Effects of a TYK2 inhibitor on IFN-signaling and responses. (A) CLL cells were cultured for 1 hour alone, or with different concentrations of AG9, before treatment with PDB (100 ng/mL), IFN-α2b (1000 U/mL), or PDB and IFN-α. After 4 hours, pSTAT1 and total STAT1 and STAT3 levels were determined by immunoblotting. STAT3 phosphorylation was abrogated by AG9, whereas STAT1 activation remained intact. Similar results were obtained with 5 different patient samples. (B) Concentrations of IL-6 (top) and IL-8 (bottom) were measured in culture supernatants after 48 hours. IL-6 production was increased by PDB and inhibited by AG9 without significantly affecting IL-8. (C) CLL cells cultured for 3 to 4 days with or without PDB and/or IFN in the presence or absence of AG9 were obtained from 5 patients and used to stimulate T cells from healthy donors to proliferate as measured 5 to 6 days later by a colorimetric assay. Averages (+ SE) from triplicate mixed lymphocyte reactions cultures (after subtracting the averages from control wells containing T cells and stimulators, alone) are shown. The different symbols represent the results obtained using individual patient stimulator cells. Prevention of IFN-mediated STAT3 phosphorylation by AG9 made CLL cells, treated concomitantly with PDB and IFN, better able to stimulate T cell proliferation. (D) The percentage of viable cells that excluded 7-AAD was determined by flow cytometry after 48 hours. The average (+ SE) of the results from 5 different patient samples are shown. The arrow indicates a statistically significant value of P < .05. (E) CLL cells from Pt. 304 were cultured with or without IFN and/or AG9 (top) and cell viability after 3 days was indicated by measuring exclusion of 7-AAD by flow cytometry. The number in each dot blot is the percentage of 7-AAD⁺ dead cells. Mean values of the forward scatter parameter for 10 other patient samples are summarized (bottom graphs). “High-risk” cells are defined on the basis of 11q− or 17p− cytogenetic lesions, over 20% CD38⁺ cells, or LDTs < 12 months. AG9 counters the increased cell size caused by IFN in aggressive CLL cells.