Background Identification of immunodominant T cell epitopes of autoantigens is crucial to understanding the pathogenesis of autoimmune diseases and developing disease-specific diagnostic and therapeutic tools. A subset of patients with systemic sclerosis (SSc) exhibit autoantibodies and CD4+ T cells specific for topoisomerase-I (topo-I), which are quantitatively associated with the presence and severity of lung fibrosis.1 Mapping of immunodominant topo-1 T cell epitopes has been difficult due to poor sensitivity, high cost of experimental protocols, and has mainly been based on in silico prediction.
Objectives To develop a new method for mapping immunodominant topo-I T cell epitopes using the natural processing and presentation of HLA-DR-restricted topo-I peptides by monocyte derived dendritic cells (MoDCs) from SSc patients.
Methods MoDCs from 6 anti-topo-I positive SSc patients were pulsed with whole topo-I protein. Following overnight exposure, immunoprecipitation was performed to isolate HLA-DR/peptide complexes. Peptides were eluted, identified by mass spectrometry, and matched to the known Topo-I sequence. These were then synthesised and used to stimulate peripheral blood mononuclear cells (PBMCs) from these patients in the presence of anti-CD40 antibody. Topo-I-reactive CD4+ T cells were identified by flow cytometry based on activation status (CD40L upregulation). PBMCs from 8 HLA-matched healthy donors and 8 additional randomly selected anti-topo-1-positive patients were stimulated using the same protocol.
Results Ten different naturally processed topo-I specific peptides were identified by mass spectrometric analysis. The median number of distinct peptides presented by each individual patient was 4 (range 1–8), with 60% being presented by two or more individuals. Interestingly, four out of six patients presented two peptides with contiguous sequences. All topo-I peptides were able to stimulate CD4+ T cells from at least one of the patients tested, and 45% of patients responded to one or both of the contiguous peptides.
Conclusions Through characterisation of naturally processed topo-1 peptides, we have identified immunodominant topo-1 epitopes capable of stimulating CD4+ T cells from patients with SSc. This method represents a cost-effective and dependable way to identify immunodominant epitopes and can provide novel targets for disease monitoring and eventually, designing peptide-targeted immunotherapy.
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