Pyridone 6

For flow cytometry and sorting experiments, antibodies to CD4 (RM4-4), NK1.1 (PK136), CD49b (DX5), CD3 (17A2), Ly6G (1A8), B220 (RA3-6B2), CD11c (N418), CD45.2 (104), CD45.1 (A20), CX3CR1 (SA011F11), IA^b (AF6-120.1), CD11b (Mac1), CD90 (G7), lineage markers (17A2/RB6-8C5/RA3-6B2/Ter-119/M1/70), and CD127 (A7R34) were purchased from BioLegend. To stimulate neutrophils in vitro and for in vivo administration, we used either recombinant mouse IFN-λ2 (purchased from Peprotech) or recombinant mouse IFN-λ2 to which polyethylene glycol was attached (provided by Bristol-Myers Squibb). Recombinant mouse IFN-β was purchased from PBL interferonsource. Recombinant human IFN-λ2 and IFN-β were purchased from Peprotech. Puromycin was purchased from Sigma. Where indicated, specific chemical inhibitors were used. To inhibit protein synthesis, cycloheximide (Sigma) was used at a concentration of 10 μg/ml. To inhibit Jak signaling, pyridone 6 (BioVision) was used. To inhibit STAT-1 signaling, fludarabine (Tocris) was used. To inhibit Jak2 signaling, either AG490 (Tocris) or 1,2,3,4,5,6-hexabromocyclohexane (HBC, Tocris) was used. To stimulate neutrophils, either E. coli LPS (Serotype O55:B5 TLR grade, purchased from Enzo) or recombinant mouse TNF (Peprotech) was used.

Cells were pre-treated with 50 nM Pyridone 6 (BioVision) or 10 μM 1,2,3,4,5,6-Hexabromocyclohexane (HBC, Tocris) for 1 h or with the indicated concentrations of baricitinib (Hycultec) or BMS-986165 (Hycultec) for 1.5 h before the indicated amounts of either IFN-α_B/D or recombinant mouse IFN-λ2 or recombinant human IFN-λ1 (68 (link)) was added to the respective culture medium containing the inhibitors.