Fig 2: Late T cell responses are positively correlated with durability of antibody titers, while early T cell responses are not associated with the peak magnitude of antibody titers(A and C) In the heatmaps, Spearman’s correlations (Benjamini-Hochberg corrected) between indicated cTfh cell populations, AIM+ CD45RA- CD4+, and cytokine+ non-naïve CD4+ T cell data and antibody titers (S protein IgG binding [area under the curve (AUC) IgG] or neutralizing antibody (Neut) are shown. The measurements that are significantly associated are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). The scale bar indicates the Spearman's correlation rho (?).(A) Correlations are performed between indicated T cell data collected at day 5 post-enrollment and indicated antibody titers at day 28 post-enrollment.(B) Scatterplots comparing MN- (left/red) and S-protein-stimulated (right/blue) IFN?-IL-21-TNF-?+-producing non-naïve CD4+ T cells collected on day 5 (MN: n = 55; S: n = 51) post-enrollment with neutralizing antibody titers collected on day 28 post-enrollment are depicted.(C) Correlations are performed between indicated T cell data collected on day 28 post-enrollment and indicated antibody titers at day 28 or 210 post-enrollment.(D) Scatterplots comparing antigen-stimulated IFN?-IL-21-TNF-?+-producing non-naïve CD4+ T cells collected on day 28 (MN: n = 81; S: n = 78) post-enrollment with neutralizing antibody titers collected on day 28 post-enrollment are shown.(E) Scatterplots comparing antigen-stimulated IFN?-IL-21-TNF-?+-producing non-naïve CD4+ T cells (left, MN: n = 48, S: n = 47) or ICOS+ cTfh cells (right, MN: n = 52, S: n = 50) collected on day 28 with neutralizing antibody titers collected on day 210 post-enrollment are shown.(B, D, and E) The neutralizing antibody titers are presented in natural logarithm, and we added +1 to allow for inclusion of participants with no neutralizing activity.The rho (?) and p values were calculated using Spearman’s correlation (Benjamini-Hochberg corrected). The lines represent the fitted linear relationship between the indicated data.See also Figures S5 and S7.

Fig 3: Kinetics of four distinct cytokine-producing SARS-CoV-2-specific CD4+ T cell populations over time and the density of CCR7 receptor expression on these populations(A and B) PBMCs from 24 COVID-19 outpatients sampled at days 5, 14, and 28 and month 4 (day 120) (n = 10 also sampled on day of enrollment [day 0]) were stimulated with MN (left) or S (right) proteins in vitro and were stained and analyzed by flow cytometry.(A) The mean and SEM of the absolute percentage of background-subtracted IFN?-IL-21-TNF-?+- (blue), IFN?+IL-21-TNF-?-- (yellow), IFN?-IL-21+TNF-?-- (red), or IFN?+IL-21-TNF-?+-producing non-naïve CD4+ T cells (green) of sequential timepoints post-enrollment are shown. These four populations were the dominant populations identified in Figure 1.(B) The relative proportion of each individual combination of background-subtracted, TNF-?-, IFN-?, and IL-21-producing non-naïve CD4+ T cells stratified by sequential days are depicted in the pie charts (mean). The p values indicated on top of the pie charts are calculated using the partial permutation test, which tests the association between MN- and S-protein stimulation of each indicated day post-enrollment. The p values in the tables indicate the significance of the associations between the different days post-enrollment calculated using the partial permutation test (left: MN-protein-stimulated T cells; right: S-protein-stimulated T cells).(C) Representative flow cytometry plots of CCR7- and CD45RA-expressing CD4+ T cells (left) with an overlay of MN-protein-specific IFN?-IL-21-TNF-?+- (blue), IFN?+IL-21-TNF-?-- (yellow), IFN?-IL-21+TNF-?--(red), or IFN?+IL-21-TNF-?+-producing non-naïve CD4+ T cells (green, right).(D) Mean fluorescence intensity (MFI) of CCR7 on MN- (left, n = 72) or S-protein-specific (right, n = 74) single positive TNF-?- (blue), IFN?- (yellow), IL-21- (red), or TNF-?- and IFN?-producing non-naïve CD4+ T cells (green) from day 28 post-enrollment.The p values were calculated using the Friedman test with Dunn’s multiple comparisons test (black line, median).See also Figures S1, S2, and S4–S6.

Fig 4: mRNA vaccination boosts IFN?+IL-21-TNF-?-- and IFN?+IL-21-TNF-?+-producing CD4+ T cells and AIM+ cTfh cellsPBMCs of COVID-19 outpatients on month 10 post-enrollment were stimulated with S or MN proteins in vitro and were stained and analyzed by flow cytometry.(A) The absolute percentage (scatterplot; black line, median) of each individual combination of TNF-?-, IFN?-, and IL-21-producing non-naïve CD4+ T cells stratified by vaccination status (closed triangle: unvaccinated, n = 20; open triangle: vaccinated, n = 20). Top panel depicts MN-protein-stimulated CD4+ T cells (red), and bottom panel depicts S-protein-stimulated CD4+ T cells (blue). p values shown above the scatterplots are calculated using the Mann Whitney test. The pie charts show the relative proportion (mean) of each individual combination of cytokine-producing non-naïve CD4+ T cells (e.g., pie slice 4 represents TNFa+IFN?-IL-21-) among the total populaton of antigen-specifc cells, stratified by antigenic stimuli. The p value shown under the pie charts is calculated using a partial permutation test.(B) The ratio between month 10 and day 20 of the absolute percentage of IFN?+IL-21-TNF-?+- (IFN + TNF, left), IFN?+IL-21-TNF-?-- (IFNsp, middle), or IFN?-IL-21-TNF-?+-producing (TNFsp, right) non-naïve CD4+ T cells stimulated with the S protein is depicted (scatterplot; black line, median).(C) The absolute percentage (left) and the ratio between month 10 and day 28 of AIM+ (CD137+/OX40+) cTfh cells (right) stimulated with the MN (red) or S (blue) proteins and stratified by vaccination status are shown.(B and C) The p values shown are calculated using the Mann Whitney test. See also Figure S8.

Fig 5: Associations between early immune response and T cell responses on day 28 post-enrollment(A) Gene-Ontology-based immune pathways (left) and plasma proteins (right) associated with the absolute percentage of SARS-CoV-2-specific IFN?-IL-21-TNF-?+ non-naïve CD4+ T cells on day 28.(B) Scatterplots showing associations between the MCP-3 on day 0 or 5 and the percentage of SARS-CoV-2-specific IFN?-IL-21-TNF-?+ non-naïve CD4+ T cells on day 28.(C) Gene-Ontology-based immune pathways (left) and plasma proteins (right) associated with the absolute percentage of ICOS+ cTfh cells on day 28.(D) Scatterplots showing associations between the MCP-3 on day 0 or 5 and ICOS+ cTfh cells on day 28.(A and C) We used regression models to test the association while controlling for the sampling time of immune pathways or proteins (day 0 or 5) and the stimulation type (MN or S) of the T cell response.(B and D) The color of the points represents the stimulating antigens. The spearman correlations (rho [?]) between MCP-3 and the T cell percentages and the corresponding p values are reported.See also Tables S2 and S3.