Tetramer-visualized gluten-specific CD4+ T cells in blood as a potential diagnostic marker for coeliac disease without oral gluten challenge

Abstract

Background: Diagnosing coeliac disease (CD) can be challenging, despite highly specific autoantibodies and typical mucosal changes in the small intestine. The T-cell response to gluten is a hallmark of the disease that has been hitherto unexploited in clinical work-up.

Objectives: We aimed to develop a new method that directly visualizes and characterizes gluten-reactive CD4+ T cells in blood, independently of gluten challenge, and to explore its diagnostic potential.

Methods: We performed bead-enrichment of DQ2.5-glia-α1a and DQ2.5-glia-α2 tetramer+ cells in the blood of control individuals, treated (TCD) and untreated patients (UCD). We visualized these cells by flow cytometry, sorted them and cloned them. We assessed their specificity by antigen stimulation and re-staining with tetramers.

Results: We detected significantly more gliadin-tetramer+ CD4+ effector memory T cells (TEM) in UCD and TCD patients, compared to controls. Significantly more gliadin-tetramer+ TEM in the CD patients than in controls expressed the gut-homing marker integrin-β7.

Conclusion: Quantification of gut-homing, gluten-specific TEM in peripheral blood, visualized with human leukocyte antigen (HLA) -tetramers, may be used to distinguish CD patients from healthy individuals. Easy access to gluten-reactive blood T cells from diseased and healthy individuals may lead to new insights on the disease-driving CD4+ T cells in CD.

Keywords: Blood test; T cells; celiac disease; diagnostic marker; diagnostics; gliadin; gluten; gluten reactivity; histology; human leukocyte antigen; immunology.

Figure 1.

Gating strategy. Flow cytometric density plots and dot plots illustrating the gating strategy for relevant gliadin-tetramer+ cells. Percentages of gated cells within each plot are shown. (a) Gating was done on single cells → lymphocytes → CD3+ cells → CD4+ cells. There were few gliadin-tetramer+ cells among CD3+ CD4- cells (lower left plot); whereas there were distinct populations of CD3+ CD4+ gliadin-tetramer+ cells (lower right plot), here shown in a TCD patient. (b) Dot plots in the left panels show CD4+ T cells in bead-enriched samples from a control individual, UCD and TCD patients binding the two different gliadin-tetramers. Tetramer+ CD4+ T cells were subdivided by the results of CD62L- and CD45RA-staining into effector memory (double negative), naïve (double positive) and central memory (CD62L+ and CD45RA-) T cells. FSC-W: Forward scatter width; FSC-A: Forward scatter areal; SSC-A: Side scatter areal; TCD: Treated coeliac disease; UCD: Untreated coeliac disease.

Figure 2.

Frequency of CD4+ gliadin-tetramer+ T cells. Number of EM, CM and N T cells binding the DQ2.5-glia-α1a-tetramer (left) and the DQ2.5-glia-α2-tetramer (right) per million total CD4+ T cells. Each participant is indicated by a closed circle. The median frequency is denoted with numbers in (a) controls, (b) TCD patients and (c) UCD patients. Frequencies below <0.01 per million are placed on the x-axis for visualization purposes. CM: Central memory; EM: effector memory; N: naïve T cells; TCD: treated coeliac disease patients; UCD: untreated coeliac disease patients.

Figure 3.

Significantly more gliadin-tetramer+ TEM in patients compared to controls. (a) Frequency of CD4+ TEM binding the DQ2.5-glia-α1-tetramer (left) and the DQ2.5-glia-α2-tetramer (right) among controls, UCD and TCD. (b) Ratio between gliadin-tetramer+ CD4+ TEM and naïve T cells (EM/N-ratio). (c) The prevalence of gliadin-tetramer+ CD4+ TEM was grouped by Marsh score in those participants where duodenal biopsies were obtained. Frequencies and ratios below <0.01 per million are placed on the x-axis for visualization purposes. Each frequency and ratio is indicated by a closed circle. P values were calculated with the Mann-Whitney U test. N: naïve T cells; TCD: treated coeliac disease patients; TEM: Effector memory T cells; UCD: untreated coeliac disease patients.

Figure 4.

Gut-homing of T cells. (a) While gliadin-tetramer+ N T cells expressed integrin-β7 at intermediate levels and the CM T cells showed no clear staining, nearly all gliadin-tetramer+ EM T cells in TCD expressed integrin-β7. Gliadin-tetramer positive and negative EM T cells in controls expressed similar amounts of integrin-β7. The DQ2.5-glia-α1a- and DQ2.5-glia-α2-tetramer were combined on one fluorochrome, to increase the number of relevant cells. (b) The percent of integrin-β7 expression on tetramer-positive and tetramer-negative cells in four TCD and four controls. (c) Tetramer+ EM in one tested TCD did not express the skin-homing CLA. Percentages of gated cells within each plot are denoted by numbers. CLA: cutaneous leukocyte-associated antigen; CM: central memory; EM: effector memory; N: naïve; TCD: treated coeliac disease patients.