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mers through cleavage of a Tyr1605-Met1606 peptide bond in the A2 domain of VWF.1,2 Functional or quantita- tive defects in ADAMTS13 levels in the circulation lead to the accumulation of high molecular weight VWF multi- mers and the formation of platelet- and VWF-rich throm- bi. Within the microvasculature, these thrombi cause mechanical fragmentation of erythrocytes inducing hemolytic anemia.1,2 In addition, the presence of hyper- adhesive VWF multimers results in platelet consumption. As a consequence, patients with TTP often present with skin petechiae due to thrombocytopenia-induced blood loss from small vessels in the skin.1,3 Additional clinical symptoms may include fever, renal failure or neurological abnormalities.1,2
In the majority of patients with TTP, the decrease in ADAMTS13 levels is due to the development of autoanti- bodies directed towards ADAMTS13. Most of these autoantibodies are composed of IgG1 and IgG4 subclass- es;4-6 these antibodies either inhibit the proteolytic func- tion of ADAMTS13 or enhance its clearance from the cir- culation.6-9 While the mechanisms responsible for the development of anti-ADAMTS13 antibodies are currently unknown, several reports have suggested that infections, pregnancy or transplantation may be considered to be risk factors for the onset of acquired TTP.10-12 The generation of high affinity antibodies against ADAMTS13 is dependent on the help of specific CD4+ T cells. Priming of antigen- specific CD4+ T cells requires presentation of ADAMTS13-derived peptides on major histocompatibili- ty complex class II (MHC-II) on professional antigen pre- senting cells.13 The MHC-II genes are highly polymorphic allowing for the selection of a broad repertoire of CD4+ T cells that is needed to combat infections. Specific MHC-II alleles have been linked to autoimmune disorders such as rheumatoid arthritis and celiac disease.14 Similarly, associ- ation studies from three different cohorts of patients with acquired TTP have identified HLA-DRB1*11 as a risk fac- tor.15-17 Conversely, the frequency of HLA-DRB1*04 was significantly lower in patients with acquired TTP, suggest- ing a protective effect of this allele.15-17 In addition to HLA-DRB1*11, higher frequencies of alleles HLA-DQB1*0315,16 and HLA-DQB1*02:0217 were found in patients with acquired TTP when compared to healthy controls. A recent study of 190 Italian TTP patients and 1255 healthy controls suggested that HLA-DQB1*05:03 was less prevalent in patients with acquired TTP.18 This study also proposed that the common single nucleotide polymorphism rs6903608, which is located between the genes encoding the alpha and beta5 chains of the HLA-DR complex, combined with HLA-DQB1*05:03 explains most of the observed association between the HLA locus and acquired TTP.18 As yet, the molecular mechanism underly- ing the observed association between polymorphic sites within the MHC II locus and acquired TTP has not been identified.
Previous observation from our laboratory has shown that monocyte-derived dendritic cells (mo-DCs) from healthy donors preferentially presented two peptides derived from the CUB2 domain of ADAMTS13.19 Both of these peptides were found to activate CD4+ T cells of patients with acquired TTP.20 In addition, CUB2 domain- derived peptide ADAMTS131239-1253 was identified as an immunodominant T-cell epitope in an HLA-DRB1 trans- genic mouse model.21 The same study revealed that ADAMTS131239-1253 reactive CD4+ T cells were present in
patients with acquired TTP as well as in peripheral blood of healthy individuals.21 As yet, the presentation of ADAMTS13-derived peptides on HLA-DQ has not been investigated. In the present work, we aimed to define the repertoire of ADAMTS13-derived peptides presented on HLA-DQ and prospectively identify putative effector and tolerated/tolerogenic T-cell epitopes using computational tools (EpiMatrix and JanusMatrix).
Methods
Materials
Recombinant full length ADAMTS13 was produced in stable transfected HEK293 cells and purified as described previously.9 Concentration of purified ADAMTS13 was determined using the Bradford assay. Lipopolysaccharide (LPS) was obtained from Sigma-Aldrich (St. Louis, USA). The hybridoma producing the HLA-DQ-specific antibody (SPV-L3)22 was a kind gift from Prof. dr. H. Spits (Academic Medical Center, Amsterdam, the Netherlands). The hybridoma producing the HLA-DR-specific monoclonal antibody (L243) was purchased from ATCC (Wesel, Germany). Antibodies were purified from culture supernatant via protein A Sepharose (GE Healthcare) and coupled to CNBr Sepharose 4B at a final concentration of 2 mg/mL (Amersham Biosciences, Buckinghamshire, UK).
Endocytosis of ADAMTS13 and affinity purification of HLA-DR and HLA-DQ
Monocytes were obtained from healthy volunteers in accor- dance with Dutch regulations after approval from the Sanquin Ethical Advisory Board, in accordance with the Declaration of Helsinki. After five days of differentiation, 5x106 immature mo- DCs were incubated with 100 nM of recombinant ADAMTS13 in Cellgro medium supplemented with 800 U/ml IL-4 and 1000 U/mL GM-CSF. After five hours of incubation, the medium was supplemented without washing with 1 mg/mL of LPS and 1% fetal calf serum to allow for mo-DCs maturation overnight. After mat- uration, the cells were detached from the plate using trisodium cit- rate and gentle pipetting. HLA-DR and HLA-DQ peptide com- plexes were purified using a modification of a previously described protocol.23 Briefly, cells were resuspended in 500 mL lysis buffer containing 10 mM Tris-HCl (pH 8.0), 0.25% octyl-b-D-glu- copyranoside, 1% sodium deoxycholate and Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific), and incubated at 4°C for 30 minutes (mins). The lysed cells were centrifuged at 20,000xg for 15 mins at 4°C. HLA-DR and HLA-DQ peptide com- plexes were purified from the supernatant by adding L243 or SPV- L3 coupled CNBr Sepharose, respectively, and incubating the tubes at 4°C overnight ‘end-over-end’, using a laboratory rotator. L243 or SPV-L3 Sepharose beads were then washed 2 times with lysis buffer and 5 times with 10 mM Tris-HCl (pH 8.0). HLA-pep- tide complexes were eluted using 500 mL 10% acetic acid for 10 mins at room temperature. Then the samples were centrifuged at 400xg for five mins at room temperature and the supernatant transferred to low-binding 1.5 mL Eppendorf tubes and heated for 15 mins at 70°C. Samples were desalted using C18 STAGE Tips (Dr. Maisch Gmbh, Amersfoort, the Netherlands).24 STAGE Tips were eluted with 60 mL of 1% formic acid/30% acetonitrile and eluates were concentrated using a SpeedVac (Savant, SPP1110, Thermo Scientific) to a final volume of 5 mL.
Mass spectrometry data analysis
Raw data files from the Orbitrap Fusion Tribid were scored against the uniprot-organism_9606_AND_keyword_kw_0181
haematologica | 2018; 103(6)