Af1021

One week after transduction, 10⁶ genetically engineered OT1 T cells were seeded in a 24-well plate in 1 ml of serum-free RPMI medium at 37 °C for 72 h. Then, the supernatant was harvested and tested for each molecule by ELISA. PD-1 IgG4 homemade-ELISA: coating antibody: anti-mouse PD-1 (R&D, AF1021, final concentration of 2 µg ml⁻¹; BioLegend, 421701). Detection antibody: anti-hIgG4-HRP (clone HP6023, Abcam; 1:2,000 dilution). IL-2v homemade ELISA: coating antibody: purified polyclonal anti-human IL-2 antibody (R&D, final concentration of 3 μg ml⁻¹ in coating buffer, BioLegend, 421701). Secondary antibody: biotin polyclonal anti-human IL-2 antibody (Invitrogen; 1:500 dilution) and streptavidin-HRP (BioLegend; 1:1,000 dilution). Supernatants from OT1 T cells transduced for expressing either the fusion molecule TIM-3–IgG4 or IL-3 variant were used as negative controls. For detection of IL-33, a commercial ELISA kit was used (LEGEND MAXTM mouse IL-33 ELISA kit, BioLegend).

GSTZ1, VEGFA, HIF-1α, PD-L1, PD-1, and CD31 were detected by IHC as previously described (50 (link)). In brief, liver tissue samples from paraffin-embedded human or mouse tumors were incubated at 4°C overnight with dilutions of the indicated primary antibodies (anti-VEGFA, ab1316, Abcam; anti–HIF-1α, ab51608, Abcam; anti–PD-L1, 13684, Cell Signaling Technology; anti–mPD-1, AF1021, R&D Systems, Bio-Techne; anti-CD31, 77699, Cell Signaling Technology). The slides were then incubated with a secondary anti-rabbit or anti-mouse IgG (ZSGB-BIO) and visualized using 3,3′-diaminobenzidine (ZSGB-BIO). Stained slides were scanned using the Motic Easy Scanner. Images were acquired using DSA assistant Lite (Motic VM V1 Viewer 2.0).
For tissue immunofluorescence staining, freshly prepared frozen liver sections were blocked with 5% goat serum for 1 hour at 25°C, then incubated with anti-CD31 (at 1:200 dilution in PBS, ab222783, Abcam) overnight at 4°C. Next, bound primary antibody was detected using an Alexa Fluor 488–conjugated goat anti-mouse IgG (A-10680, Invitrogen), and the nuclei were stained with DAPI (Roche, 1:200). The samples were then mounted with Fluoromount-G (Southern Biotech), and immunofluorescence images were acquired using a confocal microscope (FV3000, Olympus). The images were further processed using ImageJ 1.49v (NIH) or OlyVIA VS200 (Olympus).