Identification of HLA-DR-bound peptides presented by human bronchoalveolar lavage cells in sarcoidosis

Abstract

Sarcoidosis is an inflammatory disease of unknown etiology, most commonly affecting the lungs. Activated CD4+ T cells accumulate in the lungs of individuals with sarcoidosis and are considered to be of central importance for inflammation. We have previously shown that Scandinavian sarcoidosis patients expressing the HLA-DR allele DRB1*0301 are characterized by large accumulations in the lungs of CD4+ T cells expressing the TCR AV2S3 gene segment. This association afforded us a unique opportunity to identify a sarcoidosis-specific antigen recognized by AV2S3+ T cells. To identify candidates for the postulated sarcoidosis-specific antigen, lung cells from 16 HLA-DRB1*0301pos patients were obtained by bronchoalveolar lavage. HLA-DR molecules were affinity purified and bound peptides acid eluted. Subsequently, peptides were separated by reversed-phase HPLC and analyzed by liquid chromatography-mass spectrometry. We identified 78 amino acid sequences from self proteins presented in the lungs of sarcoidosis patients, some of which were well-known autoantigens such as vimentin and ATP synthase. For the first time, to our knowledge, we have identified HLA-bound peptides presented in vivo during an inflammatory condition. This approach can be extended to characterize HLA-bound peptides in various autoimmune settings.

Figure 1. RP-HPLC chromatogram of HLA-DR–bound peptides purified from BAL cells using the mAb L243 and recorded at 214 nm.

Figure 2. Total ion current of peptides eluted from HLA-DR molecules and separated by RP-HPLC.

Individual peptides were subjected to fragmentation in a collision cell.

Figure 3. Examples of MS/MS spectra.

Collision-induced decay (CID) mass spectra of the natural eluted (upper) and the synthetic (lower) peptide DSLPLVDTHSKRTLL ((M + 2H)²⁺=847.9, vimentin_429–443).