Igg control clone 2a3

Individual mice were injected s.c. with 2–5 × 10⁶ Lewis lung cancer cells (LLC1), or by injecting a total of 1 × 10⁵ LLC1-shLkb1-luc cells in 100 μL PBS-matrigel mixture into the left chest (orthotopic tumor model). Seven days after injection, the tumor-bearing mice were randomly separated into four groups. Anti-PD-1 antibody (200 µg/mouse, clone RMP1-14, BioXCell) was injected i.p. three times a week (days 1, 3, and 5 of a 7-day cycle). For CDK4/6 inhibitor (palbociclib, Selleck, S1116, 100 mg/kg), mice were treated p.o. on days 1, 2, and 3 of a 7-day cycle. The combination group was treated with both anti-PD-1 antibody and CDK4/6 inhibitor, and the control group was injected with IgG control (clone 2A3, BioXcell). The tumors were measured in two dimensions (length and width), and volume (V) was calculated as V = length × width² × 0.5. Two weeks after treatment, tumors were collected and processed for infiltrating lymphocyte isolation or immunohistochemistry. The vast majority of tumors did not exceed the 2000 mm³ permitted by our animal protocol. In some cases, this limit was exceeded on the last day of measurement and the mice were immediately euthanized.

For in vivo neutrophil depletion, 300 μg anti-L6G clone 1A8 (BioXcell) mAb or control IgG clone 2A3 (BioXcell) were administered intraperitoneally at days −2, 0 and +2, +4, +6 post transcervical challenge. For neutralization of signaling through the IFN-α/β receptor, mice were treated intraperitoneally with 500 μg per mouse of anti-IFNAR1 clone MAR1-5A3 or control IgG clone MOPC-21 (BioXcell) in phosphate-buffered saline (PBS) at days −1 and +1, +3, +5 post transcervical challenge. For neutralization of IFN-γ, 500 μg anti-IFN-γ clone XMG1.2 mAb or control IgG clone TNP6A7 (BioXcell) were administered intraperitoneally at days −1 and +2, +5 post transcervical challenge.