recombinant il 23  (Proteintech)

Journal: bioRxiv

Article Title: Homozygosity for rare or common hypomorphic IL23R variants confers a predisposition to tuberculosis in humans

doi: 10.64898/2026.03.23.713554

Figure Lengend Snippet: (A) STAT3 activity induced in response to IL-23 (10 ng/mL) by the IL23R variants used for the transient transfection of HEK293T cells, assessed with the luciferase assay. White dots represent negative controls (NT, not transfected and EV, empty vector); green dots, WT condition; red dots, LOF variant; gray dots, homozygous variants from the general population. (B) Western blot of pSTAT3 after stimulation with IL-23 (1 ng/mL) or IFN-α (1 ng/mL) in HEK293T cells transiently transfected with IL23R variants. The graph shows pSTAT3/STAT3 band density as a percentage of that in stimulated WT conditions. At least two independent experiments were performed. (C) CADD /MAF graph for the homozygous IL23R LOF variants previously described (red triangle) in the MSMD cohort, and the missense variants (dots) from the general population and our in-house cohort. The four hypomorphic variants are presented in different colors, whereas the isomorphic variants are in shown in gray. MSC, mutation significance cutoff with a 99% confidence interval. CADD, Combined annotation-dependent depletion. (D) Localization of coding and non-coding LOF (red) or hypomorphic (orange, yellow, blue and violet) IL23R variants across IL-23R protein domains.

Article Snippet: Cells were incubated in the presence or absence of live BCG, at a multiplicity of infection of 1, with or without recombinant human IL-12 (5 ng/mL, R&D), recombinant human IL-18 (25 ng/mL, R&D) and/or recombinant human IL-23 (100 ng/mL, 1290-IL R&D Systems), or IFN-γ (5000 IU/mL).

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Variant Assay, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Homozygosity for rare or common hypomorphic IL23R variants confers a predisposition to tuberculosis in humans

doi: 10.64898/2026.03.23.713554

Figure Lengend Snippet: (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 HEK293T cells transiently transfected with N-terminal NanoLuciferase-tagged versions of the wildtype 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 5-7 independent experiments conducted in triplicate wells. Statistical significance was determined in a paired t-test (*** P <0.001, * P <0.5). Data for NLuc IL-23R C115Y were obtained in a previous study. (B) HEK293T 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 60x NA1.42 oil immersion objective, a 0.5x tube lens, and a C9100-23B IMAGE EMX2 camera (Hamamatsu, Japan), with an exposure time of 20 s (gain of 25). (C) HEK293T 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 (non-specific 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 wildtype vs. NLuc IL-23R R381Q; n = 5 independent experiments performed in triplicate for NLuc IL-23R wildtype vs. NLuc IL-23R G300V; n = 5 independent experiments performed in duplicate for NLuc IL-23R wildtype vs. NLuc IL-23R G149R or NLuc IL23R L372F). (D) Representative results for pSTAT3 detection by western blotting after IL-23 stimulation (10 ng/mL) in EBV-B cell lines from healthy donors (HD: green), IL23R R381Q/R381Q (P12, P17, and P4: violet), IL23R G300V/G300V (P18, P19, and 20: orange) and IL23R LOF/LOF ( IL-23R -/- : red) patients. (E) Representative graph of multiple independent western-blot experiments showing pSTAT3/STAT3 band density. If there were several measurements on the same EBV-B cell lines, this is indicated in parentheses. (G) MFI of pSTAT3 normalized against non-stimulated T-blast cells from healthy donors (green dots), IL23R G149R/G149R (yellow dots), IL23R G300V/G300V (orange dots), IL23R R381Q/R381Q (purple dots), 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 to HD or WT as appropriate. * p < 0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: Cells were incubated in the presence or absence of live BCG, at a multiplicity of infection of 1, with or without recombinant human IL-12 (5 ng/mL, R&D), recombinant human IL-18 (25 ng/mL, R&D) and/or recombinant human IL-23 (100 ng/mL, 1290-IL R&D Systems), or IFN-γ (5000 IU/mL).

Techniques: Transfection, Microscopy, Variant Assay, Labeling, Concentration Assay, Fluorescence, Binding Assay, Western Blot, Control

Journal: bioRxiv

Article Title: Homozygosity for rare or common hypomorphic IL23R variants confers a predisposition to tuberculosis in humans

doi: 10.64898/2026.03.23.713554

Figure Lengend Snippet: (A) Dotplot of the expression of lineage- and state-specific marker genes across predicted immune cell types. (B) Heatmap analysis of the GSEA leading-edge genes for the Hallmark IFN-γ response gene set common to IL-23R-deficient and IL-12Rβ1-deficient classic monocytes. Normalized Z -transformed pseudobulk read counts are shown. (C) The fold-change in IFNG mRNA levels following IL-23 stimulation in leukocytes from 3 IL23R R381Q/R381Q , 2 IL23R G300V/G300V , one IL23R G149R/G149R and 2 TYK2 P1104A/P1104A , 1 IL12RB1- /- and 2 IL23R -/- patients was normalized against housekeeping genes across immune cells. Bulk RNA-seq pseudobulk expression counts for IFNG were normalized by this factor to obtain IFNG.HKG values. The delta score is the difference in normalized expression between IL-23 stimulation and non-stimulation (NS) conditions (IL-23 − NS), reflecting the magnitude of gene induction upon stimulation. (D) Two-dimensional plots of IL-23–induced delta IFNG expression. The fold-change difference in IFNG mRNA levels following IL-23 stimulation in leukocytes from IL23R R381Q/R381Q , IL23R G300V/G300V , IL23R G149R/G149R and TYK2 P1104A/P1104A patients relative to controls is shown on the x -axis. The y -axis shows the same parameter for IL23R -/- patients as a comparison. The color of the circles indicates the median fold-change difference (IL-23 versus NS) in normalized IFNG mRNA levels in controls for the corresponding subsets. MAIT, NK, and Vδ2 + γδT cells are highlighted.

Article Snippet: Cells were incubated in the presence or absence of live BCG, at a multiplicity of infection of 1, with or without recombinant human IL-12 (5 ng/mL, R&D), recombinant human IL-18 (25 ng/mL, R&D) and/or recombinant human IL-23 (100 ng/mL, 1290-IL R&D Systems), or IFN-γ (5000 IU/mL).

Techniques: Expressing, Marker, Transformation Assay, RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: Homozygosity for rare or common hypomorphic IL23R variants confers a predisposition to tuberculosis in humans

doi: 10.64898/2026.03.23.713554

Figure Lengend Snippet: Percent intracellular IFN-γ + induction was monitored by spectral flow cytometry in MAIT (A) , Vδ2 + γδ T cells (B) and NK cells (C) after stimulation with IL-18 (200 ng/mL) and IL-23 (1 ng/mL), alone or in combination for 24 h, or with PMA/ionomycin stimulation for 6 h for frozen PBMCs. The percentages of IFN-γ + and TNF + cells were monitored as a control. The statistical significance of differences was assessed in unpaired Mann-Whitney U tests for comparisons of each variant with HD or WT as appropriate. * p < 0.05, ** p <0.01, *** p <0.001, **** p <0.0001. ( D ) IFN-γ levels in the supernatant were assessed after the stimulation of thawed PBMCs of the indicated genotypes for 24 h with IL-23 (1 ng/mL), IL-18 (200 ng/mL), or both (E) and for 48 h with IL-23 (100 ng/mL), IL-18 (25 ng/mL), or both. The production of IFN-γ and TNF after PMA-ionomycin stimulation was monitored as a control. Nonparametric Mann-Whitney U tests were used for analysis, with * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: Cells were incubated in the presence or absence of live BCG, at a multiplicity of infection of 1, with or without recombinant human IL-12 (5 ng/mL, R&D), recombinant human IL-18 (25 ng/mL, R&D) and/or recombinant human IL-23 (100 ng/mL, 1290-IL R&D Systems), or IFN-γ (5000 IU/mL).

Techniques: Flow Cytometry, Control, MANN-WHITNEY, Variant Assay