Our work paves the way towards the characterization of the ADAMTS13-specific CD4+ T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS131239C1253-loaded HLA-DR tetramers. Introduction Thrombotic thrombocytopenic purpura Ozenoxacin (TTP) is a rare Ozenoxacin and severe autoimmune disease characterized by the occurrence of IgG autoantibodies against the metalloprotease A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13th member (ADAMTS13).1C3 ADAMTS13 cleaves multimers of von Willebrand factor, a glycoprotein involved in hemostasis. 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 ADAMTS131239C1253 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 ADAMTS131239C1253-loaded HLA-DR tetramers. Introduction Thrombotic thrombocytopenic purpura (TTP) is a rare and severe autoimmune disease characterized by the occurrence of IgG autoantibodies against the metalloprotease A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13th member (ADAMTS13).1C3 ADAMTS13 cleaves multimers of von Willebrand factor, a glycoprotein involved in hemostasis. Inhibition of ADAMTS13 by IgG leads to an accumulation of hyper-adhesive von Willebrand factor multimers causing microthrombi that occlude the lumen of the capillaries in the microcirculation, thus inducing red cell hemolysis and ischemia of downstream organs. TTP is thus characterized by a combination of microangiopathic hemolytic anemia, peripheral thrombocytopenia and organ failure of variable severity with typically neurological involvement.4 The physiopathological mechanisms underlying TTP and responsible for the synthesis of PIK3C2A anti-ADAMTS13 antibodies, and particularly the mechanisms involved in the loss of tolerance of the immune system towards ADAMTS13, are poorly understood. Polyclonal anti-ADAMTS13 antibodies are directed against different domains of ADAMTS13.5 In most patients, anti-ADAMTS13 antibodies are of the IgG isotype with a predominance of the IgG4 subclass.6 IgG from all patients recognize immunodominant B-cell epitopes located in the spacer domain of ADAMTS13.7 The B-cell epitopes have been proposed to be located between the 660C661 and 665 amino-acids.8 The fact that anti-ADAMTS13 antibodies are of the IgG isotype, of high affinity and have undergone affinity maturation, strongly suggests the requirement of CD4+ T-cell help in the development of the disease.9 Besides, the HLA-DRB1*11 (DR11) haplotype was independently identified as a strong risk factor by three research groups.10C12 However, Ozenoxacin while CD4+ T cells are thought to play a major role, the specificity Ozenoxacin and the properties of the CD4+ T lymphocytes involved in the pathogenesis of TTP have not been studied. Importantly, the HLA restriction hints at the existence of immunodominant peptides in ADAMTS13. Na?ve CD4+ T-cell activation is initiated by the interaction of the T-cell receptor (TCR) with a peptide/MHC class II complex on professional antigen-presenting cells. Extracellular antigens are endocytosed, degraded into peptides in the early endosome and loaded onto MHC class II heterodimer molecules. Sorvillo using overlapping 15-mer peptides that span the whole ADAMTS13 sequence. Altogether, 99 15-mer peptides were predicted to be strong binders to HLA-DRB1*01:01 with binding scores below 10 (i.e., with a probability of being good binders greater than 90%). Some of the predicted peptides shared common HLA-DRB1*01:01-binding core sequences (9-mer peptides). When considering only unique core sequences and after exclusion of two peptides located in the prodomain of ADAMTS13, the list came down to 15 9-mer core peptides (Table 1). The peptides were synthesized at greater than 80% purity (GL Biochem, Shanghai, China) and included the 9-mer core sequences with addition of the three residues from the N-terminal end and the three residues of the C-terminal end. Individual peptides were solubilized at 1 mg/mL in dimethylsulfoxide/water. Recombinant full-length human ADAMTS13 (rhADAMTS13) was a kind gift from Baxter Ozenoxacin (Vienna, Austria).18 Table 1. Affinity of ADAMTS13-derived peptides for HLA-DRB1*01:01 molecules. Open in a separate window HLA-peptide-binding assays HLA-DR molecules were purified from homozygous Epstein-Barr virus cell lines by affinity-chromatography using the monomorphic monoclonal antibody L243. The binding to HLA-DR molecules was assessed by competitive enzyme-linked immunosorbent assay (ELISA), using an automated workstation, as previously reported.19,20 Briefly, HLA heterodimers were incubated with a biotinylated indicator peptide and serial dilutions of competitor peptides. As reference, the unlabeled form of biotinylated reporter peptide was used as an internal control. After 24 h incubation at 37C, samples were neutralized with 450 mM Tris HCl (pH 7.5) (Sigma, St Quentin-Fallavier, France), 0.3% bovine serum albumin (Sigma), and 1 mM n-dodecyl -D-maltoside buffer (Sigma) and applied to 96-well MaxiSorp ELISA plates (Nunc.