Be0297

Cytotoxicity assays were performed as previously described^{32 (link),33 (link)}. In brief, 100,000 murine splenocytes were stimulated with Dynabeads™ Mouse T-Activator CD3/CD28 and cultured in the presence or absence of 10 µg/mL of either vdPVR-Fc, mutant vdPVR-Fc, human IgG1 isotype control (BE0297; BioXCell) or anti-CTLA4 (Yervoy; Bristol-Myers Squibb). Following a 4-h incubation period at 37°C, supernatants were collected, and cell cytotoxicity assayed using a lactate dehydrogenase kit (ab65393; Abcam). Percent cytotoxicity was calculated with the following formula: cytotoxicity (%) = ((Test Sample − Low Control)/(High Control − Low Control)) × 100.

The functionality of CAR-iMACs was assessed by co-culturing them with luciferase-expressing tumor cells in a 96-well plate (WHB-96-2). CAR-iMACs and 2 × 10³ tumor cells were mixed in a total volume of 100 μL RPMI 1640 complete medium, and the number of cells was determined based on the indicated E:T ratio. After co-culturing for 24 or 48 h, 25 μL of 33 mg/mL D-Luciferin (GoldBio, 115114-35-9) was added to each well, and luminescent signals were analyzed using a microplate reader (TECAN, SPARK).
For the 4-OI supplement assay, iMACs were pre-treated with 4-OI (MCE, HY-112675, 250 μM) or DMSO (Sigma-Aldrich, 41639) control for 3 h before challenging them with LPS (InvivoGen, tlrl-eblps) plus human IFN-γ (PeproTech, 300-02-100UG) (50 ng/mL each). The iMACs were then co-cultured with luciferase-expressing tumor cells.
To test the function of NRF2, SFN (MCE, HY-13755, 10 μM) was added to the co-culture system.
To test the function of cytokines, the supernatant was collected after iMACs were co-cultured with HO-8910 cells for 24 h (E:T = 10:1). Then human IgG1 isotype control (BioXcell, BE0297), neutralizing antibody (10 μg/mL) of IFN-γ (BioXcell, BE0235), or TNF-α (BioXcell, SIM0006) was added to the supernatant, and the cytotoxicity activity was measured by luciferase assay.