(A) C57BL/6 mice (n = 8–11) were injected i.p. with 107 CFUs of S. aureus (LAC USA300) GAS (M49), or PBS on day 0, and re-injected on day 21 with 107 CFUs of either S. aureus or GAS. Bacteria from spleen and kidneys were recovered after 24 hr. PBS/S. aureus (Sa) injected with PBS on day 0 and then injected with SA on day 21. Dashed lines indicate limit of detection.

(B) C57BL/6 mice (n = 10) were injected i.p. with S. aureus (107), GAS (5 × 106), or PBS. The mice were inoculated after 21 days with S. aureus (4 × 107) or GAS (2 × 107) and monitored for survival.

(C) C57BL/6 mice (n = 5) were infected i.p. once, twice (day 0 and 21), or four times (day 0, 21, 28, and 35) with S. aureus (107). Surviving CFUs were enumerated 24 hr after the last infection.

(D) Mice (n = 5–6) were infected as in (A). Splenocytes were harvested 7 days after the last infection, stimulated with phorbol 12-myristate 13-acetate, ionomycin, and brefeldin A for 4 hr, and stained for surface CD4 and T cell receptor β (TCR-β), and intracellular IFN-γ and IL-17A. Shown are fluorescence-activated cell sorting plots and graphs of IFN-γ and IL-17A expression by CD4+TCR-β+ cells. Results are representative of two experiments.

(E) Mice (n = 8–11) were infected as in (A), and splenocytes harvested 7 days after the last infection were stimulated with heat-killed S. aureus or GAS (MOI = 10) for 48 hr. Supernatants were analyzed by ELISA. Data are representative of two experiments. See also Figure S1

For (A), (C), and (D), each data point represents an individual mouse, bars denote median and dashed lines indicate the limit of detection. In (E), data are shown as mean (bars) and SEM (error bars). Data analysis was performed using Mann-Whitney U test for (A and D), ANOVA for (C), log rank test for (B) and Student's t test for (E). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant.