Tetramer visualization of gut-homing gluten-specific T cells in the peripheral blood of celiac disease patients

Abstract

Tetramers of MHC-peptide complexes are used for detection and characterization of antigen-specific T cell responses, but they require knowledge about both antigenic peptide and the MHC restriction element. The successful application of these reagents in human diseases involving CD4+ T cells is limited. Celiac disease, an intestinal inflammation driven by mucosal CD4+ T cells recognizing wheat gluten peptides in the context of disease-associated HLA-DQ molecules, is an ideal model to test the potential clinical use of these reagents. We investigated whether gluten-specific T cells can be detected in the peripheral blood of celiac disease patients using DQ2 tetramers. Nine DQ2+ patients and six control individuals on a gluten-free diet were recruited to the study. Participants consumed 160 g of gluten-containing bread daily for 3 days. After bread-challenge, gluten-specific T cells were detectable in the peripheral blood of celiac patients but not controls both directly by tetramer staining and indirectly by enzyme-linked immunospot. These T cells expressed the beta(7) integrin indicative of gut-homing properties. Most of the cells had a memory phenotype, but many other phenotypic markers showed a heterogeneous pattern. Tetramer staining of gluten-specific T cells has the potential to be used for diagnosis of celiac disease.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Direct detection of gluten-specific T cells by tetramer staining. Blood was drawn from study participants before and after a short bread challenge. PBMC were separated followed by positive selection of CD4+ T cells. Recovered cells were than stained for tetramers and also CD11c or CD14 to exclude contaminating monocytes from the analysis. (A) Example of dot plot in flow cytometry. Tetramer staining of CD4+ cells with the DQ2-control tetramer, the DQ2-αI tetramer, and the DQ2-αII tetramer. (Upper) Staining on day 0. (Lower) Staining with the same tetramers on day 6 after bread challenge. Cells in the upper right quadrant of the plots were considered as tetramer-positive cells. (B) Summary of data: Tetramer-positive cells in 106 CD4†T cells of celiac disease patients (filled circles) and DQ2+ (open circles) and DQ2− (open squares) controls on day 0 and day 6. The dashed line shows the cut-off value for positive staining. Tetramer staining was considered positive when the number of tetramer-binding T cells was higher than the average +3x SD (mean +3 SD) of the control individuals on a gluten-free diet. (C) Tetramer staining of CD4+ T cells from healthy DQ2+ (filled circles) and DQ2− (open circles) individuals on a gluten-containing diet. The dashed line shows the same cut-off value as in B.

Fig. 2.

Phenotype of gluten-specific T cells. CD4+ T cells were separated from peripheral blood after a short bread challenge and stained for CD11c, tetramers, and various markers of activation and differentiation. The plots shown are gated for CD11c− DQ2-αI tetramer-positive cells. (A) Expression of the activation marker CD69 and CD25 on gluten-specific T cells. A representative example is shown for both markers. (B) Expression of the β7 integrin and CD95 by T cells staining positive for the DQ2-αI tetramer. A representative example is shown for both markers. (Left) Plot showing only DQ2-αI tetramer-positive cells. (Right) Plot depicting all T cells of this patient. CD4+ cells are red, and DQ2-αI tetramer-positive cells are black. The DQ2-αI tetramer-positive cells show high expression of both β7 integrin and CD95.

Fig. 3.

Differentiation stage of gluten-specific T cells. (A) CD45RA and CD45RO expression varies among patients. In some patients, most DQ2-αI tetramer-positive cells are CD45RO+CD45RA− memory cells (Left), whereas in others, cells positive for the same tetramer have low expression of both markers (Right). (B) Expression of CD62L, CCR7, and CD27 also varies, indicating varying stages of differentiation of the gluten-specific T cells in the peripheral blood after gluten challenge. (Right) Cells from a patient with mostly effector memory cells. (Left) Another patient with more heterogeneous gluten-specific T cells. (C) Most DQ2-αI tetramer-positive cells, which are CD62L+, also express CCR7 and CD27.

Fig. 4.

IFN-γ ELISPOT on PBMC from treated celiac patients and controls before and after a short-term bread challenge. PBMC (4 × 105) were incubated with various antigens in quadruplicate wells of microtiter plates that had been coated with an anti-IFN-γ antibody. The responses against the synthetic deamidated 33-mer peptide (LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF) are shown before and after the bread challenge in the celiac disease patients and the DQ2+ (filled circles) and DQ2− (open circles) controls. SFU, spot-forming unit. Negative control values are subtracted.

Fig. 5.

Relationship between IFN-γ-producing cells and DQ2-αI tetramer staining on day 6. (A) The number of IFN-γ-producing cells determined by ELISPOT strongly correlates with the number of cells stained with DQ2-αI tetramer (r2 = 0.85). (B) The number of IFN-γ-producing cells determined by ELISPOT negatively correlates with the proportion of CD45RA+ cells among DQ2-αI tetramer-positive cells.