Figure 4.

The effect of MTF-VAC and VAC on mice spleens before and after influenza virus challenge. Groups of mice were immunized 7 days before a lethal virus challenge with MTF-VAC, Vac, MTF, or PBS. At day 0, 10LD₅₀ of the 415742Md virus was inoculated through the intranasal route. Spleen samples were collected on day 0 and 2 dpi. Flow cytometry was applied for assessing frequencies of T_FH cells with staining of Zombie Violet live/dead dye, anti-mouse CD3ε, anti-mouse CD4, anti-mouse PD-1, and anti-mouse CXCR5 antibodies. Frozen sections were prepared from 2 dpi spleen samples and stained with anti-mouse IgD and anti-mouse GL7 antibodies. The stained tissue slides were examined under the fluorescent microscope Olympus BX53. B cell follicles expressing IgD⁺GL7⁻ or IgD⁺GL7⁺ were counted with randomly renamed photos to avoid bias. (A) Schedule of influenza vaccine, virus challenge, and spleen sample collection. (B) Gating strategy and representative flow cytometry profiles of splenic T_FH cells obtained from mice. (C) T_FH cell frequencies in mice spleens assessed by flow cytometry. (D) Percentage of GL7⁺ B cell follicles (GC) among all B cell follicles (IgD⁺) in spleen. (E) Representative immunofluorescence staining of mouse spleen. MTF-VAC, miltefosine (0.2 mg) + vaccine group; Vac, vaccine only group; MTF, miltefosine (0.2 mg) only group; PBS, PBS only group. Data collected from 8 mice per group (3 independent experiments) for (B,C). Data were collected from 3 mice per group (1 independent experiment) for (D,E). * for p < 0.05; ** for p < 0.01; *** for p < 0.001, **** for p < 0.0001; calculated by two-way ANOVA followed by a Tukey’s multiple comparison test. Error bars represent standard error of the mean (SEM).