Figure 4.

Quantitative and qualitative analyses of the long-lasting induction of Mav CFA-specific multifunctional CD4⁺ T cells after CFA+GLA-SE or CFA+GLA-SE/CDG immunization in a murine model of chronic progressive Mav-PI at 10 weeks post-infection. At 10 weeks post-infection, the mice were sacrificed, and lung cells harvested from each immunized group (n = 6) and naïve group (n = 4) were stimulated by GolgiPlug and GolgiStop with or without 10 µg/ml CFA at 37°C for 9 h. The frequencies of Mav CFA-specific IFN-γ⁺IL-2⁺, IFN-γ⁺IL-17A⁺ or IFN-γ⁺TNF-α⁺-expressing CD4⁺CD44⁺CD62 L⁻ T cells were assessed after staining of intracellular cytokines and are presented as (a) pseudocolor dot plots and (b) summary bar graphs. (c) The percentages of total CD4⁺CD44⁺CD62 L⁻ T cells with differential production of IFN-γ, IL-2, IL-17A and TNF-α in response to CFA stimulation among lung single cells were determined among groups and are presented as bar graphs. (d) The values of the proportions of quadruple- (4+, crimson), triple- (3+, orange), double- (2+, yellow), and single-function (1+, light gray) CD4⁺CD44⁺CD62 L⁻ T cells expressing IFN-γ, IL-2, IL-17A and TNF-α in each immunized group and naïve group are illustrated as pie charts. Statistically significant differences among all groups in (b) and (c) were determined by one-way ANOVA with Tukey’s multiple comparison test, and the results are presented as the mean values along with the S.Ds. *p <.05, **p <.01, ****p <.0001 and n.s.: not significant. The asterisks in (c) represent significant differences between groups: black, control group vs. CFA+GLA-SE/CDG group; red, CFA+GLA-SE group vs. CFA+GLA-SE/CDG group. The representative results are shown from a single in vivo experiment. CFA, culture filtrate antigen; Control, GLA-SE immunization alone; GLA-SE, glucopyranosyl lipid a adjuvant formulated in a stable oil-in-water emulsion; GLA-SE/CDG, GLA-SE plus cyclic-di-GMP.