Myeloperoxidase

Oxidation was induced by incubating protein-coated microtiter plates with 2 μg/mL of myeloperoxidase (Sigma-Aldrich) and 50 mM H₂O₂ (Merck) in PBS (Medicago) for 2h at 37°C. Citrullination was induced by incubating protein-coated microtiter plates with 500 mU/mL human recombinant PAD4 enzyme, in the presence of reducing agent (10 mM dithiothreitol from Fermentas, Thermo Fischer Scientific) in 10 mM Tris buffer (Saveen and Werner AB) with 10 mM CaCl₂ (Merck, pH 7.4), for 2h at 37°C. Carbamylation was induced by incubating protein-coated microtiter plates with 0.1 M KCNO (Sigma-Aldrich) in PBS for 4 or 6 h at 37°C.

All reagents used in this work were analytical or higher grade. Graphite, C₁₈PMH (poly(maleic anhydride-alt-1-octadecene)), hydrazine monohydrate, myeloperoxidase, N-acetyl-l-cysteine, and Chelex 100 resin were purchased from Sigma-Aldrich. MAL-PEG_5k-NHS, DSPE-PEG_2k-NH₂, and Boc-NH-PEG_3k-NH₂ were purchased from Creative PEGworks (Winston Salem, NC, USA). S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) was purchased from Macrocyclics, Inc. (Dallas, TX, USA). H₂DCFDA, Lysotracker, LysoSensor DND 189, and DQ-OVA were purchased from Thermo Fisher Scientific. Antigen peptides, including Adpgk (CSSASMT-NMELM), M27 (LCPGNKYEM), and M30 (CSSVDWEN-VSPELNSTDQ), were synthesized by RS Synthesis (Louisville, KY, USA). Murine class B CpG ODN 1826 was purchased from Integrated DNA Technologies (Coralville, IA, USA). Anti-mouse PD-1 IgG (clone: RMP1–14) was purchased from BioXcell. All other chemicals and reagents were purchased from Thermo Fisher Scientific.