title: T-cell repertoires in refractory coeliac disease.
abstract: Refractory coeliac disease (RCD) is a potentially hazardous complication of coeliac disease (CD). In contrast to RCD type I, RCD type II is a precursor entity of enteropathy-associated T-cell lymphoma (EATL), which is associated with clonally expanding T-cells that are also found in the sequentially developing EATL. Using high-throughput sequencing (HTS), we aimed to establish the small-intestinal T-cell repertoire (TCR) in CD and RCD to unravel the role of distinct T-cell clonotypes in RCD pathogenesis.
  
  
  
  DNA extracted from duodenal mucosa specimens of controls (n=9), active coeliacs (n=10), coeliacs on a gluten-free diet (n=9), RCD type I (n=8), RCD type II (n=8) and unclassified Marsh I cases (n=3) collected from 2002 to 2013 was examined by TCRβ-complementarity-determining regions 3 (CDR3) multiplex PCR followed by HTS of the amplicons.
  
  
  
  On average, 106sequence reads per sample were generated consisting of up to 900 individual TCRβ rearrangements. In RCD type II, the most frequent clonotypes (ie, sequence reads with identical CDR3) represent in average 42.6% of all TCRβ rearrangements, which was significantly higher than in controls (6.8%; p<0.01) or RCD type I (6.7%; p<0.01). Repeat endoscopies in individual patients revealed stability of clonotypes for up to several years without clinical symptoms of EATL. Dominant clonotypes identified in individual patients with RCD type II were unique and not related between patients. CD-associated, gliadin-dependent CDR3 motifs were only detectable at low frequencies.
  
  
  
  TCRβ-HTS analysis unravels the TCR in CD and allows detailed analysis of individual TCRβ rearrangements. Dominant TCRβ sequences identified in patients with RCD type II are unique and not homologous to known gliadin-specific TCR sequences, supporting the assumption that these clonal T-cells expand independent of gluten stimulation.
authors.affiliation: Institute of Pathology, Charité-University Medicine, Berlin, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Ritter
authors.affiliation: Institute of Pathology, Charité-University Medicine, Berlin, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Zimmermann
authors.affiliation: Institute of Pathology, Charité-University Medicine, Berlin, Germany.
authors.forename: Korinna
authors.initials: K
authors.lastname: Jöhrens
authors.affiliation: Institute of Pathology, Charité-University Medicine, Berlin, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Mende
authors.affiliation: Institute of Pathology, Charité-University Medicine, Berlin, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Seegebarth
authors.affiliation: Department of Gastroenterology, Infectious Diseases and Rheumatology, Charité-University Medicine, Berlin, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Siegmund
authors.affiliation: HS Diagnomics GmbH, Berlin, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Hennig
authors.affiliation: Center for Tumor Medicine, Charité-University Medicine, Berlin, Germany.
authors.forename: Kremena
authors.initials: K
authors.lastname: Todorova
authors.affiliation: Institute of Pathology, University of Würzburg, and Comprehensive Cancer Center Mainfranken (CCCMF), Würzburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Rosenwald
authors.affiliation: Department of Gastroenterology, Infectious Diseases and Rheumatology, Charité-University Medicine, Berlin, Germany.
authors.forename: Severin
authors.initials: S
authors.lastname: Daum
authors.affiliation: Institute of Pathology, Charité-University Medicine, Berlin, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hummel
authors.affiliation: Department of Gastroenterology, Infectious Diseases and Rheumatology, Charité-University Medicine, Berlin, Germany.|Berlin Institute of Health, Berlin, Germany.|Berlin-Brandenburg School for Regenerative Therapies, Berlin, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schumann
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000378:metabolism; D016022:Case-Control Studies; D002446:Celiac Disease / Q000145:classification / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism*; D003937:Diagnosis, Differential; D055050:Diet, Gluten-Free / Q000379:methods; D004386:Duodenum / Q000473:pathology; D005260:Female; D019674:Genes, T-Cell Receptor beta / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007166:Immunosuppressive Agents / Q000627:therapeutic use; D007413:Intestinal Mucosa / Q000473:pathology; D007421:Intestine, Small / Q000473:pathology; D008297:Male; D008875:Middle Aged; D011237:Predictive Value of Tests; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D007166:Immunosuppressive Agents
keywords: COELIAC DISEASE; LYMPHOMA; MOLECULAR PATHOLOGY; SMALL INTESTINE; T-CELL RECEPTOR