Sim0010

Lung cancer cell lines were transplanted into syngeneic wild-type C57BL/6 male mice or humanized mouse models by subcutaneous injection into the right flanks at 6 weeks of age. Tumors formed until volumes were ~ 150-200mm³ as measured by digital calipers before treatments were administered. Mice were treated weekly with 200 µg of anti-mouse PD-1 mAb (BE0146, Bio X cell) or anti-human PD-1 mAb (SIM0010, clone Pembrolizumab, Bio X Cell) by intraperitoneal injection. Control mice received solvent at a volume equal to the drug dosage at the indicated drug concentrations or 200 µg of isotype control (Bio X Cell). Tumor growth measurement were performed with calipers on the days as indicated in figures. Tumor sizes were calculated by using the formula: V = (S²× L)/2, where V is the volume, S is the shortest diameter, and L is the longest diameter.

Prior to initiating the coculture, macrophages were labeled with CellTrace Violet (Invitrogen C34557) and target cells were labeled with 1:1000 diluted CellTrace CFSE (Invitrogen C34554) or CellTrace Yellow (Invitrogen C34567) for 30 minutes at 37 °C. In some conditions, macrophages were pre-treated with 2μM cytochalasin D (Cayman Chemical 11330), 10μg/mL anti-PD1 antibody (BioXCell SIM 0010) or 10μg/mL human IgG4 isotype control antibody (BioXCell CP147) prior to co-culture with target cells. Macrophages and target cells were cocultured at a 5:1 E:T ratio for 3 hours at 37 °C in ultra-low attachment 96-well plates (Corning 7007), unless otherwise indicated. Cells were stained for viability with Zombie NIR (BioLegend 423106) and in some experiments with APC-PD-1 antibody (BioLegend 329908). Data was acquired on SONY SA3800 spectral flow cytometer and analyzed using FlowJo software.