Be0090

Female C57BL/6 mice aged 6–8 weeks were purchased from the Center of Experimental Animals of the Third Military Medical Univercity (TMMU). Nude mice were purchased from VitalStar Biotechnology Co. Ltd. (Beijing, China). The mouse handling protocols were approved by the Institutional Animal Care and Use Committee of TMMU. To establish tumor models, B16F10 cells (2 × 10⁵ in 100 µL of PBS) were subcutaneously inoculated into the right flank of 6–8-week-old female C57BL/6 mice or nude mice. When the tumors became palpable, tumor volume was monitored twice per week. In immunotherapeutic models, 2 × 10⁵ B16F10 cells were subcutaneously inoculated into the right flank of 6–8-week-old female C57BL/6 mice. Mice received 200 µg of intraperitoneal anti-PD-L1 monoclonal antibody (10F.9G2, Be0101, BioXcell) or the equivalent isotype control antibody (BioXcell, BE0090) on days 4, 7, and 10.
For radiation-combined immunotherapeutic models, 2 × 10⁵ B16F10 cells were subcutaneously inoculated into the right legs of C57BL/6 mice. When the tumors reached approximately 50 mm³, the mice were locally irradiated using the Varian Trilogy Stereotactic System at a single dose of 20 Gy. On the same day, 200 µg of anti-PD-1 monoclonal antibody (10F.9G2, Be0101, BioXcell) or equivalent isotype control antibody (BioXcell, BE0090) was injected intraperitoneally every three days three times.

Anti-PD-L1 (1.5 mg; rat IgG2b isotype; clone 10F.9G2; BioXCell; BE0101) or isotype control (1.5 mg; anti-keyhole limpet hemocyanin; clone LTF-2; BioXCell; BE0090) antibodies were administered intraperitoneally (i.p.).
For CCR2 blockade, the anti-CCR2 monoclonal antibody MC21 [29 (link)], was injected i.p. (400 μg) every 4 days.

Female BALB/c mice were purchased from Guangdong Medical Lab Animal Center and housed in a specific pathogen‐free mouse facility. Murine A20 lymphoma cells³¹ (1×10⁶/100 μl), derived from BALB/c mice, were injected subcutaneously into the flank. When the tumour was palpable, cohorts of mice were intraperitoneally injected with IgG (BioXCell, BE0090), 300 μg of anti‐CD70 (BioXCell, BE0022), 200 μg of anti‐PD‐L1 (BioXCell, BE0101), or a combination of these antibodies every 3 days for a total of 4 times. Tumour growth was evaluated by measuring the tumour volume every 3 days. When the mice were sacrificed, the tumours were harvested for further investigation. The institutional animal care and use committee of Sun Yat‐Sen University approved the study.

Mice were treated with anti-CD8⁺ antibody {2.43 (BE0061, Bio X Cell) or control IgG (200 μg per mouse) [rat IgG2b (BE0090, Bio X Cell)]}. Antibodies were injected (intraperitoneally) every 3 days starting 1 day before tumor cell inoculation. Depletion efficiency was assessed using flow cytometry of blood samples collected on day 8 after tumor inoculation.