Fig. 4

GLS-1027 prevents the development of Th17 T cells. Purified, naïve human CD3 T cells from peripheral blood mononuclear cells (PBMC) by depleting B cells, NK cells, monocytes, platelets, dendritic cells, granulocytes, and erythrocytes. Isolated CD3⁺ T cells were bead- and antibody-free and incubated with 10 U/ml of IL-2, 10 ng/ml of IL-1β, 10 ng/ml of IL-23, 1ug/ml of anti-IL-4, 1ug/ml of anti-IFN-γ and anti-CD3/CD28 activation beads at a ratio of 1 bead per cell plus IL1-β 10ng/ml. In some cultures, GLS-1027 was added at a concentration of 1µM or 10µM at the same time as the Th17-inducing cytokines. (A) On day 6, the cells were stained with antibodies to CD4 and intracellular IL-17A and then evaluated by flow cytometry. Percentage of CD4⁺/IL-17⁺ cells indicated in the upper right quadrant of plots. (B) Mean Fluorescent Intensity (MFI) of IL-17 in CD4⁺ T cells. (C-D) Concentration of IL-17 and IL-23 in media of differentiated CD4.⁺ T cells; GLS-1027 concentration was 10µM. A non-parametric two-tailed t-test (Mann–Whitney) was used for statistical analysis, and the relevant p values are indicated. Error bars in all panels indicate a standard deviation among 3 replicates per condition. The experiment was repeated twice with similar results are shown in this representative experiment