Fig. 2. Tfh13 cells are induced in WT mice during allergic sensitization.

WT mice were immunized and boosted i.n. with LPS or Alternaria extract and NP16-OVA. (A) Day 8 sera from boosted mice were analyzed for high-affinity IgE by ELISA with NP7-BSAcoated plates. (B) Evans blue dye extravasation quantification after PCA with day 8 post-boost sera and NP7-BSA challenge. (C) Cd4^CreBcl6^fl/fl (T-Bcl6^−/−) or control Bcl6^fl/fl mice were immunized and boosted with Alternaria extract and NP16-OVA. Eight days later, high-affinity IgE was quantitated using NP4-BSA by ELISA. (D) 3D uniform manifold approximation and projection (UMAP) embedding of the single–cell expression profiles of n=3002 single Tfh cells sorted from WT C57BL/6 mice immunized i.n with Alternaria extract and NP-OVA. Leiden community detection on the cell-cell k=10 nearest neighbor graph segregates cells into seven clusters, five of which were identifiable on the basis expression of previously known markers: 1, Tfh2; 2, type 1 IFN T cell population; 3, proliferating T cells; 4, Tfh13 cells and 6, Tfr cells. The circle identifies cluster 4, a cluster of Il13⁺ Tfh cells (n=39). (E) Violin plots showing the expression of distinctive marker genes of Il13⁺ Tfh cluster (Il4, Il13, Gata3). (F to H) Intracellular expression of IL-4, IL-13, and IL-5 in day 8 MedLN Tfh cells induced after primary immunization with LPS or Alternaria and NP-OVA depicted as flow cytometry (F) plots and bar graphs for IL4 and IL-13 (G) and IL-5 (H). (I) IL-21 expression in Tfh cells from IL-21-TWIK reporter mice at day 8 after immunization, depicted as histogram overlay (left) and bar graphs (right). (J) GATA3 expression in Th2 cells from bronchioalveolar lavage fluid of mice immunized with Alternaria and NP-OVA, and Tfh cells from MedLN of mice immunized with LPS or Alternaria and NPOVA. Data are depicted as histogram overlay (left) and bar graphs (right). Each symbol indicates an individual mouse. Numbers in flow plots indicate percentages. Error bars indicate SEM. Dotted lines in bar graphs represent background readings of sera from naïve mice. Statistical tests: Kruskal-Wallis H test (A and C); Student’s t test (B, G and J); ANOVA (I); Mann–Whitney U test (H) *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant. Data representative of two or three independent experiments with three to five mice per group (A to C, and F to J). Data representative of three biological replicates (D and E).