Fig. 4: Tfh1 and Tfh2 cells are functionally distinct.

a C57BL/6 mice were immunized in the footpad with OVA emulsified in IFA (IFA) or incorporated with CpG in nanoparticles (NP-CpG). On day 11, Tfh (CD4⁺CD25⁻CXCR5⁺PD1⁺) and B cells (CD19⁺CD4⁻) were isolated from draining LNs by flow cytometry and co-cultured. b After 5 days of culture, cytokines in the culture medium were quantified by multiplex assays. Data representative from two experiments (culture triplicates performed with cells obtained from 10 immunized mice per group), each dot representing one replicate and bars representing mean values, analyzed by Student’s t-test: **P < 0.01, ***P < 0.001. c, d Representative dotplots (c) and quantification of IgG2a⁺ and IgG1⁺ isotype-switched B cells (d) at the end of the co-cultures (stimulation with OVA), analyzed by flow cytometry. Data from one experiment (culture triplicates performed with cells obtained from 10 immunized mice per group), each dot representing one replicate and bars representing mean values, analyzed by Student’s t-test: **P < 0.01, ***P < 0.001. e Evaluation of gene expression by RT-qPCR in Tfh cells isolated from mice 11 days after immunization under type-1 and type-2 conditions as described in a. The cells from 10 mice immunized with each adjuvant were pooled, and the resulting cDNA was 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.