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Approach for Identifying Human Leukocyte Antigen (HLA)-DR Bound Peptides from Scarce Clinical Samples
- Source :
- Molecularcellular proteomics : MCP. 15(9)
- Publication Year :
- 2016
-
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.
- Subjects :
- 0301 basic medicine
Adult
Male
Sarcoidosis
T cell
Cell
Human leukocyte antigen
Biology
Biochemistry
Analytical Chemistry
03 medical and health sciences
0302 clinical medicine
Antigen
medicine
HLA-DR
Humans
Antigen-presenting cell
Molecular Biology
HLA-DR Antigen
Cells, Cultured
medicine.diagnostic_test
Research
HLA-DR Antigens
Middle Aged
030104 developmental biology
Bronchoalveolar lavage
medicine.anatomical_structure
Immunology
Female
Peptides
Bronchoalveolar Lavage Fluid
030215 immunology
Protein Binding
Subjects
Details
- ISSN :
- 15359484
- Volume :
- 15
- Issue :
- 9
- Database :
- OpenAIRE
- Journal :
- Molecularcellular proteomics : MCP
- Accession number :
- edsair.doi.dedup.....73330406605024cc5bbf13f27562496f