Study subjects were diagnosed with Job Syndrome (AD-HIES, DN STAT3) using a diagnostic scoring system comprising immunological and non-immunological features (27) and identification of STAT3 mutations by Sanger sequencing. Patients with STAT1 GOF mutations (N658H, M202I, and P326S) were included as controls. The study protocol was approved by the Ethics Committee of the Hospitales Universitarios Virgen Macarena and Virgen del Rocío (0243-N-19). Specific informed consent forms were signed from all patients, family members and healthy volunteers at each Spanish participating center (Seville, Malaga, Valencia and Madrid). Experiments were always performed using a healthy control sample treated in the same way than samples from patients, including those that were send from other hospitals.
Whole blood stimulation, cellular staining and flow cytometry.
Fresh heparinized whole blood samples from patients with DN STAT3 or STAT1 GOF mutations and healthy controls were transferred (100µL) to polystyrene round-bottom tubes (Falcon). Cells were then stimulated with IFNγ (400 UI/mL; Imukin, Horizon Pharma) or IL6 (100ng/m; Peprotech) for 15 minutes, or with IFNα (100ng/mL; PBL Assay Science) for 30 minutes at 37°C in the presence of different concentrations of ruxolitinib (0.1 µM, 0.5 µM or 1 µM; Selleckchem), or vehicle (Dimethyl Sulfoxide; Pan Reac AppliChem). The cell suspensions were then incubated (15 minutes, room temperature) with 2 mL (1X) of lysis buffer (e-Bioscience, Invitrogen) and washed twice with RPMI 1640 (Biowest). For intracellular staining, an initial permeabilization step was performed. One-hour incubation with ice-cold methanol was followed by 2 washes with phosphate buffered saline (PBS) and 2% fetal bovine serum (FBS; South America, Biowest) to remove any residual methanol. After three washing steps, cells were incubated with the following monoclonal antibodies for 1 hour at 4°C: anti-human (h)CD14-FITC (clone M5E2, Becton Dickinson), anti-hCD3-APC-H7 (clone SK7, BD), anti-hCD4-BV711 (clone SK3, Biolegend) and anti-hCD8-PE-Cy7 (clone SK1, Biolegend), anti-hSTAT1 N-terminus-Alexa Fluor 647 (clone 1/STAT1, BD), anti-hSTAT1 (pTyr701)-PerCP-Cy5.5 (clone 4A, BD), anti-hSTAT3-PE (clone M59-50, BD) and anti-hSTAT3 (pTyr705)-BV421 (clone 13A3-1, Biolegend). Isotypes for mouse IgG1k1- Alexa Fluor 647 and IgG2a,k- PerCP-Cy5.5 (BD) were used as controls. Stained cells were washed twice (PBS/2%FBS), re-suspended in paraformaldehyde 1% (PFA, Sigma) and stored in dark at 4°C until analysis. Data were collected using the BD LSR Fortessa™ (Becton Dickinson) including FACS DIVA (v8.0) software and analyzed with the FlowJo (v. 10.7.0, Treestar, Ashland, OR, USA) software package.
Peripheral blood mononuclear cells (PBMCs) from DN STAT3 and STAT1 GOF patients and healthy donors were isolated by density-gradient centrifugation using BD Vacutainer cell preparation tubes. PBMCs were then left unstimulated or stimulated with IFNα (100ng/mL; PBL Assay Science) with or without ruxolitinib (1µM) for 30 minutes at 37°C. Cells were lysed using RIPA buffer (NaCl 150mM, NP-40 Calbiochem 1%, DCO 0.5%, SDS 0.1%, Tris HCl 50mM pH 7.5) containing 1% proteinase inhibitors and phosphatase inhibitors (Sigma Aldrich) on ice for 10 minutes. Lysates were then centrifuged at 15000 rpm for 15 minutes at 4°C. Protein concentration was quantified using Pierce BCA- Protein assay kit (Thermofisher). Samples were diluted in Laemmli buffer (Sigma Aldrich) and heated to 95°C for 5 minutes. Proteins were separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to PVDF membranes (Cytiva Amersham Hybond PVDF Membranes). Membranes were blocked in 200 mM Tris, 1500 mM NaCl (pH 7.6), 0.1% Tween 20, 5% serum bovine albumin and (T-TBS-albumin, Applichem), for 30 minutes at room temperature. To detect STAT1 proteins the membranes were incubated over night at 4°C with antibodies directed against STAT1 (Cell Signaling 9172), pSTAT1 (Py701; Cell Signaling 9167) or β-actin (Cell Signaling 4967). Membranes were washed with T-TBS and incubated for 1 hour at room temperature with polyclonal horseradish peroxidase (HRP)-conjugated secondary anti-rabbit IgG (Cell Signaling 7074). Immunoreactivity was assessed by chemiluminescence reaction using the enhanced chemiluminescence (ECL) blocking detection system (BioRad). Densitometry was performed using the automated digitizing software (Image J, NIH, Bethesda, USA). All bands were normalized to relative protein levels using β-actin as housekeeping protein.
PBMCs stimulation for transcriptomic analysis and chemokine secretion assays.
Fresh isolated PBMC were re-suspended in RPMI culture media (Biowest), supplemented with L-Glutamine (300 mg/L), penicillin (100 U/ml)/streptomycin (100 µg/ml; Gibco) and 10% FBS. PBMCs (2x106 cells/well, 12-well plate) were rested for 1 hour and stimulated with IFNγ (400 UI/mL; Imukin, Horizon Pharma) for 4 hours at 37°C, 5% CO2 in the presence of different concentrations of ruxolitinib (0.1 µM, 0.5 µM or 1 µM; Selleckchem), or vehicle (Dimethyl Sulfoxide; Pan Reac AppliChem). One unstimulated sample was included for each patient and healthy control to assess the basal state.
Determination of mRNA levels by quantitative reverse transcription polymerase chain reaction (RT-qPCR).
Unstimulated and IFNγ-stimulated PBMCs were harvested and subjected to total RNA extraction using the RNeasy mini kit (Qiagen) following manufacturer's instructions. Complementary (c) DNA was generated from 100 ng of RNA using the reverse transcription kit (Applied Biosystems). Relative STAT1, CXCL10, PD-L1, SOCS1 and SOCS3 mRNA levels were determined using the Gene Expression Assays (Hs01013996, Hs00171042, Hs00204257, Hs00705164 and Hs02330328, respectively; Thermofisher) and TaqMan Gene Expression Master Mix (Applied Biosystems) following manufacturer’s instructions. β-actin (Hs01060665; Thermofisher) was used as endogenous control. PCR conditions consisted of polymerase activation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 sec, and annealing/extension at 60°C for 1 minute. Relative mRNA levels were analyzed using the comparative 2-ΔΔct method.
Enzyme-linked immunosorbent assay (ELISA).
IFN-inducible CXCL10 protein levels were determined in PBMCs culture supernatant by ELISA (Thermofisher) following manufacturer’s instructions. CXCL10 levels obtained from patients’ cell supernatants were compared to the levels of the same-day healthy control.
Data from all experiments acquired on the same flow cytometer instrument, using the same settings, were analyzed in raw values of geometric mean of fluorescence (gMFI). Data were expressed either as direct gMFI of STAT1 and pSTAT1 levels or as percentage of the same day healthy donor’s level. RT-qPCR data from patients were also normalized with the respective same day healthy donor’s level. Graphs were performed using Prism software (version 8, GraphPad software). Statistical analysis was performed using the software RStudio Team (2021). Normality for quantitative variables was evaluated using Shapiro-Wilk. For inferential statistics Wilcoxon and Kruskal-Wallis tests were used. P-values lower than 0.05 were considered statistically significant.