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. 2014 Sep 18;9(9):e107638. doi: 10.1371/journal.pone.0107638

Figure 3. Specific recall Th17 responses were induced in SrtA/CTB-immunized mice by heterologous GAS strains.

Figure 3

Mice were immunized i.n. three times with SrtA, or CTB, or SrtA and CTB, and challenged with live M1 strain 10 days after the last immunization. (A) 24 hr after challenge CFUs retained in NALT were determined. Data are geometric means of two independent experiments. (B) Mice were immunized and challenged as in (A). Three days after challenge, mice were euthanized and NALT cell suspensions were stained with FITC-CD4 followed by intracellular staining with PE-IL-17 and APC-IFN-γ and analyzed by FACS. Data are means ± SEM of three mice per group. * P≤0.05. (C) NALTs from immunized (SrtA/CTB) and PBS control mice were taken one day after challenge. Total RNA was isolated from NALTs and Il17a and Ifnγ m-RNA levels were determined by RT-PCR. Data were expressed as 2-ΔΔCt (means ± SEM of three mice per group, ** P≤0.01, *** P≤0.001). (D) Mice were immunized as in Fig. 2 and 10 days after the last immunization each group of mice were challenged with strain M1, M28 or M49. Control mice received PBS, SrtA or live M1 strain. Three days after challenge NALTs were taken and NALT cells were stained and analyzed by flow cytometry as in (B). (E) Quantification of CD4+ IL-17+ T cells as in (D). Data are presented as the means ± SEM (n = 3) from two independent experiments. * P≤0.05 (compared to PBS/PBS group).