Figure 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-1^neg 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-1^neg 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 Tfh^D3 splenic cells.

(H) Representative flow plots showing IL-21 and IFNγ production by non-GC Tfh^D3 and GC Tfh^D3 cells 3 days after splenocyte stimulation.

(I) Percentage of IL-21- and/or IFNγ-positive cells among non-GC Tfh^D3 cells and GC Tfh^D3.

(J) Gating strategy for analysis of Bcl6 expression in CD4⁺ T cells and histograms showing Bcl6 mean fluorescence intensity for GC Tfh^D3, non-GC Tfh^D3, 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.