Om rats immunized with HLA-B27 and stimulated in vitro with Chlamydia-treated cells from HLA-B27 transgenic rats resulted within the generation of Chlamydia-specific CD8 T-cells (27). Additionally, splenocytes from HLA-B27 transgenic rats immunized with HLA-B27 created S1PR3 Agonist Molecular Weight HLA-B27-directed autoreactivity upon exposure to C. trachomatis in vitro (28). The immunological relationship involving Chlamydia and HLA-B27 revealed by these research was suggestive of molecular mimicry among bacterial and self-derived HLA-B27-restricted epitopes. In spite of troubles in substantiating molecular mimicry as a mechanism of autoimmunity (29), it played a crucial part in the pathogenesis of Chlamydia-induced autoimmune myocarditis in mice (30). Hence, there’s a sound basis to search for HLA-B27-restricted chlamydial T-cell epitopes and their feasible connection to self-derived HLAB27 ligands (31). Predictive binding and proteasomal cleavage algorithms were employed to localize putative chlamydial epitopes. The candidates had been tested for recognition by particular CTL from transgenic mice or HLA-B27 ReA individuals (32) or employed for producing B27 tetramers to detect peptide-specific T-cells (33). These research identified some HLA-B27-restricted epitopes for which distinct CTL may be identified in Chlamydia-infected ReA sufferers. On the other hand, because of the intrinsic cross-reactivity of T-cells (34), recognition of a synthetic peptide in vitro does notSEPTEMBER six, 2013 VOLUME 288 NUMBERguarantee that this peptide is the actual immunogenic epitope in vivo. The direct biochemical identification of endogenous chlamydial T-cell epitopes from infected cells has been accomplished only in the mouse method (35, 36). It really is hardly feasible in humans, resulting from the extremely low amounts of bacterial epitopes on infected cells, the difficulties related with operating with huge amounts of Chlamydia-infected human cells, and, especially, the down-regulation of MHC-I expression and PDE3 Modulator Species induction of apoptosis by C. trachomatis (19, 37). Therefore, we created an alternative method involving the stable expression of chlamydial fusion proteins on HLA-B27 human cells. Endogenously processed chlamydial peptides, including a predicted T-cell epitope, have been identified by comparing the HLA-B27-bound peptidomes from transfected and untransfected cells. These research (38, 39) have been determined by comparative MALDI-TOF MS and concerned three chlamydial proteins containing sequences very homologous to recognized human-derived HLA-B27 ligands or from which synthetic peptides have been recognized by CTL from ReA patients: DNA primase (DNAP) (CT794), Na -translocating NADH-quinone reductase subunit A (NQRA) (CT634), and pyrroloquinoline-quinone synthase-like protein (PqqC) (CT610). In two diverse research, determined by a predictive look for HLA-B27-restricted chlamydial ligands in ReA sufferers (32, 33), a sequence from ClpC protein, spanning residues 75, was recognized as a synthetic peptide by CD8 T-cells from many people, suggesting that this epitope could possibly be immunodominant. Here we used MS approaches of higher sensitivity and accuracy to investigate the endogenous processing and presentation of this and other HLA-B27-restricted peptides from ClpC and also other chlamydial proteins. Molecular dynamics simulations have been also carried out to analyze the relationship in between chlamydial and homologous human-derived B27 ligands in the conformational level.EXPERIMENTAL PROCEDURESClpC Gene Constructs–Enhanced GFP (EGFP)-ClpC fusion proteins were gene.