For quantification of payload expression, A549 cells were infected 24 h after seeding (1 × 105 cells per well of a 24-well plate) at an MOI of 10 TU/cell for single and triple payload-containing viruses. A549 cells infected with a mixture of viruses were infected with the same total MOI of 10 TU/cell, i.e., an MOI of 3.33 TU/cell per single payload vector. Infected cells and cell supernatant were harvested 72 h after infection. Infected cells were stored at −80°C before mRNA levels were quantified (see below). Cytokine secretion was determined from supernatants using a mouse IL-12-p70 sandwich ELISA assay (Invitrogen, 88-7121) for IL-12 and a mouse IL-2 sandwich ELISA kit (Invitrogen, 88-7024) for IL-2, following the manufacturer’s instructions. RMP1-14 expression was quantified via an antigen capture ELISA. A protein-binding 96-well plate (Thermo Fisher Scientific, 44-2404-21) was coated with a 20 nM murine PD-1 (Amsbio, AMS.PD1-M82F4-25UG) solution at 4°C overnight (100 μL/well). Following blocking with 1× casein (Merck, B6429), the cell supernatant or a standard of recombinantly produced and purified RMP1-14 antibodies were applied. Following three wash steps with PBS + 0.1% Tween 20, an alkaline phosphatase-coupled anti-mouse κ-LC detection antibody (SouthernBiotech, 1050-04) was added. After wash, para-nitrophenylphosphate (p-NPP) was used as a substrate, and absorbance at 406 nm was measured with an Infinite M1000 microplate reader (Tecan). Antibody concentrations were determined from a standard curve of recombinantly produced RMP1-14 protein using a sigmoidal four parameter fit.

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