The ADAMTS131239-1253 peptide is a dominant HLA-DR1-restricted CD4+ T-cell epitope

Abstract

Acquired thrombotic thrombocytopenic purpura is a rare and severe disease characterized by auto-antibodies directed against "A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13^th member" (ADAMTS13), a plasma protein involved in hemostasis. Involvement of CD4⁺ T cells in the pathogenesis of the disease is suggested by the IgG isotype of the antibodies. However, the nature of the CD4⁺ T-cell epitopes remains poorly characterized. Here, we determined the HLA-DR-restricted CD4⁺ T-cell epitopes of ADAMTS13. Candidate T-cell epitopes were predicted in silico and binding affinities were confirmed in competitive enzyme-linked immunosorbent assays. ADAMTS13-reactive CD4⁺ T-cell hybridomas were generated following immunization of HLA-DR1 transgenic mice (Sure-L1 strain) and used to screen the candidate epitopes. We identified the ADAMTS13^1239-1253 peptide as the single immunodominant HLA-DR1-restricted CD4⁺ T-cell epitope. This peptide is located in the CUB2 domain of ADAMTS13. It was processed by dendritic cells, stimulated CD4⁺ T cells from Sure-L1 mice and was recognized by CD4⁺ T cells from an HLA-DR1-positive patient with acute thrombotic thrombocytopenic purpura. Interestingly, the ADAMTS13^1239-1253 peptide demonstrated promiscuity towards HLA-DR11 and HLA-DR15. Our work paves the way towards the characterization of the ADAMTS13-specific CD4⁺ T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS13^1239-1253-loaded HLA-DR tetramers.

Figure 1.

Identification of HLA-DR1-restricted T-cell epitopes using ADAMTS13-specific CD4⁺ T-cell hybridomas. (A) Anti-ADAMTS13 IgG titers in Sure-L1 mice immunized with rhADAMTS13. The serum from an HLA-DR1-transgenic Sure-L1 mouse immunized with rhADAMTS13 was incubated in serial dilutions on ADAMTS13-coated ELISA wells. ADAMTS13-bound IgG were detected using horseradish peroxidase-coupled polyclonal goat anti-mouse IgG and substrate. Results are expressed in arbitrary units as optical density measured at 492 nm. (B) Activation of ADAMTS13-specific CD4⁺ T-cell hybridomas. A representative ADAMTS13-specific CD4⁺ T-cell hybridoma (clone 2G10δ) was incubated with AAPC^DR1 (10:1 ratio) and ADAMTS13-derived peptides as listed in Table 1. Stimulation indices represent the ratio of IL-2 secreted by the T cells measured by ELISA upon incubation with ADAMTS13 peptides over IL-2 secreted in the absence of peptide. Means±SD are from two independent experiments. (C) Delineation of the core peptide for ADAMTS13-specific CD4⁺ T-cell hybridomas. A representative ADAMTS13-specific CD4⁺ T-cell hybridoma (clone 2G10δ) was incubated with AAPC^DR1 and overlapping 15-mer peptides spanning the 1235–1256 peptide sequence. Stimulation indices were assessed as explained above.

Figure 2.

The ADAMTS13^1239–1253 peptide is an immunodominant T-cell epitope both for Sure-L1 mice as well as for a patient with TTP. (A) Stimulation by ADAMTS13-derived peptides of spleen and lymph node cells from Sure-L1 mice immunized with rhADAMTS13. Cells (3×10⁵/well) were incubated with each individual peptide (10 μg/mL), with the pool of peptides (Peptide mix) or with rhADAMTS13. Proliferation was assessed by incorporation of tritiated thymidine. Proliferation indices are defined as the ratio of cpm of stimulated cells versus cpm of control cells, and are expressed as means±SD for two Sure-L1 mice. (B) The ADAMTS13^1239–1253 peptide is processed and presented to T cells by human antigen-presenting cells. A representative ADAMTS13-specific CD4⁺ T-cell hybridoma (clone 1F5γ) was incubated with immature dendritic cells from a healthy HLA-DR1⁺ blood donor and with the ADAMTS13^1239–1253 peptide, with rhADAMTS13 or with the ADAMTS13^111–125 peptide. Stimulation indices represent the ratio of secretion of IL-2 by T cells stimulated in the presence of antigen versus the secretion of IL-2 by T cells incubated with Mo-DC alone. Means±SD are from two independent experiments. Similar results were obtained with other ADAMTS13-CD4⁺ T-cell hybridoma clones (not shown). The peptide incubated alone or with DR1⁵⁺ artificial antigen-presenting cells failed to activate the T cells (not shown). (C) ADAMTS13 epitope specificity of CD4⁺ T-cell lines from a patient with acquired TTP. CD4⁺ T-cell lines were generated after stimulation with pooled ADAMTS13-derived peptides as defined in Table 1. T cells (3×10⁵/well) were then incubated with AAPC^DR1 (3×10⁴ cells/well) alone or pulsed with individual peptide (10 μg/mL), or with the peptide pool (Peptide mix). The number of cells producing interferon-γ was then assessed by ELISPOT and is expressed as the mean number of spot-forming cells (SFC) per million cells calculated in the case of peptide-stimulated T cells minus the SFC obtained in the case of unstimulated cells. Means±SD are from two independent experiments. *The 111–125 ADAMTS13-derived peptide that did not bind to HLA-DRB1*01:01 was used as a negative control.

Figure 3.

The ADAMTS13^1239–1253 peptide is an immunodominant T-cell epitope for HLA-DRB1*11 individuals. CD4⁺ T-cell lines from HLA-DRB1*11 TTP patients at (A) acute phase or (B) in remission phase of the disease, or from (C) a HLA-DRB1*11 healthy donor, were stimulated with the ADAMTS13-derived peptides identified as immunodominant for HLA-DRB1*01. T cells (3×10⁵ cells/well) were incubated with AAPC^DR11 (3×10⁴ cells/well) alone or pulsed with individual peptide (10 μg/mL), or with the peptide pool (Peptide mix). The number of cells producing interferon-γ was then assessed by ELISPOT and is expressed as mean number of spot-forming cells (SFC) per million cells calculated in the case of peptide-stimulated T-cells minus the SFC obtained in the case of unstimulated cells. *The 111–125 ADAMTS13-derived peptide is used as a negative control, without detectable binding to HLA-DRB1*11:01 (data not shown).