Fig 2: GM-CSF-DC-activated CD4+ T cells display a Tfh1 cytokine profile. (A) T cell cytokine quantification (using CBA, except IL-21 and CXCL13, which were quantified using ELISAs) after co-culture of naive CD4+ T cells with Medium-DCs (Med DC), GM-CSF-DCs or LPS-DCs. Mean±s.e.m. from n=5. (B) Intracellular FACS staining for T cell cytokines after co-culture with the indicated DCs. Plots represent cells gated in CD4+ live cells for one representative donor. The percentage of cells in each gate is indicated on the plots. (C) Percentages of IL-21+, IL-21+ TNF-α+, IL-21+ IFN-γ+, and IL-21− IFN-γ− cells as shown in B. Mean±s.e.m. from n=6. (D) IL-21 production in CD4+ T cells measured by FACS (left) and t-SNE analysis of IL-21+ cells, identifying two clusters with different expression levels of IFN-γ (middle) and TNF-α (right) for one representative donor. *P<0.05; **P<0.01; ns, not significant (two-tailed paired Student's t-test).

Fig 3: CD40-dependent Tfh1 polarization by GM-CSF-DCs. (A) PD-1High CXCR5+ cells gated in total CD4+ T cells differentiated by either ICOS-LHigh or ICOS-LLow DCs incubated with blocking antibodies targeting CD40 or ICOS-L, or with the isotype control. One representative experiment is shown. Percentage of cells in the PD-1high CXCR5+ population is shown on the plots. (B) Percentages of PD-1High CXCR5+ cells from data as shown in A (aCD40, anti-CD40 blocking; aICOS-L, anti-ICOS-L blocking). Mean±s.e.m. from n=9. (C,D) Intracellular FACS staining for T cell cytokines induced either by (C) ICOS-LHigh or (D) ICOS-LLow DCs incubated with blocking antibodies against CD40 or ICOS-L, or with the isotype control, as indicated. One representative experiment is shown. IFN-γ and IL-4 production is gated within IL-21+ TNF-α+ cells. The percentage of cells in each gate is shown on the plots. (E–G) Percentages of (E) IL-21+ TNF-α+ cells, (F) IFN-γ+ IL-4− cells gated in IL-21+ TNF-α+ cells and (G) IL-4+ IFN-γ− cells gated in IL-21+ TNF-α+ cells from data as shown in C and D. Mean±s.e.m. from n=8. *P<0.05; **P<0.01; ***P<0.001; ns, not significant (two-tailed paired Student's t-test).

Fig 4: S-specific T-cell proliferation in response to ancestral spike peptides in pHCs and pSOTRs.a Representative gating of cell trace violet-labeled, S-specific, proliferating daughter T cells in response to ancestral SARS-CoV-2 S peptides. b Proliferating S-specific CD4+ and CD8+ T cells (% memory) in pHC, pSOTR M, and pSOTR B groups at the time of waning immunity. No significant relationships. Boxplots were used to summarize data (median, 1st–3rd quartiles (IRQ), whiskers represent minimum and maximum values). c Proliferating CD4+ and CD8+ T cells (% memory) in pSOTR B at days 0, 14, and 300. Kruskal-Wallis test, *p < 0.05, ns = not significant. Boxplots were used to summarize data (median, 1st–3rd quartiles (IRQ), whiskers represent minimum and maximum values). d Heatmap depicting correlations between proliferation of S-specific CD4+ T cells and cytokine production in pHCs and pSOTRs. The “cytokine” category combines TNF, IL-21, IL-2, and IFN-γ production. Pearson correlation coefficients are depicted in each square. Higher coefficient represents greater correlation.

Fig 5: Limited Pf-malaria antigen-specific expression patterns of cTFH defining cytokines and transcription factors for adults.Heatmaps of Wilcoxon paired two-tailed t-test p-values are shown for cTFH meta-clusters comparing (a) PfSEA-1A or (b) Pf-GARP vs unstimulated peripheral blood mononuclear cells (PBMCs) from adults (n=13) for each cytokine (IFNγ, IL-4, and IL-21) and transcription factors (Bcl6 and cMAF). The color scale indicates the significance of the p-value: white (nonsignificant, p>0.05), yellow (0.05>p>0.02), orange (0.02>p>0.005), and red (highly significant, p<0.005). The down arrow indicates a decrease of expression from unstimulated to stimulated condition, whereas no arrow indicates an increase of expression from unstimulated to stimulated condition. The only cTFH meta-clusters that expressed transcription factors were in Group 3 (purple box). Bar plots indicate the mean with standard deviation (SD) of the median fluorescence intensity (MFI) of (c) Bcl6 and (d) cMAF for the cTFH meta-clusters showing significant statistical differences between PfSEA-1A and PfGARP stimulations. The p-values from Wilcoxon paired two-tailed t-tests are indicated.