CD19+CD38hiCD24hi B cells were depleted, and the depleted B cells and undepleted B cells were both collected using flow cytometry sorting. children, 4 healthy adults, and 12 MG patients by flow cytometry. However, the percentage of CD19+ IL-10+ cells was highest in healthy children (~8%), followed by healthy adults (~3%), and was lowest in MG patients (~0.5%). CD19+CD24hiCD38hi B cells exerted immunosuppressive functions in healthy people but were refractory in MG patients. Moreover, p-STAT3 downstream of CD40 may be impaired in CD24hiCD38hi B cells from the peripheral blood of MG patients. primer sequences were 5-ATAACTGCACCCACTTCCCA-3 and 5-TCATTTCCGATAAGGCTTGG-3. The PCR conditions were as follows: 30 s at 95C, followed by 40 cycles for 5 s at 95C, 30 s at 60C, and 15 s at 95C. Cell Signaling Cells were then stimulated by incubation with 5 mg/ml purified stimulatory mouse -antihuman CD40 mAb (5 g/ml) (clone: 5C3, Biolegend, San Diego, USA) for 30 min on ice in the dark. Cells were washed, fixed, and permeabilized (Phosflow? Sigma-1 receptor antagonist 3 Fix buffer/Perm Buffer III, BD, USA) according to the manufacturer’s instructions. Cells were washed with ice-cold PBS, resuspended in warm PBS at a density of 0.5C1 106 cells/100 l, incubated with Alexa Fluor? 647 mouse anti-STAT3 antibody (20 l/test) (clone: pY705) (BD, USA) for up to 60 min at RT, washed and resuspended in Permwash buffer (BD, USA). Histology Sigma-1 receptor antagonist 3 Thymi of MG patients and controls were collected and fixed in 4% paraformaldehyde (pH 7.4) for 8 h. After dehydration in 30% sucrose, the thymi were embedded in Tissue OCT Medium (Sakura, Torrance, USA) and cut into 10-m sections with air drying. To evaluate routine histopathological findings, some sections were stained with hematoxylinCeosin and examined by light microscopy according to standard protocols. For immunofluorescence, other Sigma-1 receptor antagonist 3 sections were blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature and then incubated with mouse antihuman CD19 MAb (1:100, MAB1794, Millipore), rat antihuman IL-10 MAb (1:100, sc-53705, Sant Cruz) in 1% BSA at 4C overnight and then incubated with the corresponding fluorochrome-conjugated secondary antibodies, namely, CY3 goat antimouse IgG (1:200, Abcam) and FITC goat antirat IgG (1:200, Abcam), in the dark for 50 min at room temperature, followed by DAPI (Invitrogen) costaining. Immunofluorescence control was directly incubated with fluorochrome-conjugated secondary antibodies. After a final wash step and mounting with fluorescent antifade mounting medium (HelixGen), the slides were examined under an Olympus FV-1000 confocal microscope. Statistics All values are expressed as the mean SEM. Depending on the normal distribution of the data, we performed analysis by Student’s 0.05; Figure 1C). Expression of IL-10 mRNA in B cells of MG patients (= 8) was significantly lower than that in B cells of healthy controls (= 8; 0.001; Figure 1D). Besides, the proportion of CD19+IL-10+ B cells in EOMG was higher than that of LOMG ( 0.05; Figure 1F). However, there was no significant difference in CD19+IL-10+ B cells between different gender and different thymus status (Figures 1G, H). Open in a separate window Figure 1 The proportion of CD19+ IL-10+ cells in peripheral blood cells of MG patients and controls. (A) Lymphocytes were gated according to forward scatter and side scatter by FCM. (B) B cells were gated according to CD19+ in lymphocytes. (D) Scatter plots show the mean percentages of CD19+ IL-10+ cells in the peripheral blood of 23 OMG patients, Rtp3 18 GMG patients, and 30 healthy individuals. (E) Expression of IL-10 mRNA by real-time quantitative analysis in the peripheral blood of 8 MG patients and 8 healthy individuals. Six of 8 MG patients were ocular myasthenia gravis. (C) Representative flow cytometry plot of CD19+ IL-10+ cell gating for patients with OMG, GMG, and healthy controls. (F) The frequency of CD19+ IL-10+ cell in different MG types according to age at onset. (G) The frequency of CD19+ IL-10+ cells in different thymus status. (H) The frequency of CD19+ IL-10+ cells in different gender. (I) The titer of anti-AChR antibody in MG patients, including 21 healthy controls, 23 OMG patients, and 18 GMG patients. (J) The proportion of CD19+ IL-10+ cells had no correlation with the level of anti-AChR antibody, and the samples were from 41 patients with MG. 0.05, ** 0.01, *** 0.001, ns, not significant. We also detected the titers of anti-AChR antibody in sera of MG patients by ELISA. The level of anti-AChR antibodies in sera MG patients was significantly higher than that.