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, 456 (7221), 534-8

The Role of HLA-DQ8 beta57 Polymorphism in the Anti-Gluten T-cell Response in Coeliac Disease

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The Role of HLA-DQ8 beta57 Polymorphism in the Anti-Gluten T-cell Response in Coeliac Disease

Zaruhi Hovhannisyan et al. Nature.

Abstract

Major histocompatibility complex (MHC) class II alleles HLA-DQ8 and the mouse homologue I-A(g7) lacking a canonical aspartic acid residue at position beta57 are associated with coeliac disease and type I diabetes. However, the role of this single polymorphism in disease initiation and progression remains poorly understood. The lack of Asp 57 creates a positively charged P9 pocket, which confers a preference for negatively charged peptides. Gluten lacks such peptides, but tissue transglutaminase (TG2) introduces negatively charged residues at defined positions into gluten T-cell epitopes by deamidating specific glutamine residues on the basis of their spacing to proline residues. The commonly accepted model, proposing that HLA-DQ8 simply favours binding of negatively charged peptides, does not take into account the fact that TG2 requires inflammation for activation and that T-cell responses against native gluten peptides are found, particularly in children. Here we show that beta57 polymorphism promotes the recruitment of T-cell receptors bearing a negative signature charge in the complementary determining region 3beta (CDR3beta) during the response against native gluten peptides presented by HLA-DQ8 in coeliac disease. These T cells showed a crossreactive and heteroclitic (stronger) response to deamidated gluten peptides. Furthermore, gluten peptide deamidation extended the T-cell-receptor repertoire by relieving the requirement for a charged residue in CDR3beta. Thus, the lack of a negative charge at position beta57 in MHC class II was met by negatively charged residues in the T-cell receptor or in the peptide, the combination of which might explain the role of HLA-DQ8 in amplifying the T-cell response against dietary gluten.

Figures

Figure 1
Figure 1. Native and deamidated gluten α2-219–242 peptides recruit distinct, yet overlapping TCR repertoires
a, Humanized HLA-DQ8 transgenic mice were immunized with native (Q) and deamidated (E) versions of the 24-amino-acid gluten peptide α2-219–242 and two E peptide analogues with a Glu residue either in position 229 (E229) or 237 (E237). The draining lymph nodes were collected 8 days after immunization and CD4+ T cells were purified to perform functional assays and derive α2-219–242 gluten-peptide-specific HLA-DQ8-restricted T-cell hybridomas. b, Purified CD4+ T cells were tested for proliferative in vitro responses with the indicated peptide at the specified concentrations in the presence of irradiated (30 Gy) spleen cells from non-immunized humanized HLA-DQ8 mice. Responses are given in c.p.m. from the mean 3H-thymidine incorporation in triplicate cultures ± s.d. n = 3 mice per each condition of immunization. The data are representative of three independent experiments for upper panels, and two independent experiments for middle and lower panels. c, A panel of α2-219–242 gluten-peptide-specific CD4+ T hybridomas was derived after immunization with native (Q ) or deamidated (E) peptide alone. Hybridoma reactivity was defined by measuring IL-2 secretion. Q and E hybridomas responded exclusively to native and deamidated peptides, respectively. Q = E, hybridomas responded equally well to native and deamidated peptides; Q >E, hybridomas responded 10–100 times better to native than to deamidated peptide; E >Q, hybridomas responded 10–100 times better to deamidated than to native peptide. The percentage of T-cell hybridomas obtained in each class is indicated. Importantly, assessing the quality of the immunization, no E-specific hybridomas were obtained after Q immunization, and conversely no Q-specific hybridomas were obtained after E immunization d, TCR β-chain variable region usage of individual hybridomas was defined by flow cytometry after immunization with Q (open circles, left panel) and E peptides (filled circles, right panel).
Figure 2
Figure 2. Negative charge at position 3 of the TCR CDR3β loop is critical for native gluten peptide reactivity
a, Generation of TCR-Vβ5.1-chain mutants from a representative Q = E hybridoma with either substitution of Glu to Gln at position 3 (Vβ5.1 (E→Q)) or Asp to Asn at position 8 (Vβ5.1 (D→N)). The mutated amino acids are indicated in bold. Wild-type (WT) Vα17 chain in association with wild-type or mutant TCR Vβ5.1 chains was retrovirally transduced in the CD4 transfected 58αβ T-cell hybridoma variant. Hybridoma transfectants expressing wild-type Vβ5.1/Vα17 (as a control, left panel), and mutated Vβ5.1(E→Q)/Vα17 (middle panel) or Vβ5.1(D→N)/Vα17 (right panel) TCR heterodimers were stimulated with either Q (filled diamonds) or E (open circles) peptide at the indicated concentrations. The IL-2 release values are the mean of triplicate cultures ± s.d. The data are representative of three independent experiments. b, A representative Q = E hybridoma was stimulated with Q (left panel) and E (right panel) peptides in the presence of wild-type HLA-DQ8 (WT DQ8, filled diamonds) or mutant HLA-DQ8 (D57 DQ8, open circles) with an Ala to Asp substitution at position β57. The level of IL-2 was measured using mouse cytotoxic T lymphocyte lines (CTLL) bioassay. The mean of duplicate cultures are shown, and data are representative of three separate experiments.
Figure 3
Figure 3. Coimmunization with native and deamidated peptides amplifies the T-cell response to deamidated peptide
Humanized HLA-DQ8 transgenic mice were immunized with Q or E gluten peptides alone, or in combination. Eight days after immunization CD4+T cells were purified and re-challenged in vitro with 32.3 μM Q or E peptides. The frequency of IFN-γ-producing cells was assessed using ELISPOT. Each bar represents the mean spot number of triplicates ± s.d. **P <0.001; data are representative of three independent experiments, each comprising three mice for each type of immunization.

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