Fig 1: 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 IFN? production by non-GC TfhD3 and GC TfhD3 cells 3 days after splenocyte stimulation.(I) Percentage of IL-21- and/or IFN?-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.