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Poster Presentations
Day 4, June 25（Wed.）
Room P (Maesato East, Foyer, Ocean Wing)
- 4P-PM-50
Sensitive methods for characterization of the HLA-DR immunopeptidome of extracellular vesicles from immune cells from bronchoalveolar lavage in respiratory disease
(1Karolinska inst, 2Karolinska Inst)
Benedikt Zohrer1, Iryna Kolosenko1, Nicole Wagner1, Magnus Skold1, Akos Vegvari2, oAsa Wheelock1
Evidence suggests that self-tolerance is breached in the lung prior to clinical onset of autoimmune disease in other organs. We recently developed a sensitive workflow for identification of the immunopeptidome presented by human leukocyte antigen DR (HLA-DR) by immune cells collected by bronchoalveolar lavage from the lung (BAL-cells). The workflow was validated in at-risk healthy smokers and newly diagnosed, treatment naïve rheumatoid arthritis (RA) patients (1), facilitating detection of 1118 to 5240 peptides per subject from as little as 6 million immune cells. 77 novel immunoeptides associated with RA, including peptides from known citrullinated and non-modified RA autoantigens (e.g. α-enolase and calreticulin) were identified. Here we expanded on this method to facilitate characterization of the immunopeptidome presented by extracellular vesicles (BALF-EVs) including exosomes. EVs and exosome were isolation by sucrose gradient from conditioned media from THP-1 cells. MHCII complexes were isolated as previously described. Peptides were identified by nano-flow LC coupled to an Orbitrap MS equipped with ion mobility device (FAIMS) in data dependent acquisition mode, peptides were identified with MSFragger (4.1), and binding was predicting using MHCMotivDecon (1.1) with the reported HLA-DR genotypes DRB1*01:01, DRB1*15:01, DRB5*01:01 for THP-1. A subset of 15 peptides were identified from BALF-EVs.