HLA class I-naturally presented synovial tissue peptides are recognized by CD8+ T lymphocytes from rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is an autoimmune disease resulting from a response driven by self-reactive CD4+ T cells that recognize autoantigenic peptides presented by antigen-presenting cells (APCs). Recent evidence suggests that CD8+ T cells are also important players in this process. This study aims to define the immunopeptidome of HLA class I molecules from RA synovial tissue (ST)- APCs and synovial fluid (SF)-pulsed monocyte-derived dendritic cells (DCs), and to prove the suitability of this approach to identify peptides recognized by CD8+ T cells from RA patients. HLA-ABC/peptide complexes were obtained from DCs generated from healthy subjects (HS), which were pulsed with a pool of RA SF (SF-DCs) or left unpulsed (UP-DCs), or obtained directly from RA ST. Isolated peptides were sequenced by mass spectrometry. The autoantigenicity of a set of ten peptides selected from this repertoire was estimated by their ability to activate CD8+ T cells from RA patients, as measured by the induction of intracellular IFN-γ expression and surface exposure of CD107a by flow cytometry. Between 107 to 663 peptides were obtained from DC samples, while over 3, 500 class I peptides were identified from each ST sample. The number of peptides was narrowed down based on prioritization steps that included the selection of sequences derived from RA-relevant, immune-related proteins, for further CD8+ T-cell stimulation assays. The frequencies of CD8+ T cells co-stained for IFN-γ and CD107a were significantly higher in RA patients than in HS in response to peptides derived from the proteins MIF, ETS1, USF1, VIM, and AHR. In the present work, we validated the use of an immunopeptidomic strategy to identify a series of novel autoantigenic CD8+ T-cell epitopes for RA, derived from synovial, mostly immune-related proteins, which may be useful for future clinical applications.