Fig. 4: Local expansion and transient and reversible anergy of CD4⁺ T cells following pulmonary infection with sAg expressing S. aureus strains.

a C57BL/6 mice and DR4-DQ8 mice were intranasally infected with either 10⁸ CFU JKD6159, or 10⁸ CFU JKD6159::seb. On day 1 post infection, the concentration of a panel of cytokines in the BALF and serum was measured using a cytometric bead array (n = 6–9 mice per group, Two-way ANOVA, Dunnett’s multiple comparison). b–d C57BL/6 mice and DR4-DQ8 mice were intranasally infected with either 10⁸ CFU JKD6159, or 10⁸ CFU JKD6159::seb. On days 2, 4 or 7 post infection, the absolute number of (b) Vβ8⁺CD4⁺ T cells (c) Vβ11⁺CD4⁺ T cells in the lung, mLN and spleen was measured by flow cytometry. The lines represent different experimental groups, and symbols represent the mean ± SEM (n = 5–28 mice per group, Two-way ANOVA, Sidak’s multiple comparison). d Representative flow cytometry profiles gated on CD4⁺ T cells in the lung showing the proportion of cells expressing Vβ8 and Vβ11 on day 4 post infection. e Normalized frequencies of Vβ TCR for bulk CD4⁺ T cells in the lung and spleen compartment in DR4-DQ8 mice intranasally infected with either 10⁸ CFU JKD6159 or 10⁸ CFU JKD6159::seb at day 4 after infection. Data are pooled from 2 experiments. C57BL/6 mice and DR4-DQ8 mice were intranasally infected with either 10⁸ CFU JKD6159, or 10⁸ CFU JKD6159::seb. Four days later the proportion of (f) total CD4⁺ T cells (g) CD4⁺Vβ8⁺ T cells or (h) CD4⁺Vβ8⁻ CD4⁺ T cells in the lung and spleen producing TNFα following a brief in vitro stimulation with PMA/ION was measured by flow cytometry. Bars represent the mean ± SEM, and symbols represent individual mice (squares represent unstimulated control) (n = 5 or 6 mice cohort, Two-way ANOVA, Tukey’s multiple comparison). i–l DR4-DQ8 mice intranasally infected with either 10⁸ CFU JKD6159 or 10⁸ CFU JKD6159::seb and treated with BrdU from days 1–3 post infection. The proportion of CD4⁺Vβ8⁺ T cells and CD4⁺Vβ8⁻ T cells in the lung on (i) day 6 and (k) day 21 post infection. Bars represent the mean ± SEM (n = 2–4 mice cohort). The proportion of CD4⁺ Vβ8⁺BrdU⁺ or BrdU⁻ T cells or CD4⁺Vβ8⁻BrdU⁺ or CD4⁺Vβ8⁻BrdU⁻ T cells in the lung on (j) day 6 or (l) day 21 producing IFNγ and/or TNFα following a brief in vitro stimulation with PMA/ION was measured by flow cytometry. Bars represent the mean ± SEM (n = 5–7 mice cohort, Two-way ANOVA, uncorrected Fisher’s LSD). *p < 0.5, **p < 0.01, ***p < 0.001, and ****p < 0.0001.