Figure 3 - available via license: Creative Commons Attribution-NonCommercial 4.0 International
Content may be subject to copyright.
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 HLADRB1*11 healthy donor, were stimulated with the ADAMTS13-derived peptides identified as immunodominant for HLA-DRB1*01. T cells (3x10 5 cells/well) were incubated with AAPC DR11 (3x10 4 cells/well) alone or pulsed with individual peptide (10 mg/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 peptidestimulated 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).
Source publication
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, 13th member" (ADAMTS13), a plasma protein involved in hemostasis. The implication of CD4+ T cells in the pathogenesis of the disease is suggested by the...
Contexts in source publication
Context 1
... then assessed whether the ADAMTS13 1239-1253 pep- tide is recognized by CD4 + T cells from patients with acquired TTP. CD4 + T-lymphocytes were isolated from the blood of a 43-year -old HLA-DRB1*01:01 male, 14 days after the onset of acute acquired TTP. ADAMTS13 activity in this patient was less than 5% of the normal value due to the presence of inhibitory antibodies directed to ADAMTS13. A CD4 + T-cell line specific for ADAMTS13-derived peptides was amplified from the patient's peripheral blood mononuclear cells as explained. After 32 days of culture, the CD4 + T-cell line produced IFN-γ, as assessed by ELISPOT, following incubation with Mo-DC from HLA-DR1 healthy donors, pulsed with the pool of ADAMTS13-derived peptides with high binding affinity for HLA-DR1, but failed to produce IFN-γ when incubated with Mo-DC alone (data not shown). We then incubated the T-cell lines with AAPC DR1 and with each individual peptide. Together with the ADAMTS13 1392-1406 peptide, the ADAMTS13 1239-1253 was the only peptide able to activate human IFN-γ-producing CD4 + T cells ( Figure 2C). Similar results were obtained when the T cells and ADAMTS13 1239-1253 peptide were incubated in the presence of HLA-DR1 Mo-DC (data not shown). Further evaluation of the ADAMTS13 1239-1253 peptide by IEDB predicted good binding scores to HLA-DR11, 15, 13, 8, 3 and 4, which was confirmed in competitive ELISA at least in the case of HLA-DR11 and HLA-DR15 (Table 2). T-cell lines were also generated from one female TTP patient in acute phase (aged 19, Figure 3A), one female TTP patient in remission phase (aged 34, Figure 3B) and one 25-year old, healthy male donor ( Figure 3C), all with the HLA- DRB1*11:01 allele. Reactivity towards the ADAMTS13 1239- 1253 peptide was detected in the case of the three individu- als. Together, our data suggest that the ADAMTS13 [1239][1240][1241][1242][1243][1244][1245][1246][1247][1248][1249][1250][1251][1252][1253] peptide is a dominant and promiscuous T-cell epitope in TTP ...
Context 2
... then assessed whether the ADAMTS13 1239-1253 pep- tide is recognized by CD4 + T cells from patients with acquired TTP. CD4 + T-lymphocytes were isolated from the blood of a 43-year -old HLA-DRB1*01:01 male, 14 days after the onset of acute acquired TTP. ADAMTS13 activity in this patient was less than 5% of the normal value due to the presence of inhibitory antibodies directed to ADAMTS13. A CD4 + T-cell line specific for ADAMTS13-derived peptides was amplified from the patient's peripheral blood mononuclear cells as explained. After 32 days of culture, the CD4 + T-cell line produced IFN-γ, as assessed by ELISPOT, following incubation with Mo-DC from HLA-DR1 healthy donors, pulsed with the pool of ADAMTS13-derived peptides with high binding affinity for HLA-DR1, but failed to produce IFN-γ when incubated with Mo-DC alone (data not shown). We then incubated the T-cell lines with AAPC DR1 and with each individual peptide. Together with the ADAMTS13 1392-1406 peptide, the ADAMTS13 1239-1253 was the only peptide able to activate human IFN-γ-producing CD4 + T cells ( Figure 2C). Similar results were obtained when the T cells and ADAMTS13 1239-1253 peptide were incubated in the presence of HLA-DR1 Mo-DC (data not shown). Further evaluation of the ADAMTS13 1239-1253 peptide by IEDB predicted good binding scores to HLA-DR11, 15, 13, 8, 3 and 4, which was confirmed in competitive ELISA at least in the case of HLA-DR11 and HLA-DR15 (Table 2). T-cell lines were also generated from one female TTP patient in acute phase (aged 19, Figure 3A), one female TTP patient in remission phase (aged 34, Figure 3B) and one 25-year old, healthy male donor ( Figure 3C), all with the HLA- DRB1*11:01 allele. Reactivity towards the ADAMTS13 1239- 1253 peptide was detected in the case of the three individu- als. Together, our data suggest that the ADAMTS13 [1239][1240][1241][1242][1243][1244][1245][1246][1247][1248][1249][1250][1251][1252][1253] peptide is a dominant and promiscuous T-cell epitope in TTP ...
Context 3
... then assessed whether the ADAMTS13 1239-1253 pep- tide is recognized by CD4 + T cells from patients with acquired TTP. CD4 + T-lymphocytes were isolated from the blood of a 43-year -old HLA-DRB1*01:01 male, 14 days after the onset of acute acquired TTP. ADAMTS13 activity in this patient was less than 5% of the normal value due to the presence of inhibitory antibodies directed to ADAMTS13. A CD4 + T-cell line specific for ADAMTS13-derived peptides was amplified from the patient's peripheral blood mononuclear cells as explained. After 32 days of culture, the CD4 + T-cell line produced IFN-γ, as assessed by ELISPOT, following incubation with Mo-DC from HLA-DR1 healthy donors, pulsed with the pool of ADAMTS13-derived peptides with high binding affinity for HLA-DR1, but failed to produce IFN-γ when incubated with Mo-DC alone (data not shown). We then incubated the T-cell lines with AAPC DR1 and with each individual peptide. Together with the ADAMTS13 1392-1406 peptide, the ADAMTS13 1239-1253 was the only peptide able to activate human IFN-γ-producing CD4 + T cells ( Figure 2C). Similar results were obtained when the T cells and ADAMTS13 1239-1253 peptide were incubated in the presence of HLA-DR1 Mo-DC (data not shown). Further evaluation of the ADAMTS13 1239-1253 peptide by IEDB predicted good binding scores to HLA-DR11, 15, 13, 8, 3 and 4, which was confirmed in competitive ELISA at least in the case of HLA-DR11 and HLA-DR15 (Table 2). T-cell lines were also generated from one female TTP patient in acute phase (aged 19, Figure 3A), one female TTP patient in remission phase (aged 34, Figure 3B) and one 25-year old, healthy male donor ( Figure 3C), all with the HLA- DRB1*11:01 allele. Reactivity towards the ADAMTS13 1239- 1253 peptide was detected in the case of the three individu- als. Together, our data suggest that the ADAMTS13 [1239][1240][1241][1242][1243][1244][1245][1246][1247][1248][1249][1250][1251][1252][1253] peptide is a dominant and promiscuous T-cell epitope in TTP ...
Similar publications
Idiopathic pulmonary arterial hypertension (IPAH) and chronic thromboembolic pulmonary hypertension (CTEPH) can result in right heart failure. We aimed to evaluate the plasma protein levels of a disintegrin and metalloproteinase with thrombospondin motifs like 4 (ADAMTSL4) and its relationship with IPAH and CTEPH. Plasma ADAMTSL4 protein levels wer...
Citations
... 11,12 Two peptides derived from the CUB-2 domain of ADAMTS13 are presented on HLA-DRB1*11 and HLA-DRB1*03 respectively and recognised by iTTP patient-derived CD4+ T cells. 13,14 CD4+ T cells are pivotal in development of iTTP as CD4+ T cell help is required in the production and affinity maturation of ADAMTS13-directed antibodies. 3 Within the CD4+T cell population, the T follicular helper (Tfh) subset are vital for supporting antibody-mediated immune responses by providing co-stimulation signals through CD40L and IL-21 production, which promote the growth, differentiation and class-switching of antigen-activated naïve B cells. ...
T follicular helper (Tfh) cells regulate development of antigen-specific B-cell immunity. We prospectively investigated B-cell and cTfh subsets in 45 immune TTP patients at presentation and longitudinally after rituximab (RTX). B-cell phenotype was altered at acute iTTP presentation with decreased transitional cells and postgerminal centre (post-GC) memory B cells and increased plasmablasts compared to healthy controls. A higher percentage of plasmablasts was associated with higher anti-ADAMTS13 IgG and lower ADAMTS13 antigen levels. In asymptomatic patients with ADAMTS13 relapse, there were increased naïve B cells and a global decrease in memory subsets, with a trend to increased plasmablasts. Total circulating Tfh (CD4+CXCR5+) and PD1+ Tfh cells were decreased at iTTP presentation. CD80 expression was decreased on IgD+ memory cells and double negative memory cells in acute iTTP. Longitudinal analysis: at repopulation after B cell depletion in de novo iTTP, post-GC and double negative memory B cells were reduced compared to pre-RTX. RTX did not cause alteration in cTfh frequency. The subsequent kinetics of naïve, transitional, memory B cells and plasmablasts did not differ significantly between patients who went on to relapse vs those who remained in remission. In summary, acute iTTP is characterised by dysregulation of B- and cTfh-cell homeostasis with depletion of post-GC memory cells and cTfh cells and increased plasmablasts. Changes in CD80 expression on B cells further suggest altered interactions with T cells.
... In studies on thrombotic thrombocytopenic purpura (TTP), several T-cell peptides were identified in ADAMTS13 (Table 4), with the ADAMTS13 1239-1253 peptide (GDMLLLWGRLTWRKM) as the single immunodominant CD4 + T-cell epitope that was bound both by HLA-DRB1*15:01 and DRB1*11:01 in ELISA and also by T-cells from a strain of mice expressing human HLA-DRB1*01:01 (104). A recent study looking into peptides presented on HLA-DR and HLA-DQ (105) identified 12 peptides presented by DR and 8 by DQ. ...
IgG4 autoimmune diseases (IgG4-AID) are an emerging group of autoimmune diseases that are caused by pathogenic autoantibodies of the IgG4 subclass. It has only recently been appreciated, that members of this group share relevant immunobiological and therapeutic aspects even though different antigens, tissues and organs are affected: glomerulonephritis (kidney), pemphigus vulgaris (skin), thrombotic thrombocytopenic purpura (hematologic system) muscle-specific kinase (MuSK) in myasthenia gravis (peripheral nervous system) and autoimmune encephalitis (central nervous system) to give some examples. In all these diseases, patients’ IgG4 subclass autoantibodies block protein-protein interactions instead of causing complement mediated tissue injury, patients respond favorably to rituximab and share a genetic predisposition: at least five HLA class II genes have been reported in individual studies to be associated with several different IgG4-AID. This suggests a role for the HLA class II region and specifically the DRβ1 chain for aberrant priming of autoreactive T-cells toward a chronic immune response skewed toward the production of IgG4 subclass autoantibodies. The aim of this review is to provide an update on findings arguing for a common pathogenic mechanism in IgG4-AID in general and to provide hypotheses about the role of distinct HLA haplotypes, T-cells and cytokines in IgG4-AID.
... Similarly to other autoimmune diseases, a genetic contribution to iTTP has been described, particularly of the human leukocyte antigen (HLA) class II locus. The HLA allele DRB1*11 has been consistently reported as a risk factor in European Caucasian populations [12][13][14][15][16], and CD4+ T-cells reactive to specific ADAMTS13 CUB2 domain-derived peptides were found in HLA-DRB1*11-positive iTTP patients [17,18]. Furthermore, in a recent case-control genetic association study, our group identified the common, intergenic HLA variant rs6903608 to be independently associated with prevalent iTTP in Italians (minor allele frequency in controls 0.47, pooled odds ratio (OR) and 95% confidence interval (CI) of discovery and replication phases 2.48, 2.03 to 3.02, P 3.95 × 10 −19 ) [16]. ...
Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare, life-threatening thrombotic microangiopathy caused by severe ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13) deficiency, recurring in 30–50% of patients. The common human leukocyte antigen (HLA) variant rs6903608 was found to be associated with prevalent iTTP, but whether this variant is associated with disease relapse is unknown. To estimate the impact of rs6903608 on iTTP onset and relapse, we performed a case-control and cohort study in 161 Italian patients with a first iTTP episode between 2002 and 2018, and in 456 Italian controls. Variation in rs6903608 was strongly associated with iTTP onset (homozygotes odds ratio (OR) 4.68 (95% confidence interval (CI) 2.67 to 8.23); heterozygotes OR 1.64 (95%CI 0.95 to 2.83)), which occurred over three years earlier for each extra risk allele (β −3.34, 95%CI −6.69 to 0.02). Of 153 survivors (median follow-up 4.9 years (95%CI 3.7 to 6.1)), 44 (29%) relapsed. The risk allele homozygotes had a 46% (95%CI 36 to 57%) absolute risk of relapse by year 6, which was significantly higher than both heterozygotes (22% (95%CI 16 to 29%)) and reference allele homozygotes (30% (95%CI 23 to 39%)). In conclusion, HLA variant rs6903608 is a risk factor for both iTTP onset and relapse. This newly identified biomarker may help with recognizing patients at high risk of relapse, who would benefit from close monitoring or intensified immunosuppressive therapy.
... GO analysis of interacting DEGs includes pathways involved in nucleotideexcision repair, nucleosome assembly, and positive regulation of interferon-beta production (Fig. 4b). Relevant genes include ADAM20, a member of the ADAM family of metalloproteases which are involved in T cell responses [62,63]; CD274, which, upon binding to its receptor, mediates changes in T cell metabolism [64]; and IRF1, a key driver of Th1 cell differentiation [65] (Fig. 4c). ...
Background:
Systemic sclerosis (SSc) is a genetically complex autoimmune disease mediated by the interplay between genetic and epigenetic factors in a multitude of immune cells, with CD4+ T lymphocytes as one of the principle drivers of pathogenesis.
Methods:
DNA samples exacted from CD4+ T cells of 48 SSc patients and 16 healthy controls were hybridized on MethylationEPIC BeadChip array. In parallel, gene expression was interrogated by hybridizing total RNA on Clariom™ S array. Downstream bioinformatics analyses were performed to identify correlating differentially methylated CpG positions (DMPs) and differentially expressed genes (DEGs), which were then confirmed utilizing previously published promoter capture Hi-C (PCHi-C) data.
Results:
We identified 9112 and 3929 DMPs and DEGs, respectively. These DMPs and DEGs are enriched in functional categories related to inflammation and T cell biology. Furthermore, correlation analysis identified 17,500 possible DMP-DEG interaction pairs within a window of 5 Mb, and utilizing PCHi-C data, we observed that 212 CD4+ T cell-specific pairs of DMP-DEG also formed part of three-dimensional promoter-enhancer networks, potentially involving CTCF. Finally, combining PCHi-C data with SSc GWAS data, we identified four important SSc-associated susceptibility loci, TNIP1 (rs3792783), GSDMB (rs9303277), IL12RB1 (rs2305743), and CSK (rs1378942), that could potentially interact with DMP-DEG pairs cg17239269-ANXA6, cg19458020-CCR7, cg10808810-JUND, and cg11062629-ULK3, respectively.
Conclusion:
Our study unveils a potential link between genetic, epigenetic, and transcriptional deregulation in CD4+ T cells of SSc patients, providing a novel integrated view of molecular components driving SSc pathogenesis.
... 58 Genetic factors such as HLA-DRB1 Ã 11 in Caucasians 51,75,189 are predisposing factors for iTTP. Interestingly, especially HLA-DRB1 Ã 11 and HLA-DRB1 Ã 03 positive donors could present CUB2-derived peptides (FINVAPHAR and ASY-LIRD) on dendritic cells or APC, 55 while reactive CD4þ T cells against those peptides have been identified in iTTP patients, 56,189 suggesting that stimulation of low-affinity self-reactive CD4þ T cells might play a role in the development of anti-ADAMTS13 autoantibodies. Other factors including hormones, cytokines, and other mediators may play a role in the loss of self-tolerance and the initiation of the immune response toward ADAMTS13. ...
Thrombotic thrombocytopenic purpura (TTP) is a rare, relapsing, and life-threatening disorder with an annual incidence of 10 cases per million people. TTP is a thrombotic microangiopathy characterized by severe thrombocytopenia, microangiopathic hemolytic anemia, and organ ischemia. The disease is caused by a severe deficiency of the enzyme ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13), which can either be acquired, mainly by autoantibodies targeting ADAMTS13, or congenital due to mutations in the ADAMTS13 gene. Thanks to the establishment of national registries worldwide, fundamental and translational research, major advances have been made on the diagnosis, treatment, and fundamental understanding of TTP, since the description of the first TTP case almost 100 years ago. The introduction of therapeutic plasma exchange in the 1970s has significantly improved patient survival, but novel diagnostic assays, targeted treatments (rituximab, caplacizumab, recombinant ADAMTS13), and the unraveling of both ADAMTS13 function and TTP pathophysiology should help to further improve the patients' quality of life. However, differential diagnosis of TTP remains challenging and still a lot of questions remain unanswered to completely understand this rare and devastating disease.
... In clinical immunology and for research purposes, AAPCs represent a convenient platform to monitor CD4 + T cell responses in patients against allergens, autoantigens, or infectious antigens, as it was shown with the antigen ADAMTS13 involved in acquired thrombotic thrombocytopenic purpura (121). In addition, since NIH/3T3-derived AAPCs are able to internalize and process whole protein antigens, and to present derived T cell epitopes, they could be useful to characterize new epitopes of therapeutic interest by mass spectrometry after HLA molecule immunoprecipitation and peptide elution. ...
CD4⁺ T cells differentiate into various T helper subsets characterized by distinct cytokine secreting profiles that confer them effector functions adapted to a variety of infectious or endogenous threats. Regulatory CD4⁺ T cells are another specialized subset that plays a fundamental role in the maintenance of immune tolerance to self-antigens. Manipulating effector or regulatory CD4⁺ T cells responses is a promising immunotherapy strategy for, respectively, chronical viral infections and cancer, or severe autoimmune diseases and transplantation. Adoptive cell therapy (ACT) is an emerging approach that necessitates defining robust and efficient methods for the in vitro expansion of antigen-specific T cells then infused into patients. To address this challenge, artificial antigen presenting cells (AAPCs) have been developed. They constitute a reliable and easily usable platform to stimulate and amplify antigen-specific CD4⁺ T cells. Here, we review the recent advances in understanding the functions of CD4⁺ T cells in immunity and in immune tolerance, and their use for ACT. We also describe the characteristics of different AAPC models and the way to improve their stimulating functions. Finally, we discuss the potential interest of these AAPCs, both as fundamental tools to decipher CD4⁺ T cell responses and as reagents to generate clinical grade antigen-specific CD4⁺ T cells for immunotherapy.
... 67 In a separate study, Gilardin et al. identified another CUB2 domain-derived peptide with sequence GDMLLLWGRLTWRKM (1239-1253) as a potential DRB1*01 and DRB1*11 restricted T-cell epitope. 68 This peptide was previously found to be presented Immune TTP: interplay between haplotypes & environment haematologica | 2018; 103(7) by APCs from a DRB1*0401/DRB1*1301 positive donor which were pulsed with 500 nM ADAMTS13. 66 In a recent study, we explored the repertoire of ADAMTS13derived peptides that were presented on HLA-DQ. ...
Although outstanding progress has been made in comprehending the pathophysiology of thrombotic thrombocytopenic purpura, our understanding of the immunopathogenesis of the disease is only in its earlier stage. Anti-ADAMTS13 auto-antibodies were shown to block proteolysis of von Willebrand factor and/or induce ADAMTS13 clearance from the circulation. However, which immune cells are involved in the production of anti-ADAMTS13 autoantibodies and hence account for the remarkable efficacy of the B-cell depleting agents in this disease, remain to be identified. The mechanisms leading to the loss of tolerance of the immune system towards ADAMTS13 involve the predisposing genetic factors of the human leukocyte antigen class II locus DRB1*11 and DQB1*03 alleles as well as the protective allele DRB1*04, and modifying factors such as ethnicity, sex and obesity. In the forthcoming years, studies will have to identify why these identified genetic risk factors are also frequently found in the healthy population although the incidence of immune-mediated thrombotic thrombocytopenic purpura is extremely low. Moreover, the development of recombinant ADAMTS13 opens a new therapeutic era in the field. Interactions of recombinant ADAMTS13 with the immune system of immune-mediated thrombotic thrombocytopenic purpura patients will require intensive investigation, especially for its potential immunogenicity. Better understanding of immune-mediated thrombotic thrombocytopenic purpura immunopathogenesis should therefore provide a basis for the development of novel therapeutic approaches to restore immune tolerance towards ADAMTS13 and thereby better prevent refractoriness and relapses in patients with immune-mediated thrombotic thrombocytopenic purpura. In this review, we address these aspects and the accompanying challenges in the field.
... 20 In addition, CUB2 domainderived peptide ADAMTS13 1239-1253 was identified as an immunodominant T-cell epitope in an HLA-DRB1 transgenic mouse model. 21 The same study revealed that ADAMTS13 1239-1253 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. ...
... 21 The same study revealed that ADAMTS13 1239-1253 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). ...
... 20 A recent study by Gilardin et al. did not identify CD4 + T cells responding to peptide ADAMTS13 1239-1253 containing the FINVAPHAR core-sequence in HLA-DRB1*11positive patients. 21 In their hands, another CUB2 domainderived peptide ADAMTS13 1239-1253 was identified as an immunodominant T-cell epitope for both DRB1*01 and DRB1*11-positive acquired TTP patients. 21 Surprisingly, the ADAMTS13 1239-1253 peptide was not identified to be presented on HLA-DR or DQ in our current study, despite the fact that several HLA-DRB1*01 and HLA-DRB1*11 positive donors were included in our cohort. ...
Formation of microthrombi is a hallmark of acquired thrombotic thrombocytopenic purpura. These microthrombi originate from insufficient processing of ultra large von Willebrand factor multimers by ADAMTS13 due to the development of anti-ADAMTS13 autoantibodies. Several studies have identified the major histocompatibility complex class II alleles HLA-DRB1*11, HLA-DQB1*03 and HLA-DQB1*02:02 as risk factors for acquired thrombotic thrombocytopenic purpura development. Previous research in our department indicated that ADAMTS13 CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR are presented on HLA-DRB1*11 and HLA-DRB1*03, respectively. Here, we describe the repertoire of ADAMTS13 peptides presented on HLA-DQ. In parallel, the repertoire of ADAMTS13-derived peptides presented on HLA-DR was monitored. Using HLA-DR- and HLA-DQ-specific antibodies we purified HLA/peptide complexes from ADAMTS13-pulsed monocyte-derived dendritic cells. Using this approach, we identified ADAMTS13-derived peptides presented on HLA-DR for all 9 samples analyzed; ADAMTS13-derived peptides presented on HLA-DQ were identified in 4 out of 9 samples. We were able to confirm the presentation of the CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR on HLA-DR. In total 12 different core-peptide sequences were identified on HLA-DR and 8 on HLA-DQ. For HLA-DR11 several potential new core-peptides were found; 4 novel core-peptides were exclusively identified on HLA-DQ. Furthermore, an in silico analysis was performed using the EpiMatrix and JanusMatrix tools to evaluate the eluted peptides, in the context of HLA-DR, for putative effector or regulatory T cell responses at the population level. The results from this study provide a basis for the identification of immuno-dominant epitopes on ADAMTS13 involved in the onset of acquired thrombotic thrombocytopenic purpura.
Background:
Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an ultra-rare autoimmune disorder caused by autoantibodies against ADAMTS13. A strong association of DRB1∗11 with iTTP and DRB1∗11-restricted T-cell epitopes in ADAMTS13 have been reported in Europeans, whereas we previously found DRB1∗08:03 as a susceptible allele in Japanese.
Objectives:
The limited information is available regarding a susceptible allele and its T-cell epitopes in Japanese patients with iTTP.
Materials and methods:
We conducted a reanalysis on iTTP-predisposing alleles using 3 distinct Japanese control groups. Subsequently, a novel human leukocyte antigen (HLA)-peptide expression assay (MHC-density assay) was used to identify the presentation of 24 ADAMTS13-derived peptides, including the regions that were identified previously by MHC-peptidome analysis and/or T-cell assays or predicted by NetMHCIIpan-4.0, to DRB1∗08:03 and DRB1∗11:01.
Results:
We reconfirmed the strong association of DRB1∗08:03 with iTTP, as well as the absence of the secondary risk alleles and protective alleles in Japanese iTTP, which altogether reveal that the HLA association pattern is completely different between the European and Japanese iTTP. MHC-density assay found the 3 ADAMTS13-derived peptides in the spacer domain as a potential strong binder to DRB1∗08:03. Moreover, 6 peptides in the metalloprotease, spacer, sixth thrombospondin-1 repeat, and CUB domains in ADAMTS13 showed increased presentation by both DRB1∗08:03 and DRB1∗11:01.
Conclusion:
Altogether, the findings of distinct HLA-DR association with iTTP across populations and the presentation of common peptides by DRB1∗08:03 and DRB1∗11:01 suggest that the same ADAMTS13-derived peptides might be presented and trigger the activation of autoreactive CD4+ T cells, leading to production of anti-ADAMTS13 autoantibodies by autoreactive B cells.
Thrombotic thrombocytopenic purpura (TTP) is an ultra-rare and fatal thrombotic disease characterized by systemic ischemic organ damage due to peripheral capillary occlusion by microthrombi. An acquired form of TTP, immune-mediated TTP (iTTP), is caused by the production of auto-antibodies against von Willebrand cleaving protease, also known as ADAMTS13. At the beginning of the 2010s, three independent groups in Europe reported that DRB1*11 was one of the strongest susceptible alleles to develop iTTP among European population. Several in silico predictions for the allele-restricted ADAMTS13 epitopes against T-cell are performed, followed by in vitro experiments to validate their functional implications, including mass spectrometry analysis of eluted peptides and T-cell assay. However, these analyses had not been performed in Japanese population so far. Here, we performed HLA typing for 52 patients with iTTP from 19 institutes across Japan, using the next-generation sequencing method. Our analysis revealed that DRB1*08:03 was associated with iTTP among the Japanese population while there were no statistical differences of the allele frequency of DRB1*11 between iTTP and healthy control. Subsequently, we predicted the strong binders for DRB1*08:03 molecules using NetMHCIIpan and found ADAMTS13 peptides in several domains had the possibility to bind to the molecule. To confirm this prediction, we performed in vitro MHC-density assay that evaluated the binding strength of the candidate peptides to DR molecules. This article reviews the current status of genetic and immunological studies on iTTP and our findings for iTTP in Japanese population.
