Cytokine recall responses from protectively immunized mice are cryptococcal specific.

BALB/c mice received an initial immunization with heat killed C. neoformans strain H99γ or C. neoformans strain H99γ and allowed to rest for 60 days. Macrophages were then isolated from the spleens of the immunized mice, as well as from naïve mice, and cultured ex vivo with LPS, heat killed Candida albicans, heat killed Staphylococcus aureus or C. neoformans strain cna1Δ for 24 h. Supernatants were collected and analyzed for IL-2 (a), IFN-γ (b) and TNF-α (c), GM-CSF (d), IL-4 (e), IL-5 (f), IL-12(p70) (g), or IL-10 (h) production. Data shown are means +/- SEM of 3 independent experiments using 20–25 mice per group. A two-way ANOVA with Tukey’s multiple comparison test was performed to compare cytokine production from each stimulus within one immunization group to media alone as well as to compare cytokine production elicited by each stimulus between the 3 immunization groups. (* p < 0.05 compared to media within the same immunization group; a, p < 0.05 compared to naïve, b, p < 0.05 compared to HKH99γ within each secondary stimulation group).