T-cell repertoires in refractory coeliac disease

Objective 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. Design 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. Results On average, 10 6 sequence 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. Conclusions 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.