Background: Sarcoidosis is a granulomatous disease most commonly affecting the lung. HLA-DR is a genetic risk factor, suggesting a role of HLA-DR antigen presentation in the pathogenesis. Therefore, the study of the HLA-DR ligands in the lung of sarcoidosis patients could help unravelling pathogenic mechanisms. This technique can be applied to immune cells obtained by bronchoalveolar lavage (BAL), primarily alveolar macrophages. However, the scarce material from BAL requires a highly sensitive workflow for HLA-DR immunopeptidomics.

Aims and objectives: To develop a sensitive HLA-DR immunopeptidomics method to facilitate characterization of individual subjects.

Methods: Solubilized HLA-DR complexes were purified using an anti-HLA-DR antibody (L243). Eluted immunopeptides were analyzed by nano-flow liquid chromatography coupled to an Orbitrap mass spectrometer collecting tandem mass spectra in data dependent acquisition mode.

Results: We identified 5872 and 2747 peptides from 10 and 12 million BAL cells respectively. Using the GibbsCluster algorithm (DTU Healthtech), the peptides formed clusters reflecting probable binding motives to the HLA-DR alleles. As expected, the peptides derive predominantly from lysosomal and extracellular matrix proteins. In a sensitivity test of our workflow, we identified more than 500 peptides from as little as four million cells of an EBV transformed B cell line using an optimized mass spectrometry method.

Conclusions: To our knowledge, this reflects the deepest analysis of the HLA-DR immunopeptidome in BAL cells to date. This sensitive method paves the way to the study of HLA-DR immunopeptides of BAL cells from individual sarcoidosis patients, and their involvement in disease pathogenesis.