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:

Article Snippet: Plasmids containing the WT human IL23R (Origene #RG211477) ORF were obtained, the tag was removed, and the variants studied here were generated by site-directed mutagenesis with specific primers and CloneAmp HiFi PCR premix (Takara).

Techniques: Variant Assay

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: Plasmids containing the WT human IL23R (Origene #RG211477) ORF were obtained, the tag was removed, and the variants studied here were generated by site-directed mutagenesis with specific primers and CloneAmp HiFi PCR premix (Takara).

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: Plasmids containing the WT human IL23R (Origene #RG211477) ORF were obtained, the tag was removed, and the variants studied here were generated by site-directed mutagenesis with specific primers and CloneAmp HiFi PCR premix (Takara).

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: Plasmids containing the WT human IL23R (Origene #RG211477) ORF were obtained, the tag was removed, and the variants studied here were generated by site-directed mutagenesis with specific primers and CloneAmp HiFi PCR premix (Takara).

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

Journal: European journal of immunology

Article Title: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice

doi: 10.1002/eji.201546095

Figure Lengend Snippet: IL-23p19 and IL-27/IL-35Ebi3 can form a heterodimer, termed IL-39. (A) Detection and characterization of human IL-39 by immunoprecipitation (IP) and Western blot (WB) analyses under reducing conditions. (B) Schematic of the cDNA constructs used to genetically engineer mouse p19 and Ebi3 proteins. hPGK: human phosphoglycerate kinase 1 promoter. (C) Detection and characterization of mouse IL-39 recombinant proteins by IP and Western blot analyses under reducing conditions. (D) Coomassie Blue gel of the recombinant mouse IL-39 characterized on nonreducing polyacrylamide gels. Arrow indicates recombinant IL-39. (A, C, and D) Blots are representative of three independent experiments.

Article Snippet: Immunoprecipitation and Western blot analysis A 5 ng/mL human IL-27/IL-35 Ebi3 subunit (Origene) and 5 ng/mL human IL-23 p19 subunit (Origene) were mixed in PBS.

Techniques: Immunoprecipitation, Western Blot, Construct, Recombinant

Journal: European journal of immunology

Article Title: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice

doi: 10.1002/eji.201546095

Figure Lengend Snippet: In vitro activated B cells expressed IL-39. (A, B) Primary B cells were sorted from 8-weeks-old female C57BL/6 mice by B220 microbeads, stimulated for 48 h with LPS, and analyzed by FACS. (A) The percentages of IL-39-expressing B cells and (B) statistical analysis of the percentage of IL-12 family cytokine subunits are shown. Isotype was used as the staining control. (C) The concentration of IL-39 in the cultured supernatant from LPS-stimulated B cells was measured by sandwich ELISA by using anti-p19 and Ebi3 antibody as coated and detected antibody, respectively. (B and C) Data are shown as mean + SEM (n = 8) from one experiment representative of three other similar experiments. (D) Detection of mouse natural IL-39 proteins in the cultured supernatant on days 3 after B cells were stimulated with LPS by IP and Western blot analyses under reducing conditions. Blots are representative of three independent experiments *p < 0.05, **p < 0.01, ***p < 0.001 (two tailed Student’s t-test).

Article Snippet: Immunoprecipitation and Western blot analysis A 5 ng/mL human IL-27/IL-35 Ebi3 subunit (Origene) and 5 ng/mL human IL-23 p19 subunit (Origene) were mixed in PBS.

Techniques: In Vitro, Expressing, Staining, Control, Concentration Assay, Cell Culture, Sandwich ELISA, Western Blot, Two Tailed Test

Journal: European journal of immunology

Article Title: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice

doi: 10.1002/eji.201546095

Figure Lengend Snippet: IL-39 induces inflammation in lupus-like Mice. (A) GL7+ B cells were sorted from 8-month-old female lupus-like MRL/lpr mice were sorted by FACS and infected with control, IL-39 subunits p19 or Ebi3-specific shRNA. On day 1 after infection, p19 and Ebi3 mRNA expression were analyzed by qPCR. (B–D) A 5 × 106 control, p19 or Ebi3-specific shRNA-infected GL7+ B220+ B cells per mouse were i.v. injected into 8-week-old female lupus-like MRL/lpr mice (six mice per group). Eight-week-old female lupus-like MRL/lpr mice into which either no cells, or transferred with control shRNA-infected GL7+ B cells, were used as no-cells transfer (None) and shRNA (Control) controls respectively. (B) On day 14 after cell transfer, spleens were harvested and photographed (representative of six spleens per experiments from three independent experiments). (C) Proteinuria was measured on day 0 and day 4 after cell transfer. (D) Absolute numbers of B220+ B and GL7+ B per spleen on days 7 after cell transfer. (A, C, and D) Data are shown as mean + SEM (n = 6) from one experiment representative of two other similar experiments. *p < 0.05, **p < 0.01 (two tailed Student’s t-test).

Article Snippet: Immunoprecipitation and Western blot analysis A 5 ng/mL human IL-27/IL-35 Ebi3 subunit (Origene) and 5 ng/mL human IL-23 p19 subunit (Origene) were mixed in PBS.

Techniques: Infection, Control, shRNA, Expressing, Injection, Two Tailed Test

Journal: European journal of immunology

Article Title: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice

doi: 10.1002/eji.201546095

Figure Lengend Snippet: IL-39 signals through IL-23R and gp130 receptor subunits and activates STAT1 and STAT3 pathways. (A) Primary B cells were sorted from 8-week-old female C57BL/6 mice by B220 microbeads, stimulated for 48 h with LPS, washed, and starved for 2 h in serum- free medium (0.5% BSA), followed by stimulation for 30 min with medium, p19, Ebi3, or IL-39. STAT activation was analyzed by Western blotting. Blots are representative of four independent experiments. (B) Primary B cells were sorted from 8-week-old female C57BL/6 mice by B220 microbeads, stimulated for 24 h with LPS in the presence of medium p19, Ebi3, or IL-39. IL-39 subunits (p19 and Ebi3) mRNA was determined using qPCR assay. Data are shown as mean + SEM (n = 8) from one experiment representative of two other similar experiments. (C) Control or IL-23R, IL-27Ra, gp130-specific shRNA-infected B cells described in Supporting Information Fig. 3C were stimulated for 2 days with LPS, washed, and starved for 2 h in serum-free medium (0.5% BSA), followed by stimulation for 30 min with IL-39. Cell lysates were separated by 8% SDS-PAGE and immunoblotted for phospho-Tyr701-STAT1 (pSTAT1) and total STAT1 (upper and left panel) or phosphor-Tyr705-STAT3(pSTAT3)andtotalSTAT3(upper and right panel). Band intensities of pSTAT1 and STAT1 or pSTAT3 and STAT3 were quantified by ImageProPlus 5.0 software. The density ratios of phosphorylated to total protein compared to control shRNA-infected group are shown as mean ± SEM (n = 3) of three independent experiments (lower panel). *p < 0.05, **p < 0.01 (two tailed Student’s t-test).

Article Snippet: Immunoprecipitation and Western blot analysis A 5 ng/mL human IL-27/IL-35 Ebi3 subunit (Origene) and 5 ng/mL human IL-23 p19 subunit (Origene) were mixed in PBS.

Techniques: Activation Assay, Western Blot, Control, shRNA, Infection, SDS Page, Software, Two Tailed Test