Ifn γ

Mouse macrophage cell line RAW 264.7 and WBC264-9C was purchased from ATCC (Virginia, USA). DMEM media (RAW 264.7) and grown in Eagle’s minimum essential medium (WBC264–9C) supplemented with 10% fetal bovine serum (FBS), sodium bi-carbonate (1.5 gm/L), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C with 5% CO₂ atmosphere according to ATCC recommendations. Cells were treated with 20 ng/mL GM-CSF (#NBP2–35066, Novus Biologicals), 20 ng/mL M-CSF (#14–8983–62, ThermoFisher), 40 ng/mL IL-4 (#574306, BioLegend), 1000 IU/mL IFNγ (#NBP2–35071, Novus Biologicals), 0.5 μg/mL lLipopolysaccharide (#00–4976–93, ThermoFisher), 25 μM cycloheximide (#0970, R&D Systems) dissolved in serum-free DMEM. All RAW 264.7 cell experiments were done with cells between passage 4 and passage 8. Cells were exposed to a brief period of serum starvation in serum-free DMEM for 3 h followed by stimulation with pro-inflammatory stimuli (LPS, GM-CSF, and IFNγ), anti-inflammatory stimuli (M-CSF and IL-4), and cycloheximide (CHX) at above-mentioned doses for stated durations.

Cytokine levels in the supernatant of cultured cells were measured by an ELISA kit according to the manufacturer’s recommendation. Values were calculated based on a standard curve of recombinant cytokines. The results are expressed as picograms per milliliter (pg/mL). IFN-β (Cat# VAL612, Novus Biologicals) and TNF-α (Cat# VAL609, Novus Biologicals) levels in the supernatant of BMDCs were quantified. For detecting the cytokines level in the supernatant of cultured splenic or pulmonary lymphocytes, cells were cultured at a concentration of 2 × 10⁶/ml and stimulated with the antigens PT (2 μg/ml), FHA (2 μg/ml), and PRN (2 μg/ml). Supernatants were removed after 3 days and stored at -20°C before testing. IFN-γ (Cat# VAL607, Novus Biologicals), TNF-a (Cat# VAL609, Novus Biologicals), and IL-2 (Cat# VAL602, Novus Biologicals) levels were measured for Th1 responses; IL-5 (Cat# KA0253, Novus Biologicals) and IL-6 (Cat# VAL604, Novus Biologicals) levels were detected for Th2 responses; and IL-17A (Cat# VAL610, Novus Biologicals) and IL-22 (Cat# M2200, R&D Systems) levels were tested for Th17 responses (33 (link), 34 (link)).

Human PCa cell lines (PC-3 and DU-145) and mouse PCa cell lines (RM-1) were purchased from the American Type Culture Collection (ATCC, USA). The cell lines were cultured in the recommended media containing 10% FBS and 1% penicillin/streptomycin. The cells were treated with IFN-γ (Novus, USA) to induce PD-L1 expression. RelB was silenced in PC-3 and DU-145 cells by transfection of a plasmid-carrying shRNA duplex targeting RelB (RiboBio Co., Ltd., China) and stable cell clones were selected using G418 (Invitrogen, USA). Additionally, to restore RelB activity in RelB-defective PC-3 cells, a RelB cDNA driven by the CMV promoter in pCMV-Script vector (Stratagene, USA) was transiently transfected into RelB-silenced cells. Furthermore, RelB was knocked out (RelB-KO) in RM-1 cells using a CRISPR/Cas9-based gene-editing system. Briefly, RM-1 cells were transfected with a Cas9-single guide RNA (sgRNA) expression plasmid targeting RelB (sgRNA, 5′-GACGAATACATTAAGGAGAA-3′), followed by puromycin selection. In addition, PD-L1 cDNA was cloned into the pcDNA plasmid and then transfected into RelB-KO RM-1 cells, followed by hygromycin B (Invitrogen) selection to generate a stable cell line.