Background & Aims

Celiac disease is characterized by HLA-DQ2/8-restricted responses of CD4+ T cells to cereal gluten proteins. A diagnosis of celiac disease based on serologic and histologic evidence requires patients to be on gluten-containing diets. The growing number of individuals adhering to a gluten-free diet (GFD) without exclusion of celiac disease complicates its detection. HLA-DQ–gluten tetramers can be used to detect gluten-specific T cells in blood of patients with celiac disease, even if they are on a GFD. We investigated whether an HLA-DQ–gluten tetramer-based assay accurately identifies patients with celiac disease.

Methods

We produced HLA-DQ–gluten tetramers and added them to peripheral blood mononuclear cells isolated from 143 HLA-DQ2.5⁺ subjects (62 subjects with celiac disease on a GFD, 19 subjects without celiac disease on a GFD [due to self-reported gluten sensitivity], 10 subjects with celiac disease on a gluten-containing diet, and 52 presumed healthy individuals [controls]). T cells that bound HLA-DQ–gluten tetramers were quantified by flow cytometry. Laboratory tests and flow cytometry gating analyses were performed by researchers blinded to sample type, except for samples from subjects with celiac disease on a gluten-containing diet. Test precision analyses were performed using samples from 10 subjects.

Results

For the HLA-DQ–gluten tetramer-based assay, we combined flow-cytometry variables in a multiple regression model that identified individuals with celiac disease on a GFD with an area under the receiver operating characteristic curve value of 0.96 (95% confidence interval [CI] 0.89–1.00) vs subjects without celiac disease on a GFD. The assay detected individuals with celiac disease on a gluten-containing diet vs controls with an area under the receiver operating characteristic curve value of 0.95 (95% CI 0.90–1.00). Optimized cutoff values identified subjects with celiac disease on a GFD with 97% sensitivity (95% CI 0.92–1.00) and 95% specificity (95% CI 0.84–1.00) vs subjects without celiac disease on a GFD. The values identified subjects with celiac disease on a gluten-containing diet with 100% sensitivity (95% CI 1.00–1.00]) and 90% specificity (95% CI 0.83–0.98) vs controls. In an analysis of 4 controls with positive results from the HLA-DQ–gluten tetramer test, 2 had unrecognized celiac disease and the remaining 2 had T cells that proliferated in response to gluten antigen in vitro.

Conclusions

An HLA-DQ–gluten tetramer-based assays that detects gluten-reactive T cells identifies patients with and without celiac disease with a high level of accuracy, regardless of whether the individuals are on a GFD. This test would allow individuals with suspected celiac disease to avoid gluten challenge and duodenal biopsy, but requires validation in a larger study. Clinicaltrials.gov no: NCT02442219.

中文翻译：

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HLA-DQ –谷蛋白四聚体血液测试可准确识别无麸质消费的有或没有腹腔疾病的患者

背景与目标

腹腔疾病的特征是CD4 + T细胞对谷蛋白的HLA-DQ2 / 8限制性应答。根据血清学和组织学证据诊断乳糜泻需要患者采用含麸质饮食。坚持无麸质饮食（GFD）却不排除乳糜泻的个体数量不断增加，这使它的检测变得复杂。HLA-DQ-麸质四聚体可用于检测乳糜泻患者血液中的麸质特异性T细胞，即使它们在GFD上也是如此。我们调查了基于HLA-DQ-麸质四聚体的测定法是否能准确识别患有乳糜泻的患者。

方法

我们生产了HLA-DQ-麸质四聚体，并将其添加到从143名HLA-DQ2.5 ⁺受试者（62名患有GFD腹腔疾病的受试者，19名没有GFD腹腔疾病的受试者）中分离的外周血单核细胞中[由于自我报告麸质敏感性]，10名患有麸质饮食的乳糜泻患者和52名假定的健康个体[对照组]。结合HLA-DQ-麸质四聚体的T细胞通过流式细胞仪进行定量。研究人员进行了实验室测试和流式细胞术门控分析，不知道样品类型，但来自患有麸质饮食的腹腔疾病受试者的样品除外。使用来自10位受试者的样品进行测试精度分析。

结果

对于基于HLA-DQ-麸质四聚体的测定，我们将流式细胞仪变量组合到一个多元回归模型中，该模型在GFD上识别出患有乳糜泻的个体，该区域的受试者工作特征曲线值小于0.96（95％置信区间[ CI] 0.89–1.00）与GFD上无腹腔疾病的受试者比较。该测定法检测到在接受麸质饮食的情况下患有腹腔疾病的个体与对照组相比，接受者操作特征曲线值下的面积为0.95（95％CI 0.90–1.00）。与使用GFD的无腹腔疾病的受试者相比，优化的临界值确定了接受GFD的腹腔疾病受试者的敏感性为97％（95％CI 0.92–1.00）和特异性为95％（95％CI 0.84–1.00）。这些值以含麸质的饮食以100％的敏感性（95％CI 1.00–1）鉴定出患有乳糜泻的受试者。00]）和90％的特异性（95％CI 0.83–0.98）。在对来自HLA-DQ-麸质四聚体试验阳性结果的4个对照的分析中，有2个患有无法识别的乳糜泻，其余2个具有在体外对麸质抗原反应的T细胞增殖。

结论

一种基于HLA-DQ-麸质四聚体的检测方法，可检测麸质反应性T细胞，从而可以高度准确地识别出患有和不患有乳糜泻的患者，而不论患者是否接受GFD检查。该测试将使疑似腹腔疾病的患者避免面筋刺激和十二指肠活检，但需要在更大的研究中进行验证。Clinicaltrials.gov编号：NCT02442219。