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. 2020 Dec 23;11:591269. doi: 10.3389/fimmu.2020.591269

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Copyright © 2020 Fu, Wang, Shen, Guo, Liu, Ye, Sun, Li, Tian and Wei

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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CD24⁺CD38^hi B cells are induced under Peg-IFNα-2b and produce plenty of IL-10. (A, B) PBMCs from HBV patients and healthy controls were cultured in complete RPMI medium 1640 with or without human Peg-IFNα-2b (100 ng/ml) for 36h. Representative density plots (A) and percentage analysis (B) showing CD24 and CD38 expression on gated B cells from HBV patients and healthy controls. n = 15 (healthy controls) and 9 (HBV patients). Paired t test. (C) ELISA analysis of IL-10 secretion in cell-free supernatants of cultured PBMCs from HBV patients before or during Peg-IFNα-2b therapy and healthy controls with or without re-stimulation by Peg-IFNα-2b (25 ng/ml) during a 32-h culture. PBMC from HBV patients were collected before PEG-IFNα-2b therapy or at 12-36 weeks after the start of Peg-IFNα-2b therapy. (D) Representative density plots and (E) percentage analysis of IL-10 expression on gated CD24⁺CD38^hi B cells and CD24^-CD38^low mature B cells from HBV patients and healthy controls. These B cells were from freshly isolated PBMC in HBV patients and healthy controls. After 4 h of stimulation with PMA and ionomycin in the presence of monensin, intracellular flow cytometry staining was done and gated as CD19⁺CD3^- cells. n = 15 (healthy controls) and 9 (HBV patients). Paired t-tests were used for the analysis in subsets from the same patient. Data are presented as Mean ± SEM, **P < 0·01, ***P < 0·005, ****P < 0·0001. (F) Flow cytometry analysis of Ki67 expression on the CD24⁺CD38^high B cells from PBMC of HBV patients without (blue) or with (red) PEG-IFNα-2b therapy for 12-24 weeks. (G) Statistics calculated by the MFI of Ki67 from PBMC of HBV patients without (blue, n = 12) or with (red, n = 10) PEG-IFNα-2b therapy for 12-24 weeks. Unpaired t test. **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are presented as mean ± SD. (H) Flow cytometry analysis of Annexin V expression on the CD24⁺CD38^high B cells from PBMC of HBV patients without (blue) or with (red) PEG-IFNα-2b therapy for 12–24 weeks. (I) Statistics calculated by the percentage of Annexin V⁺ Breg cells from PBMC of HBV patients without (blue, n = 12) or with (red, n = 10) PEG-IFNα-2b therapy for 12–24 weeks. Unpaired t test. **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are presented as mean ± SD.