Isolation of HLA-DR-naturally presented peptides identifies T-cell epitopes for rheumatoid arthritis

Ann Rheum Dis. 2022 Aug;81(8):1096-1105. doi: 10.1136/annrheumdis-2021-220371. Epub 2022 Apr 22.


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.

Keywords: Arthritis, Rheumatoid; Autoimmunity; Synovial fluid; T-Lymphocyte subsets.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arthritis, Rheumatoid*
  • Epitopes, T-Lymphocyte*
  • HLA-DR Antigens
  • Humans
  • Leukocytes, Mononuclear
  • Peptides


  • Epitopes, T-Lymphocyte
  • HLA-DR Antigens
  • Peptides