Fig 2: Patient fibroblasts show different OSMR glycosylation and impaired signaling downstream of OSM. (A) Endogenous OSMR expression in fibroblast lysates from P1 and three healthy controls (HCs) with or without PNGase treatment. (B) Quantification of OSMR band intensities relative to GAPDH from A. (C and D) OSMR production of dermal fibroblasts from P1 and HCs treated with 25 µg/ml protein synthesis inhibitor (cycloheximide, CHX; 1M) for 24 h, and (D) quantification of WT OSMR bands and variant OSMR band intensities relative to GAPDH from C. Experiments shown in A and C were repeated twice with dermal fibroblasts from the patient and three HCs each. (E and F) Dermal fibroblasts from P1 and three HCs were stimulated with OSM (5 ng/ml) for 30 min. Whole-cell lysates were harvested for WB for the visualization of pSTAT3, STAT3, pSTAT5, STAT5, pERK1/2, ERK1/2, and GAPDH and quantification of WB band intensities shown in F. (G–J) Serum-starved dermal fibroblasts from P1 and three HCs were stimulated with IL-6 (100 ng/ml), IL-31 (100 ng/ml), or IL-11 (100 ng/ml) for 30 min. Whole-cell lysates were harvested for WB (G and I) for the visualization of pSTAT3, STAT3, and GAPDH and band intensities quantitated (H and J). (K) Dermal fibroblasts from P1 and HCs were stimulated with OSM (10 ng/ml) for 2 or 6 h. Total RNA was harvested for expression analysis by RT-PCR after 2 h for IL-6 and after 6 h for IRF7, CXCL10, and ICAM1. Data are representative of three independent experiments performed in quadruplicates. Statistics were calculated using the unpaired t test and comparing pooled samples from controls to patient. UT, untreated. * = P < 0.05, ** = P < 0.01. Source data are available for this figure: SourceData F3.

Fig 3: Examination of OSMR surface expression on T and B cells. (A) Representative flow plots of OSMR (APC) expression of ex vivo–unstimulated CD4+ CD8+ T cells from one of two healthy controls (HCs) and one P1. (B) Histograms of 5 days anti-CD3/CD28-stimulated CD4+ CD8+ T cells from P1 showing the OSMR-APC channel. (C) PBMCs (500,000 cells) from HCs were incubated with CpG (ODN 2006, 2 µg/ml) alone (left panel) or CpG (2 µg/ml), BAFF (10 ng/ml), and IL-21 (10 ng/ml) (right panel) in the BCM for various days as shown. The vertical axis of the flow plots shows the progression of CD27, and the horizontal axis of the flow plots shows the OSMR expression.

Fig 4: Identification of biallelic OSMR variants in a patient with HIES. (A) Characteristic facies with coarse features and poor dentition. (B) Computed tomography of the chest with findings consistent with CCPA. A single pulmonary cavity (arrowheads) containing a “fungal ball” is present in the right upper lobe: the transverse picture is seen from below and the X ray is looking at the patient from the front, ie inverted right/left. Upper part: Axial view. Bottom part: Coronal view. (C) Table with genetic information on the OSMR variant identified in P1. (D and E) Immunoblot of OSMR in NHDFs from healthy controls (HCs) and patient (P1), bands were quantified by ImageJ (E). (F and G) FC of surface expressed OSMR in NHDFs from three healthy controls (HCs) and patient (P1) (F) with the mean of fluorescence intensities (MFI) plotted (G). Similar results from western blot and FC have been observed twice prior. Statistics were calculated using the unpaired t test. ** = P < 0.01. Source data are available for this figure: SourceData F1.

Fig 5: Functional evaluation of predicted potentially deleterious homozygous missense OSMR variants from the general population (overexpressed in OSMR- and LIFR-deficient HEK293T cells). (A) All homozygous OSMR variants from gnomAD v4.1 plotted as CADD score versus MAF. The red dot refers to the variant of P1. Dotted line represents the MSC (>19.3) with 95% confidence interval. A total of 11 homozygous missense OSMR variants predicted to be potentially deleterious (CADD score >20) from gnomAD v4.1 are listed in the table with the variant identified in P1 shown in red. (B) Localization of the predicted deleterious OSMR variants in different domains of the OSMR protein with the variant in P1 shown in red. CBD, cytokine binding domain. (C) The surface receptors OSMR and LIFR known to bind OSM were knocked out in HEK293 cells by the CRISPR/Cas9 technique. KO efficiencies of two SCCs were determined by EditCo’s ICE Analysis Tool (52) and shown as indels. (D and E) Functional validation of nucleofected HEK293T cells with CRISPR guides targeting AAVS1 (control) or OSMR and LIFR from C. Cells were stimulated for 30 min by either OSM (50 ng/ml), IL-6 (100 ng/ml), or IL-11 (100 ng/ml) prior to harvest of whole-cell lysates for WB for the visualization of pSTAT3, STAT3, and GAPDH and quantification of WB band intensities shown in E. (F and G) OSMR-LIFR double KO HEK293 cells (SCC 2) overexpressing GFP, OSMR WT, or various OSMR variants from gnomAD by transfection. Cells were left untreated (UT) or stimulated with 50 ng/ml OSM for 30 min prior to harvest of whole-cell lysates for WB for the visualization of pSTAT3, STAT3, OSMR, GFP, and GAPDH, with quantification of WB band intensities shown in G. (H and I) OSMR-LIFR double KO HEK293 cells (SCC 2) overexpressing GFP, OSMR WT, or the OSMR P1 variant V436D by transfection. Cells were left untreated (UT) or stimulated with OSM (50 ng/ml), IL-6 (100 ng/ml), IL-11 (100 ng/ml), or IL-31 (100 ng/ml) for 30 min prior to harvest of whole-cell lysates for WB for the visualization of pSTAT3, STAT3, and GAPDH and quantification of WB band intensities shown in I. Source data are available for this figure: SourceData F2.