Binding of the F8 moiety to its cognate antigen was tested by ELISA. Briefly, wells of an F96 Maxisorp nunc-immuno plate (Thermo Fisher) were coated with EDA and blocked with 2% milk powder in phosphate-buffered saline (PBS). F8-LIGHT and positive control F8 in small immune protein format (SIPF8) were added at different concentrations in wells, followed by incubation with polyclonal antibodies against human kappa light chains (Dako, A0192) and by HRP-conjugated protein A (GE Healthcare, NA9120V). Colorimetric reaction was started by adding BM Blue POD substrate (Roche, 11442066001) and quenched with 1 M H2SO4. Absorbance at 450 nm was measured with a Spectra Max Paradigm multimode plate reader (Molecular Devices). In vitro activity of the LIGHT moiety was tested with a cytotoxicity assay on HT-29 cells, which have been shown to be sensitive to LIGHT in the presence of human IFN gamma (hIFNγ).50 Briefly, F8-LIGHT and recombinant hIFNγ (Biolegend, 570202) at given concentrations were added to wells containing 40000 HT-29 cells in McCoy’s 5A (Modified) Medium (Thermo Fisher, 16600082) supplemented with 10% fetal bovine serum (Thermo Fisher, 10270106) and 1x Antibiotic-Antimycotic (Thermo Fisher, 15240062). After incubation for 72 hours at 37°C, 5% CO2, cells were detached using Trypsin-EDTA solution (Thermo Fisher, 25200056), re-suspended in PBS containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA and stained with 7-AAD (Biolegend, 420404) for 5ʹ at 4°C. The percentage of living cells was determined by flow cytometry analysis using a CytoFLEX S (Beckman Coulter). Data were analyzed with FlowJo software (FlowJo, LLC, version 10).

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