Anti pd l1 mab

As reported previously for similar cytotoxic assays (20 (link), 21 (link)), 3LL cells were incubated for 15 min at 37°C with 5 μM CFSE in PBS (CFSE^hi 3LL), whereas ID8 reference cells were labeled with 0.5 μM CFSE (CFSE^lo ID8), and then washed extensively. CFSE^hi 3LL and CFSE^lo ID8 were incubated in 24-well plate, respectively. Where indicated, CD103+CD8+ T cells and CD103−CD8+ T cells were added at the ratio of 10:1 to CFSE^hi 3LL, respectively, and cultured with rat IgG isotype control or anti-4-1BB mAb (10 μg/ml; BioXCell) or anti-PD-L1 mAb (10 μg/ml; BioXCell) or the combination of anti-4-1BB mAb (10 μg/ml) and anti-PD-L1 mAb (10 μg/ml) in duplicate. After 8 h, CFSE^hi 3LL were collected by trypsin and moved into 1.2-mL FACS tubes. Before analysis, CFSE^lo ID8 were quantified as the same number of CFSE^hi 3LL without T cells, then mixed with each group of CFSE^hi 3LL. Killing of 3LL cells by CD103+CD8+ T cells or CD103−CD8+ T cells was calculated as 100% × (1-RF_eff/RF_ctrl). RF represents the relative frequency of remaining CFSE^hi 3LL cells to CFSE^lo ID8 reference cells; RF_ctrl represents the RF value in groups without CD103+CD8+ T cells or CD103−CD8+ T cells; and RF_eff represents the RF value in groups with CD103+CD8+ T cells or CD103−CD8+ T cells (21 (link)). Representative results from one of two performed experiments are shown.

The animal experiments were approved by the Ethics Committee of Xiangya Hospital (Central South University) and strictly followed the “3R” principle of experimental animals (Ethics code: 201,803,363). B16-F10 cells were collected, washed with PBS three times, and resuspended in cold serum-free medium. B16-F10 melanoma cells (5 × 10⁵ cells, 100 µL RPMI-1640 medium) were injected into the right flank of 6- to 8-week-old female C57BL/6 mice (Shanghai SLAC Laboratory Animal). When the tumors were visible, the tumor-bearing mice were randomly grouped for intraperitoneal injection of AIL, anti-PD-L1 mAb (Bio X Cell, USA), IgG isotype control (IgG2a) (Bio X Cell, USA), or PBS (vehicle control) for 9–11 days. Tumor diameters were measured with a digital caliper every other day, and tumor volume was calculated according to the formula V = (length × width²)/2. The tumors were immediately collected for flow cytometry analysis.

The sEVs were collected and purified by ultracentrifugation^{2 (link)}. Briefly, before cells were transfected, the serum-containing cell culture medium was removed. After nsEP, the cells were cultured in an exosome-free culture medium for 48 h. Then, the cell-culture supernatants were centrifuged at 2000×g for 10 min to remove debris, and large vesicles and apoptotic bodies were removed by centrifugation at 10,000×g for 30 min. The final sEV fraction was then purified after ultracentrifugation at 100,000×g for 2 h. To prepare imsEVs, CD64-sEVs were incubated with anti-CD71 mAb (Bio X Cell) and anti-PD-L1 mAb (Bio X Cell) (1:1:3, w/w/w, CD64-sEV by protein mass) for 2 h at 37 °C. Subsequently, free antibodies were removed by ultracentrifugation at 100,000×g for 2 h.