Discriminative T-cell receptor recognition of highly homologous HLA-DQ2–bound gluten epitopes

Celiac disease (CeD) provides an opportunity to study the specificity underlying human T-cell responses to an array of similar epitopes presented by the same human leukocyte antigen II (HLA-II) molecule. Here, we investigated T-cell responses to the two immunodominant and highly homologous HLA-DQ2.5–restricted gluten epitopes, DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF). Using HLA-DQ2.5–DQ2.5-glia-α1a and HLA-DQ2.5–DQ2.5-glia-ω1 tetramers and single-cell αβ T-cell receptor (TCR) sequencing, we observed that despite similarity in biased variable-gene usage in the TCR repertoire responding to these nearly identical peptide–HLA-II complexes, most of the T cells are specific for either of the two epitopes. To understand the molecular basis of this exquisite fine specificity, we undertook Ala substitution assays revealing that the p7 residue (Leu/Gln) is critical for specific epitope recognition by both DQ2.5-glia-α1a– and DQ2.5-glia-ω1–reactive T-cell clones. We determined high-resolution binary crystal structures of HLA-DQ2.5 bound to DQ2.5-glia-α1a (2.0 Å) and DQ2.5-glia-ω1 (2.6 Å). These structures disclosed that differences around the p7 residue subtly alter the neighboring substructure and electrostatic properties of the HLA-DQ2.5–peptide complex, providing the fine specificity underlying the responses against these two highly homologous gluten epitopes. This study underscores the ability of TCRs to recognize subtle differences in the peptide–HLA-II landscape in a human disease setting

Peptide-MHC class I and class II tetramers: From flow to mass cytometry

To develop better vaccines and more targeted treatments for cancer and autoimmune disorders, the disease‐specific T cells and their cognate antigens need to be better characterized. For more than two decades, peptide‐major histocompatibility complex (pMHC) tetramers and flow cytometry have been the gold standard for detection of CD8+ and CD4+ T cells specific to antigens in the context of MHC class I and class II, respectively. Nonetheless, more recent studies combining such reagents with mass cytometry, that is, cytometry by time of flight (CyTOF), have offered far more comprehensive profiling of antigen‐specific T‐cell responses. In addition, mass cytometry has enabled ex vivo screening of CD8+ T‐cell reactivities against hundreds of MHC class I restricted candidate epitopes. MHC class II molecules, on the other hand, have been challenging to combine with mass cytometry as they are more complex and bind with lower affinities to cognate T‐cell receptors than MHC class I molecules. In this review, I discuss how techniques originally developed to improve the staining capacity of pMHC tetramers in flow cytometry led to the successful combination of such reagents with mass cytometry. Especially, I will highlight very recent advances facilitating the combination with pMHC class II tetramers. Together, these mass cytometry‐based studies can help develop more targeted treatments for cancer and autoimmune disorders

Therapeutic and Diagnostic Implications of T Cell Scarring in Celiac Disease and Beyond

Few therapeutic and diagnostic tools specifically aim at T cells in autoimmune disorders, but are T cells a narrow target in these diseases? Lessons may be learned from celiac disease (CeD), one of the few autoimmune disorders where the T cell driving antigens are known, i.e. dietary gluten proteins. T cell clonotypes specific to gluten are expanded, persist for decades and express a distinct phenotype in CeD patients. Cells with this phenotype are increased also in other autoimmune conditions. Accordingly, disease-specific CD4+ T cells form an immunological scar in CeD and probably other autoimmune disorders. We discuss approaches how such T cells may be targeted for better treatment and diagnosis via their antigen specificity or via their expression of characteristic phenotypic markers

Phenotype-Based Isolation of Antigen-Specific CD4+ T Cells in Autoimmunity: A Study of Celiac Disease

The pathogenic immune response in celiac disease (CeD) is orchestrated by phenotypically distinct CD4+ T cells that recognize gluten epitopes in the context of disease-associated HLA-DQ allotypes. Cells with the same distinct phenotype, but with elusive specificities, are increased across multiple autoimmune conditions. Here, whether sorting of T cells based on their distinct phenotype (Tphe cells) yields gluten-reactive cells in CeD is tested. The method′s efficiency is benchmarked by parallel isolation of gluten-reactive T cells (Ttet cells), using HLA-DQ:gluten peptide tetramers. From gut biopsies of 12 untreated HLA-DQ2.5+ CeD patients, Ttet+/Tphe+, Ttet−/Tphe+, and Ttet−/Tphe− cells are sorted for single-cell T-cell receptor (TCR)-sequencing (n = 8) and T-cell clone (TCC)-generation (n = 5). The generated TCCs are TCR sequenced and tested for their reactivity against deamidated gluten. Gluten-reactivity is observed in 91.2% of Ttet+/Tphe+ TCCs, 65.3% of Ttet−/Tphe+ TCCs and 0% of Ttet−/Tphe− TCCs. TCR sequencing reveals clonal expansion and sequence sharing across patients, features reflecting antigen-driven responses. The feasibility to isolate antigen-specific CD4+ T cells by the sole use of phenotypic markers in CeD outlines a potential avenue for characterizing disease-driving CD4+ T cells in autoimmune conditions

HLA-DQ-Gluten Tetramer Blood Test Accurately Identifies Patients With and Without Celiac Disease in Absence of Gluten Consumption

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

Single-cell TCR sequencing of gut intraepithelial γδ T cells reveals a vast and diverse repertoire in celiac disease

A hallmark of celiac disease (CeD), a chronic condition driven by cereal gluten exposure, is increase of gut intraepithelial γδ T cells. This may indicate pathogenic involvement of γδ T cells and existence of disease-specific γδ T-cell receptors (TCRs) recognizing defined antigen(s). We performed high-throughput and paired γδ TCR sequencing of single intraepithelial γδ T cells of untreated CeD patients (n = 8; 1821 cells), CeD patients treated with a gluten-free diet (n = 5; 436 cells) and controls (n = 7; 1068 cells). We found that CeD patients, both untreated and treated, had larger and more diverse γδ TCR repertoires, more frequent usage of TRDV1 and TRDV3 and different patterns of TCRγ/TCRδ-pairing compared with controls. Although we observed no public CDR3δ sequences, there were several public CDR3γ sequences—many of which were shared by not only the CeD patients, but also by the controls. These public CDR3s were characterized by few N/P nucleotide insertions with germline and near-germline configuration, hence being easy to generate. Previous findings of CeD-specific CDR3 motifs were not replicated. Thus, being unable to raise evidence for CeD-specific γδ TCRs in this first large, paired γδ TCR single-cell sequencing study, we project challenges for identification of CeD-relevant γδ TCR ligands

Generation of circulating autoreactive pre-plasma cells fueled by naive B cells in celiac disease

Summary: Autoantibodies against the enzyme transglutaminase 2 (TG2) are characteristic of celiac disease (CeD), and TG2-specific immunoglobulin (Ig) A plasma cells are abundant in gut biopsies of patients. Here, we describe the corresponding population of autoreactive B cells in blood. Circulating TG2-specific IgA cells are present in untreated patients on a gluten-containing diet but not in controls. They are clonally related to TG2-specific small intestinal plasma cells, and they express gut-homing molecules, indicating that they are plasma cell precursors. Unlike other IgA-switched cells, the TG2-specific cells are negative for CD27, placing them in the double-negative (IgD−CD27−) category. They have a plasmablast or activated memory B cell phenotype, and they harbor fewer variable region mutations than other IgA cells. Based on their similarity to naive B cells, we propose that autoreactive IgA cells in CeD are generated mainly through chronic recruitment of naive B cells via an extrafollicular response involving gluten-specific CD4+ T cells

TCR sequencing of single cells reactive to DQ2.5-glia-α2 and DQ2.5-glia-ω2 reveals clonal expansion and epitope-specific V-gene usage

CD4+ T cells recognizing dietary gluten epitopes in the context of disease-associated human leukocyte antigen (HLA)-DQ2 or HLA-DQ8 molecules are the key players in celiac disease pathogenesis. Here, we conducted a large-scale single-cell paired T-cell receptor (TCR) sequencing study to characterize the TCR repertoire for two homologous immunodominant gluten epitopes, DQ2.5-glia-α2 and DQ2.5-glia-ω2, in blood of celiac disease patients after oral gluten challenge. Despite sequence similarity of the epitopes, the TCR repertoires are unique but shared several overall features. We demonstrate that clonally expanded T cells dominate the T-cell responses to both epitopes. Moreover, we find V-gene bias of TRAV26, TRAV4, and TRBV7 in DQ2.5-glia-α2 reactive TCRs, while DQ2.5-glia-ω2 TCRs displayed significant bias toward TRAV4 and TRBV4. The knowledge that antigen-specific TCR repertoire in chronic inflammatory diseases tends to be dominated by a few expanded clones that use the same TCR V-gene segments across patients is important information for HLA-associated diseases where the antigen is unknown

Circulating CD103+ γδ and CD8+ T cells are clonally shared with tissue-resident intraepithelial lymphocytes in celiac disease

Gut intraepithelial γδ and CD8+ αβ T lymphocytes have been connected to celiac disease (CeD) pathogenesis. Based on the previous observation that activated (CD38+), gut-homing (CD103+) γδ and CD8+ αβ T cells increase in blood upon oral gluten challenge, we wanted to shed light on the pathogenic involvement of these T cells by examining the clonal relationship between cells of blood and gut during gluten exposure. Of 20 gluten-challenged CeD patients, 8 and 10 had increase in (CD38+CD103+) γδ and CD8+ αβ T cells, respectively, while 16 had increase in gluten-specific CD4+ T cells. We obtained γδ and αβ TCR sequences of >2500 single cells from blood and gut of 5 patients, before and during challenge. We observed extensive sharing between blood and gut γδ and CD8+ αβ T-cell clonotypes even prior to gluten challenge. In subjects with challenge-induced surge of γδ and/or CD8+ αβ T cells, as larger populations of cells analyzed, we observed more expanded clonotypes and clonal sharing, yet no discernible TCR similarities between expanded and/or shared clonotypes. Thus, CD4+ T cells appear to drive expansion of clonally diverse γδ or CD8+ αβ T-cell clonotypes that may not be specific for the gluten antigen