Key interactions in the trimolecular complex consisting of the rheumatoid arthritis-associated DRB1*04:01 molecule, the major glycosylated collagen II peptide and the T-cell receptor

Objectives Rheumatoid arthritis (RA) is an autoimmune disease strongly associated with the major histocompatibility complex (MHC) class II allele DRB1*04:01, which encodes a protein that binds self-peptides for presentation to T cells. This study characterises the autoantigen-presenting function of DRB1*04:01 (HLA-DRA*01:01/HLA-DRB1*04:01) at a molecular level for prototypic T-cell determinants, focusing on a post-translationally modified collagen type II (Col2)-derived peptide. Methods The crystal structures of DRB1*04:01 molecules in complex with the peptides HSP70(289-306), citrullinated CILP982-996 and galactosylated Col2(259-273) were determined on cocrystallisation. T cells specific for Col2(259-273) were investigated in peripheral blood mononuclear cells from patients with DRB1*04:01-positive RA by cytofluorometric detection of the activation marker CD154 on peptide stimulation and binding of fluorescent DRB1*0401/Col2(259-273) tetramer complexes. The cDNAs encoding the T-cell receptor (TCR) alpha-chains and beta-chains were cloned from single-cell sorted tetramer-positive T cells and transferred via a lentiviral vector into TCR-deficient Jurkat 76 cells. Results The crystal structures identified peptide binding to DRB1*04:01 and potential side chain exposure to T cells. The main TCR recognition sites in Col2(259-273) were lysine residues that can be galactosylated. RA T-cell responses to DRB1*04:01-presented Col2(259-273) were dependent on peptide galactosylation at lysine 264. Dynamic molecular modelling of a functionally characterised Col2(259-273)-specific TCR complexed with DRB1*04:01/Col2(259-273) provided evidence for differential allosteric T-cell recognition of glycosylated lysine 264. Conclusions The MHC-peptide-TCR interactions elucidated in our study provide new molecular insights into recognition of a post-translationally modified RA T-cell determinant with a known dominant role in arthritogenic and tolerogenic responses in murine Col2-induced arthritis.