Objective Rheumatoid arthritis (RA) immunopathogenesis revolves around the presentation of poorly characterised self-peptides by human leucocyte antigen (HLA)-class II molecules on the surface of antigen-presenting cells to autoreactive CD4 +T cells. Here, we analysed the HLA-DR-associated peptidome of synovial tissue (ST) and of dendritic cells (DCs) pulsed with synovial fluid (SF) or ST, to identify potential T-cell epitopes for RA.
Methods HLA-DR/peptide complexes were isolated from RA ST samples (n=3) and monocyte-derived DCs, generated from healthy donors carrying RA-associated shared epitope positive HLA-DR molecules and pulsed with RA SF (n=7) or ST (n=2). Peptide sequencing was performed by high-resolution mass spectrometry. The immunostimulatory capacity of selected peptides was evaluated on peripheral blood mononuclear cells from patients with RA (n=29) and healthy subjects (n=12) by flow cytometry.
Results We identified between 103 and 888 HLA-DR-naturally presented peptides per sample. We selected 37 native and six citrullinated (cit)-peptides for stimulation assays. Six of these peptides increased the expression of CD40L on CD4 +T cells patients with RA, and specifically triggered IFN-γ expression on RA CD4 +T cells compared with healthy subjects. Finally, the frequency of IFN-γ-producing CD4 +T cells specific for a myeloperoxidase-derived peptide showed a positive correlation with disease activity.
Conclusions We significantly expanded the peptide repertoire presented by HLA-DR molecules in a physiologically relevant context, identifying six new epitopes recognised by CD4 +T cells from patients with RA. This information is important for a better understanding of the disease immunopathology, as well as for designing tolerising antigen-specific immunotherapies.
- Arthritis, Rheumatoid
- Synovial fluid
- T-Lymphocyte subsets
Data availability statement
All data relevant to the study are included in the article. All data relevant to the study are included in the article or uploaded as online supplemental information.
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Handling editor Josef S Smolen
Contributors JCA, DC and JM participated in the conception and design of the study. JM, MC, DC, JCA, OA, JA, DJ and EJ worked on analysis and interpretation of data. JM, DC and JCA participated in manuscript preparation and redaction. JM, MC and EJ performed experiments and data acquisition. LS, ON and MC participated in the recruitment of subjects for this study. JCA acts as guarantor.
Funding This study was funded by the following grants: Fondecyt 1181853, Fondef-IDeA ID15I10080; Fondef-IDeA ID15I20080, Fondef-IDeA ID18I10243, and REDES 180028, from ANID, Chile; and by Project RTI2018-097414-B-I00 from the Spanish Ministry of Science. Doctoral training of JM was supported by ANID-PFCHA/National Doctoral Scholarship 2018/No 21181538.
Competing interests JCA reported five grants from Agencia Nacional de Investigación y Desarrollo (ANID), Chile, as financial support for this study; DJ reported one grant from the Spanish Ministry of Science; and JM reported a scholarship for her Doctoral training from ANID, during the conduct of the study.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.
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