Impaired expression and/or conformation of IL-23R and responses to IL-23 in patient cell lines. (A) Emitted luminescence measured in the presence of the NanoLuc inhibitor is expressed as a percentage of that measured in the absence of the inhibitor (100%) in HEK cells transiently transfected with N-terminal NanoLuciferase-tagged versions of the WT or variants of the IL-23 receptor (NLuc IL-23R, NLuc IL-23R R381Q, NLuc IL-23R G300V, NLuc IL-23R G149R, NLuc IL-23R L372F, or NLuc IL-23R C115Y) and IL-12Rβ1 (4:1 ratio). The data shown are the mean ± SEM from five to seven independent experiments conducted in triplicate wells. Statistical significance was determined in a paired t test (***P < 0.001, **P < 0.01 and *P < 0.05). Data for NLuc IL-23R C115Y were obtained in a previous study (Lay et al., 2023). (B) HEK cells transiently transfected as described in A were imaged with an Olympus LuminoView 200 wide-field luminescence microscope. Representative luminescence images show the signal originating from the N-terminal NLuc tag of each IL-23R variant following the addition of furimazine (final dilution 1:400). Data from three independent experiments are shown. The scale bar represents 50 μm. Luminescence images were acquired with a 60× NA1.42 oil immersion objective, a 0.5× tube lens, and a C9100-23B IMAGE EMX2 camera (Hamamatsu, Japan), with an exposure time of 20 s (gain of 25). (C) HEK cells transiently transfected as in A were treated with various concentrations of fluorescently labeled IL-23 (IL-23–TAMRA) in the presence or absence of unlabeled IL-23. The NanoLuciferase substrate furimazine was then added (final concentration: 7.7 μM), and emitted luminescence and fluorescence were simultaneously detected with a BMG PHERAstar FS. BRET ratios were calculated by subtracting fluorescence from luminescence. The specific binding of IL-23–TAMRA was calculated by subtracting BRET ratios determined in the presence of unlabeled IL-23 (nonspecific binding) from those obtained in its absence. The data shown are the mean ± SEM (n = 4–5 independent experiments performed in triplicate wells for NLuc IL-23R WT versus NLuc IL-23R R381Q; n = 5 independent experiments performed in triplicate for NLuc IL-23R WT versus NLuc IL-23R G300V; n = 5 independent experiments performed in duplicate for NLuc IL-23R WT versus NLuc IL-23R G149R or NLuc IL23R L372F). (D) Representative results for pSTAT3 detection by western blotting after IL-23 stimulation (10 ng/ml and/or 1 ng/ml) in EBV–B cell lines from healthy controls (HD: green), IL23R^R381Q/R381Q (P12, P17, and P4: violet), IL23R^G300V/G300V (P18, P19, and P20: orange), IL23R^G149R/G149R (P24 and P25: yellow), and IL23R^LOF/LOF (IL-23R^−/−: red) patients. (E) Representative graph of multiple independent western blot experiments showing pSTAT3/STAT3 band density. The number of cell per genotype is indicated in the figure (n = ). If there were several measurements on the same EBV–B cells, this is indicated in parentheses. Statistical significance was assessed in unpaired Mann–Whitney U tests comparing each variant with HD or WT as appropriate (****P < 0.0001). (F) MFI of pSTAT3 normalized against non-stimulated T cell blasts from healthy controls (green dots), IL23R^G149R/G149R (yellow dots), IL23R^G300V/G300V (orange dots), IL23R^R381Q/R381Q (purple dots), and IL-12Rβ1– and IL-23R–deficient patients (blue and red dots, respectively) in the presence or absence of various concentrations of IL-23. IFNα2 stimulation (1 ng/ml) was used as a control. Statistical significance was assessed in unpaired Mann–Whitney’s U tests comparing each variant with HD or WT as appropriate (****P < 0.0001). Source data are available for this figure: SourceData F2.