Fig. 7: GCs induced upon type-1 or -2 immunization are enriched in Tfh1 or Tfh2 cells.

a Mice were immunized in the footpad with OVA emulsified in IFA (IFA) or incorporated with CpG in nanoparticles (NP-CpG), as described in Fig. 1, and the expression of selected genes by Tfh cells sorted 11 days after immunization was evaluated by RT-qPCR. The cells from 10 mice immunized with each adjuvant were pooled and tested in duplicate, both from C57BL/6 and Balb/c mouse strains. 2^−ΔCT values were determined in reference to the Actb housekeeping gene of the same sample and then normalized to the average 2^−ΔCT values obtained for mice immunized with the other adjuvant. b The expression of selected genes was further evaluated by flow cytometry in Tfh cells from C57BL/6 mice immunized in a similar manner. Representative histograms and quantification of median fluorescence intensity (MFI) of Tfh cells (CD4⁺Foxp3⁻CD25⁻CXCR5⁺PD1⁺) in the draining LNs from mice immunized with IFA (orange) or NP-CpG (blue). Data from one experiment (n = 4), each dot representing one sample and bars representing mean values, analyzed by Student’s t-test: **P < 0.01, ***P < 0.001. c Representative raw image of stained draining LN of mice, 11 days following immunization with IFA or NP-CpG as described, alongside example segmentation of CD4⁺ cells within B cell follicles (left panels, scale bars, 30 μm). Representative probe staining for Cebpb and CXCR3 are shown for each group (right panels, arrows show probe staining). d The proportion of GC CD4⁺ T cells positive for each marker was quantified for images processed as in c and quantified. Data from one experiment (n = 3), each dot representing one replicate and bars representing mean values, analyzed by Student’s t-test: **P < 0.01, ***P < 0.001, ****P < 0.0001. e Model representing the specialization of Tfh cells under type-1 or -2 conditions. Type-1 adjuvants or LCMV infection drives specialization of the majority of Tfh cells towards Tfh1, and a minority of Tfh2 cells. This Tfh1 cell specialization favors IgG2a isotype switching. Conversely, type-2 adjuvants or Helminths’ infection drive mostly Tfh2 specialization, with a small percentage of Tfh1 cells and a predominant production of IgG1.