Patient 4 (submandibular lymph node abscess, 6 weeks) was a 1-year-old girl with AR CGD (NCF1 delta GT). She was diagnosed with a bilateral submandibular fistulising lymph node abscess. Surgical drainage revealed the presence of Burkholderia cepacia. DHR test showed 17-25% of cells with residual NADPH oxidase function on a low level, as the shift of DHR-positive cells (DeltaGeoMean MFI) corresponded to only 1.9% of the shift observed in healthy control cells.
A deep frozen control lymph node sample of a non-CGD patient presenting recurrent lymph node hyperplasia was kindly provided by the tissue biobank of the Institute of Pathology and Molecular Pathology of University Hospital Zurich.
Tissues were transported and homogenized as described in the Materials and Methods section "cytokine and chemokine analysis." To avoid sampling errors, >50% of tissue was processed for flow cytometry analysis and cytokine quantification. Tissue-derived cells and ex vivo-primed macrophages were blocked with FcR blocking reagent (MACS, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and stained with the antibodies as follows. Anti-human CD14 PC7 (clone RMO52), anti-human CD209 (DC-SIGN)-PE (clone AZND1), anti-human CD14 PC7 (clone RMO52) were from Beckman Coulter International S.A., Nyon, Switzerland. Anti-human CD15 V500 (HI98), anti-human CD57 FITC (HNK-1), anti-human CD56 PE (B159 RUO), anti-human CD8 V450 (RPA-T8) and anti-human CD8 V450 (RPA-T8) were from BD Bioscience, Eysins, Switzerland. Anti-human CD3 Alexa Fluor  700 (UCHT1), anti-human Valpha24Jalpha18 TCR PE-Cyanine7 (6B11), and anti-human CD206 (MMR) eFluor  450 (clone 19.2) were from eBioscience, Eysins, Switzerland. Anti-human CD4 PerCP (OKT4), anti-human CD19 Brilliant Violet 605 (HIB19), anti-human CD163 PerCP/Cy 5.5 (clone GHI/61) anti-human CD86 Brilliant Violet 605TM (clone IT2.2) were from BioLegend, Lucerna-Chem AG, Luzern, Switzerland. Anti-human Mer APC (clone 125518) were from R&D Systems, Bio Techne AG, Zug, Switzerland. Cells were washed, fixed with 2% formaldehyde in PBS, measured by GalliosTM flow cytometer and analyzed by Kaluza software (Beckman Coulter International S.A., Nyon, Switzerland).
Volume of pus samples were determined and diluted to separate cells from supernatant by centrifugation. Solid tissues were kept in PBS on ice for up to 1 h until >50% of samples were weighed and homogenized with a syringe piston on a Petri-dish. Remaining tissue was removed by filtration (70 mum cell strainer). For flow cytometry analysis, cells were separated from supernatant by centrifugation. Supernatant was stored at -80 C until cytokine quantification in duplicates by 18plex Immunoassay (Cat. No. EPX180-12165-901, eBioscience, Thermo Fisher Scientific AG, Basel, Switzerland) and MILLIPLEX  MAP KIT (Cat. No. HCYTOMAG-60K, MILLIPORE, Merck AG, Zug, Switzerland) according to manufacturers' instructions for quantification of G-CSF, GM-CSF, IFN-gamma, MDC, TNFalpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-18, IL-21, IL-22, IL-23, and IL-27. Cytokines concentrations are expressed in ng/ml of undiluted tissue.
Paraffin-embedded slides were dewaxed and stained with rabbit anti-human IL-18 (LS-C313164, Lifespan Biosciences, LabForce AG, Muttenz, Switzerland) and mouse anti-human CD68 (clone 514H12, Novocastra Laboratories Ltd., Newcastle upon Tyne, UK), followed by donkey anti-rabbit FITC (711-095-152, Jackson ImmunoResearch Ltd., MILAN ANALYTICA AG, Rheinfelden, Switzerland), donkey anti-mouse Cy3 (715-165-150, Jackson ImmunoResearch Ltd., MILAN ANALYTICA AG, Rheinfelden, Switzerland) followed by mouse anti-FITC Alexa488 (200-542-037, Jackson ImmunoResearch Ltd., MILAN ANALYTICA AG, Rheinfelden, Switzerland) and 4',6-Diamidin-2-phenylindol (DAPI) staining conducted by Sophistolab AG (Muttenz, Switzerland).
Slides were analyzed by confocal microscopy (model TCD SP8, Leica Geosystems Holdings AG, Glattbrugg, Switzerland) with original magnification x40. Acquired images were processed using ImageJ.
Immunohistochemistry was perfomed by Sophistolab AG (Muttenz, Switzerland) on Leica BondMax instruments using Refine HRP-Kits (Leica DS98000, Leica Microsystems Newcastle Ltd., Newcastle upon Tyne, UK) according to manufacturer's instructions. Paraffin-slides were dewaxed, penetrated, and stained with rabbit anti-human IFN-gamma (LS-B7487, Lifespan Biosciences, LabForce AG, Muttenz, Switzerland), visualized by HRP Refine Kit (DS9800, Leica Microsystems GmbH, Wetzlar, Germany). Images were then analyzed with a bright-field microscope (Axiovert S100TV, Carl Zeiss Vision Swiss AG, Feldbach, Switzerland).
Monocytes were isolated from mononuclear cells after FICOLL (Ficoll-Paque PLUS, GE Healthcare AG, Glattbrugg, Switzerland) density gradient centrifugation, followed by magnetic bead-based positive selection (CD14 human microbeads, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to manufacturer's description. Cells were cultured (2.8 x 105 cells/well) in PRIMARIA 24 wells plate (Becton Dickinson AG, Allschwil, Switzerland) in RPMI supplemented with Octaplas SD 10% (Octapharma AG, Lachen, Switzerland), 2 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES, and M-CSF 100 ng/ml (research grade, MACS, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) for 7 days.
Macrophages were either kept in culture for 24 h in a fresh differentiation medium (M0 macrophages), or stimulated with IFN-gamma (50 ng/ml, e-Bioscience, Thermo Fisher Scientific AG, Basel, Switzerland) and LPS (10 ng/ml, Sigma-Aldrich GmbH, Buchs, Switzerland) for M1 priming, IL-4 (20 ng/ml, ProSpec-Tany TechnoGene Ltd., Ness Ziona, Israel) for M2a, in wells pre-coated with human IgG (5 mug/cm2, Sigma-Aldrich GmbH, Buchs, Switzerland), and supplemented with LPS (100 ng/ml, Sigma-Aldrich GmbH, Buchs, Switzerland) for M2b, or supplemented with IL-10 (100 ng/ml, Sanofi Genzyme, Vernier, Switzerland) and M-CSF (100 ng/ml, MACS, Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany) for M2c. For cytokine/chemokine analysis, supernatants were centrifuged, and aliquots were stored at -80 C. After 24 h of stimulation, macrophages were detached with EDTA and analyzed by flow cytometry analysis.
Supernatants from 24 h un-primed M0, or from 24 h M1, M2a, and M2c primed macrophages were removed, cells were washed with PBS without detachment, and re-primed for 72 h by incubation with priming stimuli (described in 24 h Priming of Macrophages). Ninty six hours after initial priming corresponding to 72 h after repriming, supernatants were centrifuged and stored at -80 C for cytokine/chemokine quantification.
Statistical analysis was performed as indicated by ANOVA with post hoc Bonferroni correction using SPSS (Version 22, IBM), and graphs were prepared using GraphPad Prism Software (Version 5.0a; GraphPad Software, La Jolla, California, USA).