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. 2022 Jul 28;23(8):1256–1272. doi: 10.1038/s41590-022-01271-6

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 4 — (a) Fractioning B cells by conventional standard gating. Plots show a representative HC-Ag, HC-N, pRD-Ag and the pRD-N subjects. (b) Major B cell subset frequencies. Frequencies of naive (CD27⁻IgD⁺), memory (CD27⁺), DN (CD27⁻IgD⁻) and plasmablast/plasmacell (CD27^hiCD38^hi) B cell populations are shown. (c) Transitional, CD38^int and CD38⁻ subsets frequencies from CD27⁻IgD⁺ gate. (d) Memory B cell subsets frequencies from CD27⁺ gate. For h-j statistical analyses were performed on Ag subjects (HC-Ag vs pRD-Ag) using Mann–Whitney U test with multiple comparison (Holm-Šídák method). Bars represent mean±s.e.m. with individual values from HC-Ag (n = 13), pRD-Ag (n = 13), HC-N (n = 3) and pRD-N (n = 1). (e) Detection of T-bet⁺ B cells. Gates show the CD21^lowT-bet⁺ B cells in a representative from HC-Ag and a pRD-Ag patient. Frequencies are indicted. (f) Gating for CD38^int, CD38⁻, CD27⁺ and DN (CD27⁻IgD⁻) B cells. Plot show gatings from a representative HC-Ag and a pRD-Ag patient. (g) T-bet expression in CD38^int, CD38⁻, CD27⁺ and DN (CD27⁻IgD⁻) B cells. Overlaid histogram plots depict the T-bet expression by CD38^int, CD38⁻, CD27⁺ and DN (CD27⁻IgD⁻) B cells in a representative HC-Ag subject (black) and a pRD-Ag patient (red) in comparison to HC total B cell (filled gray). Expression levels are indicated as gMFI values. (h) T-bet expression in CD38^int, CD38⁻, CD27⁺ and CD27⁻IgD⁻ populations. Expression of FcRL4, FcRL5, CD85j and CXCR3 ABC-specific markers (i), CD95 (j) and BAFF-R (k) in CD38^int, CD38⁻, CD27⁺ and CD27⁻IgD⁻ B cells. Plots for e-g were obtained from a single experiment performed simultaneously on HC-Ags (n = 10) and pRD-Ag patients (n = 4). Data for h was obtained from a single experiment performed simultaneously on HC-Ags (n = 10) and pRD-Ag patients (n = 4). Data for i-k (except for CXCR3) was obtained from a single experiment performed simultaneously on HC-Ags (n = 13) and pRD-Ag patients (n = 5). Data for i CXCR3 panel was obtained from a single experiment performed simultaneously on HC-Ags (n = 6) and pRD-Ag patients (n = 4). Data for each graph are normalized and shown as in Fig. 1e and shown as mean±s.e.m. with individual values depicted. Statistical analyses were performed using two-sided unpaired Student’s t-test.

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