Fig 2: Single-cell analysis of B lymphocytes and BCR repertoire reveal plasmablast expansion and clonality differences between genders.a, UMAP visualization of 74,019 cells in the B cell lineage identified from gene expression data. b, Dot plots of gene (top) and surface protein (bottom) expression for populations shown in a. c, Bar plot of the mean proportion of cell types shown in a. d, Proportion of total IgA and IgA2 in plasmablast and plasma cells based on BCR data. Kruskal–Wallis test with Benjamini–Hochberg correction. e, Coordinated changes between TFH and B cells assessed by differential correlation analysis (empirical P = 0.1). Shown is the Pearson correlation (± bootstrap s.e.m.) between TFH proportions and plasmablast or plasma cell (combined); only significant trends are shown. f, GSEA of MSigDB hallmark signatures in naive B cells, switched memory B cells and plasmablasts for asymptomatic/symptomatic COVID-19 versus healthy groups. Size of circles indicate (absolute) normalized enrichment score (NES). GSEA (permutation) nominal P < 0.05 and FDR < 0.25 denoted by non-gray colored dots. EMT, epithelial–mesenchymal transition; UV, ultraviolet. g, Dot plots of genes related to TNF signaling and BCR signaling in naive B cells, switched memory B cells and plasmablasts. Size of circles indicates the percentage of cells expressing the gene, and color gradient corresponds to increasing mean expression value. h, Scatterplot of clonotype size by node closeness centrality gini indices with marginal histograms indicating the distribution. Each dot represents an individual. i, BCR overlap incidence plot. Nodes correspond to individual donors colored by (inner ring) severity and (outer ring) site from which samples were collected. Edges indicate if at least one cell from each individual displayed an identical combination of heavy and light-chain V and J gene usage with CDR3 similarity allowance (=85%). j, Clonotype size (left) and node closeness centrality gini indices (right) separated by gender. Mann–Whitney U test with Benjamini–Hochberg correction between the gender groups within each severity status. Color of adjusted P values indicates the gender group with the higher mean value. The box portion of the box plots extends from the 25th to 75th percentiles, whiskers extend from the smallest to largest values, and the middle line corresponds to the median. NS, not significant.

Fig 3: Single-cell analysis of B lymphocytes proportion and gene set differences across COVID-19 severity.a, Heatmap of mean gene set enrichment scores of (top) adult peripheral blood B cell signatures32 and (bottom) bone marrow B cell signatures68. Row enrichment value is scaled from 0-1 and colour gradient corresponds to increasing mean enrichment score. b, (Top) Kruskal-Wallis test results with Benjamini-Hochberg false discovery correction for cell type proportion differences in plasmablast/plasma cells between severities. Significance is denoted by Benjamini-Hochberg corrected P < 0.05 (red text). (Bottom) Cell type abundance counts were modelled as a function of severity. Hypothesis testing was performed using quasi-likelihood F-test comparing asymptomatic to symptomatic covid, for either a linear or quadratic trend across severities. Differentially abundant cell types were determined using a 10% false discovery rate (FDR). c, (Left) Bar plot showing the mean proportion of plasmablast/plasma cells expressing IgA, IgD, IgE, IgG or IgM, based on V(D)J information. (Right) Proportion of IgM, IgA1, total IgG, IgG1, and IgG2 subclass in plasmablast and plasma cells. Kruskal-Wallis test results with Benjamini-Hochberg false discovery correction for cell type proportion differences in plasmablast/plasma cells between severities showed no significant differences. n = 10, 6, 21, 24, 11, 11 (IgM), n = 18, 9, 21, 27, 12, 9 (IgA1), n = 16, 8, 23, 27, 13, 8 (IgG (all)), n = 11, 6, 22, 25, 11, 8 (IgG1) and n = 8, 4, 16, 23, 9, 7 (IgG2) biologically independent patients/samples for healthy, asymptomatic, mild, moderate, severe and critical respectively. d, GSEA of pathways in B cell subsets for asymptomatic/symptomatic COVID versus healthy. Size of circles indicate (absolute) normalised enrichment score (NES). Pathways were considered statistically significant if GSEA (permutation) nominal P < 0.05 and FDR < 0.25 (denoted by non-grey coloured dots; boundary lines in the middle marks FDR = 0.25). EMT, Epithelial-mesenchymal transition. e, Dot plots of TNF signalling molecules, activating and inhibitory BCR signaling molecules (5’ gene expression data) in immature B cells, non-switched memory B cells, ‘exhausted’ B cells and plasma cells. Size of circles indicate percent of cells expressing the gene and colour gradient corresponds to increasing mean expression value.