Fig 2: Characterization of the B cell response against SARS-CoV-2 in acute (M0) and convalescent (M6) S-CoV patients(A) UMAP and clustering of 41,083 B cells analyzed by scRNA-seq from four S-CoV patients at M0 (left panel) and M6 (right panel) (see Table S2). Upper square in both panels shows the results of increased clustering resolution for the “Activated” cell cluster.(B) Relative cluster distribution at M0 and M6 for all sorted cells (top panel) and cells falling in the “Activated” cluster at initial clustering resolution. Bar indicates mean with SEM.(C) Feature plots showing scaled normalized counts for CD27 and CD38 barcoded antibodies as well as S and G2/M signature scores in all cells.(D) Feature plots showing scaled normalized counts for CD21, CD71, CD38, CD11c, CD95, CD27, and CD307e (FcRL5) barcoded antibodies in cells from the “Activated” cluster.(E) UMAP of all cells at M0 or M6, with cells belonging to one of the top 10% antibody-secreting cell (ASC) clones highlighted (light blue). Cells stained by the barcoded-anti-His/His-tagged SARS-CoV-2 S protein combination (barcoded-SARS-CoV-2 S stained cells) are also highlighted (red when members of one of the top 10% ASC clones at M0 [“shared”], dark blue otherwise).(F) Circus plots showing clonal relationships between cells from different UMAP clusters and time points. Blue lines indicate clonal relationships with one of the top 10% ASC clones at M0 and gray line all other clonal relationships.(G) Histograms showing the number of mutations in VH genes for cells belonging to one of the top 10% ASC clones at M0 according to their barcoded-SARS-CoV-2 S staining (top = unstained; bottom = stained).(H) Graph showing the frequencies of cells at the M0 time point harboring 0–1 (white), 2–9 (gray), or =10 (black) mutations in their VH genes for all sorted cells according to their cluster of origin (left) as well as for cells stained by the barcoded-SARS-CoV-2 S protein and/or members of the top 10% ASC clones at M0, as defined in (E).Repeated measures (RM) two-way ANOVA and Sidak’s multiple comparison tests (B). ****p < 0.0001, ***p < 0.001, *p < 0.05. See also Figure S2 and Table S2.

Fig 3: Phenotypic evolution of the SARS-CoV-2 S-specific B cell response up to 6 months post-infection in M- and S-CoV patients(A) UMAP projections of concatenated multi-parametric FACS analysis of CD19+IgD− cells from all S-CoV (n = 15) and M-CoV (n = 16) patients analyzed over time in our cohort (Table S1). His-tagged labeled SARS-CoV-2 S-specific cells are overlaid as red dots.(B) Unsupervised clustering (FlowSOM) was performed on the concatenated FACS dataset based on IgD, CD71, CD27, CD38, CD11c, CD19, and CD21 fluorescence intensity. 95% density contours for each identified cluster are overlaid in the UMAP projection.(C) Cluster distribution for all analyzed SARS-CoV-2 S-specific cells in identified clusters at indicated time point. Bars indicate mean with SEM.(D) Proportion of SARS-CoV-2 S-specific CD19+IgD−CD27+CD38− MBCs at M0, M3, and M6 in S-CoV and M-CoV patients, as compared to six pre-pandemic healthy donor (HD) controls. Each dot represents one patient, and bars indicate mean with SEM. Dashed line indicates the mean + 2 SD of the detected frequency of SARS-CoV-2 S-specific MBCs in HD.(E) Proportion of S-specific MBCs over time post symptom onset in S-CoV (black dots, dark blue line) and M-CoV (white dots, light blue line) patients. Lines indicates linear regression (R2 = 0.051, p value = 0.02 for S-CoV, and R2 = 0.018, ns, for M-CoV, Pearson correlation). Colored area between dashed lines indicates error bands. Red dots and lines represent data obtained for the four S-CoV patients analyzed as part of our scRNA-seq dataset.(F) Representative dot plot showing CD71 and CD19 expression in CD19+IgD−CD38− B cell at indicated time points from two representative S-CoV and M-CoV patients. SARS-CoV-2 S-specific cells are overlaid as red dots. Gating strategy for ABCs and classical MBCs according to CD71 expression is further displayed.(G) Proportion of SARS-CoV-2 S-specific cells displaying an ABC (CD19+CD27+IgD−CD71+) (left) or MBC (CD19+CD27+IgD−CD71low/int) (right) phenotype at indicated time points. Bars indicate mean with SEM.(H) Proportion of activated MBCs over time post symptom onset in S-CoV (black dots, dark blue line) and M-CoV (white dots, light blue line) patients. Lines represents the linear regression. Colored area between dashed lines indicates error bands. R2 and p value with Pearson correlation.(I) Correlation between the frequency of SARS-CoV-2 S-specific MBCs at M6 (expressed as % of CD27+IgD− B cells) and of ABCs at M0 in all M-CoV and S-CoV patients.RM two-way ANOVA and Sidak’s multiple comparison tests (C). ANOVA and two-tailed Mann-Whitney tests (D and G). Linear regression with Pearson correlation analysis (I). ∗∗∗∗p < 0.0001, ∗∗∗p < 0.01, ∗∗p < 0.01, ∗p < 0.05. See also Figure S3 and Table S3.

Fig 4: Phenotypic characterization of total and SARS-CoV-2 S-specific B cell populations in convalescent COVID-19 patients, Related to Figure 3(A) Cluster distribution of all analyzed CD19+IgD- B cell across FlowSOM-identified clusters for S-CoV (n = 15) and M-CoV (n = 16) donors (Table S1) at all three time points (M0, M3 and M6).(B) UMAP of concatenated down-sampled cells from all 83 samples analyzed colored according to the scaled fluorescence intensity for IgD, CD71, CD27, CD38, CD11c, CD19, CD21 expression.(C) Heatmap representing the mean fluorescent intensity for IgD, CD71, CD27, CD38, CD11c, CD19, CD21 in each identified FlowSOM cluster.(D–F) Absolute number of antibody secreting cells (PC+PB) (D), CD19+CD27+IgD- MBCs (E), and ABCs (F) at indicated time points in S-CoV and M-CoV patients. Bars indicate the mean with SEM. Dashed line indicates the threshold based on healthy donors (mean + 2 SD)(G and H) Percentage (G) and absolute numbers (H) of SARS-CoV-2 spike-specific CD19+CD27+IgD- MBCs at indicated time points in S-CoV and M-CoV patients.(I and J) Percentage (I) and absolute number (J) of SARS-CoV-2 spike-specific cells among CD27-IgD- DN cells. (G and I) Each dot represents a patient and lines connect the different time points for each patient. Dashed line indicates the threshold based on healthy donors (mean + 2 SD). (H and J) Bars indicate the mean with SEM.(K) Representative dot plot for T-bet staining in CD19+CD38- B cells from COVID-19 patients.(L) Percentage of intra-cellular T-Bet staining in SARS-CoV-2 spike-specific CD19+IgD-CD27+CD38- cells from S-CoV (n = 2) and M-CoV (n = 3) patients at indicated time points. Bar indicate the mean with SEM.(M–O) Correlation between the number of SARS-CoV-2 spike-specific CD19+CD27+IgD- MBCs at M6 and age at diagnosis (M), maximum oxygen flow during hospitalization (liters/minutes, only in S-CoV) (N) and maximum CRP during hospitalization (mg/L, available only in S-CoV) (O) using linear regression.(P) Number of spike-specific CD19+CD27+IgD- MBCs at M6 according to the initial disease severity (S-CoV and M-CoV) and the sex of the patient (Women, W, blue and Men, M, green). Bars indicate the mean with SEM.(Q) Plot showing the number of spike-specific CD19+CD27+IgD- cells in the 21 S-CoV patients analyzed at M6 colored according to the treatment received during initial hospitalization. Standard of care included oxygen, low dose anticoagulant, antibiotic if needed and symptomatic treatments.RM two-way ANOVA and Sidak’s multiple comparison tests (A). ANOVA and two-tailed Mann-Whitney tests (D–F, H, J, L). Linear regression with Pearson correlation analysis (M-0). ∗∗∗∗p < 0.0001, ∗∗p < 0.01, ∗p < 0.05.