Fig 2: TNF signaling promotes Tfh-B cell interactions(A and B) PBMC from young adults were freshly isolated and sorted for coculture of T cell subsets (naive, gated as CD4+CD45RA+CD27+; CXCR5− memory, non-naive CD4+CXCR5−; cTfh, non-naive CD4+CXCR5+PD-1+) with autologous naive B cells (CD3−CD19+CD27loIgD+). Supernatant IgM (A) and IgG1 (B) were measured after 7 days, as shown for the following conditions: unstimulated (gray), SEB alone (0.5 μg/mL, green), SEB with recombinant human TNF (125 ng/mL, tan), or SEB with α-TNF antibodies (2 μg/mL, sienna) (1-way repeated-measures ANOVA with Holm-Sidak’s test; n = 12 per group).(C and D) Correlation shown for the TNFRSF1A in ICOS+CD38+ cTfh at day 7 compared with the H1N1-specific (C) hemagglutinin inhibitory (HAI) titer (n = 14) or H3N2-specific (D) HAI titer (n = 14) in young (orange) and elderly (purple) adults.(E) Correlation of apoptosis and NF-κB gene set GSVA scores for ICOS+CD38+ cTfh at day 7 for young (orange, n = 6) and elderly (purple, n = 8) adults.(F) Pearson coefficients for BCL family member genes compared to GSVA scores for TNF-NF-κB signaling in ICOS+CD38+ cTfh at day 7 for young (x axis) and elderly (y axis) adults. Genes classified as either pro-survival (dark blue) or apoptotic (sea green).

Fig 3: Infection prior to vaccination corresponds with sustained CD21−CD11c+ MBCs and superior SARS-CoV-2 variant-neutralizing antibody(A) Representative gating on RBD-specific antigen-experienced (ag-exp.) B cells for activated MBCs (CD21−CD11c+) from N and PI PBMCs at the indicated time points.(B and C) (B) Percent and number of activated MBCs and (C) percent and number of IgG+ classical MBCs (CD21+CD27+) of RBD-specific ag-exp. B cells in N and PI PBMCs at the indicated time points.(D) Percent neutralization AUC of SARS-CoV-2 (WA-1) by plasma from N and PI individuals at the indicated time points by PRNT.(E) Percent neutralization of SARS-CoV-2(Delta, Δ) and SARS-CoV-2(Omicron, o) spike-pseudotyped virus by plasma from N and PI individuals at the indicated time points (left). Percent neutralization area under the curve (AUC) (right). Dashed line indicates the average percent neutralization of plasma from N individuals pre-vaccination. Data in (B) and (C) are represented both longitudinally (left) and by cross-group comparisons (right). Statistics determined by Wilcoxon matched-paired signed rank test for longitudinal analyses and multiple unpaired Mann-Whitney test for group analyses: not significant (ns), ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, and ∗∗∗∗p < 0.0001. Error bars represent mean and SD. Pre-vaccination (Pre), 1 week post two-dose COVID-19 mRNA vaccination (1w PV2), 3 months post two-dose vaccination (3m PV2), 2 weeks post third vaccination dose (2w PV3). See also Figure S3.

Fig 4: Mild COVID-19 Induces a Sustained Enrichment of RBD-Specific IgG+ Memory B Cells(A) Representative gating of live CD3–CD14–CD16– cells for SARS-CoV-2 RBD-specific cells (RBD tetramer+decoy tetramer–) from CoV2+ and HC PBMCs at V1 and V2.(B) Number of RBD-specific B cells (RBD tetramer+decoy tetramer–CD20+) per 1 × 106 PBMCs.(C) Representative flow cytometry plots and number of RBD-specific PBs (RBD tetramer+decoy tetramer-CD20-CD138hi) (na = could not be calculated because all values 0).(D) Representative gating of RBD-specific B cells for naive B cells (CD21+CD27-) and MBCs (CD21+CD27+/CD21–CD27+/CD21–CD27– populations outlined in green).(E) Proportion of RBD-specific B cells that are naive (CD21+CD27-), classical MBCs (CD21+CD27+), or activated MBCs (CD21-CD27+/-), statistics for the proportion that are MBCs.(F) Number of RBD-specific MBCs (classical and activated).(G) Representative gating of RBD-specific MBCs for BCR isotype (IgD, IgM, IgA, and IgG) expression.(H) Proportion of RBD-specific MBCs expressing the BCR isotypes IgD, IgM, IgA, and IgG. Statistics are for the proportion that are IgG+.(I) Number of RBD-specific IgG+ MBCs.(J) Representative gating of RBD-specific MBCs for T-bet expression and number of RBD-specific T-bet+ MBCs.Statistics for unpaired data determined by 2-tailed Mann-Whitney tests and, for paired data, by 2-tailed Wilcoxon signed-rank tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value < 0.04. Error bars represent mean and SD. Data from 2 experiments per visit. See also Figure S5.

Fig 5: Mild COVID-19 Induces a Sustained Enrichment of RBD-Specific IgG+ Memory B Cells, Related to Figure 2(A) Representative flow cytometry gates for phenotyping RBD-specific B cells from PBMCs in Figure 2 set on total B cells from a healthy control (HC) (surface stain, top; intracellular stain, bottom).(B) Number of RBD-specific IgD+, IgM+ and IgA+ MBCs (CD20+RBD tetramer+decoy tetramer- CD27+CD21+/CD27+CD21-/CD27-CD21-) from healthy control (HC) and SARS-CoV-2-recovered (CoV2+) PBMCs at Visit 1 (V1) and Visit 2 (V2).Statistics for unpaired data determined by two-tailed Mann-Whitney tests and, for paired data, by two-tailed Wilcoxon signed-rank tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value < 0.02. Error bars represent mean and SD. Data from two experiments per visit.