Yvonne Kooy-Winkelaar

T-cell receptor recognition of HLA-DQ2-gliadin complexes associated with celiac disease

Celiac disease is a T cell–mediated disease induced by dietary gluten, a component of which is gliadin. 95% of individuals with celiac disease carry the HLA (human leukocyte antigen)-DQ2 locus. Here we determined the T-cell receptor (TCR) usage and fine specificity of patient-derived T-cell clones specific for two epitopes from wheat gliadin, DQ2.5-glia-α1a and DQ2.5-glia-α2. We determined the ternary structures of four distinct biased TCRs specific for those epitopes. All three TCRs specific for DQ2.5-glia-α2 docked centrally above HLA-DQ2, which together with mutagenesis and affinity measurements provided a basis for the biased TCR usage. A non–germline encoded arginine residue within the CDR3β loop acted as the lynchpin within this common docking footprint. Although the TCRs specific for DQ2.5-glia-α1a and DQ2.5-glia-α2 docked similarly, their interactions with the respective gliadin determinants differed markedly, thereby providing a basis for epitope specificity.

Biased T Cell Receptor Usage Directed against Human Leukocyte Antigen DQ8-Restricted Gliadin Peptides Is Associated with Celiac Disease

Celiac disease is a human leukocyte antigen (HLA)-DQ2- and/or DQ8-associated T cell-mediated disorder that is induced by dietary gluten. Although it is established how gluten peptides bind HLA-DQ8 and HLA-DQ2, it is unclear how such peptide-HLA complexes are engaged by the T cell receptor (TCR), a recognition event that triggers disease pathology. We show that biased TCR usage (TRBV9(∗)01) underpins the recognition of HLA-DQ8-α-I-gliadin. The structure of a prototypical TRBV9(∗)01-TCR-HLA-DQ8-α-I-gliadin complex shows that the TCR docks centrally above HLA-DQ8-α-I-gliadin, in which all complementarity-determining region-β (CDRβ) loops interact with the gliadin peptide. Mutagenesis at the TRBV9(∗)01-TCR-HLA-DQ8-α-I-gliadin interface provides an energetic basis for the Vβ bias. Moreover, CDR3 diversity accounts for TRBV9(∗)01(+) TCRs exhibiting differing reactivities toward the gliadin epitopes at various deamidation states. Accordingly, biased TCR usage is an important factor in the pathogenesis of DQ8-mediated celiac disease.

Gluten-Specific T Cells Cross-React between HLA-DQ8 and the HLA-DQ2α/DQ8β Transdimer

Because susceptibility to celiac disease is associated strongly with HLA-DQ2 (DQA1*05/DQB1*02) and weakly with HLA-DQ8 (DQA1*03/DQB1*03), a subset of patients carries both HLA-DQ2 and HLA-DQ8. As a result, these patients may express two types of mixed HLA-DQ2/8 transdimers (encoded by DQA1*05/DQB1*03 and DQA1*03/DQB1*02) in addition to HLA-DQ2 and HLA-DQ8. Using T cells from a celiac disease patient expressing HLA-DQ8trans (encoded by DQA*0501/DQB*0302), but neither HLA-DQ2 nor HLA-DQ8, we demonstrate that this transdimer is expressed on the cell surface and can present multiple gluten peptides to T cell clones isolated from the duodenum of this patient. Furthermore, T cell clones derived from this patient and HLA-DQ2/8 heterozygous celiac disease patients respond to gluten peptides presented by HLA-DQ8trans, as well as HLA-DQ8, in a similar fashion. Finally, one gluten peptide is recognized better when presented by HLA-DQ8trans, which correlates with preferential binding of this peptide to HLA-DQ8trans. These results implicate HLA-DQ8trans in celiac disease pathogenesis and demonstrate extensive T cell cross-reactivity between HLA-DQ8 and HLA-DQ8trans. Because type 1 diabetes is strongly associated with the presence of HLA-DQ8trans, our findings may bear relevance to this disease as well.

Identification of a potential physiological precursor of aberrant cells in refractory coeliac disease type II

Objective Refractory coeliac disease type II (RCDII) is a severe complication of coeliac disease (CD) characterised by aberrant intraepithelial lymphocytes (IELs) of unknown origin that display an atypical CD3−CD7+icCD3+ phenotype. In approximately 40% of patients with RCDII these lymphocytes develop into an invasive lymphoma. In the current study we aimed to identify the physiological counterpart of these cells. Design RCDII cell lines were compared with T-cell receptor positive (TCR+) IEL (T-IEL) lines by microarray analysis, real-time quantitative PCR and flow cytometry. This information was used to identify cells with an RCDII-associated phenotype in duodenal biopsies from non-refractory individuals by multicolour flow cytometry. Results RCDII lines were transcriptionally distinct from T-IEL lines and expressed higher levels of multiple natural killer (NK) cell receptors. In addition to the CD3−CD7+icCD3+ phenotype, the RCDII lines were distinguishable from other lymphocyte subsets by the absence of CD56, CD127 and CD34. Cells matching this surface lineage-negative (Lin−) CD7+CD127−CD34− phenotype expressed a functional interleukin-15 (IL-15) receptor and constituted a significant proportion of IELs in duodenal specimens of patients without CD, particularly children, and were also found in the thymus. In patients without CD, the Lin−CD7+CD127−CD34− subset was one of four subsets within the CD3−CD7+icCD3+ population that could be distinguished on the basis of differential expression of CD56 and/or CD127. Conclusion Our studies indicate that the CD3−CD7+icCD3+ population is heterogeneous and reveal the existence of a Lin− subset that is distinct from T, B, NK and lymphoid tissue inducer cells. We speculate that this IL-15 responsive population represents the physiological counterpart of aberrant cells expanded in RCDII and transformed in RCDII-associated lymphoma.

Defective synthesis or association of T-cell receptor chains underlies loss of surface T-cell receptor–CD3 expression in enteropathy-associated T-cell lymphoma

Enteropathy-associated T-cell lymphoma, an often fatal complication of celiac disease, can result from expansion of aberrant intraepithelial lymphocytes in refractory celiac disease type II (RCD II). Aberrant intraepithelial lymphocytes and lymphoma cells are intracellularly CD3epsilon(+) but lack expression of the T-cell receptor (TCR)-CD3 complex on the cell surface. It is unknown what causes the loss of TCR-CD3 expression. We report the isolation of a cell line from an RCD II patient with the characteristic phenotype of enteropathy-associated T-cell lymphoma. We demonstrate that in this cell line the TCR-alpha and -beta chains as well as the CD3gamma, CD3delta, CD3epsilon, and zeta-chains are present intracellularly and that assembly of the CD3gammaepsilon, CD3deltaepsilon, and zetazeta-dimers is normal. However, dimerization of the TCR chains and proper assembly of the TCR-CD3 complex are defective. On introduction of exogenous TCR-beta chains, but not of TCR-alpha chains, assembly and functional cell surface expression of the TCR-CD3 complex were restored. Defective synthesis of both TCR chains was found to underlie loss of TCR expression in similar cell lines isolated from 2 additional patients. (Pre)malignant transformation in RCD II thus correlates with defective synthesis or defective association of the TCR chains, resulting in loss of surface TCR-CD3 expression.

Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

Background Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP). Methods Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays. Findings The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data. Conclusion Currently available digestive enzyme supplements are ineffective in degrading immunogenic gluten epitopes.

Determinants of Gliadin-Specific T Cell Selection in Celiac Disease

In HLA-DQ8–associated celiac disease (CD), the pathogenic T cell response is directed toward an immunodominant α-gliadin–derived peptide (DQ8-glia-α1). However, our knowledge of TCR gene usage within the primary intestinal tissue of HLA-DQ8+ CD patients is limited. We identified two populations of HLA-DQ8-glia-α1 tetramer+ CD4+ T cells that were essentially undetectable in biopsy samples from patients on a gluten-free diet but expanded rapidly and specifically after antigenic stimulation. Distinguished by expression of TRBV9, both T cell populations displayed biased clonotypic repertoires and reacted similarly against HLA-DQ8-glia-α1. In particular, TRBV9 paired most often with TRAV26-2, whereas the majority of TRBV9− TCRs used TRBV6-1 with no clear TRAV gene preference. Strikingly, both tetramer+/TRBV9+ and tetramer+/TRBV9− T cells possessed a non–germline-encoded arginine residue in their CDR3α and CDR3β loops, respectively. Comparison of the crystal structures of three TRBV9+ TCRs and a TRBV9− TCR revealed that, as a result of distinct TCR docking modes, the HLA-DQ8-glia-α1 contacts mediated by the CDR3-encoded arginine were almost identical between TRBV9+ and TRBV9− TCRs. In all cases, this interaction centered on two hydrogen bonds with a specific serine residue in the bound peptide. Replacement of serine with alanine at this position abrogated TRBV9+ and TRBV9− clonal T cell proliferation in response to HLA-DQ8-glia-α1. Gluten-specific memory CD4+ T cells with structurally and functionally conserved TCRs therefore predominate in the disease-affected tissue of patients with HLA-DQ8–mediated CD.

DNAM-1 Mediates Epithelial Cell-Specific Cytotoxicity of Aberrant Intraepithelial Lymphocyte Lines from Refractory Celiac Disease Type II Patients

In refractory celiac disease (RCD), intestinal epithelial damage persists despite a gluten-free diet. Characteristic for RCD type II (RCD II) is the presence of aberrant surface TCR-CD3− intraepithelial lymphocytes (IELs) that can progressively replace normal IELs and eventually give rise to overt lymphoma. Therefore, RCD II is considered a malignant condition that forms an intermediate stage between celiac disease (CD) and overt lymphoma. We demonstrate in this study that surface TCR-CD3− IEL lines isolated from three RCD II patients preferentially lyse epithelial cell lines. FACS analysis revealed that DNAM-1 was strongly expressed on the three RCD cell lines, whereas other activating NK cell receptors were not expressed on all three RCD cell lines. Consistent with this finding, cytotoxicity of the RCD cell lines was mediated mainly by DNAM-1 with only a minor role for other activating NK cell receptors. Furthermore, enterocytes isolated from duodenal biopsies expressed DNAM-1 ligands and were lysed by the RCD cell lines ex vivo. Although DNAM-1 on CD8+ T cells and NK cells is known to mediate lysis of tumor cells, this study provides, to our knowledge, the first evidence that (pre)malignant cells themselves can acquire the ability to lyse epithelial cells via DNAM-1. This study confirms previous work on epithelial lysis by RCD cell lines and identifies a novel mechanism that potentially contributes to the gluten-independent tissue damage in RCD II and RCD-associated lymphoma.

The composition and differentiation potential of the duodenal intraepithelial innate lymphocyte compartment is altered in coeliac disease

Objective Coeliac disease (CD), a gluten-induced enteropathy, alters the composition and function of duodenal intraepithelial T cells. The intestine also harbours four types of CD3-negative intraepithelial lymphocytes (IELs) with largely unknown function: CD56−CD127−, CD56−CD127+, CD56+CD127− and CD56+CD127+. Here we aimed to gain insight into the potential function of these innate IELs in health and disease. Design We determined the phenotypes, relative abundance and differentiation potential of these innate IEL subsets in duodenal biopsies from controls and patients with CD or patients with refractory CD type II (RCDII). Results Hierarchical clustering analysis of the expression of 15 natural killer and T cell surface markers showed that innate IELs differed markedly from innate peripheral blood lymphocytes and divided innate IEL subsets into two main branches: a CD127− branch expressing high levels of interleukin (IL) 2/15Rβ but no IL-21R, and a CD127+ branch with the opposite phenotype. While CD was characterised by the contraction of all four innate IEL subsets, a selective expansion of CD56−CD127− and CD56−CD127+ innate IEL was detected in RCDII. In vitro, in the presence of IL-15, CD56−CD127− IEL from controls and patients with CD, but not from patients with RCDII, differentiated into functional natural killer and T cells, the latter largely dependent on notch-signalling. Furthermore, compared with non-coeliac controls, CD56−CD127− IEL from patients with CD expressed more intracellular CD3ε and CD3γ and gave more pronounced T cell differentiation. Conclusions Thus, we demonstrate previously unappreciated diversity and plasticity of the innate IEL compartment and its loss of differentiation potential in patients with RCDII.

CD4 T-cell cytokines synergize to induce proliferation of malignant and nonmalignant innate intraepithelial lymphocytes.

Significance Refractory celiac disease type II (RCDII) is a severe variant of celiac disease, an autoimmune disorder of the small intestine caused by inflammatory T-cell responses to gluten, a common food protein. Typical of RCDII is the presence of aberrant lymphocytes in the duodenal epithelium, which often give rise to a lethal lymphoma. A single growth factor promoting the expansion of aberrant cells has been identified: epithelial cell-derived IL-15. The experiments described in this paper identify three additional growth factors—TNF, IL-2, and IL-21—produced by gluten-specific T cells. Thus, these findings suggest a potential mechanism for the contribution of gluten-specific T cells to RCDII. Refractory celiac disease type II (RCDII) is a severe complication of celiac disease (CD) characterized by the presence of an enlarged clonal population of innate intraepithelial lymphocytes (IELs) lacking classical B-, T-, and natural killer (NK)-cell lineage markers (Lin−IELs) in the duodenum. In ∼50% of patients with RCDII, these Lin−IELs develop into a lymphoma for which no effective treatment is available. Current evidence indicates that the survival and expansion of these malignant Lin−IELs is driven by epithelial cell-derived IL-15. Like CD, RCDII is strongly associated with HLA-DQ2, suggesting the involvement of HLA-DQ2–restricted gluten-specific CD4+ T cells. We now show that gluten-specific CD4+ T cells isolated from CD duodenal biopsy specimens produce cytokines able to trigger proliferation of malignant Lin−IEL lines as powerfully as IL-15. Furthermore, we identify TNF, IL-2, and IL-21 as CD4+ T-cell cytokines that synergistically mediate this effect. Like IL-15, these cytokines were found to increase the phosphorylation of STAT5 and Akt and transcription of antiapoptotic mediator bcl-xL. Several small-molecule inhibitors targeting the JAK/STAT pathway blocked proliferation elicited by IL-2 and IL-15, but only an inhibitor targeting the PI3K/Akt/mTOR pathway blocked proliferation induced by IL-15 as well as the CD4+ T-cell cytokines. Confirming and extending these findings, TNF, IL-2, and IL-21 also synergistically triggered the proliferation of freshly isolated Lin−IELs and CD3−CD56+ IELs (NK-IELs) from RCDII as well as non-RCDII duodenal biopsy specimens. These data provide evidence implicating CD4+ T-cell cytokines in the pathogenesis of RCDII. More broadly, they suggest that adaptive immune responses can contribute to innate IEL activation during mucosal inflammation.