Fig 2. TLR7 agonist in the presence of IL-12 stimulates T cells directly leading to IFNγ production.

(A) Spleen cells obtained from C57BL/6, MyD88^fl/flxLCK^CRE and MyD88^fl/fl x WTmice were incubated in the presence of different TLR agonists as indicated for 5 days. Supernatants were analyzed for the presence of IFNγ by ELISA. Bar graphs represent concentration of IFNγ in the culture supernatants. (B) Spleen cells from MyD88^fl/fl xWT or MyD88^fl/flxLCK^CRE mice were incubated in the presence of different TLR agonists as indicated for 18h. Cells were surface stained and intacellularly stained for IFNγ. Bar graphs represent percentage of CD4 or CD8 T cells which are positive for IFNγ. (C) TLR7^-/- or conjenically marked WT (B6.SJL) splenocytes were incubated either separately (black bars) or mixed at 1:1 ratio in the presence (R848) or absence (media) of TLR7 agonist. IFNγ production was assessed by intracellular staining and the summary of three independent experiments is shown. Bars represent the means +/- SEM. (D) Splenocytes were incubated as indicated for 18h, IFNγ production by CD4 and CD8 T cells in response to indicated stimulations was assessed by intracellular staining. Bar graphs indicate percentage of IFNγ+ cells among CD4 and CD8 T cells. (E) WT or IL-18^-/- splenocytes were incubated with R848 for 18h, IFNγ production was assessed by intracellular staining and the summary of three independent experiments is shown. Bars represent the means +/- SEM (similar results were obtained for CD8 T cells—not shown). (F) Naïve and memory CD4 and CD8 T cells were flow sorted as CD4 (or CD8) positive, CD19^-, CD44+ (for memory) and CD44^- (for naïve). IFNγ production by sorted T cells in response to indicated stimulations was assessed by intracellular staining. Bar graph represent percentage of IFNγ+ sorted memory of naïve CD4 T cells (similar data was obtained for CD8 T cells—not shown). All data are representative of three or more independent experiments.