Fig 2: Reconstitution of patient fibroblasts with WT IFNAR2 restores IFNAR2 signaling, ISG induction and viral control. (A and B) Primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the heterozygous mother of P1 (Het) were transduced with lentiviral vectors encoding EGFP, IFNAR2 WT, or IFNAR2 p.Ser53Pro. 2 d after transduction, cells were pretreated with IFNß 100 IU/ml for either (A) 30 min before being lysed and harvested for whole cell lysates for immunoblotting or (B) 6 h before being lysed and harvested for total RNA for RT-qPCR of MX1 and IFIT1 relative to GAPDH (mean ± SD of n = 3 independent repeats; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant; two-way ANOVA with Tukey’s test for multiple comparisons). For A, one representative immunoblot of three independent experiments is shown. (C and D) SV40-immortalized dermal fibroblasts from P1 and a healthy control were transduced with lentiviral vectors encoding EGFP or IFNAR2 WT. 2 d after transduction, cells were pretreated with IFNß 100 IU/ml for 24 h before infection with HSV1 (KOS strain) at an MOI of 1. At 24 h p.i., supernatants were harvested and titrated for (C) TCID50 (geometric mean ± SD of n = 3 independent replicates; ****, P < 0.0001; ns, non-significant; two-way ANOVA with Tukey’s test for multiple comparisons) and the cells were lysed for Western blotting for the HSV1 protein VP5, IFNAR2, and GAPDH as loading control (D). Source data are available for this figure: SourceData F5.

Fig 3: Identification of a novel homozygous IFNAR2 variant leading to IFNAR2 deficiency. (A) IFNAR2 protein sequence with known deleterious variants marked. (B) Pedigrees of affected individuals originating from Greenland (P1), Canada (P2 and P3), and Alaska (P4 and P5). Double lines refer to consanguinity. (C) Principal component analysis of 4,630 Greenlandic individuals combined with three super populations from the 1000 Genomes Project. (D) Local ancestry haplotypes of admixed and p.Ser53Pro IFNAR2 heterozygous Greenlandic individuals colored according to ancestry. Each heterozygous individual is represented with two haplotypes, one for the p.Ser53Pro haplotype (top) and one for the WT haplotype (bottom). The fraction of Inuit ancestry of the WT and p.Ser53Pro haplotype is estimated at the location of the variant. (E) IFNAR2 cell surface expression analyzed by flow cytometry in PBMCs and distinct cell subsets of healthy controls (C1–C5) and patients (P1 and P2). Representative of a single experiment in P1 and P2 PBMC. (F) Immunoblotting showing IFNAR2 expression in whole-cell lysates of PBMCs from P1 compared to three healthy controls (C1–C3). GAPDH was used as loading control. Not repeated due to limited patient material. (G) RT-qPCR of whole-cell RNA lysates of PBMCs evaluating IFNAR2 mRNA levels relative to TBP for P1 compared to three healthy controls (pooled, mean ± SD of n = 3 biological replicates; **, P < 0.01, Welch’s t test). Representative of a single experiment in P1 PBMC. Source data are available for this figure: SourceData F1.

Fig 4: In vitro functional modelling of the IFNAR2 p.Ser53Pro mutant. (A) EBV-transformed B cells from P2 and a healthy control (C1) were treated with IFNa2b 1,000 IU/ml for 30 min. Whole cell lysates were harvested for immunoblotting and visualization of STAT1, pSTAT1 and GAPDH as loading control. Representative of n = 2 repeat experiments. (B) Graphical summary of experimental strategy. IFNAR2-deficient dermal fibroblasts were reconstituted with WT or mutant IFNAR2 lentiviral constructs or an empty vector control. (C) IFNAR2-deficient dermal fibroblasts were reconstituted with WT or mutant IFNAR2 lentiviral constructs or an empty vector control and protein lysates prepared for immunoblotting for IFNAR2, GFP, and GAPDH loading control. Representative of n = 3 independent experiments. (D) IFNAR2-deficient dermal fibroblasts were reconstituted with WT or mutant IFNAR2 lentiviral constructs or an empty vector control. After overnight stimulation with IFNa2b or IFN? (1,000 IU/ml) protein lysates were prepared for immunoblotting for the ISG products MX1, USP18, RSAD2 and ISG15 alongside GAPDH loading control. Representative of n = 3 independent experiments. (E) Primary IFNAR2-deficient fibroblasts, stably reconstituted with empty vector, WT IFNAR2 or p.Ser53Pro IFNAR2, were pre-treated with IFNa2b or IFN? (1,000 IU/ml) overnight, prior to infection with HSV1 (17 + strain, MOI 0.01). At 48 h after infection, lysates were prepared for immunoblotting for HSV gD, alongside MX1, RSAD2, ISG15 and GAPDH as loading control. Representative of n = 3 independent experiments. (F) Primary IFNAR2-deficient fibroblasts, stably reconstituted with empty vector, WT IFNAR2 or p.Ser53Pro IFNAR2, were pre-treated with IFNa2b or IFN? (1,000 IU/ml) overnight, before infection with a cytopathic dose of EMCV. At 24 h after infection, cell viability was assessed in an imaging based live cell viability assay (mean ± SD of n = 3 independent experiments, one-way ANOVA with Šidák’s multiple comparisons test; ****, P < 0.0001; ns, non-significant). Source data are available for this figure: SourceData FS3.

Fig 5: Defective signaling and ISG induction downstream of IFNAR2 p.Ser53Pro. (A) PBMCs from P1 and three healthy controls (C1–C3) were treated with IFNß 100 IU/ml for 5, 15, or 30 min. Whole-cell lysates were harvested for Western blotting for the visualization of pSTAT1, STAT1, and GAPDH, which was used as the loading control. Not repeated due to limited patient material. (B) Primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the heterozygous mother of P1 (Het) were treated with IFNß 100 IU/ml for 30 min. Whole-cell lysates were harvested for immunoblotting and the visualization of IFNAR2, STAT1, pSTAT1, and GAPDH as the loading control. One representative immunoblot of n = 3 independent experiments is shown. (C) Primary IFNAR2-/- dermal fibroblasts reconstituted with lentiviruses expressing GFP, WT, or p.Ser53Pro IFNAR2 were treated with IFNa2b or IFN? 1,000 IU/ml for 30 min. Whole-cell lysates were harvested for immunoblotting and visualization of pJAK1, JAK1, pSTAT2, STAT2, STAT1, pSTAT1, and GAPDH as the loading control. One representative immunoblot of n = 3 independent experiments is shown. (D) PBMCs from P1 and three healthy controls (C1–C3) were treated with IFNß 100 IU/ml for 6 h. Total RNA was purified for RT-qPCR of IFIT1 and IRF7 relative to TBP (mean ± SD of n = 3 independent experiments; ****, P < 0.0001, ns, non-significant; two-way ANOVA with Šidák’s test for multiple comparisons). (E) Primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the mother of P1 (Het) were treated with IFNß 100 IU/ml for 6–24 h. Total RNA was purified for RT-qPCR of MX1 and IFIT1 relative to TBP (mean ± SD of n = 3 independent experiments; ****, P < 0.0001; ns, non-significant; two-way ANOVA with Dunnett’s test for multiple comparisons). (F) Primary dermal fibroblasts from P1 or a healthy control (C1) were treated with IFNa2b 1,000 IU/ml overnight. Whole-cell lysates were prepared for immunoblotting and visualization of MX1, USP18, RSAD2, ISG15, and a-tubulin (ATUB) as the loading control. One representative immunoblot of n = 3 independent experiments is shown. Source data are available for this figure: SourceData F2.