Abstract
Chlamydia trachomatis triggers reactive arthritis, a spondyloarthropathy linked to the human major histocompatibility complex molecule HLA-B27, through an unknown mechanism that might involve molecular mimicry between chlamydial and self-derived HLA-B27 ligands. Chlamydia-specific CD8(+) T-cells are found in reactive arthritis patients, but the immunogenic epitopes are unknown. A previous screening of the chlamydial genome for putative HLA-B27 ligands predicted multiple peptides that were recognized in vitro by CD8(+) T-lymphocytes from patients. Here stable transfectants expressing bacterial fusion proteins in human cells were generated to investigate the endogenous processing and presentation by HLA-B27 of two such epitopes through comparative immunoproteomics of HLA-B27-bound peptide repertoires. A predicted T-cell epitope, from the CT610 gene product, was presented by HLA-B27. This is, to our knowledge, the first endogenously processed epitope involved in HLA-B27-restricted responses against C. trachomatis in reactive arthritis. A second predicted epitope, from the CT634 gene product, was not detected. Instead a non-predicted nonamer from the same protein was identified. Both bacterial peptides showed very high homology with human sequences containing the HLA-B27 binding motif. Thus, expression and intracellular processing of chlamydial proteins into human cells allowed us to identify two bacterial HLA-B27 ligands, including the first endogenous T-cell epitope from C. trachomatis involved in spondyloarthropathy. That human proteins contain sequences mimicking chlamydial T-cell epitopes suggests a basis for an autoimmune component of Chlamydia-induced HLA-B27-associated disease.
Highlights
Chlamydia trachomatis triggers reactive arthritis, a spondyloarthropathy linked to the human major histocompatibility complex molecule HLA-B27, through an unknown mechanism that might involve molecular mimicry between chlamydial and self-derived HLA-B27 ligands
Identification of Endogenously Processed HLA-B27-restricted T-cell Epitopes from C. trachomatis in Human Cells— The following strategy (Fig. 1) was originally developed in a previous study from our laboratory [16] and was here adapted to investigate the endogenous processing and presentation of HLA-B27 ligands from C. trachomatis recognized by CD8ϩ T-cells from reactive arthritis (ReA) patients [6]. 1) B*2705-C1R cells were used as recipients to generate stable transfectants expressing bacterial fusion proteins
Expression of Chlamydial Fusion Proteins in B*2705-C1R Cells—We focused on the CT634 and CT610 genes, coding for the NADH-quinone reductase subunit A (NQRA) and PqqC proteins, respectively [27], which contain sequences recognized in vitro as exogenously added synthetic peptides by CD8ϩ T-cells from ReA patients [6]
Summary
ReA, reactive arthritis; MHC, major histocompatibility complex; CTL, cytotoxic T-lymphocyte; GFP, green fluorescent protein; NQRA, Naϩ-translocating NADH-quinone reductase subunit A; PqqC, pyrroloquinoline-quinone synthase-like protein; htMDM, high throughput mass data manager; s/n, signal-to-noise ratio; IFN, interferon; fr., fraction; E3, ubiquitin-protein isopeptide ligase; TCR, T-cell antigen receptor. In a previous study [6] predictive algorithms were used to screen the whole genome of C. trachomatis for nonamer peptide sequences containing the HLA-B*2705 binding motif and a high probability of being generated by proteasomal cleavage This led to identifying multiple sequences that, when used as synthetic peptides in vitro, stimulated CD8ϩ T-cells from patients with Chlamydia-induced ReA. Because of the intrinsic cross-reactivity of T-cells [21, 22], it is conceivable that synthetic peptides recognized in vitro may be different from the natural epitopes generated by endogenous processing of the chlamydial proteins that elicit the HLA-B27-restricted T-cell responses in ReA patients To investigate this issue we focused on two predicted epitopes [6]. The endogenous processing and presentation of the predicted epitopes or other peptides from the same bacterial protein were analyzed by comparative immunoproteomics analysis of the B*2705bound peptide repertoires from transfected and untransfected cells and sequencing of peptides differentially presented on the bacterial protein transfectant
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