Autoimmune rheumatologic diseases
A subset of patients with systemic sclerosis (SSc) have autoantibodies and CD4+ T cells specific for the autoantigen topoisomerase-I (Topo-I), which are associated with the presence, progression and severity of lung fibrosis. Identification of immunodominant Topo-I epitopes is critical for understanding disease pathogenesis and developing autoantigen-specific diagnostic and therapeutic tools. Existing data are limited by the low precision of in silico epitope prediction and the poor sensitivity of autoantigen-specific T cell detection assays. Thus, we developed a novel method which couples the specificity of natural antigen processing to the sensitivity of CD154 upregulation for measuring antigen-specific T cell responses. After pulsing with whole Topo-I protein monocyte-derived dendritic cells obtained from 6 anti-Topo-I antibody positive SSc patients, HLA-DR/peptide complexes were isolated by immunoprecipitation. Bound peptides were eluted and identified by mass spectrometry. The 10 Topo-I peptides detected were located mainly in the core (III) and linker domain of the molecule. Peptide overlap among patients existed despite differences in their HLA-DR haplotype, with 8 out of 10 epitopes being presented by two or more subjects. All Topo-I peptides were able to stimulate significant CD154 upregulation by CD4+ T cells from at least one anti-Topo-I positive patient when compared to subsequently tested HLA-matched healthy controls(n=14). In conclusion, our approach allowed successful identification of naturally processed autoreactive Topo-I peptides and showed that a restricted set of immunodominant Topo-I epitopes is shared by SSc patients carrying diverse HLA-DR alleles.