Figure 5.

The LYP inhibitor 8b increases T cell activation. (A) Compound 8b increases activation of ZAP-70 in human T cells. JTAg cells were pre-incubated with 15 µM 8b (red graphs) or DMSO alone (blue graphs) for 30 min at 37°C, followed by stimulation with increasing concentrations of C305 supernatant (18.75 µg/ml, solid lines; 37.5 µg/ml, dashed lines; 75 µg/ml, long-dashed lines) or left unstimulated (shaded graphs) for 2 min at 37°C. Graphs show cell fluorescence after staining with an AlexaFluor-488-conjugated anti-phospho-ZAP-70 (Y319) antibody. Median fluorescence intensity (MFI) of each sample is shown. (B) Compound 8b increases CD69 expression on human T cells. JTAg cells were incubated with 15 µM 8b (red graph, MFI=616) or DMSO alone (blue graph, MFI=451) for 4.5 hours at 37°C. Graphs show cell fluorescence after staining with a FITC-conjugated anti-CD69 antibody. (C–D) Compound 8b increases the activation of primary mouse T cells. Thymocytes from Nur77^GFP mice were incubated with 15 µM 8b (red graph) or DMSO alone (blue graph) for 3.5 hours at 37°C. (C) Graphs show cell fluorescence after staining with a FITC-conjugated anti-CD69 antibody (MFI of 8b–treated sample=758; MFI of DMSO-treated sample=381). (D) Graphs show Nur77 expression as assessed by GFP cell fluorescence (MFI of 8b–treated sample=1120; MFI of DMSO-treated sample=604). Histograms from all 8b–treated samples in this figure were assessed compared to histograms from the respective DMSO-treated samples using the Kolmogorov-Smirnov test, and the distributions were found to be distinct with 99.9% confidence. Data in this figure are representative of 2 independent experiments with similar results.