Ifn α2b

For stimulation with type I IFN, IFN-α-2b (11105-1, PBL, Piscataway, NJ, USA) was used. A VeriKine™ Human IFN-Alpha ELISA kit was used to detect type I IFN (PBL IFN Source, Piscataway, NJ, USA) from supernatants of AGS cells, and human IFN-γ was detected by ELISA using the Quantikine™ kit (R&D Systems, Minneapolis, MN, USA).

Human CD4⁺IL-10⁺ T cells were induced in vitro as per previously described protocols [21 (link)]. Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy donor blood samples (Central Blood Bank in Yantai, China) using gradient separation with Ficoll-Hypaque (1.077 g/mL; Solarbio, Beijing, China). PBMCs were cultured using the RPMI-1640 medium (HyClone, Boston, MA) and incubated at 37°C, and naive CD4⁺ T cells were separated using a naive CD4⁺ T cell isolation kit (human; Miltenyi Biotec, San Diego, CA) according to the manufacturer’s instructions.
Naive CD4⁺ T cells were treated with or without the Nrf2 inhibitor-ML385 (10 μM, MCE, NJ, USA) for 12 h, and then naive CD4⁺ T cells were cultured with or without hPMSCs. Anti-human CD3ε and anti-human CD28 mAb (1 μg/mL, Life Technologies, Waltham, MA, USA), recombinant human (rh) IL-2 (200 U/mL, Proteintech, Wuhan, China), IL-10 (100 U/mL, Proteintech), IL-27 (63 U/μL, Proteintech), and IFN-α2b (2.09 U/μL, PBL Assay Science, NJ, USA) were added to the coculture system. After 3 days, the cells were stimulated using PMA/ionomycin (1.25 μg/mL, 0.25 mg/mL, MultiSciences, Hangzhou, China) and BFA/monensin (0.75 mg/mL, 0.25 mg/mL, MultiSciences) for 5 h and subjected to surface staining and intracellular staining procedures.

l-KYN and QUNA were purchased from Sigma-Aldrich (St. Louis, MO). l-TRP was purchased from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). l-KYN sulfate (ring-D4, 3, 3-D2) (was purchased from Cambridge Isotope Laboratories (Tewksbury, MA). Hippuric acid-d5 were purchased from Toronto Research Chemicals (Toronto, Canada). All other solvents and reagents used were commercially available LC/MS or HPLC grade.
Phorbol 12-myristate 13-acetate (PMA) was obtained from Sigma-Aldrich. The recombinant human cytokines used in this study and their providers were as follows: interferon (IFN)-α 2a and IFNα-2b, PBL Assay Science (Piscataway, NJ); IFNγ and interleukin (IL)-4, Thermo Fisher Scientific (Waltham, MA); IFNβ, IL-1β, and tumor necrosis factor α (TNFα), R&D systems (Minneapolis, MN); IL-6 and IL-10, BioLegend (San Diego, CA). All cytokines were prepared at 10 µg/mL in phosphate-buffered saline (PBS) supplemented with 10% fetal bovine serum (FBS) and stored at − 80 °C.

A549 WT and ISG15 KO cells were co-transfected with two Herc5 or Ifnar1-targeting gRNA CRISPR/Cas9-GFP plasmids, respectively. HEK293 WT cells were transfected with three Isg15-targeting gRNA CRISPR/Cas9-GFP plasmids (Supplementary Table S1) (Horizon Cambridge, UK). After 72 h, cells were sorted by FACS (FACSMelody, BD) and single-cell derived clones were initially screened by PCR genotyping. Additionally, both HERC5 and ISG15/IFNAR1 (dKO) clones were functionally tested by assessing their ISGylation profile and expression of ISGs, respectively, after IFNα priming. Briefly, A549 WT and HERC5 and dKO clones were primed with IFNα2b (100 IU/ml) (PBL Assay Science) for 24 h and the expression of ISG15-conjugates and IFIT3 was analyzed by Western blot. HEK293 cells were primed with IFNα2b (1000 IU/ml) for 8 h, total RNA was isolated and Isg15 mRNA expression was assessed by RT-qPCR.