Ifn λ2

Huh7, HepG2, U87, U373, A549, 293T, BHK-21, B16, BNL, and NIH3T3 cells were maintained in high glucose DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 1× MEM nonessential amino acids, and 10 mM HEPES (Invitrogen). Jurkat cells were cultured in complete RPMI 1640 medium. Cryopreserved PHHs from HIV, HBV, and HCV-free donors were purchased from BD Biosciences. Fresh PHHs were also obtained from the Cellular and Molecular Physiology core facility of the Yale Liver Center. Primary human astrocytes and HCMV-GFP were kindly provided by Dr. Anthony van den Pol (Yale University). Primary human T lymphocytes were originally obtained from the New York Blood Bank, and the CD4⁺ population was purified and kindly provided by Dr. Walther Mothes (Yale University). CD4⁺ T cells were cultured in complete RPMI 1640 medium supplemented with 100 U/ml IL-2 and 100 ng/ml IL-7 and stimulated with 10 µg/ml PHA every 72 h. Cells were treated with recombinant human IFN-α2a, IFN-β, human IFN-λ1, IFN-λ2, IFN-λ3 (R&D Systems), murine IFN-λ2 (PeproTech), or universal type I IFN (hIFN-αA/D) (PBL Interferon Source). DNMT inhibitor 5azadC and HDAC inhibitors SAHA, TSA, sodium butyrate, MS-275, apicidin, tubacin, and nicotinamide were purchased from Selleckchem.

ELISAs for analysis of human IL-8, IFN-β, IFN-λ1, and IFN-λ2 (all from R&D Systems) were performed according to the manufacturer’s protocol.

Naive mice were intranasally administered recombinant mouse IL-13 (15 µg/35 µL; Biolegend), IL-5 (15 µg/35 µL; Biolegend) or a low or high dose (0.12 µg/50 µL and 10 µg/50 µL ,respectively) of lipopolysaccharide (LPS) (Escherichia coli, 0111:B4; Sigma-Aldrich), with samples collected 24 h later. HDM-sensitised and challenged Tlr7^−/− mice, which also received live RV1B, were intranasally administered either recombinant mouse IFNα2 (10 000 IU/50 µL; eBioscience), IFNβ1 (10 000 IU/50 µL; Biolegend), IFNλ2 (1 µg/50 µL; R&D Systems)14 (link) or a vehicle control (phosphate buffered saline) on day 18 (2 h following RV1B infection). Mice were sacrificed 24 h postinfection and samples collected.

Cells were seeded in 12-well plates and treated for 8 hours in biological quadruplicates with IFNα (0.5 ng/ml, R&D Systems), IFNβ (0.5 ng/ml, GenScript), IFNγ (1 ng/ml, R&D Systems), IFN-λ1 (5 ng/ml, R&D Systems), IFN-λ2 (60 ng/ml, R&D Systems), and custom IFN-λ3 (20 ng/ml) and IFN-λ4 (50 ng/ml) (25 (link)), with no treatment used as a negative control.