br Introduction Rheumatoid arthritis RA is a chronic
Introduction Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease that can be divided in at least two subsets based on the presence of antibodies to citrullinated proteins (ACPAs) . Importantly, such ACPA target proteins [, , , ] also represent candidate autoantigens for CD4+ T cell responses, as further substantiated by the strong genetic association between ACPA-positive RA and the so called shared epitope HLA-DR Cholera Toxin [, , ]. Indeed, previous studies of peripheral blood from ACPA-positive RA patients have demonstrated autoreactive CD4+ T cells against several citrullinated self-proteins [, , , , ]. Alpha-enolase represents an interesting autoantigen in RA as the presence of autoantibodies against citrullinated α-enolase is preferentially linked to HLA-DRB1*04 , which is the most studied HLA class II allele in the context of RA. Although α-enolase is considered a ubiquitous protein, it is citrullinated and overexpressed in the inflamed synovium , and antibodies to the immunodominant citrullinated B cell epitope CEP-1 are present in approximately half of all ACPA-positive patients [14,15]. These citrullinated α-enolase-specific autoantibodies are highly specific for RA [4,14] and significantly enriched in synovial fluid . CD4+ T cells play an important role in RA by secreting pro-inflammatory cytokines and activating B cells, which contribute to the inflammatory perpetuation. A major technical hurdle, regarding the characterization of T cells that specifically recognize potential RA-specific autoantigens, is related to the fact that autoantigen-specific T cells are generally present in very low frequencies [, , , , ]. Through recent improvements in the field of HLA class II/peptide tetramers, based on bead enrichment, it is now possible to assess rare antigen-specific CD4+ T cells directly ex vivo without prior in vitro expansion [13,, , ]. Furthermore, several T cell epitopes have been identified based on the notion that the P4 pocket of RA-associated HLA-DR alleles cannot accommodate a positively charged arginine side chain but can fit a neutral citrulline . However, a growing list of citrulline-specific T cell epitopes have demonstrated that the citrulline can also be located in positions contributing to TCR contact [13,26,27]. We have recently scanned the entire α-enolase protein for T cell epitopes and demonstrated the existence of several peptides for which the citrulline modification is not confined to the classical P4 position in the HLA binding cleft . Since this alternative repertoire of T cell epitopes has been less studied, we have here focused on the α-enolase peptide KRIAKAVNEKSCNCL, spanning residue 326 to 340 which comprises an arginine residue at the N-terminus. We have previously demonstrated the suitability of this epitope for HLA-tetramer studies aiming at characterization of specific autoreactive T cell populations . Moreover, we have demonstrated in peptide competition assays that both peptide versions, native eno326-340 and modified cit-eno326-340 have similar binding affinities to HLA-DRB1*04:01 . This provided us with a unique opportunity to assess the contribution of the non-conventionally located arginine/citrulline residue of the peptide to T cell receptor (TCR) recognition. To achieve this, we determined the crystal structures of the HLA-DRB1*04:01/eno326-340 and HLA-DRB1*04:01/cit-eno326-340 complexes at 1.33 Å and 1.35 Å resolution, respectively, and demonstrated the existence of both specific as well as cross-reactive T cells. Strikingly, only cit-α-eno326-340 specific T cells were found to be enriched in the RA joint implicating their involvement in the HLA-restricted immune response driving joint inflammation.
Material and methods
Discussion HLA-DRB1*04:01 is the most studied RA-associated HLA class II molecule. In the present study we have determined the crystal structures of HLA-DRB1*04:01 in complex with a 15-mer peptide derived from an RA candidate autoantigen, i.e. peptide 326–340 of α-enolase, in both its native and citrullinated forms. Whereas the native peptide is an abundantly available self-peptide, the citrullinated version represents a neo-antigen not necessarily present during thymic T cell selection. This is an important distinction since the posttranslational citrulline modification did not alter peptide binding to HLA, but instead would bias TCR binding based on contacts made with either the positively charged side chain of the arginine or the neutrally charged citrulline residue.