Figure 4.

Inhibition of STAT4 reverses T1-IFN induced cytotoxicity. IGRP-CTL avatars were transfected with Accell siRNAs specific for STAT1, STAT4, or nontargeting (NT) controls for 72 h. A and B: RT-PCR analysis to assess gene silencing was performed for STAT1 (A) and STAT4 (B). All values were normalized relative to 18S mRNA expression. C–E: IGRP-CTL siRNA transfectants were primed with T1-IFN and assessed for GZMB expression by flow cytometry. Representative histograms (C) and mean fluorescence intensities (MFI) for GZMB (D) are plotted. E: Viability analysis on siRNA-transfected CTLs is plotted. F–I: IGRP-CTL avatars were pretreated with lisofylline (LSF) and analyzed for pSTAT4 activation by Western blot (F) and phospho-flow cytometry (G). LSF-treated CTLs were primed with IFNα and analyzed for GZMB expression by flow cytometry. Representative histograms (H) and MFI (I) for GZMB are shown. For representative Western blots and histograms (C, F, and H), all lanes or lines within a panel are from a single donor. Data plotted in A, B, D, E, G, and I are mean ± SEM; T cells from each donor (3 donors for A and B; at least 5 donors for D, E, and G; and at least 7 donors for I) were weighted equally. There were at least three separate experiments for each donor. Statistical significance was determined with a nonparametric paired t test with Wilcoxon post-test analysis. *P < 0.05; **P < 0.01; ***P < 0.001 for comparison to control; †P < 0.05; ††P < 0.01 for comparison within IFNα dosage group. tSTAT, total STAT.