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

Abstract

words):Objective: Autoimmune responses to DNA topoisomerase-I (TOP1) are found in a subset of patients with scleroderma at high risk for interstitial lung disease (ILD) and mortality. Anti-TOP1 antibodies (ATA) are associated with specific HLA-DRB1 alleles, and the frequency of HLA-DR-restricted TOP1-specific CD4+ T cells is associated with the presence, severity, and progression of ILD.Although this strongly implicates the presentation of TOP1 peptides by HLA-DR in scleroderma pathogenesis, the processing and presentation of TOP1 has not been studied. Methods:We developed a natural antigen processing assay (NAPA) to identify putative CD4+ T cell epitopes of TOP1 presented by monocyte-derived dendritic cells (mo-DCs) from six ATA-positive patients with scleroderma. Mo-DCs were pulsed with TOP1 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 CD4+ T cell activation in 11 ATA-positive and 11 ATA-negative scleroderma patients. Results:We found that a common set of 10 TOP1 epitopes was presented by mo-DCs from scleroderma patients with diverse HLA-DR variants. Sequence analysis revealed shared peptidebinding motifs within the HLA-DRβ chains of ATA-positive patients and a subset of TOP1 epitopes with distinct sets of anchor residues capable of binding to multiple different HLA-DR variants. The NAPA-derived epitopes elicited robust CD4+ T cell responses in 73% (8/11) ATA-positive patients, and the number of epitopes recognized correlated with ILD severity (p=0.025). Conclusion:These findings mechanistically implicate presentation of a convergent set of TOP1 epitopes in the development of scleroderma.

Borrelia burgdorferi-Induced Changes in the Class II Self-Immunopeptidome Displayed on HLA-DR Molecules Expressed by Dendritic Cells

“…The observation that B. burgdorferi -induced changes led to robust expression of HLA-DR on the surface of monocyte-derived dendritic cells in a time- and dose-dependent manner led us to reason that the HLA-DR associated self-immunopeptidome was altered, and formed the premise for this study. Using the natural processing and presentation capabilities of mo-DCs, we isolated sufficient peptide-HLA-DR complexes ( 13 ) to define the self-immunopeptidome at steady state, and its variation during stimulation with the TLR-2 ligand lipoteichoic acid or with live B. burgdorferi . In all conditions, we isolated peptides that varied in length (seven to 65 amino acids long) ( 25 ) averaging 15 residues, the optimal length of MHC class II presented antigens.…”

“…We performed a natural antigen processing assay (NAPA) as described previously ( 13 ). Briefly, immature mo-DCs were seeded in 6-well plates at a density of 1 × 10 6 cells in 1 mL of RPMI 1640 and 10% fetal bovine serum, supplemented with 2 mM L-glutamine and 20 μM ß-mercaptoethanol in duplicate.…”

“…Immature mo-DCs were stimulated with 1 μg/mL purified LTA or live B. burgdorferi strains A3 or B31-5A19 at a multiplicity of infection of 10 bacteria per cell for 24 h. Cells were incubated at 37°C, 5% CO 2 , and >95% humidity. At the end of the incubation period, naturally processed and presented peptides were isolated from HLA-DR molecules by immunoprecipitation using a natural antigen processing assay ( 13 ). Briefly, cells were harvested and pelleted at 1,200 rpm for 10 min at 4°C.…”

“…Peripheral blood mononuclear cells (PBMCs) were isolated from donor leukopacks by Ficoll-Paque density gradient centrifugation from ∼100 mL of leukocytes isolated by leukapheresis. CD14 MicroBeads (MACS Miltenyi Biotec) were used for CD14 + selection according to manufacturer's instructions and isolated monocytes were differentiated into mo-DCs in Mo-DC Differentiation Medium (MACS Miltenyi Biotec) at 10 6 cells/ml for 7 days as described previously (13). Cells were incubated at 37 • C, 5% CO 2 , and >95% humidity.…”

Isolation of HLA-DR-naturally presented peptides identifies T-cell epitopes for rheumatoid arthritis

“…Nevertheless, those approaches have mainly focused on proteins previously described as autoantibody targets and have not taken into consideration the restrictions imposed by antigen-processing and peptide-loading onto HLA molecules. These limitations can be circumvented by the isolation of peptide/HLA complexes (pHLA) and further sequencing of naturally presented peptides (NPPs) by liquid chromatography coupled to mass spectrometry (LC-MS/MS), a technology that has already identified several autoimmunity-associated T-cell epitopes,17–19 as well as post-translationally modified NPPs 20 21…”