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. 2021 Nov 3;12:697725. doi: 10.3389/fimmu.2021.697725

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Copyright © 2021 Fu, Wang, Yu, Lin, Huang, Liu, Zhao, Lu, Xu, Liu, Liu, Wang and Chu

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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The expression levels of IgA and TGF-β/Smad pathway are upregulated in human CD11b⁺ B cells in vitro. (A) Representative staining of CD19 (green), CD11b (white), IgA (red), and 4′, 6-diamidino-2-phenylindole (DAPI) (blue) in the colon tissue of ulcerative colitis (UC) patients. The area density of each marker was calculated. Average data were collected from 20 random fields per patient, n = 6. (B) The relative area density of CD11b and IgA was identified. (C) Total PBMCs were purified and stimulated with CpG-ODN (2 μg/mL) and recombinant human IL-10 for 72 h in vitro. Cells were stained for CD11b detection and analyzed using flow cytometry. (D) The percentage of IgA⁺ B cells in CD11b⁺ and CD11b⁻ B cells was analyzed using flow cytometry. (E) The percentage of TGF-β⁺ B cells in CD11b⁺ and CD11b⁻ B cells was analyzed using flow cytometry. (F) The culture supernatant was harvested to determine the production of IgA (upper panel) via ELISA. The relative frequencies of CD19⁺CD11b⁺ cells and sIgA level were measured (lower panel). (G) The culture supernatant was harvested to determine the production of TGF-β (upper panel) via ELISA. The relative frequencies of CD19⁺CD11b⁺ cells and TGF-β level were measured (lower panel). The PBMCs from healthy donors were purified and subsequently stimulated by CpG and TGF-β for 0, 30, and 120 min. Expression of TGF-βRII (H) and the phosphorylation level of Smad2/3 (I) were analyzed using flow cytometry. **P < 0.01; ***P < 0.001. Data represent the mean of more than three independent experiments.