Background/Purpose:

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. Mapping of immunodominant Topo-I T cell epitopes has been difficult due to poor sensitivity, high cost of current protocols, and has mainly been based on in silico prediction or overlapping peptide libraries. Existing data are limited by the low precision of these approaches and the poor sensitivity of detection assays. We present 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 identified by mass spectrometry, and matched to the Topo-I sequence. The peptides were then synthesized 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/CD154 upregulation). PBMCs from 8 additional randomly selected anti-Topo-I + patients and 6 anti-Topo-I negative controls (anti-centromere or anti-RNA-Polymerase-III) were stimulated using the same protocol.

Results:

Ten different naturally processed Topo-I peptides were identified. The peptides detected were located mainly in the core region of the molecule. The median number of distinct peptides presented by each individual patient was 4 (range 1-8). 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 CD154 upregulation by CD4+ T cells from at least one of the 14 anti-Topo-I positive patients tested. These T cell responses were significantly higher than that of 6 randomly selected anti-Topo-I negative SSc controls (p<0.001).

Conclusion:

Through characterization of naturally processed peptides, we have identified immunodominant Topo-I epitopes capable of stimulating CD4+ T cells from anti-Topo-I positive patients with SSc. Additionally, our approach showed that a restricted set of immunodominant Topo-I epitopes is presented by SSc patients carrying diverse HLA-DR alleles. 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.