Fig 2: Age promotes pre-Tfh cell differentiation in humans. Flow plots showing the frequency of CXCR5+PD-1+ cells amongst CD4+ following 4 days in vitro activation of naïve CD4+ T cells taken from younger (17–39 yrs) and older (>60 yrs) donors in the presence/absence IL-12 and TGFß (a). Percentage of CXCR5+PD-1+ following activation of naïve CD4+ T cells from younger and older donors in the absence (b) or presence (c) of IL-12 and TGFß. Percentage of CXCR5+PD1+ cells following activation of naïve CD4+ T cells from older donors with or without neutralising antibodies to IL-12, Activin A and TGFß (d). Bar graphs showing percentage of cells expressing pSTAT3 (e, f), and pSTAT5 (g, h) on day 3 of culture in presence/absence of IL-12 and TGFß in donors of the indicated ages. RT-PCR determination MAF (i), BCL6 (j), FOXP3 (k) and CXCL13 (l) following 4 days activation of naïve CD4+ T cells from younger and older donors in the absence of polarising cytokines. Flow plots (m-o) and bar graphs (n-o) showing percentage of IFN? and IL-21 expressing cells following PMA and ionomycin mediated restimulation after 4 days in vitro culture of naïve CD4+ T cells from younger and older donors in the absence of IL-12 and TGFß (m-o). Bar graph showing levels of IgG produced by B cells following co-culture with CD4+ T cells from day four cultures from young and older donors, in the presence/absence of Tfh-polarising cytokines (p). Each symbol is representative of an independent biological replicate, and the height of the bar represents the mean. Statistics were calculated using Mann–Whitney U test *p < 0.05, **p < 0.005, ***p < 0.0005

Fig 3: Dimensionality reduction and clustering analysis of SARS-CoV-2 S-specific CD4+AIM+ T cells(A) Dimensionality reduction and phenograph-derived clustering (k = 40) overlaid for total CD4+AIM+(CD154+CD69+) T cells pooled from all participants (left). Heatmap expression of the indicated parameters over UMAP space (right).(B–P) All cells from the indicated clusters (CL) are represented in corresponding color from UMAP plot in (A) overlayed on all CD4+AIM+ T cells and showing expression of select parameters (IL-2, IFN-?, IL-10, CXCR5, CD127, CD25, IL-4, and IL-21). Enumeration of each cluster is per 1 × 106 non-naive (nn) CD4+ T cells in SARS-CoV-2 naive (blue) and SARS-CoV-2 previously infected (red) participants. Pre-vaccination (Pre), 1 week post two-dose COVID-19 mRNA vaccination (1w PV2), 3 months post two-dose vaccination (3m PV2). Significance was determined by multiple unpaired Mann-Whitney test for group analyses and by Wilcoxon matched-paired signed rank test for longitudinal analyses: not significant (ns), *p < 0.05, **p < 0.01.