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Immunogenic HLA-DR-Presented Self-Peptides Identified Directly from Clinical Samples of Synovial Tissue, Synovial Fluid, or Peripheral Blood in Patients with Rheumatoid Arthritis or Lyme Arthritis.

ABSTRACT: Human leukocyte antigen-antigen D related (HLA-DR) molecules are highly expressed in synovial tissue (ST), the target of the immune response in chronic inflammatory forms of arthritis. Here, we used LC-MS/MS to identify HLA-DR-presented self-peptides in cells taken directly from clinical samples: ST, synovial fluid mononuclear cells (SFMC), or peripheral blood mononuclear cells (PBMC) from five patients with rheumatoid arthritis (RA) and eight with Lyme arthritis (LA). We identified 1593 non-redundant HLA-DR-presented peptides, derived from 870 source proteins. A total of 67% of the peptides identified in SFMC and 55% of those found in PBMC were found in ST, but analysis of SFMC/PBMC also revealed new antigen-presented peptides. Peptides were synthesized and examined for reactivity with the patients' PBMC. To date, three autoantigens in RA and four novel autoantigens in LA, presented in ST and/or PBMC, were shown to be targets of T- and B-cell responses in these diseases; ongoing analyses may add to this list. Thus, immunoprecipitation and LC-MS/MS can now identify hundreds of HLA-DR-presented self-peptides from individual patients' tissues or fluids with mixed cell populations. Importantly, identification of HLA-DR-presented peptides from SFMC or PBMC allows testing of more patients, including those early in the disease. Direct analysis of clinical samples facilitates identification of novel immunogenic T-cell epitopes.

SUBMITTER: Wang Q

PROVIDER: S-EPMC5766322 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Immunogenic HLA-DR-Presented Self-Peptides Identified Directly from Clinical Samples of Synovial Tissue, Synovial Fluid, or Peripheral Blood in Patients with Rheumatoid Arthritis or Lyme Arthritis.

Wang Qi Q Drouin Elise E EE Yao Chunxiang C Zhang Jiyang J Huang Yu Y Leon Deborah R DR Steere Allen C AC Costello Catherine E CE

Journal of proteome research 20161107 1

Human leukocyte antigen-antigen D related (HLA-DR) molecules are highly expressed in synovial tissue (ST), the target of the immune response in chronic inflammatory forms of arthritis. Here, we used LC-MS/MS to identify HLA-DR-presented self-peptides in cells taken directly from clinical samples: ST, synovial fluid mononuclear cells (SFMC), or peripheral blood mononuclear cells (PBMC) from five patients with rheumatoid arthritis (RA) and eight with Lyme arthritis (LA). We identified 1593 non-red ...[more]

PMID: 27726376

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Project description:HLA-DR molecules are highly expressed in synovial tissue (ST), the target of the immune response in chronic inflammatory arthritides, including in rheumatoid arthritis (RA) and Lyme arthritis (LA). In the current study, we identified HLA-DR-presented self-peptides (T cell epitopes) in ST, synovial fluid mononuclear cells (SF), and peripheral blood mononuclear cells (PB) from five patients with RA and eight with LA. Altogether, from 22 samples, 1,532 non-redundant HLA-DR-presented peptides were identified that were derived from 778 source proteins. Moreover, as the study progressed, application of newer, high sensitivity LC-MS instruments resulted in the identification of larger numbers of HLA-DR-presented peptides. Surprisingly, the source proteins for HLA-DR-presented peptides were as likely to have intracellular as extracellular locations. Although the number of peptides identified was greater in ST than in SF or PB, 68% of the peptides identified in SF were found in ST, and 55% of the peptides in PB were found in ST. Two RA patients had the same HLA-DR genotype (DRB1*0401 and 0101), and 44% of the peptides identified in these two patients were the same. In contrast, among the patients with RA or LA who had no shared HLA-DR alleles, only 6% of the peptides were the same. In the RA patients, HLA-DR-presented peptides were identified from source proteins that are thought to serve as potential autoantigens: collagen, enolase, fibrinogen, fibronectin, immunoglobulin and vimentin. Interestingly, peptides from these same source proteins were also commonly found in LA patients. However, citrullinated T cell epitopes were not identified in any patient. Thus, LC-MS/MS techniques are now sensitive enough to identify large numbers of T cell epitopes from tissue or fluids in individual patients. Importantly, analysis of SF or PB allowed us to identify a broader range of HLA-DR-presented self-peptides from individual patients and from patients seen earlier in the disease.

Project description:BACKGROUND:Rheumatoid arthritis and osteoarthritis are two common musculoskeletal disorders that affect the joints. Despite high prevalence rates, etiological factors involved in these disorders remain largely unknown. Dissecting the molecular aspects of these disorders will significantly contribute to improving their diagnosis and clinical management. In order to identify proteins that are differentially expressed between these two conditions, a quantitative proteomic profiling of synovial fluid obtained from rheumatoid arthritis and osteoarthritis patients was carried out by using iTRAQ labeling followed by high resolution mass spectrometry analysis. RESULTS:We have identified 575 proteins out of which 135 proteins were found to be differentially expressed by ?3-fold in the synovial fluid of rheumatoid arthritis and osteoarthritis patients. Proteins not previously reported to be associated with rheumatoid arthritis including, coronin-1A (CORO1A), fibrinogen like-2 (FGL2), and macrophage capping protein (CAPG) were found to be upregulated in rheumatoid arthritis. Proteins such as CD5 molecule-like protein (CD5L), soluble scavenger receptor cysteine-rich domain-containing protein (SSC5D), and TTK protein kinase (TTK) were found to be upregulated in the synovial fluid of osteoarthritis patients. We confirmed the upregulation of CAPG in rheumatoid arthritis synovial fluid by multiple reaction monitoring assay as well as by Western blot. Pathway analysis of differentially expressed proteins revealed a significant enrichment of genes involved in glycolytic pathway in rheumatoid arthritis. CONCLUSIONS:We report here the largest identification of proteins from the synovial fluid of rheumatoid arthritis and osteoarthritis patients using a quantitative proteomics approach. The novel proteins identified from our study needs to be explored further for their role in the disease pathogenesis of rheumatoid arthritis and osteoarthritis.Sartaj Ahmad and Raja Sekhar Nirujogi contributed equally to this article.

Dataset Information

Immunogenic HLA-DR-Presented Self-Peptides Identified Directly from Clinical Samples of Synovial Tissue, Synovial Fluid, or Peripheral Blood in Patients with Rheumatoid Arthritis or Lyme Arthritis.

Publications

Immunogenic HLA-DR-Presented Self-Peptides Identified Directly from Clinical Samples of Synovial Tissue, Synovial Fluid, or Peripheral Blood in Patients with Rheumatoid Arthritis or Lyme Arthritis.

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