HLA-DQ Molecules Research Articles

Interchain disulfide engineering enables the efficient production of functional HLA-DQ-Fc fusion proteins

HLA-DQ molecules drive unwanted alloimmune responses after solid-organ transplants and several autoimmune diseases, including Type1 Diabetes and celiac disease. Biologics with HLA molecules as part of the design are emerging therapeutic options for these allo- and autoimmune conditions. However, the soluble α and β chains of class II HLA molecules do not dimerize efficiently without their transmembrane domains, which hinders their production. In this study, we examined the feasibility of inter-chain disulfide engineering by introducing paired cysteines to juxtaposed positions in the α and β chains of HLA-DQ7, encoded by HLA-DQA1*05:01 and HLA-DQB1*03:01 respectively. We identified three variant peptide-HLA-DQ7-Fc fusion proteins (DQ7Fc) with increased expression and production yield, namely Y19C-D6C (YCDC), A83C-E5C (ACEC), and A84C-N33C (ACNC). The mutated residues were conserved across all HLA-DQ proteins and had limited solvent exposure. Further characterizations of the YCDC variant showed that the expression of the fusion protein is peptide-dependent; inclusion of a higher-affinity peptide correlated with increased protein expression. However, high-affinity peptide alone was insufficient for stabilizing the DQ7 complex without the engineered disulfide bond. Multiple DQ7Fc variants demonstrated expected binding characteristics with commercial anti-DQ antibodies in two immunoassays and by a cell-based assay. Lastly, DQ7Fc variants demonstrated dose-dependent killing of DQ7-specific B cell hybridomas in a flow cytometric, complement-dependent cytotoxicity assay. These data support inter-chain disulfide engineering as a novel approach to efficiently producing functional HLA-DQ molecules and potentially other class II HLA molecules as candidate therapeutic agents.

A Tool for the Assessment of HLA-DQ Heterodimer Variation in Hematopoietic Cell Transplantation

BackgroundWhen optimizing transplants, clinical decision-makers consider HLA-A, -B, -C, -DRB1 (8 matched alleles out of 8), and sometimes HLA-DQB1 (10 out of 10) matching between the patient and donor. HLA-DQ is a heterodimer formed by the β chain product of HLA-DQB1 and an α chain product of HLA-DQA1. In addition to molecules defined by the parentally-inherited cis haplotypes, α-β trans-dimerization is possible between certain alleles, leading to unique molecules and a potential source of mismatched molecules. Recently, researchers uncovered that clinical outcome after HLA-DQB1-mismatched unrelated donor HCT depends on the total number of HLA-DQ molecule mismatches and the specific α-β heterodimer mismatch. ObjectiveOur objective in this study is to develop an automated tool for analyzing HLA-DQ heterodimer data and validating it through numerous datasets and analyses. By doing so, we provide an HLA-DQ heterodimer tool for DQα-DQβ trans-heterodimer evaluation, HLA-DQ imputation, and HLA-DQ-featured source selection to the transplant field. Study designIn our study, we leverage 352,148 high-confidence, statistically-phased (via a modified expectation-maximization algorithm) HLA-DRB1∼DQA1∼DQB1 haplotypes, 1,052 pedigree-phased HLA-DQA1∼DQB1 haplotypes, and 13,663 historical transplants to characterize HLA-DQ heterodimers data. ResultsUsing our developed QLASSy (HLA-DQA1 and HLA-DQB1 Heterodimers Assessment) tool, we first assessed the data quality of HLA-DQ heterodimers in our data for trans-dimers, missing HLA-DQA1 typing, and unexpected HLA-DQA1 and HLA-DQB1 combinations. Since trans-dimers enable up to four unique HLA-DQ molecules in individuals, we provide in-silico validations for 99.7% of 275 unique trans-dimers generated by 176,074 U.S. donors with HLA-DQA1 and HLA-DQB1 data. Many individuals lack HLA-DQA1 typing, so we developed and validated high-confidence HLA-DQ annotation imputation via HLA-DRB1 with >99% correct predictions in 23,698 individuals. A select few individuals displayed unexpected HLA-DQ combinations. We revisited the typing of 61 donors with unexpected HLA-DQ combinations based on their HLA-DQA1 and HLA-DQB1 typing and corrected 22 out of 61 (36%) cases of donors through data review or retyping and used imputation to resolve unexpected combinations. After verifying the data quality of our datasets, we analyzed our datasets further: we explored the frequencies of observed HLA-DQ combinations to compare HLA-DQ across populations (for instance, we found more high-risk molecules in Asian/Pacific Islander and Black/African American populations), demonstrated the effect of HLA-DQA1 and HLA-DQB1 mismatching on HLA-DQ molecular mismatches, and highlighted where donor selections could be improved at the time of search for historical transplants with this new HLA-DQ information (where 51.9% of G2-mismatched transplants had lower-risk, G2-matched alternatives). ConclusionWe encapsulated our findings into a tool that imputes missing HLA-DQA1 as needed, annotates HLA-DQ (mis)matches, and highlights other important HLA-DQ data to consider for the present and future. Altogether, these valuable datasets, analyses, and a culminating tool serve as actionable resources to enhance donor selection and improve patient outcomes.

A microbiota derived metabolite and pediatric celiac disease pathogenesis

Abstract Celiac Disease (CD) is an autoimmune condition triggered by gluten ingestion and defined by small intestine inflammation leading to villous atrophy in genetically predisposed individuals. 20-40% of the general population possess the predisposing HLA-DQ2 or HLA-DQ8 alleles, however only 1-2% of the population develops CD, indicating that there are additional environmental factors at play in the pathogenesis of the disease. In a prospective cohort study using fecal and plasma samples from children at ages 2.5 and 5 years, before the onset of celiac disease (CD progressors), we found that CD progressors have an altered gut microbiome, plasma metabolome and cytokine profile years before diagnosis. IgA sequencing revealed an increased IgA response and unique microbial targets of IgA. One metabolite increased in CD progressors is taurodeoxycholic acid (TDCA), a secondary bile acid produced by gut microbes. Treating C57BL/B6 mice with TDCA caused villous atrophy as well as upregulated CD4+T-cells in intraepithelial lymphocytes and natural killer cells in the lamina propria (LP). TDCA increased the expression of Qa-1 and NKG2D on T-cells. The DQ8-Dd-villin-IL-15tg mouse model of CD possesses the predisposing HLA-DQ8 molecule and overexpresses IL-15 in the LP. Treating these mice with TDCA caused the villi/crypt ratio to decrease in the treatment group of pooled villi. Our results indicate that TDCA and TDCA-producing bacteria could potentially contribute to the pathogenesis of CD.

HLA-DQB1*05 subtypes and not DRB1*10:01 mediates risk in anti-IgLON5 disease.

Anti-IgLON5 disease is a rare and likely underdiagnosed subtype of autoimmune encephalitis. The disease displays a heterogeneous phenotype that includes sleep, movement and bulbar-associated dysfunction. The presence of IgLON5-antibodies in CSF/serum, together with a strong association with HLA-DRB1*10:01∼DQB1*05:01, supports an autoimmune basis. In this study, a multicentric human leukocyte antigen (HLA) study of 87 anti-IgLON5 patients revealed a stronger association with HLA-DQ than HLA-DR. Specifically, we identified a predisposing rank-wise association with HLA-DQA1*01:05∼DQB1*05:01, HLA-DQA1*01:01∼DQB1*05:01 and HLA-DQA1*01:04∼DQB1*05:03 in 85% of patients. HLA sequences and binding cores for these three DQ heterodimers were similar, unlike those of linked DRB1 alleles, supporting a causal link to HLA-DQ. This association was further reflected in an increasingly later age of onset across each genotype group, with a delay of up to 11 years, while HLA-DQ-dosage dependent effects were also suggested by reduced risk in the presence of non-predisposing DQ1 alleles. The functional relevance of the observed HLA-DQ molecules was studied with competition binding assays. These proof-of-concept experiments revealed preferential binding of IgLON5 in a post-translationally modified, but not native, state to all three risk-associated HLA-DQ receptors. Further, a deamidated peptide from the Ig2-domain of IgLON5 activated T cells in two patients, compared with one control carrying HLA-DQA1*01:05∼DQB1*05:01. Taken together, these data support a HLA-DQ-mediated T-cell response to IgLON5 as a potentially key step in the initiation of autoimmunity in this disease.

Tolerance-inducing therapies in coeliac disease - mechanisms, progress and future directions.

Coeliac disease is an autoinflammatory condition caused by immune reactions to cereal gluten proteins. Currently, the only available treatment for the condition is a lifelong avoidance of gluten proteins in the diet. There is an unmet need for alternative therapies. Coeliac disease has a strong association with certain HLA-DQ allotypes (DQ2.5, DQ2.2 and DQ8), and these disease-associated HLA-DQ molecules present deamidated gluten peptides to gluten-specific CD4+ T cells. The gluten-specific CD4+ T cells are the drivers of the immune reactions leading to coeliac disease. Once established, the clonotypes of gluten-specific CD4+ T cells persist for decades, explaining why patients must adhere to a gluten-free diet for life. Given the key pathogenic role of gluten-specific CD4+ T cells, tolerance-inducing therapies that target these T cells are attractive for treatment of the disorder. Lessons learned from coeliac disease might provide clues for treatment of other HLA-associated diseases for which the disease-driving antigens are unknown. Thus, intensive efforts have been and are currently implemented to bring an effective tolerance-inducing therapy for coeliac disease. This Review discusses mechanisms of the various approaches taken, summarizing the progress made, and highlights future directions in this field.

Impact of HLA-DQ Heterodimer Group (G1/G2) Mismatch on Relapse and Survival Following Haploidentical Donor Transplantation

The role of HLA-DQ matching in transplant outcomes after post-transplant cyclophosphamide (PTCy)-based haploidentical donor transplantation (HIDT) remains unclear. DQB1 alleles can be grouped into group 1 (DQB1*02/03/04) or group 2 (DQB1*05/06) based on the ability of their corresponding β chains to form stable heterodimers with α chains of specific DQA1 alleles (Petersdorf et al. Blood 2022). For transplant recipients expressing a G1G1 or G2G2 genotype, additional HLA-DQ diversity is achieved through successful DQα-β transdimerization from the other parental chromosome, resulting in up to 4 unique HLA-DQ molecules. We hypothesized that an HLA-DQ group G1/G2 mismatch (mm) would reduce relapse and improve survival following PTCy-based HIDT, and that the effect may be more significant in G1G1 or G2G2 (vs. G1G2) recipients due to a higher number of heterodimer mismatches. A total of 242 consecutive patients with ALL (n=71), AML (n=125) or MDS (n=46) receiving a PTCy-based HIDT from a single institution were analyzed (median age 51 (19,80), 38% non-White, 32% DRI high/v. high). PBSC was the graft source in 87%, and conditioning was myeloablative in 57%. Recipient HLA-DQ group genotype was G1G2, G1G1, and G2G2 in 50%, 29% and 21%, respectively. Donor-recipient HLA-DQ alleles were G1/G2 group matched in 49% of all transplant recipients (49% and 48% in G1G2 and G1G1/G2G2 recipients, respectively). Baseline patient, disease and transplant characteristics were well matched in the HLA-DQ G1/G2 group matched vs. mm transplants. Median follow-up was 62 [23, 199] months. In univariate analysis, 5-year DFS was 56% vs. 43%, p=0.12 for HLA-DQ group mm vs. matched, respectively. Corresponding rates of relapse were 31% vs. 41%, p=0.18. As predicted, the protective effect of a group mm was stronger in recipients with a G1G1 or G2G2 (vs. G1G2) genotype (see figure). In G1G1/G2G2 recipients, 5-yr DFS (55% vs. 34%, p=0.017) and relapse (29% vs. 49%, p=0.038) was improved for HLA-DQ group mm vs. matched, respectively. In multivariable analysis, controlled for disease risk index, regimen intensity, donor age, and sex match, an HLA-DQ G1/G2 group mm was associated with significantly improved OS (HR 0.64, p=0.022), DFS (HR 0.65, p=0.020) and relapse risk (HR 0.60, p=0.021), an effect even more pronounced when restricted to G1G1/G2G2 recipients - OS (HR 0.48, p=0.006), DFS (HR 0.46, p=0.002) and relapse (HR 0.41, p=0.003). In contrast, there was no beneficial effect of an HLA-DQ group mm in transplant recipients with a G1G2 genotype. In summary, an HLA-DQ G1/G2 group mm significantly decreases relapse risk and improves survival in the half of HIDT recipients characterized by a G1G1 or G2G2 genotype. Our results may have considerable importance for donor selection in these patients. Furthermore, this finding reinforces the existing literature, suggesting that HLA-DQ matching follows a different paradigm than other class II antigens.

O30 Site-directed mutagenesis of HLA-DQ molecules reveals the amino acids crucial for binding to human monoclonal HLA-specific antibody

O30 Site-directed mutagenesis of HLA-DQ molecules reveals the amino acids crucial for binding to human monoclonal HLA-specific antibody

HLA DQ protein changes the cell surface distribution pattern of HLA proteins as monitored by Förster resonance energy transfer and high-resolution electron microscopy.

Peptide presentation by MHC class I and MHC class II molecules plays important roles in the regulation of the immune response. One factor in these displays is the density of antigen, which must exceed a critical threshold for the effective activation of T cells. Nonrandom distribution of MHC class I and class II has already been detected at the nanometer level and at higher hierarchical levels. It is not clear how the absence and reappearance of some protein molecules can influence the nonrandom distribution. Therefore, we performed experiments on HLA II-deficient bare lymphocyte syndrome (BLS1) cells: we created a stable transfected cell line, tDQ6-BLS-1, and were able to detect the effect of the appearance of HLA-DQ6 molecules on the homo and heteroassociation of different cell surface molecules by comparing Förster resonance energy transfer (FRET) efficiency on transfected cells to that on nontransfected BLS-1 and JY human B-cell lines. Our FRET results show a decrease in homoassociation FRET between HLA I chains in HLA-DQ6-transfected tDQ6-BLS-1 cells compared with the parent BLS-1 cell line and an increase in heteroassociation FRET between HLA I and HLA II (compared with JY cells), suggesting a similar pattern of antigen presentation by the HLA-DQ6 allele. Transmission electron microscopy (TEM) revealed that both HLA class I and class II molecules formed clusters at higher hierarchical levels on the tDQ6-BLS-1 cells, and the de novo synthesized HLA DQ molecules did not intersperse with HLA class I islands. These observations could be important in understanding the fine tuning of the immune response.

HLA-DQ antibodies in alloimmunity, what makes them different?

De novo HLA-DQ antibodies are the most frequently observed after solid-organ allotransplantation; and are associated with the worse adverse graft outcomes compared with all other HLA antibodies. However, the biological explanation for this observation is not yet known. Herein, we examine unique characteristics of alloimmunity directed specifically against HLA-DQ molecules. While investigators attempted to decipher functional properties of HLA class II antigens that may explain their immunogenicity and pathogenicity, most early studies focused on the more expressed molecule - HLA-DR. We here summarize up-to-date literature documenting specific features of HLA-DQ, as compared to other class II HLA antigens. Structural and cell-surface expression differences have been noted on various cell types. Some evidence suggests variations in antigen-presenting function and intracellular activation pathways after antigen/antibody interaction. The clinical effects of donor-recipient incompatibility at HLA-DQ, the risk of generating de novo antibodies leading to rejection, and the inferior graft outcomes indicate increased immunogenicity and pathogenicity that is unique to this HLA antigen. Clearly, knowledge generated for HLA-DR cannot be applied interchangeably. Deeper understanding of features unique to HLA-DQ may support the generation of targeted preventive-therapeutic strategies and ultimately improve solid-organ transplant outcomes.

Association of Genetic Variants of HLA-DQA1 with Bullous Pemphigoid Induced by Dipeptidyl Peptidase-4 Inhibitors

Bullous pemphigoid (BP) is the most common autoimmune blistering disorder. Several factors, including an antidiabetic (dipeptidyl peptidase-4 inhibitor [DPP-4i]), have been reported to trigger BP. To identify the genetic variants associated with BP, GWAS and HLA fine-mapping analyses were conducted. The 21 cases of noninflammatory BP induced by DPP-4i (i.e., DPP-4i-induced noninflammatory BP) and 737 controls (first cohort) and the 8 cases and 164 controls (second cohort) were included in the GWAS. Combining GWAS satisfied the genome-wide significant association of HLA-DQA1 (chromosome 6, rs3129763 [T/C]) with the risk of DPP-4i-induced noninflammatory BP (allele T carrier of 72.4% [21 of 29] in cases vs. 15.3% [138 of 901] in controls; dominant model, OR= 14, P= 1.8× 10-9). HLA fine mapping revealed that HLA-DQA1∗05 with serine at position 75 of HLA-DQα1 (Ser75) had the most significant association with the combined cohort of DPP-4i-induced noninflammatory BP (79.3% [23 of 29] cases vs. 16.1% [145 of 901] controls; dominant model, OR= 21, P= 2.0× 10-10). HLA-DQα1 Ser75 polymorphism was located inside the functional pocket of HLA-DQ molecules, suggesting the impact of HLA-DQα1 Ser75 on DPP-4i-induced noninflammatory BP.

Machine learning reveals limited contribution of trans-only encoded variants to the HLA-DQ immunopeptidome

Human leukocyte antigen (HLA) class II antigen presentation is key for controlling and triggering T cell immune responses. HLA-DQ molecules, which are believed to play a major role in autoimmune diseases, are heterodimers that can be formed as both cis and trans variants depending on whether the α- and β-chains are encoded on the same (cis) or opposite (trans) chromosomes. So far, limited progress has been made for predicting HLA-DQ antigen presentation. In addition, the contribution of trans-only variants (i.e. variants not observed in the population as cis) in shaping the HLA-DQ immunopeptidome remains largely unresolved. Here, we seek to address these issues by integrating state-of-the-art immunoinformatics data mining models with large volumes of high-quality HLA-DQ specific mass spectrometry immunopeptidomics data. The analysis demonstrates highly improved predictive power and molecular coverage for models trained including these novel HLA-DQ data. More importantly, investigating the role of trans-only HLA-DQ variants reveals a limited to no contribution to the overall HLA-DQ immunopeptidome. In conclusion, this study furthers our understanding of HLA-DQ specificities and casts light on the relative role of cis versus trans-only HLA-DQ variants in the HLA class II antigen presentation space. The developed method, NetMHCIIpan-4.2, is available at https://services.healthtech.dtu.dk/services/NetMHCIIpan-4.2.

Rank Wise Effect of HLA-DQ5 Explains Risk for the Development of Anti-IgLON5 Disease

Objective To better characterize the genetic association between human leukocyte antigen (HLA) and anti-IgLON5 disease and to explore auto-antigen binding to associated HLA molecules and their functional involvement in pathophysiology. Background Anti-IgLON5 disease is a rare, but likely underdiagnosed type of autoantibody encephalitis with a heterogeneous clinical phenotype, including sleep, movement and brainstem dysfunction. Its pathophysiology remains elusive, although dominant association with HLA-DRB1*10:01-DQB1*05:01 strongly supports an autoimmune basis. Design/Methods A multicentric cohort of 62 patients and 433 controls matched by principal component analysis was included. Genome-wide association analysis was performed with 4-digit resolution HLA imputation and selected 8-digit resolution validation typing. A generalized logistic model was used to determine the association with individual alleles and haplotype counts to establish haplotype associations. Furthermore, we computationally predicted binding of IgLON5-derived peptides to risk-associated HLA-molecules. Results Our results indicate a rank wise effect of HLA-DQA1*01:05∼DQB1*05:01 (heterozygotes: OR 46.6), HLA-DQA1*01:01∼DQB1*05:01 (homozygotes: OR 26.9; heterozygotes: OR 2.5) and HLA-DQA1*01:04-∼DQB1*05:03 (homozygotes: OR 30.9; heterozygotes: OR 5.6), in order of descending relative risk predisposition. Differences between encoded heterodimers are minimal (a few amino acids outside the main HLA sequence binding region), suggesting a common function. Computational binding predictions support similar, high binding affinity for IgLON5275-283 in a post-translationally modified (N-deglycosylated) form by all three of these HLA-DQ molecules and other common binders. In contrast, association analysis suggests that effects of HLA-DR are likely explained by linkage disequilibrium. Conclusions This study is the, so far, largest genetic study on anti-IgLON5 disease. Our results strongly suggest HLA-DQ over HLA-DR association, with higher reactivity against post-translationally modified versus physiological peptides, in line with reduced T cell priming against these epitopes. Further studies should address the functional implications of these HLA associations.

OR24-2 Cutting-edge translational strategies in type 1 diabetes

Abstract Background and purpose of the study The overall goal of this study is to develop precision-targeted therapies for type 1 diabetes (T1D) by blocking the presentation of pancreatic antigens to T cells using an innovative translational approach. There is currently no curative therapy for T1D and the only available treatment is insulin replacement, which – although being life-saving – remains problematic and can be associated with often fatal high or low glucose levels. We hypothesize that the presentation of pathogenic pancreatic peptides to T cells within the HLA-DQ8 pocket is a key trigger in T1D, and that blocking the peptide binding to this pocket can be harnessed to treat/prevent the autoimmune response targeting beta cells in T1D. We propose to block antigen presentation to treat/prevent autoimmunity in T1D using retro-inverso D-amino acid peptides (RIPs). To test our hypothesis, we used the following tools: recombinant human HLA-DQ8, human B cells homozygous for HLA-DQ8, humanized mice expressing human HLA-DQ8, NOD mice, and peripheral blood mononuclear cells (PBMCs) isolated from new onset HLA-DQ8 T1D patients. All of them represent exquisite translational instruments to evaluate in vitro, ex vivo, and in vivo the functional role of our RIPs in preventing beta cell destruction. Summary of results We have identified a RIP (RI-CT) that inhibits InsB: 9-23 binding to recombinant HLA-DQ8 molecule, as well as its binding to HLA-DQ8 expressed on a human B-cell line. Specifically, RI-CT averted T-cell activation in a mixed lymphocyte reaction containing human DQ8 cells loaded with InsB: 9-23 peptide and murine T-cells expressing a human TCR specific for the InsB: 9-23–DQ8 complex. These results were also confirmed in humanized transgenic B6-DQ8 mice (KO for murine MHC-II) both ex vivo and in vivo, as shown by decreased production of pro-inflammatory cytokines (including IL-2 and IFN-γ) and reduced lymphocyte proliferation. Moreover, RI-CT prevents autoimmune diabetes in NOD mice as demonstrated by a significant decrease in pancreatic immune cell infiltration in treated mice versus controls. Importantly, at 25 weeks of age 70% of RI-CT treated mice were protected from the development of diabetes, while as expected 80% of control NOD mice had developed the disease. Interestingly, RI-CT injection also altered T-cell subpopulations in NOD mice significantly decreasing CD8 expression and significantly increasing Foxp3 levels in treated mice. Of note, RI-CT significantly inhibits InsB: 9-23-mediated lymphocyte activation in peripheral blood mononuclear cells isolated from new onset DQ8-T1D patients. Conclusions In summary, we discovered a RIP that blocks InsB: 9-23 binding to HLA-DQ8 and its presentation to T-cells averting beta cells destruction, and that delays T1D in NOD mice. These data set the stage for using our approach of blocking antigen presentation by RIP as a novel therapeutic approach for autoimmune diseases in general. Presentation: Monday, June 13, 2022 11:15 a.m. - 11:30 a.m.

The Immunobiology and Pathogenesis of Celiac Disease.

Among human leukocyte antigen (HLA)-associated disorders, celiac disease has an immunopathogenesis that is particularly well understood. The condition is characterized by hypersensitivity to cereal gluten proteins, and the disease lesion is localized in the gut. Still, the diagnosis can be made by detection of highly disease-specific autoantibodies to transglutaminase 2 in the blood. We now have mechanistic insights into how the disease-predisposing HLA-DQ molecules, via presentation of posttranslationally modified gluten peptides, are connected to the generation of these autoantibodies. This review presents our current understanding of the immunobiology of this common disorder that is positioned in the border zone between food hypersensitivity and autoimmunity.

A Mouse Model of Celiac Disease.

The design and use of mouse models that reproduce key features of human diseases are critical to advance our understanding of the pathogenesis of autoimmune diseases and to test new therapeutic strategies. Celiac disease is a unique organ-specific autoimmune-like disorder occurring in genetically susceptible individuals carrying HLA-DQ2 or HLA-DQ8 molecules who consume gluten. The key histological characteristic of the disease in humans is the destruction of the lining of the small intestine, a feature that has been difficult to reproduce in immunocompetent animal models. This unit describes the DQ8-Dd -villin-IL-15 transgenic mouse model of CeD, which was engineered based on the knowledge acquired from studying CeD patients' intestinal samples, and which represents the first animal model that develops villous atrophy in an HLA- and gluten-dependent manner without administration of any adjuvant. We provide detailed protocols for inducing and monitoring intestinal tissue damage, evaluating the cytotoxic properties of intraepithelial lymphocytes that mediate enterocyte lysis, and assessing the activation of the enzyme transglutaminase 2, which contributes to the generation of highly immunogenic gluten peptides. Detailed protocols to prepare pepsin-trypsin digested gliadin (PT-gliadin) or chymotrypsin-digested gliadin (CT-gliadin), which allow antibody detection against native or deamidated gluten peptides, are also provided in this unit. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of celiac-like disease in DQ8-Dd -villin-IL-15tg mice Basic Protocol 2: Histological assessment of villous atrophy Support Protocol 1: Morphometric assessment of villous/crypt ratio Support Protocol 2: Evaluation of epithelial cells renewal Support Protocol 3: Evaluation of the density of intraepithelial lymphocytes Basic Protocol 3: Analysis of cytotoxic intraepithelial lymphocytes Basic Protocol 4: Transglutaminase 2 activation and measurement of antibodies against native and deamidated gluten peptides Support Protocol 4: Preparation of CT-gliadin Support Protocol 5: Preparation of PT-gliadin.

OA24 Predicting drug immunogenicity to tumour necrosis factor inhibitors in patients with rheumatoid arthritis

Abstract Background/Aims Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease that irreversibly damages synovial joints. Tumour necrosis factor inhibitors (TNFi) may be prescribed for patients with moderate to severe RA. However, approximately 40% of patients do not respond well to TNFi, with a lack of effectiveness underpinned by immunogenicity (development of anti-drug antibodies) in some patients. A recent well-powered study by Sazonovs et al. (2020) reported a strong genetic association between the HLA-DQA1 locus and immunogenicity to TNFi in a cohort of Crohn's disease patients treated with either infliximab or adalimumab. The current study aimed to investigate the association between HLA alleles, primarily HLA-DQA1, and immunogenicity to adalimumab in RA patients. Methods Anti-adalimumab antibody titres were measured using radioimmunoassay in serum samples from RA patients following six months of treatment. Genetic datasets were generated using the Illumina OmniExpress genotyping array and, following standard quality control filtering, variants were imputed using SNP2HLA statistical software. Following imputation, filtering was performed to exclude variants in linkage disequilibrium or with low minor allele frequency. Logistic regression models were developed to explore the association between HLA alleles and amino acids and immunogenicity (all models were adjusted for sex). Amino acid positions of interest were annotated using P2Rank; a ligand binding site prediction tool. Results A total of 171 HLA alleles and 339 amino acid positions were analysed in 226 RA patients. The strongest association observed between HLA alleles and immunogenicity was for HLA-DQB1*02 (coefficient = 0.87; 95% CI, 0.31-1.43; p value = 2.10E-3), followed by HLA-DQA1*05 (coefficient = 0.91; 95% CI, 0.29-1.53; p value = 3.95E-3). The strongest associated amino acid positions were amino acids 66 and 67 within the ligand binding domain of the HLA-DQB1 protein. Conclusion In keeping with previous findings in Crohn's disease, variants within HLA-DQ molecules confer the strongest evidence for immunogenicity risk in RA patients receiving adalimumab. RA patients carrying these variants could be redirected to receive etanercept, an effective alternative to monoclonal antibodies that is not immunogenic. Further well-powered studies in RA are now warranted to replicate these findings. Disclosure M. Alshehri: None. N. Nair: None. C. Yap: None. J.D. Isaacs: None. K. Hyrich: Honoraria; Abbvie. A.W. Morgan: None. A.G. Wilson: None. A. Barton: None. D. Plant: None.

Celiac Disease.

Celiac Disease.

Celiac Disease Defined by Over-Sensitivity to Gliadin Activation and Superior Antigen Presentation of Dendritic Cells

The autoimmune condition, Celiac Disease (CeD), displays broad clinical symptoms due to gluten exposure. Its genetic association with DQ variants in the human leukocyte antigen (HLA) system has been recognised. Monocyte-derived mature dendritic cells (MoDCs) present gluten peptides through HLA-DQ and co-stimulatory molecules to T lymphocytes, eliciting a cytokine-rich microenvironment. Having access to CeD associated families prevalent in the Czech Republic, this study utilised an in vitro model to investigate their differential monocyte profile. The higher monocyte yields isolated from PBMCs of CeD patients versus control individuals also reflected the greater proportion of dendritic cells derived from these sources following lipopolysaccharide (LPS)/ peptic-tryptic-gliadin (PTG) fragment stimulation. Cell surface markers of CeD monocytes and MoDCs were subsequently profiled. This foremost study identified a novel bio-profile characterised by elevated CD64 and reduced CD33 levels, unique to CD14++ monocytes of CeD patients. Normalisation to LPS stimulation revealed the increased sensitivity of CeD-MoDCs to PTG, as shown by CD86 and HLA-DQ flow cytometric readouts. Enhanced CD86 and HLA-DQ expression in CeD-MoDCs were revealed by confocal microscopy. Analysis highlighted their dominance at the CeD-MoDC membrane in comparison to controls, reflective of superior antigen presentation ability. In conclusion, this investigative study deciphered the monocytes and MoDCs of CeD patients with the identification of a novel bio-profile marker of potential diagnostic value for clinical interpretation. Herein, the characterisation of CD86 and HLA-DQ as activators to stimulants, along with robust membrane assembly reflective of efficient antigen presentation, offers CeD targeted therapeutic avenues worth further exploration.

T cell receptor recognition of hybrid insulin peptides bound to HLA-DQ8

HLA-DQ8, a genetic risk factor in type I diabetes (T1D), presents hybrid insulin peptides (HIPs) to autoreactive CD4+ T cells. The abundance of spliced peptides binding to HLA-DQ8 and how they are subsequently recognised by the autoreactive T cell repertoire is unknown. Here we report, the HIP (GQVELGGGNAVEVLK), derived from splicing of insulin and islet amyloid polypeptides, generates a preferred peptide-binding motif for HLA-DQ8. HLA-DQ8-HIP tetramer+ T cells from the peripheral blood of a T1D patient are characterised by repeated TRBV5 usage, which matches the TCR bias of CD4+ T cells reactive to the HIP peptide isolated from the pancreatic islets of a patient with T1D. The crystal structure of three TRBV5+ TCR-HLA-DQ8-HIP complexes shows that the TRBV5-encoded TCR β-chain forms a common landing pad on the HLA-DQ8 molecule. The N- and C-termini of the HIP is recognised predominantly by the TCR α-chain and TCR β-chain, respectively, in all three TCR ternary complexes. Accordingly, TRBV5 + TCR recognition of HIP peptides might occur via a ‘polarised’ mechanism, whereby each chain within the αβTCR heterodimer recognises distinct origins of the spliced peptide presented by HLA-DQ8.

Interplay Between Gluten, HLA, Innate and Adaptive Immunity Orchestrates the Development of Coeliac Disease.

Several environmental, genetic, and immune factors create a “perfect storm” for the development of coeliac disease: the antigen gluten, the strong association of coeliac disease with HLA, the deamidation of gluten peptides by the enzyme transglutaminase 2 (TG2) generating peptides that bind strongly to the predisposing HLA-DQ2 or HLA-DQ8 molecules, and the ensuing unrestrained T cell response. T cell immunity is at the center of the disease contributing to the inflammatory process through the loss of tolerance to gluten and the differentiation of HLA-DQ2 or HLA-DQ8-restricted anti-gluten inflammatory CD4+ T cells secreting pro-inflammatory cytokines and to the killing of intestinal epithelial cells by cytotoxic intraepithelial CD8+ lymphocytes. However, recent studies emphasize that the individual contribution of each of these cell subsets is not sufficient and that interactions between these different populations of T cells and the simultaneous activation of innate and adaptive immune pathways in distinct gut compartments are required to promote disease immunopathology. In this review, we will discuss how tissue destruction in the context of coeliac disease results from the complex interactions between gluten, HLA molecules, TG2, and multiple innate and adaptive immune components.