Recombinant murine il 18

For rIL33, mice were treated with intraperitoneal (i.p.) injections of 500 ng of carrier-free recombinant murine IL33 (R&D Systems) in sterile PBS daily for 7 days, and then every 2 days thereafter as previously described^{36 (link)}. For rIL18, mice were treated with i.p. injection of 2 μg of carrier-free recombinant murine IL-18 (R&D Systems) in sterile PBS at days 3, 7, 11, and 15 after tumor inoculation as previously described^{42 (link)}. The chimeric anti-mouse PD-1 antibody (4H2) used in this study engineered as a mouse IgG1 isotype monoclonal antibody (mAb) was shown to bind to CHO transfectants expressing PD-1 and block binding of PD-L1 and PD-L2 to these cells. The affinity of 4H2 for mouse PD-1, determined by surface plasmon resonance using PD-1-Fc, was 4.68×10⁻⁹ M. The antibody was produced and purified at Bristol Myers Squibb (BMS). Each batch was certified to have <0.5 EU/mg endotoxin and be of >95% purity. All dosing solutions were prepared in PBS. Mice were treated with i.p. injection of 250 μg anti-PD1 every 2 days. Transient reduction in tumor size but subsequent regrowth while on continuous αPD-1 treatment was defined as a partial response. No reduction in tumor size while on continuous αPD-1 was defined as resistance.

iSell^Tomato mice were infected with AdV and CD45⁺CD62L⁻CD4⁺Tomato⁺ IE T cells were sorted 10 days post infection. Sorted T cells were cultured in RPMI 1640 (Gibco), 10% FBS (Sigma), 1% Pen/Strep (Gibco), 1% L-glutamine (Gibco), 1% Sodium Pyruvate (Gibco), 2% Non-essential Amino Acids (Gibco), 2.5% 1M HEPES (Gibco), 50μM 2-Mercaptoethanol (Sigma), 10 ng/ml recombinant murine IL-2 (R&D) and 5 ng/ml recombinant murine IL-7 (R&D) at 10–20.000 cells/well in a 96-well round bottom plate (Corning). T cells were stimulated with 10 ng/ml recombinant murine IL-12 (R&D), 10 ng/ml recombinant murine IL-15/IL-15Rα complex (ThermoFisher) and 10 ng/ml recombinant murine IL-18 (R&D). Supernatants were collected 24-hour post stimulation and IFN-γ was measured with IFN-γ ELISA (Invitrogen) by following manufacturer’s instructions.