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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. 7 — (a) IGH-V-D-J gene usage from CD38^int, CD38⁻ and CD27⁺ B cells. Heat maps represent the frequency of each V_H, -D_H and -J_H gene by subjects, HC-N (n = 1), HC-Ag (n = 5), pRD-N (n = 1), pRD-Ag (n = 5-6) shown as mean±s.e.m. with individual values. Subject IDs are depicted on the left side of each heat map; and compartments as CD38^int, CD38⁻ and CD27⁺ B cells depicted with blue, red, and green on the left. Ig V_H, -D_H and -J_H genes are listed on the bottom by their chromosomal order. Color scales represent gene frequencies for each family separately. d.: distal genes, p.: proximal genes. Statistical analysis was performed using two-sided unpaired t-test with multiple comparison (Holm-Šídák method) to compare V_H, -D_H and -J_H gene usage between HC-Ag and pRD-Ag donors and P values are indicated when reached significance. Plot inserts shows changes in gene usages in CD38⁻ and CD27⁺ cell compartments compared to the values of HC-Ags in CD38^int compartment (horizontal black line) for HCs (gray) and pRD patients (red), infant donors (triangles), and antigen-experienced donors (circles). (b) “Hedgehog” plots – targeted hot- and coldspot sequence motifs. Frequencies of WRCY/RGYW (red), SYC/GRS (blue), WA/TW (green) and neutral (gray) mutations are visualized. (c) Enumeration of different SHM hot- and coldspots in CD38^int, CD38⁻ and CD27⁺ B cells. Graph shows mutation frequency in different SHM hot- and coldspots from HC-N (n = 1), HC-Ag (n = 5), pRD-N (n = 1), pRD-Ag (n = 5) shown as mean±s.e.m. with individual values. (d) IgG surface expression. Contour plots show the surface expression of IgG and IgM of a representative HC-Ag (dark gray) and a pRD-Ag patient (red) overlayed from their CD38^int, CD38⁻ and CD27⁺ B cells separately. (e) Frequency of B cells based on IgM and IgG surface marker expression. Graphs show frequency of IgM⁺ IgG⁻, IgM⁺ IgG^dim, IgM⁻ IgG⁺, IgM⁻ IgG⁻ from CD38^int, CD38⁻ and CD27⁺ B cells and shown as mean±s.e.m. with individual values. Statistical analysis was performed on Ag-experienced subjects using Mann–Whitney U test. Data for d-e was obtained from HC-N (n = 3), HC-Ag (n = 15), pRD-N (n = 1), pRD-Ag (n = 11).

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