Fig. 2: J8-Lipo-DT-PHAD-induced Th17 cells.

BALB/c mice (n = 5 mice/group; female, 4–6 weeks old) were immunized i.n. on days 0, 21, and 42 with PBS, J8-Lipo-DT-PHAD, J8-Lipo-DT, Lipo-DT, Lipo-DT-PHAD, J8-Lipo-PHAD, J8-Lipo, Lipo-PHAD or Lipo. Six weeks post-last-immunization mice were euthanized, and lungs excised. Lung cells were stimulated with J8-DT (0.05 mg/well) or media for 16 h and IL-17A⁺ CD4⁺ cells were quantified using intracellular cytokine staining (ICS). Flow cytometry contour plot representing the gating strategy used to identify IL-17-A⁺CD4⁺ (Th17) cells from PBS and J8-Lipo-DT-PHAD immunized mice (a). A boxplot (min/max; medium with SEM) representing the frequency of IL-17A⁺ from CD4⁺ cells (b). Data are normalized to the media stimulation control (% IL-17A⁺ cells from the J8-DT stimulated group minus % IL-17A⁺ cells from the media stimulated group [to remove background]). Statistical analysis was performed using a nonparametric, unpaired Mann–Whitney U test (one-tailed) (*p < 0.05, **p < 0.01) to compare the different immunization cohorts to J8-Lipo-DT-PHAD. c, d Secreted IL-17A from immune cells isolated from lung digests following intranasal vaccination and ex-vivo stimulation. BALB/c mice (n = 3/group; female, 4–6 weeks old) were immunized as above. Lung (c) and spleen (d) cells were stimulated ex-vivo with J8-DT (0.05 mg/well) or media. At 72 h post-stimulation, supernatants were isolated and levels of secreted IL-17A were assessed in culture supernatants using a cytometric bead array. Data are normalized to the media stimulation control and represented as mean ± SEM. Statistical analysis was performed using two-way ANOVA, corrected for multiple comparisons using Sidak’s multiple comparison test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Experiments were repeated once to confirm the results. Source data are provided as a Source Data file.