Upon assembly of HLA tetramers loaded with either of
Upon assembly of HLA tetramers loaded with either of the two peptide versions, we could query the T cell repertoires of RA subjects and healthy individuals for the presence of cognate T tnf alpha inhibitors recognizing the respective peptide-HLA complexes. We found no significant difference in frequencies of T cells recognizing the native eno326-340 between peripheral blood of RA patients and healthy controls, implicating that such cells are usually not fully eliminated during negative selection, but rather part of the normal repertoire. T cells recognizing the native peptide further lacked a memory phenotype and signs of expansion, thus implying that they are naïve (i.e. have not encountered their cognate antigen(s)). In contrast, elevated frequencies of T cells specific for the citrullinated version of the α-enolase peptide in complex with HLA-DRB1*04:01 were observed in RA synovial fluid. These were primarily of memory phenotype indicating that they have been previously activated and expanded. Rheumatoid arthritis manifests in the joints both as synovitis, i.e. the growth of the synovial lining layer, and as joint swelling with exudates in the synovial joint space. We retrieved most of the data on synovial fluid, the edema of inflamed joints, but also had the possibility to assess inflamed synovial tissue. Although direct tissue staining is still not available for HLA class II tetramers, digesting synovial tissues and assaying single cell suspensions makes it still possible to interrogate the RA joint. As a proof of principle, we could demonstrate the presence of auto-reactive CD4+ T cells in synovial tissue. Two findings in this study are of particular interest. First, T cells reactive to HLA-DRB1*04:01 in complex with either the native α-enolase-derived eno326-340 or the post translational modified version cit-eno326-340 are part of the normal circulating T cell repertoire. Secondly, citrulline-reactive memory T cells were not only found elevated in synovial fluid but also in peripheral blood, which is compatible with the notion that the primary activation of auto-reactive T cells may take place outside the joints, for example in the gums [40,41] or in the lungs [8,42,43]. These memory T cells may subsequently migrate to the joints in response to some ‘local insult’ , where they may be reactivated since extracellular citrullination is abundant during any kind of inflammation. These effector memory T cells may then contribute to the maintenance of an inflammatory milieu. Moreover, our structural data allow the dissection of cross-reactivity and the possibility to predict TCR interactions. The vast majority of crystal structures of ternary TCR-peptide-HLA class II complexes indicate that the α-chain of the TCR is landing in the vicinity of the first anchor residue [, , ]. The residue preceding the first anchor position is always protruding towards the TCR and during classical TCR-landing is typically located between the CDR3α and CDR1α loops. Our crystal structures demonstrate that the native and citrullinated versions of the peptide adopt nearly identical conformations throughout the entire length of the binding cleft of HLA-DRB1*04:01. This explains the similar binding affinities of the two peptide versions to HLA-DRB1*04:01 and hence that the arginine/citrulline residue serves as a TCR contact; it is therefore not surprising that citrullination may expand a new fraction of the T cell repertoire. Furthermore, the partial cross-reactivity, which we also functionally detected, could be taken to suggest the existence of three different types of TCRs: One type of TCR recognizes both peptides, i.e. without making direct contact with the citrulline or arginine, while the other two TCR types interact directly to either the arginine or citrulline, hence exhibiting a charge preference only for the positive arginine or the neutral citrulline. It will be of interest to investigate in further detail to which extent cross-reactive and single-reactive T cells give different functional responses (e.g. cytokine secretion or proliferation) in response to the native or citrullinated peptide [, , ] and to what extent single-reactive T cells exhibit different TCR usage.