IL18BP c.15-16del is loss-of-function. (A) Pedigree and familial segregation of the mutations and schematic representation of IL-18BP. (B) CADD vs. MAF graph for homozygous IL18BP variants from gnomAD and Bravo and the patients’ allele. (C) CoNeS score of IL-18BP. (D) Representative western blot for secreted IL-18BP (His-tag) in non-concentrated supernatant (10 μg protein/lane) (left) and for intracellular IL-18BP (His-tag) in lysates (20 μg protein/lane) (right) from transiently transfected COS7 cells. Data are representative of five independent experiments. (E) Relative luciferase activity (Firefly to Renilla signal ratio) in HEK293 cells transiently transfected with NF-κB–Firefly luciferase + IL-18RAP + Renilla luciferase plasmids upon 12-h stimulation with recombinant IL-18 (5 ng/ml) and/or recombinant WT IL-18BP-His (100 ng/ml) or concentrated supernatant (100 μg/ml of total protein) from COS7 cells transiently transfected with empty, WT, or mutant IL-18BP–tagged constructs. Relative luciferase activity was normalized against non-stimulated (NS) cells, for which the value was set to 1. (F) IFN-γ production detected by ELISA on the supernatant of KG-1 cells stimulated for 24 h with recombinant TNF (20 ng/ml), recombinant IL-18 (20 ng/ml), and/or recombinant WT IL-18BP-His (100–200 ng/ml) or concentrated supernatant (175 μg/ml of total protein) from COS7 cells transiently transfected with empty, WT, or mutant IL-18BP–tagged constructs. (E and F) Data are presented as the mean ± SEM of five independent experiments. All IL-18BP constructs and supernatants were generated as previously described (2). WT, wild-type; M, mutated; E?, unknown genotype; SP, Signal peptide; P, patient; MAF, minor allele frequency; HOM, homozygous; IEI, Inborn errors of Immunity; AD, autosomal dominant. Source data are available for this figure: SourceData F1.