Lft 2

CD4 and/or CD8 T cells were depleted by i.p. administration of monoclonal antibodies YTS191 (anti-CD4, BioXcell) and YTS169 (anti-CD8, BioXcell) on days -3 and -1 of infection. Isotype depletion control was done using the antibody LFT-2 (BioXcell). On each day, 300 μg of the respective antibody was administered. The efficiency of the depletion was verified on the day of infection (day 0) by flow cytometry and was > 98%. Specific depletion of CD11b⁺ and CD11c⁺ cells in DTR chimeric mice was accomplished by i.p injection of 0.2 μg of diphtheria toxin (DT) on days -1, 0, 1 and 2 of infection.

CD4 and CD8 T cells were depleted via intraperitoneal administration of anti-CD4 (YTS191, BioXcell) and anti-CD8 (YTS169.4, BioXcell) depleting antibodies three and one day prior to infection (total of 300 μg per application). A control group received an isotype control antibody (LFT-2, BioXcell) at the same time and dose.

The LLC cell line (provided by G Molema, UMCG, the Netherlands) syngeneic to C57bl/6 mice, as well as the C51 and CT26 mouse colon carcinoma cell lines (Philogen S.p.A.) syngeneic to Balb/c mice, were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Lonza), supplemented with 10% fetal bovine serum (FBS) in a humidified 5% CO₂ chamber at 37°C. L19–IL2 (Philogen S.p.A.) was diluted in sterile phosphate-buffered saline (PBS) to 200 µg/mL. For in vivo IC inhibition, rat antibodies directed against cytotoxic T lymphocyte associated protein 4 (anti-CTLA-4, 9D9), programmed death 1 (anti-PD-1, RMP1-14) or its ligand (anti-PD-L1, 10F.9G2) and rat IgG2b control (LFT-2) were diluted in sterile PBS to 2.5 mg/mL (BioXcell). For in vivo cell depletion, rat anti-CD8α (YTS 169.4), rat anti-NK1.1 (PK136), and rat IgG2b control (LTF-2) were diluted in sterile PBS to 2.5 mg/mL (BioXcell).