Icrf44

Venous blood was drawn from IgAV patients and HBDs and collected into heparin-containing tubes. Whole blood immunophenotyping was performed using 7-Color Immunphenotyping kit (Miltenyi Biotec). Briefly, 100 μl of whole blood was incubated with 10 μl immunophenotyping reagent for 10 min in the dark, at 4 °C. After incubation, erythrocytes were lysed using Red Blood Lysing Solution (Miltenyi Biotec). Neutrophil phenotyping was performed in 50 μl of whole blood, incubated for 30 min at 4 °C in the dark with mouse anti-human antibodies to CD16 (conjugated to PE, clone eBioCB16, eBioscience), CD62L (conjugated to PE-Cyanine 5, clone DREG56, eBioscience), and CD11b (conjugated to APC, clone ICRF44, eBioscience). After incubation, samples were lysed, using Whole Blood Lysing Reagent Kit (Beckman Coulter). All samples were analyzed using flow cytometer MACSQuant Analyzer 10 (Miltenyi Biotec). Analysis of flow cytometry data was performed using MACSQquantify (Analysis Software version 2.8) and FlowLogic (Flow Cytometry Analysis Package, version 700.0a).

Murine cells were stained using standard protocols for the surface markers CD45 (clone 30-F11), CD11b (M1/70, eBioscience), Ly6C (HK1.4, eBioscience), Ly6G (RB6-8C5, eBioscience), F4-80, (BM8, eBioscience), MHCII (M5/114.15.2, BD), CD86 (GL1, eBioscience), CD4 (GK1.5, eBioscience), CD8 (53-6.7, eBioscience), CD25 (PC61.5, eBioscience), and LIVE/DEAD Fixable Near-IR Dead Cell Stain. Intracellular staining for Arginase 1 (R&D) (polyclonal sheep IgG), iNOS (CXNFT, eBioscience), TNFa (MP6-XT22, eBioscience) was performed on cells fixed/permeabilized with the Transcription Factor Staining Buffer Set (eBioscience). Human MDSCs were stained for the surface markers CD11b (ICRF44, eBioscience), CD33 (WM-53, eBioscience), HLA-DR (L234, BioLegend), CD4 (OKT4, eBioscience), CD8 (HIT8A, eBioscience). Cytokine production was assessed after 3 hours of PMA + Ionomycin stimulation. Cells were analyzed on the BD Symphony and sorted on the BD FACS Aria.