Figure 6.

HSPCs from PCA2-infected mice are trained for proinflammatory cytokine production and protect mice against a virulent C. albicans secondary infection. Intracellular detection by flow cytometry of TNF-α and IL-6 in pre-gated Lin^– c-Kit⁺ cells from (A) total RBC-lysed bone marrow cell or splenocyte cultures isolated from 7-day PCA2-infected or uninfected mice and stimulated with LPS for 6 h with brefeldin A for the final 4 h or (B) total RBC-lysed bone marrow cell or splenocyte cultures isolated from 7-day PCA2-infected or uninfected mice i.p. infected for 24h with 30 million of ATCC 26555 yeasts and stimulated with PMA/ionomycin and brefeldin A for 6 h. Dot plots indicating the % of cytokine-producing LKS⁺ and LKS^– cells are shown, as well as the total cell numbers and the MFI of cytokine-producing LKS⁺ and LKS^– cells in the bone marrow and spleen. Data are presented as mean plus SD of 3-4 mice, and statistical significance was assessed by Student’s t test (*P < 0.05, **P < 0.01 and ***P < 0.001). (C) 5 million c-Kit⁺ or 30 million c-Kit^– cells were isolated from the bone marrow or spleen of 7-day PCA2-infected mice and adoptively transferred to uninfected mice, 3 days later mice were i.p. infected with 30 million of ATCC 26555 yeasts and CFUs were measured in the kidneys after 4 additional days. Non-adoptively transferred mice served as controls. (D) Fungal burden in the kidneys expressed as CFUs per gram of tissue. Data shown is pulled from 2 independent experiments (n = 8-12 per experiment). Statistical significance was assessed by one way ANOVA followed by Dunnett’s test for multiple comparisons. See also Supplementary Figure 5 .