title: Approach for Identifying Human Leukocyte Antigen (HLA)-DR Bound Peptides from Scarce Clinical Samples.
abstract: Immune-mediated diseases strongly associating with human leukocyte antigen (HLA) alleles are likely linked to specific antigens. These antigens are presented to T cells in the form of peptides bound to HLA molecules on antigen presenting cells, e.g. dendritic cells, macrophages or B cells. The identification of HLA-DR-bound peptides presents a valuable tool to investigate the human immunopeptidome. The lung is likely a key player in the activation of potentially auto-aggressive T cells prior to entering target tissues and inducing autoimmune disease. This makes the lung of exceptional interest and presents an ideal paradigm to study the human immunopeptidome and to identify antigenic peptides.Our previous investigation of HLA-DR peptide presentation in the lung required high numbers of cells (800 × 10(6) bronchoalveolar lavage (BAL) cells). Because BAL from healthy nonsmokers typically contains 10-15 × 10(6) cells, there is a need for a highly sensitive approach to study immunopeptides in the lungs of individual patients and controls.In this work, we analyzed the HLA-DR immunopeptidome in the lung by an optimized methodology to identify HLA-DR-bound peptides from low cell numbers. We used an Epstein-Barr Virus (EBV) immortalized B cell line and bronchoalveolar lavage (BAL) cells obtained from patients with sarcoidosis, an inflammatory T cell driven disease mainly occurring in the lung. Specifically, membrane complexes were isolated prior to immunoprecipitation, eluted peptides were identified by nanoLC-MS/MS and processed using the in-house developed ClusterMHCII software. With the optimized procedure we were able to identify peptides from 10 × 10(6) cells, which on average correspond to 10.9 peptides/million cells in EBV-B cells and 9.4 peptides/million cells in BAL cells. This work presents an optimized approach designed to identify HLA-DR-bound peptides from low numbers of cells, enabling the investigation of the BAL immunopeptidome from individual patients and healthy controls in order to identify disease-associated peptides.
authors.affiliation: From the ‡Respiratory Medicine Unit, Department of Medicine and Center for Molecular Medicine, Solna, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden; §Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden;
authors.forename: Tina
authors.initials: T
authors.lastname: Heyder
authors.affiliation: From the ‡Respiratory Medicine Unit, Department of Medicine and Center for Molecular Medicine, Solna, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden;
authors.forename: Maxie
authors.initials: M
authors.lastname: Kohler
authors.affiliation: §Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden;
authors.forename: Nataliya K
authors.initials: NK
authors.lastname: Tarasova
authors.affiliation: ¶Division of Medical Inflammation Research, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden;
authors.forename: Sabrina
authors.initials: S
authors.lastname: Haag
authors.affiliation: §Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden;
authors.forename: Dorothea
authors.initials: D
authors.lastname: Rutishauser
authors.affiliation: From the ‡Respiratory Medicine Unit, Department of Medicine and Center for Molecular Medicine, Solna, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden;
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Rivera
authors.affiliation: ‖Rheumatology Unit, Department of Medicine, Solna, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Charlotta
authors.initials: C
authors.lastname: Sandin
authors.affiliation: ‖Rheumatology Unit, Department of Medicine, Solna, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Sohel
authors.initials: S
authors.lastname: Mia
authors.affiliation: ‖Rheumatology Unit, Department of Medicine, Solna, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Vivianne
authors.initials: V
authors.lastname: Malmström
authors.affiliation: From the ‡Respiratory Medicine Unit, Department of Medicine and Center for Molecular Medicine, Solna, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden;
authors.forename: Åsa M
authors.initials: ÅM
authors.lastname: Wheelock
authors.affiliation: From the ‡Respiratory Medicine Unit, Department of Medicine and Center for Molecular Medicine, Solna, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden;
authors.forename: Jan
authors.initials: J
authors.lastname: Wahlström
authors.affiliation: ¶Division of Medical Inflammation Research, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden;
authors.forename: Rikard
authors.initials: R
authors.lastname: Holmdahl
authors.affiliation: From the ‡Respiratory Medicine Unit, Department of Medicine and Center for Molecular Medicine, Solna, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden;
authors.forename: Anders
authors.initials: A
authors.lastname: Eklund
authors.affiliation: §Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden;
authors.forename: Roman A
authors.initials: RA
authors.lastname: Zubarev
authors.affiliation: From the ‡Respiratory Medicine Unit, Department of Medicine and Center for Molecular Medicine, Solna, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden;
authors.forename: Johan
authors.initials: J
authors.lastname: Grunewald
authors.affiliation: §Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; ‖Rheumatology Unit, Department of Medicine, Solna, Karolinska Institutet, Stockholm, Sweden jimmy.ytterberg@ki.se.
authors.forename: A Jimmy
authors.initials: AJ
authors.lastname: Ytterberg
mesh_terms: D000328:Adult; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology / Q000276:immunology*; D002478:Cells, Cultured; D005260:Female; D006684:HLA-DR Antigens / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding; D012507:Sarcoidosis / Q000276:immunology / Q000628:therapy*
chemical_list: D006684:HLA-DR Antigens; D010455:Peptides