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, 106 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.

中文翻译：

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难治性乳糜泻中的 T 细胞库

目的难治性乳糜泻（RCD）是乳糜泻（CD）的潜在危险并发症。与 I 型 RCD 不同，II 型 RCD 是肠病相关 T 细胞淋巴瘤 (EATL) 的前体实体，它与在顺序发展的 EATL 中也发现的克隆性扩增 T 细胞有关。使用高通量测序 (HTS)，我们旨在建立 CD 和 RCD 中的小肠 T 细胞库 (TCR)，以阐明不同 T 细胞克隆型在 RCD 发病机制中的作用。从对照 (n=9)、活跃的腹腔动物 (n=10)、无麸质饮食的腹腔动物 (n=9)、RCD I 型 (n=8) 的十二指肠粘膜标本中提取的设计 DNA，2002 年至 2013 年收集的 RCD II 型（n=8）和未分类的 Marsh I 病例（n=3）通过 TCRβ-互补决定区 3（CDR3）多重 PCR 和扩增子的 HTS 进行检查。结果 平均每个样本产生 106 个序列读数，由多达 900 个单独的 TCRβ 重排组成。在 RCD II 型中，最常见的克隆型（即具有相同 CDR3 的序列读数）平均占所有 TCRβ 重排的 42.6%，显着高于对照（6.8%；p<0.01）或 RCD I 型（6.7%） ；p<0.01)。个别患者的重复内窥镜检查显示克隆型的稳定性长达数年，而没有 EATL 的临床症状。在 RCD II 型个体患者中鉴定的显性克隆型是独特的，患者之间不相关。CD相关，麦胶蛋白依赖性 CDR3 基序只能在低频率下检测到。结论 TCRβ-HTS 分析揭示了 CD 中的 TCR，并允许对单个 TCRβ 重排进行详细分析。在 RCD II 型患者中鉴定的显性 TCRβ 序列是独特的，与已知的麦胶蛋白特异性 TCR 序列不同源，支持这些克隆性 T 细胞独立于麸质刺激而扩增的假设。