HLA Genotype and Probiotics Modify the Association Between Timing of Solid Food Introduction and Islet Autoimmunity in the TEDDY Study.

OBJECTIVE: To study the interaction among HLA genotype, early probiotic exposure, and timing of complementary foods in relation to risk of islet autoimmunity (IA). RESEARCH DESIGN AND METHODS: The Environmental Determinants of Diabetes in the Young (TEDDY) study prospectively follows 8,676 children with increased genetic risk of type 1 diabetes. We used a Cox proportional hazards regression model adjusting for potential confounders to study early feeding and the risk of IA in a sample of 7,770 children. RESULTS: Any solid food introduced early (<6 months) was associated with increased risk of IA if the child had the HLA DR3/4 genotype and no probiotic exposure during the 1st year of life. Rice introduced at 4-5.9 months compared with later in the U.S. was associated with an increased risk of IA. CONCLUSIONS: Timing of solid food introduction, including rice, may be associated with IA in children with the HLA DR3/4 genotype not exposed to probiotics. The microbiome composition under these exposure combinations requires further study.

Biology and Natural History of Type 1 Diabetes Mellitus.

Type 1 diabetes mellitus is a clinical condition characterized by insufficient insulin production due to progressive loss of pancreatic islet ß-cells mediated by an autoimmune response. This deregulation of the immune system is caused by the action of genetic, epigenetic, and environmental factors in varying combinations for each individual. Although the inflammation of the islets with immune cell infiltration, known as insulitis, is an important element in pathogenesis, other factors are necessary for disease initiation. Associations with variants of HLA and other genes related to immune system function, mainly haplotypes HLA-DR3-DQ2 and HLA-DR4-DQ8, are more evident. The influence of polymorphisms and epigenetic modifications, as well as the microbiome, is convincing proof of the existence of a complex interaction between genetic, immune, and environmental factors in the etiology and pathogenesis of this metabolic disorder. Loss of selftolerance to autoimmunity is a critical point in the development of the disease, and regulatory T cells play a key role in this process. Thus, any failure of these cells, either due to an insufficient number or altered expression of cytokines and transcription factors, may be the trigger for the onset of the disease. The protective action of regulatory T cells is controlled by gene expression that is modulated by epigenetic modifications, including the dysregulation of noncoding RNAs. This review takes an updated approach to the natural history of type 1 diabetes, focusing on the factors involved in the etiology and pathogenesis.

The relationship between islet autoantibody status and the genetic risk of type 1 diabetes in adult-onset type 1 diabetes.

AIMS/HYPOTHESIS: The reason for the observed lower rate of islet autoantibody positivity in clinician-diagnosed adult-onset vs childhood-onset type 1 diabetes is not known. We aimed to explore this by assessing the genetic risk of type 1 diabetes in autoantibody-negative and -positive children and adults. METHODS: We analysed GAD autoantibodies, insulinoma-2 antigen autoantibodies and zinc transporter-8 autoantibodies (ZnT8A) and measured type 1 diabetes genetic risk by genotyping 30 type 1 diabetes-associated variants at diagnosis in 1814 individuals with clinician-diagnosed type 1 diabetes (1112 adult-onset, 702 childhood-onset). We compared the overall type 1 diabetes genetic risk score (T1DGRS) and non-HLA and HLA (DR3-DQ2, DR4-DQ8 and DR15-DQ6) components with autoantibody status in those with adult-onset and childhood-onset diabetes. We also measured the T1DGRS in 1924 individuals with type 2 diabetes from the Wellcome Trust Case Control Consortium to represent non-autoimmune diabetes control participants. RESULTS: The T1DGRS was similar in autoantibody-negative and autoantibody-positive clinician-diagnosed childhood-onset type 1 diabetes (mean [SD] 0.274 [0.034] vs 0.277 [0.026], p=0.4). In contrast, the T1DGRS in autoantibody-negative adult-onset type 1 diabetes was lower than that in autoantibody-positive adult-onset type 1 diabetes (mean [SD] 0.243 [0.036] vs 0.271 [0.026], p<0.0001) but higher than that in type 2 diabetes (mean [SD] 0.229 [0.034], p<0.0001). Autoantibody-negative adults were more likely to have the more protective HLA DR15-DQ6 genotype (15% vs 3%, p<0.0001), were less likely to have the high-risk HLA DR3-DQ2/DR4-DQ8 genotype (6% vs 19%, p<0.0001) and had a lower non-HLA T1DGRS (p<0.0001) than autoantibody-positive adults. In contrast to children, autoantibody-negative adults were more likely to be male (75% vs 59%), had a higher BMI (27 vs 24 kg/m2) and were less likely to have other autoimmune conditions (2% vs 10%) than autoantibody-positive adults (all p<0.0001). In both adults and children, type 1 diabetes genetic risk was unaffected by the number of autoantibodies (p>0.3). These findings, along with the identification of seven misclassified adults with monogenic diabetes among autoantibody-negative adults and the results of a sensitivity analysis with and without measurement of ZnT8A, suggest that the intermediate type 1 diabetes genetic risk in autoantibody-negative adults is more likely to be explained by the inclusion of misclassified non-autoimmune diabetes (estimated to represent 67% of all antibody-negative adults, 95% CI 61%, 73%) than by the presence of unmeasured autoantibodies or by a discrete form of diabetes. When these estimated individuals with non-autoimmune diabetes were adjusted for, the prevalence of autoantibody positivity in adult-onset type 1 diabetes was similar to that in children (93% vs 91%, p=0.4). CONCLUSIONS/INTERPRETATION: The inclusion of non-autoimmune diabetes is the most likely explanation for the observed lower rate of autoantibody positivity in clinician-diagnosed adult-onset type 1 diabetes. Our data support the utility of islet autoantibody measurement in clinician-suspected adult-onset type 1 diabetes in routine clinical practice.

Low copy numbers of complement C4 and C4A deficiency are risk factors for myositis, its subgroups and autoantibodies.

BACKGROUND: Idiopathic inflammatory myopathies (IIM) are a group of autoimmune diseases characterised by myositis-related autoantibodies plus infiltration of leucocytes into muscles and/or the skin, leading to the destruction of blood vessels and muscle fibres, chronic weakness and fatigue. While complement-mediated destruction of capillary endothelia is implicated in paediatric and adult dermatomyositis, the complex diversity of complement C4 in IIM pathology was unknown. METHODS: We elucidated the gene copy number (GCN) variations of total C4, C4A and C4B, long and short genes in 1644 Caucasian patients with IIM, plus 3526 matched healthy controls using real-time PCR or Southern blot analyses. Plasma complement levels were determined by single radial immunodiffusion. RESULTS: The large study populations helped establish the distribution patterns of various C4 GCN groups. Low GCNs of C4T (C4T=2+3) and C4A deficiency (C4A=0+1) were strongly correlated with increased risk of IIM with OR equalled to 2.58 (2.28-2.91), p=5.0×10-53 for C4T, and 2.82 (2.48-3.21), p=7.0×10-57 for C4A deficiency. Contingency and regression analyses showed that among patients with C4A deficiency, the presence of HLA-DR3 became insignificant as a risk factor in IIM except for inclusion body myositis (IBM), by which 98.2% had HLA-DR3 with an OR of 11.02 (1.44-84.4). Intragroup analyses of patients with IIM for C4 protein levels and IIM-related autoantibodies showed that those with anti-Jo-1 or with anti-PM/Scl had significantly lower C4 plasma concentrations than those without these autoantibodies. CONCLUSIONS: C4A deficiency is relevant in dermatomyositis, HLA-DRB1*03 is important in IBM and both C4A deficiency and HLA-DRB1*03 contribute interactively to risk of polymyositis.

Phase III, randomised, double-blind, placebo-controlled, multicentre trial to evaluate the efficacy and safety of rhGAD65 to preserve endogenous beta cell function in adolescents and adults with recently diagnosed type 1 diabetes, carrying the genetic HLA DR3-DQ2 haplotype: the DIAGNODE-3 study protocol.

INTRODUCTION: Type 1 diabetes (T1D) is an autoimmune disease leading to the destruction of the insulin-producing beta cells resulting in insulin deficiency and hyperglycaemic. Today, no approved therapy exists to halt this detrimental immunologic process. In a recent phase 2b study, intralymphatic administration of recombinant human glutamic acid decarboxylase 65 kDa (rhGAD65) adsorbed to Alhydrogel adjuvant to individuals recently diagnosed with T1D and carrying the HLA DR3-DQ2 haplotype showed promising results in preserving endogenous insulin secretion, confirming the results of a large meta-analysis of three randomised placebo-controlled trials of subcutaneous rhGAD65. The aim of the current precision medicine phase 3 study is to determine whether intralymphatic administration of rhGAD65 preserves insulin secretion and improves glycaemic control in presumed responder individuals with recently diagnosed T1D carrying HLA DR3-DQ2. METHODS AND ANALYSIS: Individuals ≥12 and <29 years recently diagnosed with T1D (<6 months) will be screened for the HLA DR3-DQ2 haplotype, endogenous insulin production estimated by fasting C-peptide and presence of GAD65 antibodies. 330 patients are planned to be randomised to 3 monthly intralymphatic injections of rhGAD65 or placebo (both accompanied by oral vitamin D supplementation), followed by 22 months of follow-up. The study is powered to detect a treatment effect in the two coprimary endpoints; change from baseline in AUC(0-120min) C-peptide levels during a mixed meal tolerance test, and change from baseline in glycaemic control estimated by haemoglobin A1c at 24 months. Secondary endpoints include effects on glucose patterns collected by masked continuous glucose monitoring, proportion of patients in partial remission and number of episodes of severe hypoglycaemia and/or diabetic ketoacidosis. ETHICS AND DISSEMINATION: The trial is approved by Ethics Committees in Poland (124/2021), the Netherlands (R21.089), Sweden (2021-05063), Czech Republic (EK-1144/21), Germany (2021361) and Spain (21/2021). Results will be published in international peer-reviewed scientific journals and presented at national and international conferences. TRIAL REGISTRATION NUMBER: EudraCT identifier: 2021-002731-32, NCT identifier: NCT05018585.

Obesity induced gut dysbiosis contributes to disease severity in an animal model of multiple sclerosis.

Background: Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the CNS. The etiology of MS is complex, and results from the interaction of multiple environmental and genetic factors. Although human leukocyte antigen-HLA alleles such as HLA-DR2 and -DR3 are considered the strongest genetic factors, the environmental factors responsible for disease predisposition are not well understood. Recently, diet and gut microbiota have emerged as an important environmental factors linked to the increased incidence of MS. Especially, western diets rich in protein and fat have been linked to the increased incidence of obesity. Numerous clinical data indicate a role of obesity and gut microbiota in MS; however, the mechanistic link between gut microbiota and obesity in the pathobiology of MS remains unclear. The present study determines the mechanisms driving MS severity in the context of obesity utilizing a high-fat diet (HFD) induced obese HLA-DR3 class-II transgenic mouse model of MS. Methods: HLA-DR3 transgenic mice were kept on a standard HFD diet or Normal Chow (NC) for eight weeks. Gut microbiota composition and functional analysis were performed from the fecal DNA of mice. Experimental autoimmune encephalomyelitis-EAE (an animal model of MS) was induced by immunization with the proteolipid protein-PLP91-110 peptide in complete Freud's Adjuvant (CFA) and pertussis toxin. Results: We observed that HFD-induced obesity caused gut dysbiosis and severe disease compared to mice on NC. Amelioration of disease severity in mice depleted of gut microbiota suggested an important role of gut bacteria in severe EAE in obese mice. Fecal microbiota analysis in HFD mice shows gut microbiota alterations with an increase in the abundance of Proteobacteria and Desulfovibrionaceae bacteria and modulation of various bacterial metabolic pathways including bacterial hydrogen sulfide biosynthetic pathways. Finally, mice on HFD showed increased gut permeability and systemic inflammation suggesting a role gut barrier modulation in obesity induced disease severity. Conclusions: This study provides evidence for the involvement of the gut microbiome and associated metabolic pathways plus gut permeability in obesity-induced modulation of EAE disease severity. A better understanding of the same will be helpful to identify novel therapeutic targets to reduce disease severity in obese MS patients.

GAD65 Antibody Epitopes and Genetic Background in Latent Autoimmune Diabetes in Youth (LADY).

Epitope-specific GAD65Abs and HLA-DR-DQ gene assays help improve the value of risk stratification in autoimmune diabetes mellitus and protect islet function. Identification and early intervention are important for latent autoimmune diabetes in youth (LADY). The aims of this study were to investigate 1) the frequencies of the epitope-specific GAD65Abs and HLA-DR-DQ genes in LADY and 2) the association between HLA-DR-DQ genes and epitope-specific GAD65Abs. Higher frequencies of GAD65-CAb and multiepitope GAD65Abs were observed in young type 1 diabetes, LADY, and old type 1 diabetes subjects than those in latent autoimmune diabetes in adult (LADA) patients. The frequencies of the specific susceptible HLA haplotype DR3, total susceptible HLA haplotypes, and high-risk genotypes were higher in type 1 diabetes and LADY patients than those in LADA patients. In contrast, type 1 diabetes and LADY patients had lower frequencies of low/no genetic risk genotypes (DRX/X) than those of LADA patients. Logistic regression analysis suggested that the susceptible HLA haplotypes were risk factors for glutamic acid decarboxylase antibody (GADA) multiepitope positivity in autoimmune diabetes mellitus. LADY may be more severe than LADA, and LADY seemed to be a transitional type of type 1 diabetes and LADA. GADA epitope and HLA-DR-DQ gene assays are important for risk stratification in autoimmune diabetes mellitus and protection of islet function.

Childhood type 1 diabetes mellitus and risk factor of interactions between dietary cow's milk intake and HLA-DR3/DR4 genotype.

Short-term breastfeeding and early exposure to dairy products into infant diets, may be critical factors for development of type 1 diabetes. In this study, we investigate whether cow's milk proteins are risk factors for type 1 diabetes in genetically susceptible individuals (HLA DR3/DR4) by using statistical analysis and in silico approach. In order to verify the potential risk of the early introduction of cow's milk, we conducted this study to validate the veracity of this hypothesis in our population. We included 121 subjects, 55 type 1 diabetics and 74 controls from the region of Tlemcen (Algeria). Thus, the in silico approach was performed to determine the molecular mimicry region between Bovine serum albumin and beta-lactoglobulin with self-Islet antigen 2 and glutamate decarboxylase 65 by determining their sequences and their 3D structures. The risk factors associated with type 1 diabetes in a genetically predisposed individual (HLA DR3/DR4) retained by the logistic model are: type 1 and type 2 diabetes inheritance, the early introduction of cow's milk before 6 months and breastfeeding less than 9 months. Besides, the epitopes of cow's milk proteins have the capacity to bind to predisposing HLA class II molecules (HLA DR3/DR4) and induce an immune reaction by the secretion of Interleukin 4 (Th2) and Interferon (Th1) which lead to the destruction of pancreatic beta cells. The early introduction of cow's milk proteins in susceptible individuals is considered as risk factors for the pathogenesis of T1DM. The in silico approach confirm that BSA and BLG share sequence and structure homology with IA2 and GAD65.Communicated by Ramaswamy H. Sarma.

Association of High-Affinity Autoantibodies With Type 1 Diabetes High-Risk HLA Haplotypes.

OBJECTIVE: Electrochemiluminescence (ECL) assays are high-affinity autoantibody (Ab) tests that are more specific than Abs detected by traditional radiobinding assays (RBA) for risk screening and prediction of progression to type 1 diabetes. We sought to characterize the association of high-risk human leukocyte antigen (HLA) haplotypes and genotypes with ECL positivity and levels in relatives of individuals with type 1 diabetes. METHODS: We analyzed 602 participants from the TrialNet Pathway to Prevention Study who were positive for at least 1 RBA diabetes-related Ab [glutamic acid decarboxylase autoantibodies (GADA) or insulin autoantibodies (IAA)] and for whom ECL and HLA data were available. ECL and RBA Ab levels were converted to SD units away from mean (z-scores) for analyses. RESULTS: Mean age at initial visit was 19.4 ± 13.7 years; 344 (57.1%) were female and 104 (17.3%) carried the high-risk HLA-DR3/4*0302 genotype. At initial visit 424/602 (70.4%) participants were positive for either ECL-GADA or ECL-IAA, and 178/602 (29.6%) were ECL negative. ECL and RBA-GADA positivity were associated with both HLA-DR3 and DR4 haplotypes (all Ps < 0.05), while ECL and RBA-GADA z-score titers were higher in participants with HLA-DR3 haplotypes only (both Ps < 0.001). ECL-IAA (but not RBA-IAA) positivity was associated with the HLA-DR4 haplotype (P < 0.05). CONCLUSIONS: ECL-GADA positivity is associated with the HLA-DR3 and HLA-DR4 haplotypes and levels are associated with the HLA-DR3 haplotype. ECL-IAA positivity is associated with HLA-DR4 haplotype. These studies further contribute to the understanding of genetic risk and islet autoimmunity endotypes in type 1 diabetes.

CD4+ T cells in the lungs of acute sarcoidosis patients recognize an Aspergillus nidulans epitope.

Löfgren's syndrome (LS) is an acute form of sarcoidosis characterized by a genetic association with HLA-DRB1*03 (HLA-DR3) and an accumulation of CD4+ T cells of unknown specificity in the bronchoalveolar lavage (BAL). Here, we screened related LS-specific TCRs for antigen specificity and identified a peptide derived from NAD-dependent histone deacetylase hst4 (NDPD) of Aspergillus nidulans that stimulated these CD4+ T cells in an HLA-DR3-restricted manner. Using ELISPOT analysis, a greater number of IFN-γ- and IL-2-secreting T cells in the BAL of DR3+ LS subjects compared with DR3+ control subjects was observed in response to the NDPD peptide. Finally, increased IgG antibody responses to A. nidulans NDPD were detected in the serum of DR3+ LS subjects. Thus, our findings identify a ligand for CD4+ T cells derived from the lungs of LS patients and suggest a role of A. nidulans in the etiology of LS.

HLA Class II Polymorphisms Modulate Gut Microbiota and Experimental Autoimmune Encephalomyelitis Phenotype.

Multiple sclerosis (MS) is an autoimmune disease of the CNS in which the interaction between genetic and environmental factors plays an important role in disease pathogenesis. Although environmental factors account for 70% of disease risk, the exact environmental factors associated with MS are unknown. Recently, gut microbiota has emerged as a potential missing environmental factor linked with the pathobiology of MS. Yet, how genetic factors, such as HLA class II gene(s), interact with gut microbiota and influence MS is unclear. In the current study, we investigated whether HLA class II genes that regulate experimental autoimmune encephalomyelitis (EAE) and MS susceptibility also influence gut microbiota. Previously, we have shown that HLA-DR3 transgenic mice lacking endogenous mouse class II genes (AE-KO) were susceptible to myelin proteolipid protein (91-110)-induced EAE, an animal model of MS, whereas AE-KO.HLA-DQ8 transgenic mice were resistant. Surprisingly, HLA-DR3.DQ8 double transgenic mice showed higher disease prevalence and severity compared with HLA-DR3 mice. Gut microbiota analysis showed that HLA-DR3, HLA-DQ8, and HLA-DR3.DQ8 double transgenic mice microbiota are compositionally different from AE-KO mice. Within HLA class II transgenic mice, the microbiota of HLA-DQ8 mice were more similar to HLA-DR3.DQ8 than HLA-DR3. As the presence of DQ8 on an HLA-DR3 background increases disease severity, our data suggests that HLA-DQ8-specific microbiota may contribute to disease severity in HLA-DR3.DQ8 mice. Altogether, our study provides evidence that the HLA-DR and -DQ genes linked to specific gut microbiota contribute to EAE susceptibility or resistance in a transgenic animal model of MS.

The High-Risk Type 1 Diabetes HLA-DR and HLA-DQ Polymorphisms Are Differentially Associated With Growth and IGF-I Levels in Infancy: The Cambridge Baby Growth Study.

OBJECTIVE: This study explored the link between HLA polymorphisms that predispose to type 1 diabetes and birth size, infancy growth, and/or circulating IGF-I in a general population-based birth cohort. RESEARCH DESIGN AND METHODS: The Cambridge Baby Growth Study is a prospective observational birth cohort study that recruited 2,229 newborns for follow-up in infancy. Of these, 612 children had DNA available for genotyping single nucleotide polymorphisms in the HLA region that capture the highest risk of type 1 diabetes: rs17426593 for DR4, rs2187668 for DR3, and rs7454108 for DQ8. Multivariate linear regression models at critical ages (cross-sectional) and mixed-effects models (longitudinal) were performed under additive genetic effects to test for associations between HLA polymorphisms and infancy weight, length, skinfold thickness (indicator of adiposity), and concentrations of IGF-I and IGF-binding protein-3 (IGFBP-3). RESULTS: In longitudinal models, the minor allele of rs2187668 tagging DR3 was associated with faster linear growth (P = 0.007), which was more pronounced in boys (P = 3 × 10-7) than girls (P = 0.07), and was also associated with increasing IGF-I (P = 0.002) and IGFBP-3 (P = 0.003) concentrations in infancy. Cross-sectionally, the minor alleles of rs7454108 tagging DQ8 and rs17426593 tagging DR4 were associated with lower IGF-I concentrations at age 12 months (P = 0.003) and greater skinfold thickness at age 24 months (P = 0.003), respectively. CONCLUSIONS: The variable associations of DR4, DR3, and DQ8 alleles with growth measures and IGF-I levels in infants from the general population could explain the heterogeneous growth trajectories observed in genetically at-risk cohorts. These findings could suggest distinct mechanisms involving endocrine pathways related to the HLA-conferred type 1 diabetes risk.

Human Leukocyte Antigen Profile Predicts Severity of Autoimmune Liver Disease in Children of European Ancestry.

BACKGROUND AND AIMS: Genetic predisposition to autoimmune hepatitis (AIH) in adults is associated with possession of human leukocyte antigen (HLA) class I (A*01, B*08) and class II (DRB1*03, -04, -07, or -13) alleles, depending on geographic region. Juvenile autoimmune liver disease (AILD) comprises AIH-1, AIH-2, and autoimmune sclerosing cholangitis (ASC), which are phenotypically different from their adult counterparts. We aimed to define the relationship between HLA profile and disease course, severity, and outcome in juvenile AILD. APPROACH AND RESULTS: We studied 236 children of European ancestry (152 female [64%], median age 11.15 years, range 0.8-17), including 100 with AIH-1, 59 with AIH-2, and 77 with ASC. The follow-up period was from 1977 to June 2019 (median 14.5 years). Class I and II HLA genotyping was performed using PCR/sequence-specific primers. HLA B*08, -DRB1*03, and the A1-B8-DR3 haplotype impart predisposition to all three forms of AILD. Homozygosity for DRB1*03 represented the strongest risk factor (8.8). HLA DRB1*04, which independently confers susceptibility to AIH in adults, was infrequent in AIH-1 and ASC, suggesting protection; and DRB1*15 (DR15) was protective against all forms of AILD. Distinct HLA class II alleles predispose to the different subgroups of juvenile AILD: DRB1*03 to AIH-1, DRB1*13 to ASC, and DRB1*07 to AIH-2. Possession of homozygous DRB1*03 or of DRB1*13 is associated with fibrosis at disease onset, and possession of these two genes in addition to DRB1*07 is associated with a more severe disease in all three subgroups. CONCLUSIONS: Unique HLA profiles are seen in each subgroup of juvenile AILD. HLA genotype might be useful in predicting responsiveness to immunosuppressive treatment and course.

The oxylipin profile is associated with development of type 1 diabetes: the Diabetes Autoimmunity Study in the Young (DAISY).

AIMS/HYPOTHESIS: Oxylipins are lipid mediators derived from polyunsaturated fatty acids. Some oxylipins are proinflammatory (e.g. those derived from arachidonic acid [ARA]), others are pro-resolving of inflammation (e.g. those derived from α-linolenic acid [ALA], docosahexaenoic acid [DHA] and eicosapentaenoic acid [EPA]) and others may be both (e.g. those derived from linoleic acid [LA]). The goal of this study was to examine whether oxylipins are associated with incident type 1 diabetes. METHODS: We conducted a nested case-control analysis in the Diabetes Autoimmunity Study in the Young (DAISY), a prospective cohort study of children at risk of type 1 diabetes. Plasma levels of 14 ARA-derived oxylipins, ten LA-derived oxylipins, six ALA-derived oxylipins, four DHA-derived oxylipins and two EPA-related oxylipins were measured by ultra-HPLC-MS/MS at multiple timepoints related to autoantibody seroconversion in 72 type 1 diabetes cases and 71 control participants, which were frequency matched on age at autoantibody seroconversion (of the case), ethnicity and sample availability. Linear mixed models were used to obtain an age-adjusted mean of each oxylipin prior to type 1 diabetes. Age-adjusted mean oxylipins were tested for association with type 1 diabetes using logistic regression, adjusting for the high risk HLA genotype HLA-DR3/4,DQB1*0302. We also performed principal component analysis of the oxylipins and tested principal components (PCs) for association with type 1 diabetes. Finally, to investigate potential critical timepoints, we examined the association of oxylipins measured before and after autoantibody seroconversion (of the cases) using PCs of the oxylipins at those visits. RESULTS: The ARA-related oxylipin 5-HETE was associated with increased type 1 diabetes risk. Five LA-related oxylipins, two ALA-related oxylipins and one DHA-related oxylipin were associated with decreased type 1 diabetes risk. A profile of elevated LA- and ALA-related oxylipins (PC1) was associated with decreased type 1 diabetes risk (OR 0.61; 95% CI 0.40, 0.94). A profile of elevated ARA-related oxylipins (PC2) was associated with increased diabetes risk (OR 1.53; 95% CI 1.03, 2.29). A critical timepoint analysis showed type 1 diabetes was associated with a high ARA-related oxylipin profile at post-autoantibody-seroconversion but not pre-seroconversion. CONCLUSIONS/INTERPRETATION: The protective association of higher LA- and ALA-related oxylipins demonstrates the importance of both inflammation promotion and resolution in type 1 diabetes. Proinflammatory ARA-related oxylipins may play an important role once the autoimmune process has begun.

Low-Risk Human Leukocyte Antigen Genes and Mild Villous Atrophy Typify Celiac Disease With Immunoglobulin A Deficiency.

OBJECTIVES: We aimed to establish if in celiac disease (CD) with immunoglobulin A deficiency (IgAD) duodenal histopathology is influenced by human leukocyte antigen (HLA)-DQB1∗02 alleles dosage. Clinical differences between patients with CD and patients with CD and IgAD (CD-IgAD) were also evaluated. METHODS: Five hundred and sixteen CD and 16 patients with CD-IgAD, enrolled over the time of 8 years, took part in this study. The severity of duodenal histopathology and frequency of CD at-risk HLA class II genes were compared in patients with CD versus patients with CD-IgAD. HLA class II genotypes were subdivided into two categories of genetic risk: high: HLA-DR3/DR7, -DR3/DR3, -DR4/DR4 -DR3/DR4 and low: HLA-DR5/DR7, -DR3/X, -DR4/X and X/X, where X means neither -DR3 nor -DR4. Then, they were compared with two types of duodenal histopathology: 0, 1, 2 and 3a of mild villous atrophy (MVA) and 3b and 3c of severe villous atrophy (SVA) according to the Marsh-Oberhuber classification. Clinical data concerning gender, number of esophagogastroduodenoscopies (EGDs) and association with other autoimmune diseases were obtained from medical records. RESULTS: In comparison with CD, CD-IgAD showed an increased frequency of MVA (P < 0.0001). Furthermore, CD-IgAD with MVA showed an increase of HLA low-risk genotypes (P = 0.036) and half HLA-DQ2 heterodimers (P = 0.0443). Interestingly, CD-IgAD demanded an increased number of EGDs to reach the diagnosis of CD (P = 0.0104) and autoimmune liver diseases were more frequent compared to CD (P = 0.0049). CONCLUSIONS: CD-IgAD is associated with MVA, low-risk HLA class II genes, an increased number of EGDs and autoimmune liver diseases.

Thyrotropin Receptor-Specific Lymphocytes in Adenovirus-TSHR-Immunized Native and Human Leukocyte Antigen-DR3-Transgenic Mice and in Graves' Disease Patient Blood.

Background: Antigen-specific lymphocytes are increasingly investigated in autoimmune diseases and immune therapies. We sought to identify thyrotropin receptor (TSHR)-specific lymphocytes in mouse models of Graves' disease, including Graves' patient-specific immunotype human leukocyte antigen (HLA)-DR3, and in frozen and thawed Graves' patient blood samples. Methods and Results: Splenic lymphocytes of adenovirus (Ad)-TSHR-immunized BALB/c mice were stimulated with TSHR-specific peptides C, D, or J. Furthermore, CD154-expressing cells were enriched, expanded in vitro, and analyzed for binding of peptide-major histocompatibility complex (MHC) II multimers ("tetramers," immunotype H2-IAd). Only peptides C and J were able to elicit increased expression/secretion of CD154 and interferon-γ, and tetramers which were loaded with peptide C resulted in antigen-specific signals in splenic lymphocytes from Ad-TSHR-immunized mice. Accordingly, TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 specifically bound to human HLA-DR3-(allele B1*03:01)-transgenic Bl/6 mouse splenic T lymphocytes. In addition, we fine-tuned a protocol to reliably measure thawed human peripheral blood mononuclear cells (PBMCs), which resulted in reliable recovery after freezing and thawing with regard to vitality and B and T cell subpopulation markers including regulatory T cells (CD3, CD4, CD25, FoxP3, CD25high, CD127low). TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 identified antigen-specific T cells in HLA-DR3-positive Graves' patients' thawed PBMCs. Moreover, stimulation-dependent release of interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha from thawed PBMCs occurred at the expected levels. Conclusions: Novel MHC II tetramers identified TSHR-specific T lymphocytes in Ad-TSHR-immunized hyperthyroid BALB/c or HLA-DR3-transgenic mice and in thawed human PBMCs from patients with Graves' disease. These assays may contribute to measure both disease severity and effects of novel immune therapies in future animal studies and clinical investigations of Graves' disease.

Cepharanthine Blocks Presentation of Thyroid and Islet Peptides in a Novel Humanized Autoimmune Diabetes and Thyroiditis Mouse Model.

Autoimmune polyglandular syndrome type 3 variant (APS3v) refers to an autoimmune condition in which both type 1 diabetes (T1D) and autoimmune thyroiditis (AITD) develop in the same individual. HLA-DR3 confers the strongest susceptibility to APS3v. Previously we reported a unique amino acid signature pocket that predisposes to APS3v. We found that this pocket is flexible and can trigger APS3v by presenting both thyroid (Tg.1571, TPO.758) and islet (GAD.492) peptides to induce autoimmune response. We hypothesized that blocking the specific APS3v-HLA-DR3 pocket from presenting thyroid/islet antigens can block the autoimmune response in APS3v. To test this hypothesis we performed a virtual screen of small molecules blocking APS3v-HLA-DR3, and identified 11 small molecules hits that were predicted to block APS3v-HLA-DR3. Using the baculovirus-produced recombinant APS3v-HLA-DR3 protein we tested the 11 small molecules in an in vitro binding assay. We validated 4 small molecule hits, S9, S5, S53 and S15, that could block the APS3v-HLA-DR3 pocket in vitro. We then developed a novel humanized APS3v mouse model induced by co-immunizing a peptide mix of Tg.1571, TPO.758 and GAD.492. The immunized mice developed strong T-cell and antibody responses to the thyroid/islet peptides, as well as mouse thyroglobulin. In addition, the mice showed significantly lower free T4 levels compared to controls. Using the APS3v mouse model, we showed that one of the 4 small molecules, Cepharanthine (S53), blocked T-cell activation by thyroid/islet peptides ex vivo and in vivo. These findings suggested Cepharanthine may have a therapeutic potential in APS3v patients carrying the specific APS3v-HLA-DR3 pocket.

Cost Effectiveness of Teplizumab for Prevention of Type 1 Diabetes Among Different Target Patient Groups.

OBJECTIVE: Teplizumab was recently shown to be the first-ever drug to prevent or delay type 1 diabetes mellitus onset in at-risk individuals, especially those with certain genetic and antibody characteristics. However, its potentially high price may pose challenges for coverage and reimbursement for payers and policymakers. Thus, it is critical to investigate the cost effectiveness of this drug for different target individuals. RESEARCH DESIGN AND METHODS: Using Markov microsimulation modeling, we compared the cost effectiveness of five options for choosing target individuals (i.e., all at-risk individuals, individuals without human leukocyte antigen (HLA)-DR3 or with HLA-DR4 allele, individuals without HLA-DR3 and with HLA-DR4 allele, individuals with anti-zinc transporter 8 (ZnT8) antibody negative, and no provision at all) at different possible prices of teplizumab. Effectiveness was measured by quality-adjusted life-years. Costs were estimated from a health system perspective. RESULTS: If the price of teplizumab is below US$48,900, treating all at-risk individuals is cost effective. However, it will be cost effective to treat only individuals without HLA-DR3 or with HLA-DR4 alleles for prices between US$48,900 and US$58,200, only individuals both without HLA-DR3 and with HLA-DR4 alleles for prices between US$58,200 and US$88,300, and only individuals with negative ZnT8 antibody status for prices between US$88,300 and US$193,700. CONCLUSIONS: Cost-effective provision of teplizumab to target individuals depends on the price of teplizumab and genetic and antibody characteristics of treated individuals. As the drug makes its way to the market, findings from this study will help inform policymakers and payers on cost-effective ways to provide this innovative but expensive drug to at-risk individuals.

Efficacy of GAD-alum immunotherapy associated with HLA-DR3-DQ2 in recently diagnosed type 1 diabetes.

AIMS/HYPOTHESIS: The aim of this study was to determine if retention of C-peptide following immunotherapy using recombinant GAD65 conjugated to aluminium hydroxide (GAD-alum) is influenced by HLA risk haplotypes DR3-DQ2 and DR4-DQ8. METHODS: HLA-dependent treatment effect of GAD-alum therapy on C-peptide retention in individuals with recent-onset type 1 diabetes was evaluated using individual-level patient data from three placebo-controlled, randomised clinical trials using a mixed repeated measures model. RESULTS: A significant and dose-dependent effect was observed in individuals positive for the genotypes that include HLA-DR3-DQ2 but not HLA-DR4-DQ8 and in the broader subgroup of individuals positive for all genotypes that include HLA-DR3-DQ2 (i.e. including those also positive for HLA-DR4-DQ8). Higher doses (three or four injections) showed a treatment effect ratio of 1.596 (95% CI 1.132, 2.249; adjusted p = 0.0035) and 1.441 (95% CI 1.188, 1.749; adjusted p = 0.0007) vs placebo for the two respective HLA subgroups. CONCLUSIONS/INTERPRETATION: GAD65-specific immunotherapy has a significant effect on C-peptide retention in individuals with recent-onset type 1 diabetes who have the DR3-DQ2 haplotype. Graphical abstract.

"Small" Intestinal Immunopathology Plays a "Big" Role in Lethal Cytokine Release Syndrome, and Its Modulation by Interferon-γ, IL-17A, and a Janus Kinase Inhibitor.

Chimeric antigen receptor T cell (CART) therapy, administration of certain T cell-agonistic antibodies, immune check point inhibitors, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) and Toxic shock syndrome (TSS) caused by streptococcal as well as staphylococcal superantigens share one common complication, that is T cell-driven cytokine release syndrome (CRS) accompanied by multiple organ dysfunction (MOD). It is not understood whether the failure of a particular organ contributes more significantly to the severity of CRS. Also not known is whether a specific cytokine or signaling pathway plays a more pathogenic role in precipitating MOD compared to others. As a result, there is no specific treatment available to date for CRS, and it is managed only symptomatically to support the deteriorating organ functions and maintain the blood pressure. Therefore, we used the superantigen-induced CRS model in HLA-DR3 transgenic mice, that closely mimics human CRS, to delineate the immunopathogenesis of CRS as well as to validate a novel treatment for CRS. Using this model, we demonstrate that (i) CRS is characterized by a rapid rise in systemic levels of several Th1/Th2/Th17/Th22 type cytokines within a few hours, followed by a quick decline. (ii) Even though multiple organs are affected, small intestinal immunopathology is the major contributor to mortality in CRS. (iii) IFN-γ deficiency significantly protected from lethal CRS by attenuating small bowel pathology, whereas IL-17A deficiency significantly increased mortality by augmenting small bowel pathology. (iv) RNA sequencing of small intestinal tissues indicated that IFN-γ-STAT1-driven inflammatory pathways combined with enhanced expression of pro-apoptotic molecules as well as extracellular matrix degradation contributed to small bowel pathology in CRS. These pathways were further enhanced by IL-17A deficiency and significantly down-regulated in mice lacking IFN-γ. (v) Ruxolitinib, a selective JAK-1/2 inhibitor, attenuated SAg-induced T cell activation, cytokine production, and small bowel pathology, thereby completely protecting from lethal CRS in both WT and IL-17A deficient HLA-DR3 mice. Overall, IFN-γ-JAK-STAT-driven pathways contribute to lethal small intestinal immunopathology in T cell-driven CRS.