Human fc block

KIR transfectants and the parental untransfected cell line were fluorescently barcoded (28 (link)) using CellTraceViolet (Life Technologies) and eFluor670 (eBiosciences) proliferation dye reagents (Supplementary Figure S1). The transfectants were pooled and incubated with human FcBlock (1:100 dilution, Miltenyi) for 10 min on ice to reduce non-specific binding of antibodies. A total of 225,000 cells were incubated with 3.3% serum in 1 × PBS with 10% FCS in 96-well-plates at 4°C for 30 min. Cells were washed twice in 1 × PBS with 2 mM EDTA and 0.5% human serum albumin followed by staining with secondary antibody. Binding of human IgG to cells was determined using flow cytometry. For each serum, the median fluorescent intensity (MFI) signal for each transfectant was divided by the MFI signal of the untransfected cells to yield a specific IgG-binding ratio. The specific IgG-binding ratio was transformed to z-scores [(X–mean_healthy)/SD_healthy] and a threshold for antibody positivity was set to 4.

For CMV-specific MHCI tetramer staining, human PBMCs (3x10⁵ cells/condition) were incubated with anti-CD8 (BD, 562428, RRID:AB_11154035), anti-CD25 (BD, 564467), anti-PD-1 (BD, 561272, RRID:AB_2744340), anti-LAG-3 (BioLegend, 369212, RRID:AB_2728373) or anti-LFA-1 (BD, 559875, RRID:AB_2129113) antibodies or respective isotype control antibodies where indicated and PE-CMV tetramer (MBL International, TB-0010–1) or PE-control tetramer (MBL International, TB-0029–1) in FACS buffer containing 1% human Fc Block (Miltenyi Biotec, 130-059-901, RRID:AB_2892112) for 30 min at 4 °C and were then washed three times. Flow cytometry analyses were performed using LSRFortessa (BD Biosciences) and data were analyzed using FlowJo version 10.8 (FlowJo LLC).

About 1 × 10⁵MM6 cells were incubated for 30 min at room temperature with antibodies for CD11b (CD11b-PECy7, eBioscience), CD14 (CD14-APC, eBioscience), CD15 (CD15-eFluor 450, eBioscience), CD33 (CD33-PE P67.6, eBioscience), FVD eFluor^® (eBioscience) or with the proper control IgGs. The optimal concentration for each antibody was adjusted according to manufacturer’s instructions. To block unspecific binding of antibodies, human Fc Block (Miltenyi) was added to the cell suspension. After washing with PBS, FACS analysis was performed using FACS Canto II (Becton Dickinson).

For detection of cell binding, AB21 was fluorescently labeled with the Alexa Fluor 647 Protein Labeling Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. 250,000 cells per well in staining buffer (PBS, 0.5% BSA or 2% FBS) were plated in 96-well plates (Falcon). Cells were first stained with fixable Live/Dead Stain (Invitrogen) and washed once in staining buffer prior to all binding assays.
To detect SIRPα binding to cells, 500 nM Alexa Fluor 647-labeled AB21 was titrated 1:4 for seven dilutions and added to cells in 100 μL volume of FACS buffer (PBS + 0.5% BSA) supplemented with a cocktail of human Fc block (Miltenyi Biotec) or mouse Fc block (Biolegend), anti-CD14 (Biolegend) for human and cynomolgus PBMCs or anti-CD11b (Biolegend) for mouse splenocytes. After a 60-min incubation on ice, cells were washed twice in staining buffer and fixed in 0.5% paraformaldehyde.
To block CD47 binding to SIRPα on PBMCs, Alexa Fluor 647 labeled CD47Fc at a concentration of 500 nM and 1:4 titration starting at 1 µM of AB21 were added to cells. After a 60-min incubation on ice, cells were washed twice in staining buffer and fixed in 0.5% formaldehyde.
Cells were analyzed on a FACS Canto II (BD Biosciences), with subsequent data analysis using Flowjo 10.7.

HSPCs (CD34+ cells) were isolated from cryopreserved fetal or juvenile bone marrow MNCs or fetal liver MNCs via a MACS magnetic enrichment column (Miltenyi, Gaithersburg, MD). At least 10,000,000 viable bone marrow MNCs or 2,000,000 liver MNCs were incubated with anti-human CD34 antibody (BD Biosciences, clone 563) and human Fc block (Miltenyi) together using 3 μL and 6 μL, respectively, per 1,000,000 cells for 20 min at room temperature. The rest of the MACS column protocol was followed according to manufacturer’s instructions (Miltenyi). The CD34-flow-through fraction was collected in addition to the CD34+ fraction. CD34 enrichment efficiency was validated with flow cytometry. Enriched fractions yielded over 90% HSPCs.