Figure 2. Cytokine-induced IFN-γ... | Rockefeller University Press

Figure 2.

$Figure 2. Cytokine-induced IFN-γ production by autoantigen-primed iNKT cells does not require concurrent TCR signaling. (A) Cultured human iNKT cells were exposed to CD1d+ APCs in medium containing 50 µM of the MEK inhibitor U0126 or in medium alone (NT). The APCs and inhibitor were then removed, the iNKT cells were incubated in medium containing IL-12 and IL-18 for 16 h, and IFN-γ secreted into the culture supernatant was quantitated by ELISA. Results are representative of four independent experiments using three different iNKT cell clones. (B) Cultured iNKT cells that had been transiently exposed to CD1d+ APCs were incubated in medium containing IL-12 and IL-18 in the presence or absence of U0126. The plot shows the amount of IFN-γ secreted in the presence of the inhibitor as a percentage of that produced by control iNKT cells stimulated in the absence of U0126 (NT). Results are representative of four independent experiments using three different iNKT cell clones. (C) Effect of CsA on IFN-γ secretion by cultured iNKT cells exposed to IL-12 and IL-18 in the presence of CD1d+ APCs (concurrent) compared with primed iNKT cells during subsequent stimulation with IL-12 and IL-18. The plot shows IFN-γ secretion by treated iNKT cells as a fraction of the amount produced by control iNKT cells that were stimulated in the absence of CsA. Results are representative of four independent experiments using three different iNKT cell clones. (D) Cultured iNKT cells were exposed to CD1d+ APCs, then the APCs were removed, and the iNKT cells were exposed to IL-12 and IL-18 in the presence of the p38 inhibitors SB203580 or SB202190 or the inactive analogue SB202474 (mock). Results are representative of four independent experiments using three different iNKT cell clones. All plots show the means of three replicate samples, with error bars indicating the standard deviations of the means (not always visible on the scales shown).$

Cytokine-induced IFN-γ production by autoantigen-primed iNKT cells does not require concurrent TCR signaling. (A) Cultured human iNKT cells were exposed to CD1d⁺ APCs in medium containing 50 µM of the MEK inhibitor U0126 or in medium alone (NT). The APCs and inhibitor were then removed, the iNKT cells were incubated in medium containing IL-12 and IL-18 for 16 h, and IFN-γ secreted into the culture supernatant was quantitated by ELISA. Results are representative of four independent experiments using three different iNKT cell clones. (B) Cultured iNKT cells that had been transiently exposed to CD1d⁺ APCs were incubated in medium containing IL-12 and IL-18 in the presence or absence of U0126. The plot shows the amount of IFN-γ secreted in the presence of the inhibitor as a percentage of that produced by control iNKT cells stimulated in the absence of U0126 (NT). Results are representative of four independent experiments using three different iNKT cell clones. (C) Effect of CsA on IFN-γ secretion by cultured iNKT cells exposed to IL-12 and IL-18 in the presence of CD1d⁺ APCs (concurrent) compared with primed iNKT cells during subsequent stimulation with IL-12 and IL-18. The plot shows IFN-γ secretion by treated iNKT cells as a fraction of the amount produced by control iNKT cells that were stimulated in the absence of CsA. Results are representative of four independent experiments using three different iNKT cell clones. (D) Cultured iNKT cells were exposed to CD1d⁺ APCs, then the APCs were removed, and the iNKT cells were exposed to IL-12 and IL-18 in the presence of the p38 inhibitors SB203580 or SB202190 or the inactive analogue SB202474 (mock). Results are representative of four independent experiments using three different iNKT cell clones. All plots show the means of three replicate samples, with error bars indicating the standard deviations of the means (not always visible on the scales shown).