Fig 2: CD40L signaling is required for migratory cDC maturation and downstream induction of TFH after intranasal 5-A-RU/MG administration(A) WT mice were treated i.n. with either PBS or 5-A-RU/MG. Three hours later, lungs were harvested for flow cytometry. MFI of CD40L expression by MAIT cells.(B) Schematic of experimental design. Twelve hours prior and 1 h after i.n. treatment, mice were given 500 μg of αCD40L intraperitoneally (i.p.).(C) Fold change MFI of CD86 on migratory cDC1 (B220− TCRβ− CD11c+ MHCIIhi SIRPα−) and cDC2 (B220− TCRβ− CD11c+ MHCIIhi SIRPα+ CD11b+). Fold change of CD69 MFI on MAIT cells (B220− TCRβ+ CD64− MR1-5-OP-RU+). Fold change is relative to the average of PBS-treated mice.(D) Schematic of experimental design. WT mice were treated i.n. with either 5 nmol OVA alone or in combination with 5-A-RU/MG, three times spaced 2 weeks apart. Five hours prior and 18 h after i.n. treatment, mice were given 500 μg of αCD40L i.p.(E) Frequency and total number of mLN TFH cells and OVA-specific TFH cells. MAIT cell frequency of total T cells and frequency of PD-1+ MAIT cells.Graphs depicted as mean ± SD. Data represent a combination of n = 2 (A) or n = 3 (C and E) individual experiments, with n = 3–8 mice per group, per experiment. Unpaired t test (A and C) or one-way ANOVA with Tukey’s multiple comparisons test (E) were performed, with ns, p ˃ 0.05; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗∗p ≤ 0.0001. See also Figure S7.

Fig 3: Analysis of lung CFU, leukocytes, and ex vivo antigen-stimulated Th intracellular cytokine production following GP-Cda1/Cda2 vaccination and/or infection.BALB/c mice were vaccinated subcutaneously thrice at biweekly intervals with GP-Cda1/Cda2. Two weeks after the last boost, the mice received a pulmonary challenge with C. neoformans. Mice were euthanized at 0 dpi (uninfected), 10 dpi, or 70 dpi. Controls included unvaccinated mice euthanized at 0 dpi or 10 dpi. Lungs were harvested and single-cell suspensions were prepared. a CFU/lung were determined. b Leukocytes at the interface of a 67 and 40% Percoll gradient were collected and counted. c–h Leukocytes were cultured in complete media supplemented with amphotericin B and stimulated with indicated antigens or left unstimulated (Unstim) for 18 h. Then the cells were collected, stained, and analyzed by polychromatic FACS, as described in Methods. c, d The numbers of CD4+ T and CD8+ T cells were calculated by multiplying the percentage of each population times the total leukocyte count. e–h The numbers of CD4+ T cells expressing the activation marker CD154, or producing the intracellular cytokines IFNγ, IL-17, and TNFα following ex vivo stimulation. Each group had five mice. In Fig. 6a, the bar represents the median of individual lung CFUs, and the dotted line represents the inoculum dosage for a challenge (2 × 104 Cryptococcus). In Fig. 6b–h, data are presented as mean ± SEM. Vac vaccinated with GP-Cda1/Cda2, Chall challenge with C. neoformans, Dpi days post infection, HK heat killed. Statistical comparisons between groups are shown in Supplementary Fig. 5.

Fig 4: Frequency of T cell subsets in splenocytes assessed by flow cytometry. Two weeks after immunization, spleen lymphocytes isolated from vaccine and control mice were stimulated in vitro with the fusion protein for 16 h. Representative dot plots are shown for (A) % IFN-γ+ cells in CD4+ T cell subset, (B) % IL-17A+cells in CD4+ T cell subset, (C) % CD154+cells in CD4+ T cell subset, (D) % IFN-γ+ cells in CD8+ T cell subset, (E) % CD107a+ cells in CD8+ T cell subset, and (F) % IFN-γ+CD107a+cells in CD8+ T cell subset. Data are shown as mean±SD, n = 5. Statistical analyses were performed by one-way ANOVA (ns, No significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).