Gluten-Free Diet Induces Rapid Changes in Phenotype and Survival Properties of Gluten-Specific T Cells in Celiac Disease.

BACKGROUND & AIMS: The treatment of celiac disease (CeD) with gluten-free diet (GFD) normalizes gut inflammation and disease-specific antibodies. CeD patients have HLA-restricted, gluten-specific T cells persisting in the blood and gut even after decades of GFD, which are reactivated and disease driving upon gluten exposure. Our aim was to examine the transition of activated gluten-specific T cells into a pool of persisting memory T cells concurrent with normalization of clinically relevant biomarkers during the first year of treatment. METHODS: We followed 17 CeD patients during their initial GFD year, leading to disease remission. We assessed activation and frequency of gluten-specific CD4+ blood and gut T cells with HLA-DQ2.5:gluten tetramers and flow cytometry, disease-specific serology, histology, and symptom scores. We assessed gluten-specific blood T cells within the first 3 weeks of GFD in 6 patients and serology in an additional 9 patients. RESULTS: Gluten-specific CD4+ T cells peaked in blood at day 14 while up-regulating Bcl-2 and down-regulating Ki-67 and then decreased in frequency within 10 weeks of GFD. CD38, ICOS, HLA-DR, and Ki-67 decreased in gluten-specific cells within 3 days. PD-1, CD39, and OX40 expression persisted even after 12 months. IgA-transglutaminase 2 decreased significantly within 4 weeks. CONCLUSIONS: GFD induces rapid changes in the phenotype and number of gluten-specific CD4+ blood T cells, including a peak of nonproliferating, nonapoptotic cells at day 14. Subsequent alterations in T-cell phenotype associate with the quiescent but chronic nature of treated CeD. The rapid changes affecting gluten-specific T cells and disease-specific antibodies offer opportunities for clinical trials aiming at developing nondietary treatments for patients with newly diagnosed CeD.

Systematic characterization of immunoglobulin loci and deep sequencing of the expressed repertoire in the Atlantic cod (Gadus morhua).

BACKGROUND: The Atlantic cod is a prolific species in the Atlantic, despite its inconsistent specific antibody response. It presents a peculiar case within vertebrate immunology due to its distinct immune system, characterized by the absence of MHCII antigen presentation pathway, required for T cell-dependent antibody responses. Thorough characterisation of immunoglobulin loci and analysis of the antibody repertoire is necessary to further our understanding of the Atlantic cod's immune response on a molecular level. RESULTS: A comprehensive search of the cod genome (gadmor3.0) identified the complete set of IgH genes organized into three sequential translocons on chromosome 2, while IgL genes were located on chromosomes 2 and 5. The Atlantic cod displayed a moderate germline V gene diversity, comprising four V gene families for both IgH and IgL, each with distinct chromosomal locations and organizational structures. 5'RACE sequencing revealed a diverse range of heavy chain CDR3 sequences and relatively limited CDR3 diversity in light chains. The analysis highlighted a differential impact of V-gene germline CDR3 length on receptor CDR3 length between heavy and light chains, underlining different recombination processes. CONCLUSIONS: This study reveals that the Atlantic cod, despite its inconsistent antibody response, maintains a level of immunoglobulin diversity comparable to other fish species. The findings suggest that the extensive recent duplications of kappa light chain genes do not result in increased repertoire diversity. This research provides a comprehensive view of the Atlantic cod's immunoglobulin gene organization and repertoire, necessary for future studies of antibody responses at the molecular level.

TCRpower: quantifying the detection power of T-cell receptor sequencing with a novel computational pipeline calibrated by spike-in sequences.

T-cell receptor (TCR) sequencing has enabled the development of innovative diagnostic tests for cancers, autoimmune diseases and other applications. However, the rarity of many T-cell clonotypes presents a detection challenge, which may lead to misdiagnosis if diagnostically relevant TCRs remain undetected. To address this issue, we developed TCRpower, a novel computational pipeline for quantifying the statistical detection power of TCR sequencing methods. TCRpower calculates the probability of detecting a TCR sequence as a function of several key parameters: in-vivo TCR frequency, T-cell sample count, read sequencing depth and read cutoff. To calibrate TCRpower, we selected unique TCRs of 45 T-cell clones (TCCs) as spike-in TCRs. We sequenced the spike-in TCRs from TCCs, together with TCRs from peripheral blood, using a 5' RACE protocol. The 45 spike-in TCRs covered a wide range of sample frequencies, ranging from 5 per 100 to 1 per 1 million. The resulting spike-in TCR read counts and ground truth frequencies allowed us to calibrate TCRpower. In our TCR sequencing data, we observed a consistent linear relationship between sample and sequencing read frequencies. We were also able to reliably detect spike-in TCRs with frequencies as low as one per million. By implementing an optimized read cutoff, we eliminated most of the falsely detected sequences in our data (TCR α-chain 99.0% and TCR ß-chain 92.4%), thereby improving diagnostic specificity. TCRpower is publicly available and can be used to optimize future TCR sequencing experiments, and thereby enable reliable detection of disease-relevant TCRs for diagnostic applications.

Overcoming Thermal Quenching in X-ray Scintillators through Multi-Excited State Switching.

X-ray scintillators have gained significant attention in medical diagnostics and industrial applications. Despite their widespread utility, scintillator development faces a significant hurdle when exposed to elevated temperatures, as it usually results in reduced scintillation efficiency and diminished luminescence output. Here we report a molecular design strategy based on a hybrid perovskite (TpyBiCl5) that overcomes thermal quenching through multi-excited state switching. The structure of perovskite provides a platform to modulate the luminescence centers. The rigid framework constructed by this perovskite structure stabilized its triplet states, resulting in TpyBiCl5 exhibiting an approximately 12 times higher (45 % vs. 3.8 %) photoluminescence quantum yield of room temperature phosphorescence than that of its organic ligand (Tpy). Most importantly, the interactions between the components of this perovskite enable the mixing of different excited states, which has been revealed by experimental and theoretical investigations. The TpyBiCl5 scintillator exhibits a detection limit of 38.92 nGy s-1 at 213 K and a detection limit of 196.31 nGy s-1 at 353 K through scintillation mode switching between thermally activated delayed fluorescence and phosphorescence. This work opens up the possibility of solving the thermal quenching in X-ray scintillators by tuning different excited states.

Three-Component Donor-π-Acceptor Covalent-Organic Frameworks for Boosting Photocatalytic Hydrogen Evolution.

Two-dimensional covalent-organic frameworks (2D COFs) have recently emerged as great prospects for their applications as new photocatalytic platforms in solar-to-hydrogen conversion; nevertheless, their inefficient solar energy capture and fast charge recombination hinder the improvement of photocatalytic hydrogen production performance. Herein, two photoactive three-component donor-π-acceptor (TCDA) materials were constructed using a multicomponent synthesis strategy by introducing electron-deficient triazine and electron-rich benzotrithiophene moieties into frameworks through sp2 carbon and imine linkages, respectively. Compared with two-component COFs, the novel TCDA-COFs are more convenient in regulating the inherent photophysical properties, thereby realizing outstanding photocatalytic activity for hydrogen evolution from water. Remarkably, the first sp2 carbon-linked TCDA-COF displays an impressive hydrogen evolution rate of 70.8 ± 1.9 mmol g-1 h-1 with excellent reusability in the presence of 1 wt % Pt under visible-light illumination (420-780 nm). Utilizing the combination of diversified spectroscopy and theoretical prediction, we show that the full π-conjugated linkage not only effectively broadens the visible-light harvesting of COFs but also enhances charge transfer and separation efficiency.

Innate lymphoid cell characterization in the rat and their correlation to gut commensal microbes.

Innate lymphoid cells (ILCs) are important for tissue immune homeostasis, and are thoroughly characterized in mice and humans. Here, we have performed in-depth characterization of rat ILCs. Rat ILCs were identified based on differential expression of transcription factors and lack of lineage markers. ILC3s represented the major ILC population of the small intestine, while ILC2s were infrequent but most prominent in liver and adipose tissue. Two major subsets of group 1 ILCs were defined. Lineage- T-bet+ Eomes+ cells were identified as conventional NK cells, while lineage- T-bet+ Eomes- cells were identified as the probable rat counterpart of ILC1s based on their selective expression of the ILC marker CD200R. Rat ILC1s were particularly abundant in liver and intestinal tissues, and were functionally similar to NK cells. Single-cell transcriptomics of spleen and liver cells confirmed the main division of NK cells and ILC1-like cells, and demonstrated Granzyme A as an additional ILC1 marker. We further report differential distributions of NK cells and ILCs along the small and large intestines, and the association of certain bacterial taxa to frequencies of ILCs. In conclusion, we provide a framework for future studies of ILCs in diverse rat experimental models, and novel data on the potential interplay between commensals and intestinal ILCs.

Stereotyped B-cell responses are linked to IgG constant region polymorphisms in multiple sclerosis.

Clonally related B cells infiltrate the brain, meninges, and cerebrospinal fluid of MS patients, but the mechanisms driving the B-cell response and shaping the immunoglobulin repertoires remain unclear. Here, we used single-cell full-length RNA-seq and BCR reconstruction to simultaneously assess the phenotypes, isotypes, constant region polymorphisms, and the paired heavy- and light-chain repertoires in intrathecal B cells. We detected extensive clonal connections between the memory B cell and antibody-secreting cell (ASC) compartments and observed clonally related cells of different isotypes including IgM/IgG1, IgG1/IgA1, IgG1/IgG2, and IgM/IgA1. There was a strong dominance of the G1m1 allotype constant region polymorphisms in ASCs, but not in memory B cells. Tightly linked to the G1m1 allotype, we found a preferential pairing of the immunoglobulin heavy-chain variable (IGHV)4 gene family with the κ variable (IGKV)1 gene family. The IGHV4-39 gene was most used and showed the highest frequency of pairing with IGKV1-5 and IGKV1(D)-33. These results link IgG constant region polymorphisms to stereotyped B-cell responses in MS and indicate that the intrathecal B-cell response in these patients could be directed against structurally similar epitopes.

Investigations on incidence and relevant factors of allergies in 5725 urban pregnant women: a cohort study in China.

BACKGROUND: Allergic diseases are highly prevalent in the women of childbearing age. As we know, the immune system could change when pregnancy, which may affect the course of allergic diseases. Meanwhile, they also can affect the course and outcome of pregnancy. The data on incidence of allergies during pregnancy is lacking and conducting clinical trials in pregnant women was limited, therefore, we observed a prebirth cohort to supplement the relevant data and strengthen concerned research conductions. OBJECTIVE: We aim to obtain the incidence of allergies in urban pregnancy and explore the relevant factors of allergic diseases in urban pregnancy. METHODS: We design a multicenter and prospective cohort in 20 institutions above municipal level which were eligible according to the study design from 14 provinces covering all-side of China. This cohort was conducted from 13+6 weeks of gestation to 12 months postpartum and in our study, we chose the prenatal part to analyze. The outcome was developing allergies during pregnancy, which were diagnosed by clinicians according to the uniform criterion from National Health Commission. All the data was collected by electronic questionnaires through tablet computers. RESULTS: The incidence of allergic diseases in urban pregnant women was 21.0% (95%CI 20.0% ~ 22.0%). From social demography data, the history of allergies of pregnant women and their parents had statistical significance(p < 0.01); For exposure to living or working environment, house decoration for less than half a year, exposure to plush toys, disinfectants, insecticides, antihistamines, glucocorticoids, antipyretic analgesics, tocolytic agent and probiotics had statistical significance (all p < 0.05); For psychological status, self-rated depression and anxiety had statistical significance (p = 0.026;p = 0.006). CONCLUSION: The incidence of allergic diseases in urban pregnant women was similar to the former study and kept a medium-high level. The history of allergies of pregnant women and their parents, house decoration time, exposure to plush toys, disinfectants, insecticides, antihistamines, glucocorticoids, antipyretic analgesics, tocolytic agents, probiotics, self-rated depression, and anxiety were relevant factors of allergic diseases during pregnancy.

A molecular basis for the T cell response in HLA-DQ2.2 mediated celiac disease.

The highly homologous human leukocyte antigen (HLA)-DQ2 molecules, HLA-DQ2.5 and HLA-DQ2.2, are implicated in the pathogenesis of celiac disease (CeD) by presenting gluten peptides to CD4+ T cells. However, while HLA-DQ2.5 is strongly associated with disease, HLA-DQ2.2 is not, and the molecular basis underpinning this differential disease association is unresolved. We here provide structural evidence for how the single polymorphic residue (HLA-DQ2.5-Tyr22α and HLA-DQ2.2-Phe22α) accounts for HLA-DQ2.2 additionally requiring gluten epitopes possessing a serine at the P3 position of the peptide. In marked contrast to the biased T cell receptor (TCR) usage associated with HLA-DQ2.5-mediated CeD, we demonstrate with extensive single-cell sequencing that a diverse TCR repertoire enables recognition of the immunodominant HLA-DQ2.2-glut-L1 epitope. The crystal structure of two CeD patient-derived TCR in complex with HLA-DQ2.2 and DQ2.2-glut-L1 (PFSEQEQPV) revealed a docking strategy, and associated interatomic contacts, which was notably distinct from the structures of the TCR:HLA-DQ2.5:gliadin epitope complexes. Accordingly, while the molecular surfaces of the antigen-binding clefts of HLA-DQ2.5 and HLA-DQ2.2 are very similar, differences in the nature of the peptides presented translates to differences in responding T cell repertoires and the nature of engagement of the respective antigen-presenting molecules, which ultimately is associated with differing disease penetrance.

Characterization of T-cell receptor transgenic mice recognizing immunodominant HLA-DQ2.5-restricted gluten epitopes.

We created a TCR transgenic mouse with CD4+ T cells recognizing the immunodominant DQ2.5-glia-ω2 gluten epitope. We show that these cells respond to deamidated gluten feed in vivo and compare them to previously published α2- and γ1-specific mice. These mice may help enlighten key aspects of celiac disease pathogenesis.

Neutral Antiaromatic Bis(1,2-dithiolene)-Chelated Nickel Complexes Bearing Multiradical Characters.

Metal-organic complexes with radical characteristics are unique species attracting immense attention in recent years due to their peculiar properties and promising applicability in a wide variety of innovative research fields. However, the reported complexes typically do not exceed diradicality. This study systematically investigates a series of square planar neutral Ni-bis(1,2-dithiolene) and Ni-bis(1,2-dioxolene) complexes with linear, branched, and macrocyclic configurations via ab initio calculations. The linear Ni-complexes display strong singlet diradical characters, while their branched counterparts can also exhibit moderate singlet multiradical characters. Importantly, the macrocyclic Ni-complexes can possess extremely strong singlet multiradical characters up to dodeca-radicality along with their global antiaromaticity and hence strong induced ring current in the presence of an external magnetic field, ascribed to the localization of unpaired α and ß electrons residing in the highest few molecular orbitals at different molecular sites, minimizing their coupling and annihilation. Our work represents the first indication in the rational design of novel multiradical neutral antiaromatic macrocyclic complexes for potential applications in molecular machines and electronic devices.

Sevoflurane ameliorates LPS-induced inflammatory injury of HK-2 cells through Sirtuin1/NF-κB pathway.

The anesthetic sevoflurane (SEV) has been shown to protect against organ's injury during sepsis. The present study intended to uncover the protective effects of SEV on sepsis-induced acute kidney injury (SI-AKI) and its possible mechanism. Human renal tubular epithelial cell HK-2 was treated with 10 µg/mL lipopolysaccharide (LPS) to construct SI-AKI cell model. LPS-induced HK-2 cells were pretreated with SEV in the absence or presence of EX527, an inhibitor of Sirtuin 1 (SIRT1), after which were the detection of cell viability, lactate dehydrogenase (LDH) release, apoptosis, inflammation, and oxidative stress. Our results demonstrated that LPS caused decreased cell viability, increased LDH release, improved cell apoptosis along with decreased expression of Bcl2 and enhanced expressions of Bax, cleaved PARP and cleaved caspase, enhanced production, and protein expressions of TNF-α, IL-6, and IL-1ß, increased generation of reactive oxygen species (ROS) and malondialdehyde (MDA), but contributed to declined activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). LPS inhibited SIRT1 and IκBα expressions but up-regulated p-NF-κB p65 and acetyl-p53 expressions as well. However, SEV pretreatment abolished all above-mentioned effects of LPS on HK-2 cells, while EX527 significantly reversed the effects of SEV. In conclusion, SEV effectively protected HK-2 cells against LPS-induced apoptosis, inflammation, and oxidative stress, and these effects may depend on the increase of SIRT1 expression, thereby inactivating NF-κB signaling.

Photocatalytic Hydrogen Production on a sp2 -Carbon-Linked Covalent Organic Framework.

Two-dimensional covalent organic frameworks (2D-COFs) have emerged as attractive platforms for solar-to-chemical energy conversion. In this study, we have implemented a gradient heating strategy to synthesize a sp2 -carbon-linked triazine-based COF, COF-JLU100, exhibiting high crystallinity, large surface area, good durability and carrier mobility for solar-driven photocatalytic hydrogen evolution. The Pt-doped COF-JLU100 demonstrated a high hydrogen evolution rate of over 100 000 µmol g-1 h-1 for water splitting under visible-light illumination (λ>420 nm). Experimental and theoretical studies corroborate that the cyano-vinylene segments in COF-JLU100 extend the π-delocalization and enable fast charge transfer and separation rates as well as good dispersion in water. Moreover, COF-JLU100 can be prepared by low-cost and easily available monomers and has excellent stability, which is desirable for practical solar-driven hydrogen production.

T cell receptor repertoire as a potential diagnostic marker for celiac disease.

An individual's T cell repertoire is skewed towards some specificities as a result of past antigen exposure and subsequent clonal expansion. Identifying T cell receptor signatures associated with a disease is challenging due to the overall complexity of antigens and polymorphic HLA allotypes. In celiac disease, the antigen epitopes are well characterised and the specific HLA-DQ2-restricted T-cell repertoire associated with the disease has been explored in depth. By investigating T cell receptor repertoires of unsorted lamina propria T cells from 15 individuals, we provide the first proof-of-concept study showing that it could be possible to infer disease state by matching against a priori known disease-associated T cell receptor sequences.

Podocyte RNF166 deficiency alleviates diabetic nephropathy by mitigating mitochondria impairment and apoptosis via regulation of CYLD signal.

Diabetic nephropathy (DN) is a major cause of renal failure in diabetic patients. RING-finger protein 166 (RNF166), composed of an N-terminal RING domain and C-terminal ubiquitin interaction motif, plays a critical role in mediating various cellular processes. However, its potential in DN has not been investigated. In the present study, we found that DN patients exhibited significantly increased expression of RNF166 in renal tissues compared with the normal individuals, and abundant RNF166 was detected in podocytes. We then showed that podocyte-conditional RNF166 knockout (RNF166cKO) markedly reduced blood glucose levels and ameliorated renal dysfunction in streptozotocin (STZ)-induced diabetic mice. Additionally, abnormal histological changes and podocyte injury were observed in STZ-induced diabetic mice, while being markedly ameliorated by RNF166cKO. Furthermore, podocyte-specific RNF166 deficiency considerably mitigated apoptosis and mitochondrial impairments in glomeruli podocytes of STZ-challenged mice through suppressing Caspase-3 cleavage and improving mitochondrial fission-associated molecules. In vitro studies further confirmed that high glucose (HG) induced mitochondrial dysfunction, along with enhanced releases of Cyto-c from mitochondria and elevated expression of cleaved Caspase-9, contributing to intrinsic apoptosis in podocytes. Intriguingly, these effects triggered by HG were dramatically ameliorated by RNF166 knockout. Mechanistically, we demonstrated that RNF166 directly interacted with cylindromatosis (CYLD), and negatively regulated CYLD expression. Notably, RNF166 knockout-attenuated mitochondrial damage and apoptosis were mainly through CYLD in podocytes upon HG stimulation. Together, all these findings provided new insights into the novel effects of RNF166 on maintaining mitochondrial function and apoptosis in podocytes during DN progression both in vivo and in vitro through interacting with CYLD, indicating that RNF166/CYLD may be an innovative therapeutic target for developing effective strategy against DN development.

Immunobiology and conflicting roles of the human CD161 receptor in T cells.

Human C-type lectin-like CD161 is a type-II transmembrane protein expressed on the surface of various lymphocytes across innate and adaptive immune systems. CD161+ T cells displayed enhanced ability to produce cytokines and were shown to be enriched in the gut. Independently of function, CD161 was used as marker of innate-like T cells and marker of IL-17-producing cells. The function of CD161 is still not fully understood. In T cells, CD161 was proposed to act as co-signalling receptor that influence T-cell receptor-dependent responses. However, conflicting studies were published demonstrating lack of agreement over the role of CD161 during T-cell activation. In this review, we outline phenotypical and functional consequences of CD161 expression in T cells. We provide critical discussion over the most pressing issues including in depth evaluation of the literature concerning CD161 putative co-signalling properties.

C-type lectin-like CD161 is not a co-signalling receptor in gluten-reactive CD4 + T cells.

C-type lectin-like CD161, a class II transmembrane protein, is a surface receptor expressed by NK cells and T cells. In coeliac disease, CD161 was expressed more frequently on gluten-reactive CD4 + T cells compared to other memory CD4 + T cells isolated from the same tissue compartment. CD161 is a putative co-signalling molecule that was proposed to act as co-stimulatory receptor in the context of signalling through TCR, but contradicting results were published. In order to understand the role of CD161 in gluten-reactive CD4 + T cells, we combined T cell stimulation assays or T cell proliferation assays with ligation of CD161 and intracellular cytokine staining. We found that CD161 ligation provided neither co-stimulatory nor co-inhibitory signals to modulate proliferation and IFN-γ or IL-21 production by gluten-reactive CD4 + T cell clones. Thus, we suggest that CD161 does not function as a co-signalling receptor in the context of gluten-reactive CD4 + T cells.

Epitope Selection for HLA-DQ2 Presentation: Implications for Celiac Disease and Viral Defense.

We have reported that the major histocompatibility molecule HLA-DQ2 (DQA1*05:01/DQB1*02:01) (DQ2) is relatively resistant to HLA-DM (DM), a peptide exchange catalyst for MHC class II. In this study, we analyzed the role of DQ2/DM interaction in the generation of DQ2-restricted gliadin epitopes, relevant to celiac disease, or DQ2-restricted viral epitopes, relevant to host defense. We used paired human APC, differing in DM expression (DMnull versus DMhigh) or differing by expression of wild-type DQ2, versus a DM-susceptible, DQ2 point mutant DQ2α+53G. The APC pairs were compared for their ability to stimulate human CD4+ T cell clones. Despite higher DQ2 levels, DMhigh APC attenuated T cell responses compared with DMnull APC after intracellular generation of four tested gliadin epitopes. DMhigh APC expressing the DQ2α+53G mutant further suppressed these gliadin-mediated responses. The gliadin epitopes were found to have moderate affinity for DQ2, and even lower affinity for the DQ2 mutant, consistent with DM suppression of their presentation. In contrast, DMhigh APC significantly promoted the presentation of DQ2-restricted epitopes derived intracellularly from inactivated HSV type 2, influenza hemagglutinin, and human papillomavirus E7 protein. When extracellular peptide epitopes were used as Ag, the DQ2 surface levels and peptide affinity were the major regulators of T cell responses. The differential effect of DM on stimulation of the two groups of T cell clones implies differences in DQ2 presentation pathways associated with nonpathogen- and pathogen-derived Ags in vivo.

Boosting Electrocatalytic Activity of 3d-Block Metal (Hydro)oxides by Ligand-Induced Conversion.

The 3d-transition-metal (hydro)oxides belong to a group of highly efficient, scalable and inexpensive electrocatalysts for widespread energy-related applications that feature easily tailorable crystal and electronic structures. We propose a general strategy to further boost their electrocatalytic activities by introducing organic ligands into the framework, considering that most 3d-metal (hydro)oxides usually exhibit quite strong binding with reaction intermediates and thus compromised activity due to the scaling relations. Involving weakly bonded ligands downshifts the d-band center, which narrows the band gap, and optimizes the adsorption of these intermediates. For example, the activity of the oxygen evolution reaction (OER) can be greatly promoted by ≈5.7 times over a NiCo layered double hydroxide (LDH) after a terephthalic acid (TPA)-induced conversion process, arising from the reduced energy barrier of the deprotonation of OH* to O*. Impressively, the proposed ligand-induced conversion strategy is applicable to a series of 3d-block metal (hydro)oxides, including NiFe2 O4 , NiCo2 O4 , and NiZn LDH, providing a general structural upgrading scheme for existing high-performance electrocatalytic systems.

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 αß 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.