in the regulation of several immune-mediated processes, including autoimmunity and responses to infectious disease and cancer. It has been demonstrated that human peripheral CD19+CD24hiCD38hi B cells possessed regulatory function and that CD19+CD5+Foxp3+B cells may be potential Breg. However, there are still few researches on how Breg functions in pSS and its relevance with disease activities and clinical features. In this research, we tried to investigate the differences among Breg in lymphocytes and CD19+B cells, the expression of CD69, CD80and BAFF receptor (BAFF-R) on B cells and Breg in the peripheral blood of patients with primary Sjogren’s syndrome (pSS), systemic lupus erythematosus (SLE) and healthy people. Then we analyzed the correlations of these items with clinical manifestations. We also attempt to identify the function of pSS peripheral Breg.Methods.34newly diagnosed pSS patients from SICCA and PUMCH were recruited. Ten newly diagnosed SLE patient and19healthy female were accepted as controls. Anti-coagulated peripheral blood was collected and mononuclear cells were isolated.PBMC were surface stained with CD19, CD24and CD38(to identify Breg), CD69, CD80and BAFF-R mAbs and then detected by flow cytometry.Clinical data was collected and disease activity was evaluated according to ESSDAI. Patients were classified into subgroups according to status of medical treatment which concluded untreated, immunosuppressive treated and other therapies treated pSS, ESSDAI scores and serum IgG titers.PBMC isolated from pSS patients with Breg depleted or nondepleted by flow cytometry sorting were then respectively cultured for72hrs with1mg/ml plate-bound CD3mAbs. Cells were then surface stained with CD4and CD19mAbs, fixated/permeabilized, and stained with IFN-y and IL-10mAbs. Alternatively, IL-10and IFN-γ were measured in the culture supernatants by ELISA. Resμlts.1. pSS patients had higher Breg percentage in CD19+B cell (2.63±2.65%) than SLE patients (0.93±1.18%, p=0.024) and healthy controls (1.32±1.32%, p=0.025). There were no significant differences among different treatment groups. The percentage of Breg in CD19+B cell of untreated pSS group (3.54±3.01%) was higher than that of SLE patients(p=0.005) and HC (p=0.001). The Breg percentage in lymphocytes of untreated pSS group (0.17±0.21%) was higher than immunosuppressive treated group (0.10±0.15%, p=0.026) and other therapies received group (0.05±0.04%, p=0.032、 SLE patients (p=0.000) and HC (p=0.004)。2. pSS patients with mouth dryness had higher Breg rate in lymphocytes (0.08±0.06%) than those without mouth dryness (0.25±0.28%, p=0.029); patients with eye dryness also had higher Breg rate in lymphocytes (0.09±0.11%) than those without eye dryness (0.19±0.25%, p=0.014). No significant difference between Breg percentages of pSS patients with ESSDAI≤6and of group with ESSDAI>6. Breg percentage in CD19+B cells of pSS patients (1.63±1.31%) with IgG>20g/L (3.82±3.34%) was lower than IgG≤20g/L group (p=0.045).3. pSS patients had higher Breg BAFF-R expression rate (87.42±28.84%) and mean fluorescence intensity (MFI,10065.20±6526.50) than those of SLE patients (43.73±48.19%, p=0.009,2295.35±2580.39, p=0.000), but lower than HC group(99.38±1.52%, p=0.001,34017.21±56136.49, p=0.010).4. There were significant differences of MFI of CD69(p=0.023), CD80(p=0.025) and BAFF-R (p=0.003) on Breg among pSS medical treatment group. The expression rate of CD69(1.75±3.06%, p=0.021), MFI of CD80(672.05±1275.36, p=0.020)on and MFI of BAFF-R (8308.75±4551.27, p=0.038) in immunosuppressive treated group were significantly lower than those of untreated patients.5. Breg ratio in lymphocytes of untreated pSS patients was negative correlated with C4(p=0.044, R=-0.589), and it was positively correlated with IgA(p=0.044, R=0.546) and IgG (p=0.029, R=0.545). Breg percentage in CD19+B lymphocytes of untreated pSS patients was negative correlated with C4(p=0.030, R=-0.624), and it was positively correlated with IgG (p=0.004, R=0.680).6. CD19+B lymphocytes in pSS patients expresse lower CD80rate (9.53±6.61%) but higher MFI (2357.23±320.30) than those in SLE patients (17.6±11.41%, p=0.033, 2071.38±218.39, p=0.006), while higher BAFF-R MFI (19567.03±7841.24) than HC group(72025.89±19532, p=0.010). No significant difference of expression rate of CD69, CD80and BAFF-R on CD19+B cells was found among treatment pSS groups. CD69expression rate on CD19+B lymphocytes in pSS patients was positively correlated with IgG (p=0.013, R=0.440), and those of BAFF-R was positively correlated with C3(p=0.030, R=0.462).7. Breg-depleted-PBMC expressed lower percentage of CD19+IL-10+cells, but higher CD4+IFN-y+Thl percentage than those of nondepleted. IL-10and IFN-γ in cultural supernatants were both lower in Breg-depleted PBMC.Conclusion. Breg percentage in peripheral CD19+B cells in pSS patients was significantly higher than HC, and it was positively correlated with serum IgG. The untreated pSS patients had higher Breg percentage in lymphocytes than immuno-suppressive treated group. Breg of pSS patients expressed higher rate but lower MFI of BAFF-R than HC. The function of Breg in pSS peripheral blood was detected and basically normal. It was suggested that Breg participated in the pathogenesis of pSS, and it might suppress the overactivated autoimmunity.