Nicoletta Sorvillo

Residues Arg568 and Phe592 contribute to an antigenic surface for anti-ADAMTS13 antibodies in the spacer domain

Background The majority of patients diagnosed with thrombotic thrombocytopenic purpura have autoantibodies directed towards the spacer domain of ADAMTS13. Design and Methods In this study we explored the epitope specificity and immunoglobulin class and immunoglobulin G subclass distribution of anti-ADAMTS13 antibodies. The epitope specificity of anti-spacer domain antibodies was examined using plasma from 48 patients with acute acquired thrombotic thrombocytopenic purpura by means of immunoprecipitation of ADAMTS13 variants containing single or multiple alanine substitutions. Using similar methods, we also determined the presence of anti-TSP2-8 and CUB1-2 domain antibodies in this cohort of patients. Results Antibody profiling revealed that anti-ADAMTS13 immunoglobulin G1 and immunoglobulin G4 predominate in plasma of patients with acquired thrombotic thrombocytopenic purpura. Analysis of anti-spacer domain antibodies revealed that Arg568 and Phe592, in addition to residues Arg660, Tyr661, and Tyr665, also contribute to an antigenic surface in the spacer domain. The majority of patients (90%) lost reactivity towards the spacer domain following introduction of multiple alanine substitutions at Arg568, Phe592, Arg660, Tyr661 and Tyr665. Anti-TSP2-8 and anti-CUB1-2 domain-directed antibodies were present in, respectively, 17% and 35% of the patients’ samples analyzed. Conclusions Immunoglobulin G directed towards a single antigenic surface comprising residues Arg568, Phe592, Arg660, Tyr661 and Tyr665 predominates in the plasma of patients with acquired thrombotic thrombocytopenic purpura.

The macrophage mannose receptor promotes uptake of ADAMTS13 by dendritic cells

ADAMTS13 is a plasma metalloproteinase that regulates platelet adhesion and aggregation by cleaving ultra-large VWF multimers on the surfaces of endothelial cells. Autoantibodies directed against ADAMTS13 prohibit the processing of VWF multimers, initiating a rare and life-threatening disorder called acquired thrombotic thrombocytopenic purpura. The formation of autoantibodies depends on the activation of CD4(+) T cells. This process requires immune recognition, endocytosis, and subsequent processing of ADAMTS13 into peptides that are presented on MHC class II molecules to CD4(+) T cells by dendritic cells (DCs). In the present study, we investigated endocytosis of recombinant ADAMTS13 by immature monocyte-derived DCs using flow cytometry and confocal microscopy. After incubation of fluorescently labeled ADAMTS13 with DCs, significant uptake of ADAMTS13 was observed. Endocytosis of ADAMTS13 was completely blocked by the addition of EGTA and mannan. ADAMTS13 endocytosis was decreased in the presence of a blocking mAb directed toward the macrophage mannose receptor (MR). Furthermore, siRNA silencing of MR reduced the uptake of ADAMTS13 by DCs. In addition, in vitro binding studies confirmed the interaction of ADAMTS13 with the carbohydrate recognition domains of MR. The results of the present study indicate that sugar moieties on ADAMTS13 interact with MR, thereby promoting its endocytosis by APCs.

Preferential HLA-DRB1*11 dependent presentation of CUB2 derived peptides by ADAMTS13 pulsed dendritic cells

Autoantibodies directed against ADAMTS13 prohibit the processing of von Willebrand factor multimers, initiating a rare and life-threatening disorder called acquired thrombotic thrombocytopenic purpura (TTP). Recently, HLA-DRB1*11 has been identified as a risk factor for the development of acquired TTP. Here, we identified ADAMTS13-derived peptides presented on MHC class II alleles from 17 healthy donors. Dendritic cells from a panel of both HLA-DRB1*11-positive and -negative donors were pulsed with ADAMTS13, and the HLA-DR-presented peptide repertoire was analyzed by mass spectrometry. Interestingly, at low antigen concentrations, HLA-DRB1*11- or DRB1*03-positive donors presented a limited number of CUB2-derived peptides. Pulsing of dendritic cells using higher concentrations of ADAMTS13 resulted in the presentation of larger numbers of ADAMTS13-derived peptides by both HLA-DRB1*11-positive and -negative donors. Although the presented peptides were derived from several ADAMTS13 domains, inspection of the peptide profiles revealed that CUB2 domain-derived peptides were presented with a higher efficiency when compared with other peptides. Remarkably, dendritic cells from DRB1*11 donors pulsed with higher concentrations of ADAMTS13-present derivatives of a single CUB2-derived peptide. We hypothesize that functional presentation of CUB2-derived peptides on HLA-DRB1*11 contributes to the onset of acquired TTP by stimulating low-affinity, self-reactive CD4+ T cells.

Essential Domains of A Disintegrin and Metalloprotease With Thrombospondin Type 1 Repeats-13 Metalloprotease Required for Modulation of Arterial Thrombosis

Objective—A disintegrin and metalloprotease with thrombospondin type 1 repeats-13 (ADAMTS13) inhibits platelet aggregation and arterial thrombosis by cleavage of von Willebrand factor. However, the structural components of ADAMTS13 required for inhibition of arterial thrombosis are not fully defined. Methods and Results—Using recombinant proteins and a murine model, we demonstrated that an ADAMTS13 variant truncated after either the eighth thrombospondin type 1 repeat or the spacer domain inhibits ferric chloride–induced arterial thrombosis in ADAMTS13−/− mice with efficacy similar to that of full-length ADAMTS13. The results obtained from monitoring thrombus formation in carotid and mesenteric arteries were highly concordant. Further analyses by site-directed mutagenesis and human monoclonal antibody inhibition assay revealed that the Cys-rich and spacer domains of ADAMTS13, particularly the amino acid residues between Arg559 and Glu664 in the spacer domain, may be critical for modulation of arterial thrombosis in vivo. Finally, the thrombosis-modulating function of ADAMTS13 and variants/mutants was highly correlated with the von Willebrand factor–cleavage activity under fluid shear stress. Conclusion—Our results suggest that the amino terminus of ADAMTS13, specifically the variable region of the spacer domain, is crucial for modulation of arterial thromboses under (patho)physiological conditions. These findings shed more light on the structure-function relationship of ADAMTS13 in vivo and may be applicable for rational design of protein- or gene-based therapy of arterial thromboses.

Humoral immune response to ADAMTS13 in acquired thrombotic thrombocytopenic purpura.

Summary.  The apparently spontaneous development of autoantibodies to ADAMTS13 in previously healthy individuals is a major cause of thrombotic thrombocytopenic purpura (TTP). Epitope mapping studies have shown that in most patients antibodies directed towards the spacer domain of ADAMTS13 are present. A single antigenic surface comprising Arg660, Tyr661 and Tyr665 that contributes to the productive binding of ADAMTS13 to unfolded von Willebrand factor is targeted by anti‐spacer domain antibodies. Antibodies directed to the carboxyl‐terminal CUB1–2 and TSP2–8 domains have also been observed in the plasma of patients with acquired TTP. As yet it has not been established whether this class of antibodies modulates ADAMTS13 activity. Inspection of the primary sequence of human monoclonal anti‐ADAMTS13 antibodies suggests that the variable heavy chain germline gene segment VH1–69 is frequently incorporated. We suggest a model in which ‘shape complementarity’ between the spacer domain and residues encoded by the VH1–69 gene segment explain the preferential use of this variable heavy chain gene segment. Finally, a model is presented for the development of anti‐ADAMTS13 antibodies in previously healthy individuals that incorporates the recent identification of HLA DRB1*11 as a risk factor for acquired TTP.

Acquired TTP: ADAMTS13 meets the immune system

The majority of the patients affected by acquired thrombotic thrombocytopenic purpura (TTP) develop autoantibodies directed towards ADAMTS13 that interfere with its von Willebrand Factor (VWF) processing activity. B cell responses have been shown to primarily target the spacer domain of ADAMTS13 thereby prohibiting the binding of ADAMTS13 to the VWF A2 domain. In this review we summarize recent knowledge gained on the immune recognition and processing of ADAMTS13 by antigen-presenting cells (APCs). HLA-DRB1*11 has been identified as a risk factor for acquired TTP. Analysis of MHC class II/peptide complexes of ADAMTS13 pulsed dendritic cells have shown that the CUB2 domain derived peptide FINVAPHAR is preferentially presented on HLA-DRB1*11. Based on these findings we propose a model for the initiation of the autoimmune reactivity against ADAMTS13 in previously healthy individuals. We hypothesize that mimicry between a pathogen-derived peptide and the CUB2 derived FINVAPHAR-peptide might contribute to the onset of acquired TTP.

Von Willebrand factor binds to the surface of dendritic cells and modulates peptide presentation of factor VIII

It has been proposed that von Willebrand factor might affect factor VIII immunogenicity by reducing factor VIII uptake by antigen presenting cells. Here we investigate the interaction of recombinant von Willebrand factor with immature monocyte-derived dendritic cells using flow cytometry and confocal microscopy. Surprisingly, von Willebrand factor was not internalized by immature dendritic cells, but remained bound to the cell surface. As von Willebrand factor reduces the uptake of factor VIII, we investigated the repertoire of factor VIII presented peptides when in complex with von Willebrand factor. Interestingly, factor VIII-derived peptides were still abundantly presented on major histocompatibility complex class II molecules, even though a reduction of factor VIII uptake by immature dendritic cells was observed. Inspection of peptide profiles from 5 different donors showed that different core factor VIII peptide sequences were presented upon incubation with factor VIII/von Willebrand factor complex when compared to factor VIII alone. No von Willebrand factor peptides were detected when immature dendritic cells were pulsed with different concentrations of von Willebrand factor, confirming lack of von Willebrand factor endocytosis. Several von Willebrand factor derived peptides were recovered when cells were pulsed with von Willebrand factor/factor VIII complex, suggesting that factor VIII promotes endocytosis of small amounts of von Willebrand factor by immature dendritic cells. Taken together, our results establish that von Willebrand factor is poorly internalized by immature dendritic cells. We also show that von Willebrand factor modulates the internalization and presentation of factor VIII-derived peptides on major histocompatibility complex class II.

Identification of N‐linked glycosylation and putative O‐fucosylation, C‐mannosylation sites in plasma derived ADAMTS13

Acquired deficiency of ADAMTS13 causes a rare and life‐threatening disorder called thrombotic thrombocytopenic purpura (TTP). Several studies have shown that aberrant glycosylation can play an important role in the pathogenesis of autoimmune diseases. N‐linked glycosylation and putative O‐fucosylation sites have been predicted or identified in recombinant ADAMTS13. However, it is not known which of these sites are glycosylated in plasma derived ADAMTS13.

CD4+ T cells from patients with acquired thrombotic thrombocytopenic purpura recognize CUB2 domain-derived peptides.

Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder resulting from the development of autoantibodies against ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). HLA-DRB1*11 provides a risk factor for developing acquired TTP. Pulsing of antigen-presenting cells from HLA-DRB1*11- and HLA-DRB1*03-positive individuals with ADAMTS13 resulted in presentation of peptides derived from the CUB2 domain of ADAMTS13 with core sequences FINVAPHAR or ASYILIRD. Here, we assessed whether FINVAPHAR- or ASYILIRD-reactive CD4(+)T cells are present in peripheral blood mononuclear cells from HLA-DRB1*11 and HLA-DRB1*03-positive subjects with acquired TTP. The presence of ADAMTS13-reactive CD4(+)T cells was addressed by flow cytometry and the expression of activation marker CD40 ligand by CD4(+)T cells. FINVAPHAR-reactive CD4(+)T cells were identified in an HLA-DRB1*11-positive patient during the acute phase of the disease whereas ASYILIRD-positive CD4(+)T cells were identified in a DRB1*03-positive patient with acquired TTP. Frequencies of CUB2 domain-reactive CD4(+)T cells ranged from 3.3% to 4.5%. Control peptides in which the anchor residues were modified did not induce activation of CD4(+)T cells. Taken together, our data provide evidence for the involvement of CUB2 domain-reactive CD4(+)T cells in the etiology of acquired TTP.

Analysis of the HLA-DR peptidome from human dendritic cells reveals high affinity repertoires and nonconventional pathways of peptide generation

Dendritic cells (DCs) are the major professional APCs of the immune system; however, their MHC‐II–associated peptide repertoires have been hard to analyze, mostly because of their scarce presence in blood and tissues. In vitro matured human monocyte‐derived DCs (MoDCs) are widely used as professional APCs in experimental systems. In this work, we have applied mass spectrometry to identify the HLA‐DR–associated self‐peptide repertoires from small numbers of mature MoDCs (∼5 × 106 cells), derived from 7 different donors. Repertoires of 9 different HLA‐DR alleles were defined from analysis of 1319 peptides, showing the expected characteristics of MHC‐II–associated peptides. Most peptides identified were predicted high binders for their respective allele, formed nested sets, and belonged to endo‐lysosomal pathway‐degraded proteins. Approximately 20% of the peptides were derived from cytosolic and nuclear proteins, a recurrent finding in HLA‐DR peptide repertoires. Of interest, most of these peptides corresponded to single sequences, did not form nested sets, and were located at the C terminus of the parental protein, which suggested alternative processing. Analysis of cleavage patterns for terminal peptides predominantly showed aspartic acid before the cleavage site of both C‐ and N‐terminal peptides and proline immediately after the cleavage site in C‐terminal peptides. Proline was also frequent next to the cut sites of internal peptides. These data provide new insights into the Ag processing capabilities of DCs. The relevance of these processing pathways and their contribution to response to infection, tolerance induction, or autoimmunity deserve further analysis.