Identification of disease-related T cell clones and arthritogenic antigens in ankylosing spondylitis

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Ankylosing spondylitis (AS) is a highly prevalent chronic inflammatory disease, which affects both the axial skeleton and peripheral joints. Its pathogenesis is unclear, but it is remarkable that significantly more than 90 percent of all patients share a particular HLA allele, namely the HLA class I allele HLA-B*27:05 (in Caucasians). HLA molecules present antigenic peptides to T cells and initiate immune responses. The second strongest genetic marker is "ERAP1", an enzyme that trims antigenic peptides into the binding cleft of HLA molecules. Amongst other hypotheses for the pathogenesis of AS, it has therefore been assumed that "arthritogenic peptides" are presented by HLA-B27 molecules to T cells that would initiate and sustain the inflammatory process. However, so far none such peptides have been identified. Here we propose to identify arthritogenic peptides by a detour via disease-related T cells. To this end, we will first study the T cell receptor (TCR) repertoire and identify clonally expanded CD8+ T cells that are putatively disease-related. Then we will express their TCRs in vitro, and use the recombinant TCRs to identify antigenic peptide antigens presented by HLA-B*27 molecules to the T cells. In preliminary studies, we have identified oligoclonal T cell expansions of antigen experienced CD45RO+ CD8+ memory T cells isolated from synovial fluid of five AS patients with peripheral arthritis by next generation sequencing of TCR alpha- and beta-chains. In all patients the expansions were limited to only a few clones and several clones from different AS patients had highly homologous TCR chains. We are planning to complete these repertoire analyses in a larger cohort and to express about 20 recombinant TCRs which will be used for in vitro antigen searches. To this end, we will use a new technology recently developed in our labs that is based on plasmid-encoded combinatorial peptide libraries and an extremely sensitive detection system. We propose to identify peptide antigens presented by HLA-B*27 to CD8+ T cells and investigate whether candidate arthritogenic human peptides have homologues in commensal microbes or pathogens. To investigate molecular properties of the peptides and their generation in vivo, we will study the role of ERAP1 in processing of the parent proteins. To study whether or not the candidate disease-related peptides are detectable in larger cohorts of AS patients, we will generate HLA-B*27-peptide tetramers and determine frequencies of antigen-specific T cells in peripheral blood and other body fluids of patients and controls and are planning to identify disease-related T cell clones in spinal biopsy samples of AS patients using PCR and HLA-B*27-peptide tetramers. Identification of arthritogenic peptides may have impact in early diagnostics and may possibly also be of therapeutic use.