Definition of Naturally Processed Peptides Reveals Convergent Presentation of Autoantigenic Topoisomerase I Epitopes in Scleroderma

Objective Autoimmune responses to DNA topoisomerase I (topo I) are found in a subset of scleroderma patients who are at high risk for interstitial lung disease ( ILD ) and mortality. Anti–topo I antibodies ( ATA s) are associated with specific HLA – DRB 1 alleles, and the frequency of HLA – DR –restricted topo I–specific CD 4+ T cells is associated with the presence, severity, and progression of ILD . Although this strongly implicates the presentation of topo I peptides by HLA – DR in scleroderma pathogenesis, the processing and presentation of topo I has not been studied. Methods We developed a natural antigen processing assay ( NAPA ) to identify putative CD 4+ T cell epitopes of topo I presented by monocyte‐derived dendritic cells (mo‐DCs) from 6  ATA ‐positive patients with scleroderma. Mo‐DCs were pulsed with topo I protein, HLA – DR –peptide complexes were isolated, and eluted peptides were analyzed by mass spectrometry. We then examined the ability of these naturally presented peptides to induce CD 4+ T cell activation in 11 ATA ‐positive and 11 ATA ‐negative scleroderma patients. Results We found that a common set of 10 topo I epitopes was presented by Mo‐DCs from scleroderma patients with diverse HLA – DR variants. Sequence analysis revealed shared peptide‐binding motifs within the HLA – DR β chains of ATA ‐positive patients and a subset of topo I epitopes with distinct sets of anchor residues capable of binding to multiple different HLA – DR variants. The NAPA ‐derived epitopes elicited robust CD 4+ T cell responses in 73% of ATA ‐positive patients (8 of 11), and the number of epitopes recognized correlated with ILD severity ( P = 0.025). Conclusion These findings mechanistically implicate the presentation of a convergent set of topo I epitopes in the development of scleroderma.