Cell lines, cell culture and antibodies
Ba/F3 (obtained from DSMZ, ACC 300, RRID: CVCL_0161), EL4 (ATCC, TIB-39, RRID: CVCL_0255), SK-BR-3 (ATCC, HTB-30, RRID: CVCL_0033), IIa1.6 (ATCC, TIB-308, RRID: CVCL_0J27) cells were cultured in RPMI 1640 medium (RPMI 1640 Glutamax; Thermo Fisher, 61870036) supplemented with 10% fetal calf serum (FCS, Bodinco), 100U/ml penicillin/streptomycin (Pen/Strep, Thermo Fisher, 15140122), and 0.2 ng/mL murine IL-3 (Immunotools, 12340035) for Ba/F3 cells. All cell lines were authenticated by the suppliers and used at low passage numbers after immediate freezing upon receipt. Additionally, they were routinely screened for mycoplasma using Myco alert Mycoplasma detection kit (Lonza, LT07-318). The retroviral vector pMX human FcγRI was generated by cloning the full-length human FCGR1A cDNA into the pMX vector backbone using standard molecular biology techniques. High-titer retrovirus was produced in packaging cells and used to transduce target cells, followed by selection and confirmation of FcγRI surface expression by flow cytometry65. All cells were transduced using amphotropic viral particles made in HEK293T cells. Puromycin selection (5 μg/ml, Merck, P8833) produced stable Ba/F3-FcγRI, Ba/F3-FcγRIIIa, Ba/F3-FcγRIIIb, EL4-FcγRI and SKBR3-FcγRI cell populations. No extra selection was needed to produce stable IIa1.6-FcγRIIa and IIa1.6-FcγRIIb. All information on antibodies used in this study are provided in Supplementary Table 3.
Production of chimeric FcγRI-specific antibodies
Seven anti-FcγRI antibodies have been generated in-house19. In short, C57BL/6 mice were immunized intravenously by cellular immunization with FcγRI expressing cells within our UMAB facility and boosted 4 times. Blood was collected and serum antibody titers for FcγRI were analyzed. The spleens were harvested 4 days after the last boost, snap-frozen and stored at −80 °C. A murine scFv antibody library was generated from total RNA of the spleen isolated from the mouse with highest serum antibody titer. Phage display was performed by two panning steps using Ba/F3-FcγRI cells. After the final panning, binding of individual phage antibodies to FcγRI was tested by whole cell ELISA using Ba/F3 and Ba/F3-FcγRI cells. The screening process was monitored by next generation sequencing (NGS) and enriched scFv clones were selected and converted into Fc-silent human IgG1 (LALAPG mutation; L234A, L235A, P329G23), via overlap extension-PCR (OE-PCR). Here, primers (Integrated DNA technologies, Supplementary Table 4) were diluted to 10 μM in sterile H20. VL and VH, and OE-PCR reactions were performed in 50 μl volumes, containing 10 μM forward and 10 μM reverse primer, 5 ng of template DNA, 10 mM dNTPS (Qiagen), 1 unit of Phusion DNA polymerase (Boike), 5X phusion HF or GC buffer (Boike). Initial denaturation was conducted at 98 °C for 30 seconds, followed by 27 cycles of 98 °C at 10 s, 55-62 °C at 20 s, 72 °C at 15 s and a final extension of 72 °C at 10 min. When required, gel purification was conducted using the nucleospin Gel and PCR clean-up kit (Macherey-nagel). For OE-PCR Reaction 1 (VL and VH), pcDNA3.4 expression vector (RRID: Addgene_221396) was used as DNA template. For OE-PCR Reaction 2 either VL or VH of interest in the Pak100 vector was used as DNA template. For OE-PCR Reaction 3 the corresponding product from OE-PCR reaction 1 and 2 was used as DNA template. The resulting sequences were ultimately transfected into ExpiCHO-S cells (ThermoFisher, A29127). Antibodies were purified using a HiTrap rProtein A FF column (GE Healthcare, 10146224) attached to the ÄKTA Start (GE Healthcare) fast protein liquid chromatography system according to the manufacturer’s protocol. The protein sequences of the antibodies are included in a related patent application (P37203NL00).
Phylogenetic tree reconstruction
Phylogenetic reconstructions of the amino acid sequences of the variable heavy and light chains were made using the function “compute” of ETE3 3.1.2 as implemented on the GenomeNet66. Alignment was performed with Clustal Omega (v1.2.4) with the default options67. A maximum-likelihood tree was inferred using PhyML (v20160115). Branch supports are the Chi2-based parametric values by the approximate likelihood ratio test68. The obtained newick files were then uploaded in the Interactive Tree Of Life (iTOL) (v6) annotation software to generate midpoint rooted phylogenetic trees69.
Chimeric domain swap receptors
All human FcγRI: mouse FcγRI domain swap chimeric receptors were constructed in the pMX_puro vector. Receptor sequences were obtained from UniProt; human FcγRI (L03418) and murine FcγRI (M31314). For the domain swap chimeric receptors, extracellular domains (EC) 1, 2, or 3 were replaced by the murine equivalent. For mECD1-hEC2-hEC3, 91.3% of hEC1 was replaced by mECD1. For hEC1-mEC2-hEC3, the whole hEC2 was replaced, including the overlap with hEC1. For hEC1-hEC2-mEC3, hEC3 could easily be replaced by mECD3.
The obtained plasmids were retrovirally transduced in HEK293T cells, the virus supernatant was harvested and added to EL4 cells. Selection was performed after 7-14 days with puromycin. To evaluate binding, 1 × 105 transduced cells were added per well, after which the antibodies were added (e.g., C01-LALAPG, C04-LALAPG, 10.1 (mIgG1), M22 (mIgG1), and hIgG1). After 1 h incubation at 4 °C, a labeled secondary staining antibody (anti-mouse IgG or anti-human IgG) was added. Binding was evaluated via flow cytometry and analyzed with BD FACSDiva (v9.0).
Flow cytometry assays
For all Fluorescence-Activated Cell Sorting (FACS) assays, 1 × 105 cells/well were added to a 96-well plate. All incubation steps were performed for 1 h at 4 °C, unless stated otherwise. In each experiment, 10 μg/ml (labeled) antibody was used, unless stated otherwise. After each incubation step, cells were washed with 100 μL FACS buffer (PBS + 0.5 % bovine serum albumin (BSA, Sigma, 10735094001) + 0.1% sodium azide (Immunosource, 40-2010-01)) and spun down at 1500 RPM for 5 min, unless stated otherwise. After final staining, cells were washed once with PBS, fixed with 1% paraformaldehyde (PFA) for 15 min at 4 °C and resuspended in FACS buffer. All measurements were performed in duplo or triplo using a FACSCanto II (BD Sciences) and accompanying software (FACS DIVA, v8) and FlowJo (v10.10). An exemplary gating strategy is provided in Supplementary Fig. 2a.
Commercial antibodies ligand binding and blocking assay
Ba/F3-FcγRI and EL4-FcγRI cells were incubated with the indicated anti-FcγRI antibodies for 1 hour. After washing, mouse or human IgG bound to the cells was detected by using APC-labeled anti-mouse IgG or AF647-labeled anti-human IgG for 1 hour. To measure the ligand blocking capacity, 5 μg/ml hIgG1-AF647 was added for 1 h after the first incubation step. Ligand blocking (%) was calculated by using the formula: Ligand blocking (%) = 100 – ((MFI sample / MFI ligand only) x 100). For 197 and 10.1, F(ab’)2 fragments were generated to test Fab-mediated interactions. Immobilized Ficin was used to prepare the F(ab’)2 fragments using Pierce Mouse IgG1 Fab and F(ab´)2 Preparation Kit (Thermo Scientific, 44980). For both antibodies, 1 mg was digested with 4 mM cysteine for 28 h at 37 °C, following manufacturer’s protocol. Purity was confirmed via SDS-PAGE gel electrophoresis (reducing condition, 4-20% gradient gel, Bio-Rad, 4561094) and stained using InstantBlue Ultrafast Protein Stain (Sigma Aldrich, ab119211).
Antibody binding assay
For antibody binding assays, 1 × 105 Ba/F3-FcγRI or EL4-FcγRI cells per well were added to a 96-well plate and pre-labeled commercial anti-FcγRI antibodies or FITC-labeled antibodies were added. For the titration experiments, cells were incubated with the different antibodies at the indicated concentrations for the specified time. For the specificity assay, IIa1.6, IIa1.6-CD32a, IIa1.6-CD32b, Ba/F3, Ba/F3-CD16a and Ba/F3-CD16b were incubated with a titration range of C01-FITC, C04-FITC, anti-CD16-PE (clone 3G8; BD Pharmingen) and anti-CD32-FITC (clone FL18.26; BD Pharmingen). For antibody binding in presence of plasma IgGs, human peripheral blood mononuclear cells (PBMCs) were isolated from donor blood (in-house donor facility, University Medical Center Utrecht) and Ba/F3-FcγRI cells were first incubated with 20 µl of 200 µg/ml intravenous immunoglobulins (IVIg) (Nanogam, Sanquin, RVG31627), and then with FITC-labeled anti-FcγRI antibodies. For cross-block assays, Ba/F3-FcγRI cells were pre-incubated with either unlabeled 10.1 or anti-FcγRI clones. Next, FITC-labeled anti-FcγRI clones or 10.1 was added to measure residual binding.
Expression and purification of C01 Fab and rhFcγRI
Codon optimized C01 heavy and light chain V gene sequences ordered from TWIST Bioscience were designed with either only the constant heavy 1 (CH1) or constant light (CL) domains but including the ultimate cysteine residues implicated in Fab conjugation. Constructs ligated into the pcDNA3.4 expression vector (RRID: Addgene_221396) were transfected into the ExpiCHO expression system (Thermo Fisher, A29133). Culture supernatants were harvested 10 days post-transfection and purified subsequently on a Kappa Select column, equilibrated in PBS and using 0.1 M glycine pH 2.5 as eluent, and a Superdex75 16/600 column in 10 mM HEPES-buffer pH 7.5 with 150 mM NaCl. Recombinant production of FcγRI ectodomains in ExpiCHO entailed the previously published construct including 19 stabilizing mutations and fusion to a C-terminal His6-tag46. Protein purification comprised of an IMAC affinity step on a 1 mL HisTrap HP column equilibrated in 25 mM HEPES-buffer pH 7.5 with 0.5 M NaCl and using wash and elution buffers containing 50 and 100 mM Imidazole pH 7.5, respectively, followed by a cation exchange step with a 1 mL ResourceS column in 25 mM MES-buffer and elution using a 0.1–1 M NaCl gradient25. As a final step prior to crystallization, the C01 Fab fragment and rhFcγRI were mixed in a 1:1 ratio to a final concentration of 3 mg/mL and purified on a Superdex200 Increase 10/300 column equilibrated in 10 mM HEPES-buffer pH 7.5 with 150 mM NaCl, to separate the complex from the individual components. All columns used in this protocol were obtained from Cytiva LifeSciences.
Crystallization and structure determination of the Fab C01-rhFcγRI complex
Crystallization trials were set up in sitting drops vapor diffusion screens by mixing 100 nl 10 mg/mL Fab C01-rhFcγRI complex in 10 mM HEPES-buffer pH 7.5 with 150 mM NaCl and 100 nl of reservoir solution, at 293 K. Fab C01-rhFcγRI crystals were grown during 2 months using the Morpheus Crystallization screen, in a condition containing the MOPS/HEPES buffer system, pH 7.5, alcohol mixture (0.12 M end conc.), and the PEG20k-PEG MME 550 precipitant mixture (30 % end concentration)70. X-Ray diffraction data to 3.2 Å resolution of two separate crystals grown in the same drop were collected at the Diamond Light Source at beamline I24, processed individually with the xia2 multiplex protocol, and subsequently merged and scaled with Aimless71,72. The structure solution by molecular replacement in Phaser using models of hFcγRI (PDB-ID: 4W4O) and mFab VH, VL, CH & CL domains (PDB: 1i8m) as search templates revealed the presence of two Fab C01-fhFcγRI complexes in the asymmetric unit. The model was further improved by manual modeling in Coot25,73,74,75. During the refinement in Refmac5, external restraints were used, that were generated by Lowrestr against experimentally determined structures of FcγRI homologs and Fab fragments, and alphafold 2 models76,77,78. Quality of the geometry was analyzed using MolProbity79, showing 94.08% favored and 5.27% allowed backbone geometries according to Ramachandran’s statistics, as well as 81.7% favored and 10.42% allowed rotamer conformations. Final Rwork/Rfree statistics are 0.261 / 0.301. Data collection and refinement statistics are listed in Supplementary Table 1. All programs were used as implemented in CCP480.
Ligand tracer assay
The binding kinetics of hIgG, 10.1, C01 and C04 to FcγRI were examined by using LigandTracer Green Technology (Ridgeview Instruments AB). SKBR3 and SKBR3-FcγRI were plated out on opposite sides of a culture dish in an elliptical shape, at a concentration of 8 × 104 cells/ml and incubated overnight at 37 °C to attach to the plate. Next, the plate was washed with culture medium and transferred to the LigandTracer apparatus. First, a baseline measurement of 15 min was performed. Subsequently, association was measured at 10 nM FITC-labeled antibody for 1 h and subsequently at 30 nM for 1 h. Then, dissociation was measured for 2 h by replacing the culture medium containing 30 nM antibody for antibody-free medium. The effective affinity of the antibodies was calculated by using a OneToOneBI fitting model in TraceDrawer (v1.9.2) software (Ridgeview Instruments AB).
Calcium release assay
For the calcium release assays, 5 × 105 Ba/F3-FcγRI cells per well were incubated with 8 μM Fluo-4 AM (Invitrogen, F14201) and 10 μM Fura-Red (Invitrogen, F3021) at 37 °C for 20 min. Cells were removed from the incubator, anti-FcγRI antibodies m22 (own production), H22 (MDX-33, Antibody System), C01 and/or C04 were added (10 μg/ml) and the cells were incubated at 37 °C for an additional 10 min. Cells were washed once with RPMI without phenol red + 1% P/S + 10% FCS and once with Hank’s Balanced salt solution (HBSS, Gibco, 15266355) containing magnesium, calcium and no phenol red, supplemented with 10% FCS. Cells were resuspended in HBSS + 10% FCS and transferred to a FACS tube in a final volume of 500 μL. The cells were left undisturbed in the dark at RT for at least 60 min. Cytosolic calcium levels were measured on FACSCanto II (BD Biosciences) as ratio of Fluo-4/Fura Red. After 30 seconds of baseline measurement, a cross-linking antibody (goat anti-mouse IgG; Jackson, 115-007-003, or rabbit anti-human IgG; Jackson, 309-006-008) was added and calcium release was measured for 4.5 min. Lastly, 2 μg/mL ionomycin (Sigma, I0634) was added as a loading control and measured for 45 seconds.
Superoxide induction
IFN-γ stimulated neutrophils were used for measuring superoxide burst by anti-FcγRI antibodies. Neutrophils from 3 healthy donors were cultured overnight with 100 U/mL IFN-γ (Immunotools, 11343534) and 100 U/ml G-CSF (Immunotools, 11343133) in RPMI with 10% FCS and 1% P/S. All next steps were performed on ice to limit superoxide burst. 197, m22, H22, 10.1, C01 and C04 were added at 10 µg/ml in 50 µl, after which 4 × 105 cells in 100 µl per well were added. Lastly 150 µM luminol (Sigma) in 50 µl was added and placed a luminometer (SpectraMax M3, Molecular Devices) set at 37 °C. Measurements of spontaneous production of superoxide were made every 40 to 60 seconds, starting immediately following luminol addition. The production of superoxide was measured in relative light units (RLU) for 20 min.
Soluble IC binding and blocking assay
Soluble IC was formed by incubating 10 µg/ml hIgG1 (Palivizumab; Synagis, 66658-230-01) with 5 µg/ml AF647-Goat F(ab’)2 anti-human IgG F(ab’)2 (Jackson, 109-605-006) for 30 min at 37 °C, followed by 10 min incubation on ice. 1 × 105 Ba/F3-FcγRI cells were added per well. Anti-FcγRI was added (25 µl per well at 5, 10 or 25 µg/ml) followed by 45 min incubation on ice. Next, hIgG1-AF647 or soluble IC was added (25 µl per well) (no washing step in between), followed by 45 min incubation on ice. Percentage binding was calculated using the following formula: Binding (%) = (sample – negative control)/(baseline – negative control)*100%. Negative control is the unstained condition. Baseline is the condition where only hIgG-AF647 or soluble IC was added and no blocking antibody.
hIgG and soluble IC displacement assay
Soluble IC were generated by incubating 10 µg/ml hIgG1 (Palivizumab; Synagis, 66658-230-01) with 5 µg/ml AF647-Goat F(ab’)2 anti-human IgG F(ab’)2 (Jackson, 109-605-006) for 30 min at 37 °C, followed by 10 min incubation on ice. Antibodies and IC were dissolved in HEPES-buffered saline + 1 mM MgCl2 + 1,25 mM CaCl2 + 0,2 ng/ml murine IL-3 or (for overnight incubation) RPMI 1640 without phenol red (Gibco, 11835030) + 10% FCS + 0,2 ng/ml murine IL-3. 1 × 105 cells (Ba/F3 or Ba/F3-FcγRI cells) were added per well. Palivizumab-AF647 (in-house labeled), or soluble IC was added (25 µl per well) followed by 45 min incubation on ice. Next, anti-FcγRI antibody was added (25 µl per well) (no washing step in between) followed by 45 min or overnight incubation at 37 °C. Subsequent preparation for measurement, analysis, and calculations were done similarly as for blocking experiments, with the exception that the negative control used in the calculation is the condition where only the AF647-Goat F(ab’)2 anti-human IgG F(ab’)2 was added. For the displacement assays, polymorphonuclear leukocytes (PMNs) or PBMCs from 3 healthy donors were isolated by diluting whole blood 1:1 with PBS and layering this mixture on Ficoll Paque Plus (GE Healthcare, 17‑1440‑03). After careful centrifuging, PBMCs were collected and monocytes were isolated via CD14 + MACS Cell Separation (Miltenyi Biotec, 130‑050‑201). The remaining serum and Ficoll layer were removed to have the PMN layer left. The cells were cultured overnight in either RPMI with human serum or FCS in a 6-wells plate at a density of 2 million/ml. Additionally, neutrophils were stimulated with 100U/ml IFN-γ (Immunotools, 11343534) and G-CSF (Immunotools, 11343133) and the monocytes with 100U/ml GM-CSF (Immunotools, 11343125).
Rosette (IgG-coated beads) assay
Dinitrophenol (DNP)-BSA (Thermo Fisher, A-8020) was coupled to Dynabeads M-450 Epoxy (4,5 µm) (Thermo Fisher, 14012D) according to manufacturer’s instructions. DNP-BSA Dynabeads were opsonized with rabbit IgG anti-DNP (Vector Laboratories, SP-0603-1). Different coating antibody concentrations were evaluated to maximize bead-to-cell binding and the best binding was obtained at 1 µg/ml, and this concentration was deemed sufficient for subsequent blocking tests (1 × 108 beads per ml antibody solution). Per well, 1 × 105 Ba/F3-CD64 cells were incubated with 50 µl of 1-100 μg/ml blocking antibody for 45 min at 4 °C. After washing, 3.5 × 105 beads were added and incubated for 1 h at 4 °C while shaking. Next, cells were fixated with 3% PFA (Klinipath) and spun down at 1500 RPM for 2 min, no brake. Rosette formation was evaluated using a bright-field microscope (EVOS, Thermo Fisher) at 20x magnification. Cells with five or more beads bound to them were defined as rosettes. Counting was performed using ImageJ v2.11 (Fiji) software. For each condition, triplicates were measured. To be able to compare different experiments, the percentage of rosettes in each condition was calculated to the baseline percentage without blocking antibody.
Patients and healthy donors
A peripheral blood sample was obtained from an ACPA-positive RA patient visiting the outpatient clinical of the department of Rheumatology at Leiden University Medical Center (LUMC, Leiden, The Netherlands). Informed consent was obtained for this individual and all protocols were approved by the ethical committee of the LUMC, the Netherlands. Samples from patients with ITP were derived from the PICI study (trial number NL66313.041.18), a cross-sectional observational study amongst patients with chronic ITP approved by the NedMEC medical ethics committee. All patients gave written informed consent. Healthy volunteers were recruited amongst hospital staff and students. Ethical permission was obtained (protocol number 18-774) and all donors provided written informed consent.
Patient-derived ACPA-IC assay
ACPAs were isolated from RA patient (see patient section) plasma by affinity chromatography using the ÄKTA Pure equipped with a self-packed HiTrap streptavidin HP 1 mL column (Cytiva, 17-5112-01) with biotinylated CCP4 peptides or biotinylated CArgP4 peptides81. The peptides were kindly provided by Dr. J. W. Drijfhout (Dept. of IHB, LUMC). Additionally, we used a recombinant non-glycosylated (NG) monoclonal ACPA-IgG1, 7E4, which were produced based on the BCR sequences from ACPA + RA patients82. In this case the sequence of 7E4 was provided by Dr Rispens, Sanquin, The Netherlands. Since ACPA express variable domain glycans, the glycan sites were mutated out by back-mutating the N-linked glycosylation sites into the germline sequence (based on IMGT). Codon optimized ACPA heavy chain and light chain V gene sequences were designed and ordered from GeneArt (Life Technologies) and ligated into a pcDNA3.1 (+) expression vector (Invitrogen, V79020). The heavy and light chain vectors were transfected into Freestyle 293-F cells (Gibco, R79007). After 5-6 days the supernatant was harvested and purified for ACPA IgG with a 1 ml HiTrap Protein G HP affinity column (GE Healthcare, 29-0485-81) according to the manufacturer’s instructions.
PMNs or PBMCs from 3 healthy donors were isolated by diluting whole blood 1:1 with PBS and layering this mixture on Ficoll Paque Plus (GE Healthcare, 17-1440-03). After careful centrifuging, PBMCs were collected, after which the remaining serum and Ficoll layer were removed to have the PMN layer left. For the monocytes, the whole PBMC population was used. For the macrophage stimulation, the PBMCs were plated in a NUNC Upcell 6-well plate (Thermo Fisher, 174901) at a density of 5 million cells/mL in 3 mL. The monocytes were left to attach to the plate for 3 h, after which the wells were washed 2 times to remove any unbound non-monocytic cell. RPMI medium with 10% FCS and 1% P/S was added, together with 50 ng/ml GM-CSF (Immunotools, 11343125) to induce differentiation of the monocytes into M1 macrophages. Every 3 days medium was replaced and fresh cytokines added. On day 7 100 U/ml IFN-γ (Immunotools, 11343534) was added to induce polarization and left on the cells for 24 h, after which the cells were ready for FACS. To lyse the erythrocytes within the PMN fraction, 1x RBC lysis buffer (Biolegend, 420302) was added twice, after which the PMNs were washed with PBS. PMNs were stimulated overnight with 100 U/ml IFN-γ (Immunotools, 11343534) and G-CSF (Immunotools, 11343133) to induce FcγRI expression. The PMNs consist mainly of neutrophils and are therefore referred to neutrophils in the main text.
Neutrophils, PBMCs or macrophages were seeded at a density of 1 × 105 cells/well, after which the blocking antibodies (C01, C04 or 10.1 F(ab’)2) or PBS (25 µl/well) were added and incubated for 30 min at 37 °C. ICs were formed using 2.5 μg/ml fluorescently labeled goat-derived polyclonal F(ab’)2-anti-hIgG-F(ab’)2 (Jackson, 109-606-006) with either 5 µg/ml recombinant monoclonal ACPA antibodies (mACPA) or with patient-derived polyclonal ACPA (pACPA) for 30 min at 37 °C and then 10 min on ice. Cells were washed with FACS buffer and 10 µg/ml mACPA-IC or pACPA-IC (10 µg/ml, 25 µl/well) was added and incubated for 1 h at 37 °C. All subsequent steps were performed on ice. For both monocytes and macrophages, cells were stained with live/dead marker eF780 (Fisher Scientific, 13539140), after which CD14-PE (BD) for 30 min at 4 °C, afterwards cells were washed and fixated in 1% PFA. For neutrophils, cells were washed with cold FACS buffer and cold staining mix was added and incubated for 30 min at 4 °C. The staining mix included CD15 (eBioscience), CD16 (FcγRIII, BD), CD32 (FcγRII, Miltenyi), and CD66b (BD) in FACS buffer. Cells were washed once more with cold FACS buffer and resuspended in cold FACS buffer and kept on ice. 15 min before measuring the sample, 7-AAD was added as a live/dead marker. Cells were first gated on live cells and then neutrophils were gated as CD15+ and CD66b + . Percentage blocking per donor is calculated using the following formula: Blocking (%) = 100 – (((sample – negative control) / (ACPA-IC only – negative control)) x 100%). Several controls were taken along to assess changes in FcγRI (clone C01), FcγRII (clone IV.3) and FcγRIII (clone 3G8) on unstimulated and IFN-γ stimulated neutrophils.
Blocking assay opsonized platelets and ITP patient-derived PBMCs
Platelet-rich plasma (PRP) was prepared from whole blood of a healthy donor and adjusted to 200.000 platelets/µL. PRP was diluted 1:1 with PBS and incubated for 30 min at 4 °C with 10 µg of GPIIbIIIa-APC (AssayLite, 11587-05061) antibody to opsonize the platelets. Meanwhile, PBMCs from six ITP patients (see patients section) were carefully thawed and plated at 2 × 105 cells per well. C01, C04 or 10.1 were added and incubated for 30 min at 37 °C to block FcγRI. After washing the PBMCs, the opsonized platelets were added at a physiological ratio of 1:250 PBMC/platelet and incubated for 1 h at 37 °C. The wells were carefully washed once with PBS, and a staining mix of anti-CD45-Pe-Cy7 (BD, 560915), anti-CD14-eF450 (eBioscience, 48‑0149‑42) and the live/dead marker eF780 (Fisher Scientific, 13539140) were added for 20 min at 4 °C. Cells were carefully washed once more, after which the cells were fixated in 1% PFA for 15 min and measured on a BDCanto II.
Generation of human immune system mice
NOD- (001976), SCID- (001303), and γc-deficient (005557) mice were originally obtained from The Jackson Laboratory. Mice lacking the fcer1 gene (FcRγ − /−) were provided by Jeffrey Ravetch (Rockefeller University)29. The NOD-SCID/γc-FcRγ − /− (NSG-FcRγ − /−) mice were generated by crossing SCID, γc-deficient, and FcRγ − /− mice onto a NOD background for a minimum of six generations, followed by intercrossing to generate NSG-FcRγ − /− animals29. In line with their immunodeficient status, the NSG-FcRγ − /− mice received acidified drinking water (pH 3.0) to reduce the risk of bacterial infection. Human immune system (humanized) NSG-FcRγ−/− mice were generated by transplanting human hematopoietic stemc cells (HSCs) into irradiated newborn mice29. Specifically, newborn NSG-FcRγ−/− mice were irradiated at the dose of 1.4 Gy and injected intravenously with 30,000–50,000 human HSCs 4–6 h after irradiation83,84, purified from umbilical cord blood with the written consent of patients and according to the clinical guidelines of the Friedrich-Alexander-University Erlangen-Nürnberg and the Klinikum Fürth (license number: 22-272B). The reconstitution efficiency and leukocyte composition were determined by flow cytometric identification of human cell subsets in whole-blood samples using a Cytek Northern Lights flow cytometer. Within alive (ZombieNIR-, Biolegend, 423105), single cells, the following populations were identified: murine leukocytes (mouse CD45+, clone A20, Biolegend) human leukocytes (human CD45+, clone HI30, Cytek), human B cells (CD19, clone HIB19, Cytek), T cells (CD3+, clone SK7, Cytek), NK cells (CD56+, clone 5.1H11, Cytek), neutrophils (CD66b+, clone G10F5, Biolegend) and monocytes (CD33+, clone P67.6, Cytek) further divided into classical (CD14+, clone M5E2, Cytek) and non-classical monocytes (CD16+, clone 3G8, Cytek). Mice with >5% human CD45+ cells were considered to be successfully humanized and used in ITP experiments.
Both male and female mice (age 12–20 weeks) were used, with groups matched for sex, age, and body weight. No sex-based analysis was performed, as the study employed a short, 4 h ITP model, where sex-specific differences are not expected and have not been reported in prior studies. All experiments were performed with male and female humanized mice at the age of 12 to 20 weeks. Mice were kept in the animal facilities of Friedrich-Alexander-University Erlangen-Nürnberg under specific-pathogen-free conditions in isolated ventilated cages. The housing environment was maintained at 20–24°C with 45–65% humidity and a 12-hour light/dark cycle. Mice were euthanized in CO2. All animal experiments were performed in accordance with the legal requirements of Germany, and were approved by the local experimental animal welfare body, Regierung von Unterfranken (54-2532.1-8/13).
In vivo ITP Induction
ITP was induced by intraperitoneal injection of 100 μg 6A6-hIgG1 antibodies (GenScript) (N = 5). To block human FcγRI, 100 µg of C01 antibody was injected intravenously 2 h prior to 6A6-hIgG1 application. PBS-treated mice (N = 4) were used as control. Platelet counts were determined before and 4 h after antibody injection, at a 1:10 dilution in PBS with an Advia 120 hematology system (Siemens). Platelet counts before antibody injection were set to 100%.
Statistical analysis
Statistical analysis was performed using GraphPad Prism v10.4.0. An unpaired Student’s t-test was used to compare the mean values between the two groups. Statistical analysis for multiple comparison was performed using one- or two-way ANOVA, with Bonferroni correction hoc test or Tukey’s test as indicated in the respective figure legend. Graphs represent mean +- SEM, unless indicated otherwise.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
