Figure 2.

Treatment with TMP778 suppresses the production of both IL-17 and IFN-γ by sensitized spleen and eye-infiltrating cells. (A) Spleen cells from B10.A mice immunized with IRBP and treated with TMP778, or the vehicle, were collected on days 7 or 14 post immunization and cultured with IRBP at the indicated concentrations. Supernatants were analyzed for the levels of IL-17 and IFN-γ, using ELISA kits. Collected data of repeated experiments (5 experiments for day 7 and 8 experiments for day 14) were normalized as detailed in the Materials and Methods section. Importantly, the figure shows that the treatment with TMP778 for 14 days reduced the response of both IL-17 (expected) and IFN-γ (unexpected). Treatment for 7 days had slight effect on IL-17 production and no effect on the levels of IFN-γ. Data are shown as +- SEM (n=4–5 mice in each group) and are pooled from 5 independent experiments for day 7 and 8 experiments for day 14). (B) Flow cytometric analysis of intracellular expression of IFN-γ and IL-17 by spleen cells of mice treated with TMP778, or the vehicle, for 7 or 14 days. Left: the scatterplots show data of representative experiments; right: the histogram bars show mean values of cells expressing IFN-γ or IL-17 among the total CD4 populations of repeated experiments. Data are shown as +- SEM (n=4–5 mice in each group of each experiment) and are pooled from 5 independent experiments with cells collected after 7 days of treatment, and from 10 experiments with cells collected after 14 days of treatment. (C) Flow cytometric analysis of intracellular expression of of IFN-γ and IL-17 by lymphoid cells collected from eyes of mice on day 14 of treatment with the vehicle (DMSO) or TMP778. The figure shows data collected in a representative experiment, while values of 4 repeated experiments are summarized in the histograms, as detailed above. * p<0.05, ** p<0.01; unpaired t test.