Fig. 1: Adjuvants leading to the production of Th1- and Th2-related antibody isotypes induce the differentiation of Tfh cells with distinct transcriptomes.

a C57BL/6 mice were immunized subcutaneously in the footpad with OVA either emulsified in IFA (IFA), admixed with CpG (CpG), or incorporated with CpG in nanoparticles (NP-CpG). Eleven days later, blood and draining lymph nodes (LNs) were collected for analysis. b ELISA quantification of anti-OVA IgG1 and IgG2a, and IgG1/IgG2a ratio in serum of the immunized mice. c, d Representative flow cytometry plots (c) and quantification of GC B cells (CD19⁺CD95⁺GL7⁺), Tfh cells (CD4⁺Foxp3⁻CD25⁻CXCR5⁺PD1⁺), and GC-Tfh cells (CD4⁺Foxp3^-CD25^-CXCR5^hiPD1^hi) (d) in the draining LNs. Data from one experiment (n = 4), each dot representing one sample and bars representing mean values, analyzed by one-way ANOVA and Tukey’s multiple comparisons tests: **P < 0.01, ***P < 0.001. e T cells from OVA-specific TCR-transgenic mice were adoptively transferred into congenic recipients, immunized on the following day with OVA associated with different adjuvants. f On day 11, OVA-specific activated Th and Tfh cells were isolated by FACS for RNA-seq according to the represented gating strategy. g Principal component analysis (PCA) from the RNA-seq datasets of Th and Tfh cells, isolated from the two strains, and three types of immunization. PC1 explained 15% of the variance, discriminating datasets from the two strains. h PC2 and PC3 have a similar impact on the variance, with PC2 segregating Tfh cells from activated non-follicular T cells, and PC3 separating the samples based on the type of adjuvant used (type-1 vs type-2).