Fig 2: Ex vivo and induced Tfh display distinct phenotypic landscapes and differentiation trajectories(A) Deep immunophenotyping was performed after 3 days of culture (n = 2 independent donors). CD4+ CXCR5+ T cell selection was based on the expression of CD45, CD8, CD11c, CD56, and CD19. The Uniform Manifold Approximation and Projection (UMAP) algorithm was used to represent the whole set of CD4+ CXCR5+ T cells in a multiparametric manner at D0 and D3 after antigenic stimulation of splenocytes from two donors.(B) Projection of 8 clusters determined by k-means on the UMAP representation of D0 and D3 CD4+ CXCR5+ T cells.(C) Heatmap representing the mean expression of 29 markers by 8 cell clusters and their relative abundance, defined among D0 and D3 CD4+ CXCR5+ T cells.(D and E) Trajectory and pseudotime analysis on total D0 (D) and D3 (E) CD4+ T cells. Tree plots (left) show cluster trajectories, cell number, and metacluster assignment, and density plots (right) show the density of pseudotime across metaclusters.

Fig 3: Naive and memory CD4+ can orient toward a Tfh cell profile(A) Four CD4+ T cell subsets are defined at day 0: (1) naive CD4+ CD45 RA+ T cells, (2) memory CD4+ CD45 RA- PD-1- T cells (MemPD-1neg), (3) memory CD4+ CD45 RA- PD-1+ T cells (MemPD-1pos), and (4) Tfh.(B) At day 0, D4+ T cell subsets were sorted according to the gating strategy presented in (A). Isolated CD4+ T cell subsets were stained with cell trace violet (CTV) and mixed back into the negative splenocyte fraction. Stimulation and culture were next performed as previously described (Figure 1A).(C) Representative flow plots of CTV tracking for each stimulated CD4+ T cell subset 3 days after antigenic stimulation (top) combined with flow cytometry analysis of CXCR5 and PD-1 expression among CTV+ cells at day 3 (bottom).(D) Percentage of CXCR5+PD-1+ cells (TfhD3) among traced CD4+ T cell subsets after 3 days of antigenic stimulation.(E) Percentage of TfhD3 cells according to the divisions of stimulated CD4+ T cell subsets.(F) Representative histograms of Bcl6 expression for TfhD3 (colored line) compared with CXCR5-PD-1+ (black line) and CXCR5-PD-1- (gray line) deriving from respective CD4+ T cell subsets naive (blue), MemPD-1neg (turquoise blue), MemPD-1pos (green), and Tfh (orange).(G) RNA sequencing was performed on CD4+ T cell subsets (Day 0) and the corresponding derived TfhD3 counterparts (n = 2). Multidimensional scaling was used to better visualize transcriptomic proximity of different CD4+ T cells.(H) Venn diagrams were used to highlight Tfh-associated genes (Table 2) among differentially expressed genes that were shared during transition from D0 CD4+ T cell subsets to their TfhD3 counterparts (D and E). Each symbol represents an individual donor. A Wilcoxon matched pairs test was performed; *, p < 0.05; **, p < 0.005.

Fig 4: Antigenic stimulation of human splenic mononuclear cells mimics the T CD4+ response of GC reaction(A) Splenic mononuclear cells (splenocytes) were stimulated with CytoStim and cultured for 3 days in the presence of cytokines (IL-7, IL-12, activin) (A). CXCR5 and PD-1 expressions among CD4+ T cells were assessed. Representative flow plots showing CXCR5 and PD-1 expression on CD4+ T cells from ex vivo splenocytes and splenocytes cultured for 3, 5, and 10 days (left) and the percentage of Tfh among CD4+ T cells (right).(B) Representative flow plots showing IL-21 production by Tfh (left) and the percentage of IL-21-positive cells among Tfh (right).(C) Representative histogram showing ICOS expression among Tfh (left) and relative expression of ICOS among Tfh (right).(D) Gating strategy allowing the identification of PD-1neg Tfh, non-GC Tfh, and GC Tfh among total CXCR5+PD-1+ cells ex vivo or after 3 days of stimulation of splenocytes or PBMCs in polarizing cytokines.(E) Percentage of total CXCR5+PD-1+ cells including PD-1neg Tfh, non-GC Tfh, and GC Tfh among CD4+ T cells ex vivo or after 3 days of culture using splenocytes or PBMCs (n = 7–14).(F and G) Mean fluorescence intensity of PD-1 (F) and CXCR5 (G) expression on ex vivo GC Tfh and GC TfhD3 splenic cells.(H) Representative flow plots showing IL-21 and IFNγ production by non-GC TfhD3 and GC TfhD3 cells 3 days after splenocyte stimulation.(I) Percentage of IL-21- and/or IFNγ-positive cells among non-GC TfhD3 cells and GC TfhD3.(J) Gating strategy for analysis of Bcl6 expression in CD4+ T cells and histograms showing Bcl6 mean fluorescence intensity for GC TfhD3, non-GC TfhD3, and CXCR5−PD-1- CD4+ T cell subsets (n = 14). Each symbol represents an individual donor. A Wilcoxon matched pairs test was performed; ∗, p < 0.05; ∗∗, p < 0.005.