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06.15 Naturally processed topoisomerase i peptides presented by dendritic cells identify immunodominant t cell epitopes in systemic sclerosis
  1. Eleni Tiniakou1,
  2. Andrea Fava1,
  3. Tara Gurh1,
  4. Francesco Boin2,
  5. Erika Darrah1
  1. 1Division of Rheumatology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
  2. 2Division of Rheumatology, University of California San Francisco, San Francisco, CA, USA

Abstract

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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