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OBJECTIVE To distinguish among predictors of seroconversion, progression to multiple autoantibodies and from multiple autoantibodies to type 1 diabetes in young children. RESEARCH DESIGN AND METHODS Genetically high-risk newborns (n = 8,502) were followed for a median of 11.2 years (interquartile range 9.3–12.6); 835 (9.8%) developed islet autoantibodies and 283 (3.3%) were diagnosed with type 1 diabetes. Predictors were examined using Cox proportional hazards models. RESULTS Predictors of seroconversion and progression differed, depending on the type of first appearing autoantibody. Male sex, Finnish residence, having a sibling with type 1 diabetes, the HLA DR4 allele, probiotic use before age 28 days, and single nucleotide polymorphism (SNP) rs689_A (INS) predicted seroconversion to IAA-first (having islet autoantibody to insulin as the first appearing autoantibody). Increased weight at 12 months and SNPs rs12708716_G (CLEC16A) and rs2292239_T (ERBB3) predicted GADA-first (autoantibody to GAD as the first appearing). For those having a father with type 1 diabetes, the SNPs rs2476601_A (PTPN22) and rs3184504_T (SH2B3) predicted both. Younger age at seroconversion predicted progression from single to multiple autoantibodies as well as progression to diabetes, except for those presenting with GADA-first. Family history of type 1 diabetes and the HLA DR4 allele predicted progression to multiple autoantibodies but not diabetes. Sex did not predict progression to multiple autoantibodies, but males progressed more slowly than females from multiple autoantibodies to diabetes. SKAP2 and MIR3681HG SNPs are newly reported to be significantly associated with progression from multiple autoantibodies to type 1 diabetes. CONCLUSIONS Predictors of IAA-first versus GADA-first autoimmunity differ from each other and from the predictors of progression to diabetes.

Antigen-specific T cells can serve as a response biomarker in non-clinical or clinical immunotherapy studies in autoimmune disease. There are protocols with optimized multimer staining methods to detect peptide (p)MHCII+ CD4+ T cells, and some qualified and validated protocols for pMHCI+ CD8+ T cells. However, no protocol is fully or partially qualified to enumerate and characterize antigen-specific pMHCII+ CD4+ T cells from patient samples. Implementing such an assay requires a desired level of specificity and precision, in terms of assay repeatability and reproducibility. In transgenic type II collagen (CII)-immunized HLA-DR1/DR4 humanized mouse models of collagen-induced arthritis (CIA), CII259-273-specific T cells dominantly expand. Therefore antigen-specific T cells recognizing this epitope presented by rheumatoid arthritis (RA)-associated risk HLA-DR allomorphs are of interest to understand disease progression and responses to immunotherapy in RA patients. Using HLA-DRB1∗04:01 or ∗01:01-collagen type II (CII)259–273 tetramers, we evaluated parameters influencing precision and reproducibility of an optimized flow cytometry–based method for antigen-specific CD4+ T cells and eight specific subpopulations with and without tetramer positivity. We evaluated specificity, precision, and reproducibility for research environments and non-regulated laboratories. The assay has excellent overall precision with %CV

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.

Dental caries being the leading health issue worldwide has no specific cure due to its multifactorial etiology and genetic susceptibility. Hence, this paper attempted to correlate the clinical and hereditary factors between mother and child, to predict the caries occurrence in child in future, and thereby implement early preventive measures.The aim of the study was to look for an association between maternal and child's human leukocyte antigen (HLA)-DR4 levels and relate it with other physiochemical factors to assess caries susceptibility in children.Saliva samples were collected from children who were in the age group of 0-6 years and their mothers by spitting method and swab method. The clinical indicators such as Decayed, Missing, and Filled Teeth, decayed, extraction needed, and filled teeth, salivary flow rate, and pH were recorded by clinical evaluation. The Streptococcus mutans count was measured by culture plate followed by colony count method, and the HLA-DR4 factor was assessed using ELISA.The results revealed a statistically significant correlation between the physiochemical factors of the mother and the child. The genetic factor in which the HLA-DR4 caries indicator was checked also has a strong association between the mother and the offspring. Thus, a strong caries prediction formula was derived through which probability of caries occurrence in the child could be determined prematurely.Thus, it can be concluded that using the clinical and genetic factors, the caries prediction can be done for the child and preventive protocol can be started before disease occurrence.

International audience; Rheumatoid arthritis (RA) is associated with HLA-DRB1 alleles expressing the "shared epitope". RA is usually preceded by the emergence of anti-citrullinated protein autoantibodies (ACPAs). ACPAs recognize citrulline residues on numerous proteins. Conversion of arginine into citrulline is performed by enzymes called peptidyl arginine deiminases (PADs). We have previously demonstrated that C3H mice immunized with PADs can produce ACPAs by a hapten-carrier mechanism. Here, we address the influence of HLA-DR alleles in this model in mice expressing RA-associated HLA-DRB1*04:01 (KO/KI*04:01) or HLA-DRB1*04:04 (KO/KI*04:04) or non RA-associated HLA-DRB1*04:02 (KO/KI*04:02) after murine PAD2 (mPAD2) immunization. Immunization with mPAD2 triggers production of ACPAs in wild type (WT) and HLA-DR4 C57BL/6 mice. Both I-Ab and HLA-DR are involved in the activation of mPAD2 specific T lymphocytes. Among HLA-DR4 mice, mice expressing RA-associated HLA-DRB1*04:01 are the best responders to mPAD2 and the best anti-citrullinated peptide antibody producers. This article is protected by copyright. All rights reserved.

OBJECTIVE: Type 1 diabetes is associated with autoantibodies to different organs that include the gut. The objective of the study was to determine the risk of developing gastric parietal cell autoimmunity in relation to other autoimmunity in individuals with a family history of type 1 diabetes. METHODS: Autoantibodies to the parietal cell autoantigen, H+ /K+ ATPase subunit A (ATP4A) was measured in 2218 first-degree relatives of patients with type 1 diabetes, who were prospectively followed from birth for a median of 14.5 years. All were also tested regularly for the development of islet autoantibodies, transglutaminase autoantibodies, and thyroid peroxidase autoantibodies. RESULTS: The cumulative risk to develop ATP4A autoantibodies was 8.1% (95% CI, 6.6-9.6) by age 20 years with a maximum incidence observed at age 2 years. Risk was increased in females (HR, 1.9; 95% CO, 1.3 - 2.8; P = .0004), relatives with the HLA DR4-DQ8/DR4-DQ8 genotype (HR, 3.4; 95% CI, 1.9 - 5.9; P < .0001) and in participants who also had thyroid peroxidase autoantibodies (HR, 3.7; 95% CI, 2.5-5.5; P < .0001). Risk for at least one of ATP4A-, islet-, transglutaminase-, or thyroid peroxidase-autoantibodies was 24.7% (95% CI, 22.6 - 26.7) by age 20 years and was 47.3% (95% CI, 41.3-53.3) in relatives who had an HLA DR3/DR4-DQ8, DR4-DQ8/DR4-DQ8, or DR3/DR3 genotype (P < .0001 vs other genotypes). CONCLUSIONS: Relatives of patients with type 1 diabetes who have risk genotypes are at very high risk for the development of autoimmunity against gastric and other organs.

We report a Human Immune System (HIS)-humanized mouse model ("DRAGA": HLA-A2.HLA-DR4.Rag1KO.IL-2 RγcKO.NOD) for COVID-19 research. DRAGA mice express transgenically HLA-class I and class-II molecules in the mouse thymus to promote human T cell development and human B cell Ig-class switching. When infused with human hematopoietic stem cells from cord blood reconstitute a functional human immune system, as well as human epi/endothelial cells in lung and upper respiratory airways expressing the human ACE2 receptor for SARS-CoV-2. The DRAGA mice were able to sustain SARS-CoV-2 infection for at least 25 days. Infected mice showed replicating virus in the lungs, deteriorating clinical condition, and human-like lung immunopathology including human lymphocyte infiltrates, microthrombi and pulmonary sequelae. Among the intra-alveolar and peri-bronchiolar lymphocyte infiltrates, human lung-resident (CD103

Accumulating evidence supports a critical role for posttranslationally modified (PTM) islet neoantigens in type 1 diabetes. However, our understanding regarding thymic development and peripheral activation of PTM autoantigen-reactive T cells is still limited. Using HLA-DR4 humanized mice, we observed that deamidation of GAD65115–127 generates a more immunogenic epitope that recruits T cells with promiscuous recognition of both the deamidated and native epitopes and reduced frequency of regulatory T cells. Using humanized HLA/T-cell receptor (TCR) mice, we observed that TCRs reactive to the native or deamidated GAD65115–127 led to efficient development of CD4+ effector T cells; however, regulatory T-cell development was reduced in mice expressing the PTM-reactive TCR, which was partially restored with exogenous PTM peptide. Upon priming, both the native-specific and the deamidated-specific T cells accumulated in pancreatic islets, suggesting that both specificities can recognize endogenous GAD65 and contribute to anti–β-cell responses. Collectively, our observations in polyclonal and single TCR systems suggest that while effector T-cell responses can exhibit cross-reactivity between native and deamidated GAD65 epitopes, regulatory T-cell development is reduced in response to the deamidated epitope, pointing to regulatory T-cell development as a key mechanism for loss of tolerance to PTM antigenic targets.

Immunogenetic as well as environmental and occupational exposures have been linked to the development of rheumatoid arthritis (RA), RA-associated lung disease, and other primary lung disorders. Importantly, various inhalants can trigger post-translational protein modifications, resulting in lung autoantigen expression capable of stimulating pro-inflammatory and/or pro-fibrotic immune responses. To further elucidate gene-environment interactions contributing to pathologic lung inflammation, we exploited an established model of organic dust extract (ODE) exposure with and without collagen-induced arthritis (CIA) in C57BL/6 wild type (WT) versus HLA-DR4 transgenic mice. ODE-induced airway infiltration driven by neutrophils was significantly increased in DR4 versus WT mice, with corresponding increases in bronchoalveolar lavage fluid (BALF) levels of TNF-⍺, IL-6, and IL-33. Lung histopathology demonstrated increased number of ectopic lymphoid aggregates comprised of T and B cells following ODE exposure in DR4 mice. ODE also induced citrullination, malondialdehyde acetaldehyde (MAA) modification, and vimentin expression that co-localized with MAA and was enhanced in DR4 mice. Serum and BALF anti-MAA antibodies were strikingly increased in ODE-treated DR4 mice. Coupling ODE exposure with Type II collagen immunization (CIA) resulted in similarly augmented pro-inflammatory lung profiles in DR4 mice (relative to WT mice) that was accompanied by a profound increase in infiltrating lung CD4+and CD8+T cells as well as CD19+CD11b+autoimmune B cells. Neither modeling strategy induced significant arthritis. These findings support a model in which environmental insults trigger enhanced post-translational protein modification and lung inflammation sharing immunopathological features with RA-associated lung disease in the selected immunogenetic background of HLA-DR4 mice.

Introduction T1DM is the most frequent form of diabetes in children. It has a multifactorial pathogenesis in which genetic, environmental and immunological factors are involved. Among genetic explanations a major role is attributed to second class HLA genes, with the greatest risk associated with the simultaneous presence of the haplotypes DR3DQ2 and DR4DQ8. Based on results obtained in other countries, the aim of this research is to verify a possible association between the haplotype DRB1 * 04: 05-DQA1 * 03-DQB1 * 02 and the onset of T1DM among Italian children with possible genotype-phenotype correlations. Greater knowledge of genes which increase or decrease susceptibility is important for genome analysis. Materials and methods 165 patients with type 1 diabetes treated at the Diabetology Unit of the Meyer Children’s University Hospital, were clinically analyzed. Data relating to age at diagnosis, pancreatic anti-beta cell autoimmunity, comorbidities with date of diagnosis and family history were retrospectively collected from medical data. A case-control study was conducted to investigate the HLA types of the patients compared to a control group of 819 Tuscan donors enrolled in the National Bone Marrow Donor Register. Typing was carried out using the Eurospital "DIABEGEN" kit, currently in use at the immunology laboratory of the Meyer Children’s University Hospital. Results Mean age at diagnosis was 9.3 years; most children (97%) had anti-pancreatic beta cell autoimmunity; the anti-insulin antibody (IAA) was more frequent among children with early clinical disease onset (0–5 years of age). From the case control comparison performed on HLA typing, it emerged that the greatest risk for the development of type 1 diabetes is conferred by the haplotypes DR3DQ2 and DR4DQ8, but in addition to these haplotypes, already known in other countries, we identified another haplotype, DR4DQ2 (DRB1 * 04: 05-DQA1 * 03-DQB1 * 02) which appears to predispose children to type 1 diabetes (p value 2.80E-08) and it is associated with early clinical disease onset (p-value = 0.002). Conclusions We report a new haplotype which increases susceptibility to type 1 diabetes among Italian children and which is associated with early clinical disease onset. Given the central role attributed to genetic factors in the pathogenesis of T1DM and to the II class HLA genes, this new haplotype ought to be recognized as a risk factor and included in tests routinely carried out to identify patients with a genetic predisposition to type I diabetes in Italy. These findings could have practical implications in research and prevention programs.

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.

BackgroundType 1 diabetes is a chronic autoimmune disease that leads to destruction of insulin-producing beta cells and dependence on exogenous insulin for survival. Some interventions have delayed the loss of insulin production in patients with type 1 diabetes, but interventions that might affect clinical progression before diagnosis are needed.MethodsWe conducted a phase 2, randomized, placebo-controlled, double-blind trial of teplizumab (an Fc receptor-nonbinding anti-CD3 monoclonal antibody) involving relatives of patients with type 1 diabetes who did not have diabetes but were at high risk for development of clinical disease. Patients were randomly assigned to a single 14-day course of teplizumab or placebo, and follow-up for progression to clinical type 1 diabetes was performed with the use of oral glucose-tolerance tests at 6-month intervals.ResultsA total of 76 participants (55 [72%] of whom were ≤18 years of age) underwent randomization - 44 to the teplizumab group and 32 to the placebo group. The median time to the diagnosis of type 1 diabetes was 48.4 months in the teplizumab group and 24.4 months in the placebo group; the disease was diagnosed in 19 (43%) of the participants who received teplizumab and in 23 (72%) of those who received placebo. The hazard ratio for the diagnosis of type 1 diabetes (teplizumab vs. placebo) was 0.41 (95% confidence interval, 0.22 to 0.78; P = 0.006 by adjusted Cox proportional-hazards model). The annualized rates of diagnosis of diabetes were 14.9% per year in the teplizumab group and 35.9% per year in the placebo group. There were expected adverse events of rash and transient lymphopenia. KLRG1+TIGIT+CD8+ T cells were more common in the teplizumab group than in the placebo group. Among the participants who were HLA-DR3-negative, HLA-DR4-positive, or anti-zinc transporter 8 antibody-negative, fewer participants in the teplizumab group than in the placebo group had diabetes diagnosed.ConclusionsTeplizumab delayed progression to clinical type 1 diabetes in high-risk participants. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT01030861.).

Typhoid fever is an acute illness in humans, caused by Salmonella typhi, a gram-negative bacterium. Outer membrane proteins of S. typhi have strong potential for its use in the development of subunit vaccine against typhoid. In the current study, peptide-based subunit vaccine was constructed from outer membrane protease E (PgtE) against S. typhi. B cell and T cell epitopes were identified at fold level with a validated three-dimensional modeled structure. T cell epitopes from PgtE (IHPDTSANY) have 99.5% binding to a maximum number of major histocompatibility complex class I and class II alleles. They also bind to the typhoid-resistant human leukocyte antigen (HLA) alleles DRB1*0401. PgtE epitopes were docked with HLA-DR4 (PDB ID: 1D5M) and a contact map was constructed. A simulation search for the binding site for full flexibility of the peptide from CABS- (C

OBJECTIVE This study explored the link between HLA polymorphisms that predispose to type 1 diabetes and birth size, infancy growth and/or circulating IGF-1 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-1 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), more pronounced in boys (P=3x10-7) than girls (P=0.07), and with increasing IGF-1 (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 respectively associated with lower IGF-1 concentrations at age 12 months (P=0.003) and greater skinfold thickness at age 24 months (P=0.003). CONCLUSIONS The variable associations of DR4, DR3 and DQ8 alleles with growth measures and IGF-1 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.

Individuals expressing HLA-DR4 bearing the shared susceptibility epitope (SE) have an increased risk of developing rheumatoid arthritis (RA). Posttranslational modification of self-proteins via citrullination leads to the formation of neoantigens that can be presented by HLA-DR4 SE allomorphs. However, in T cell-mediated autoimmunity, the interplay between the HLA molecule, posttranslationally modified epitope(s), and the responding T cell repertoire remains unclear. In HLA-DR4 transgenic mice, we show that immunization with a Fibβ-74cit69-81 peptide led to a population of HLA-DR4Fibβ-74cit69-81 tetramer+ T cells that exhibited biased T cell receptor (TCR) β chain usage, which was attributable to selective clonal expansion from the preimmune repertoire. Crystal structures of pre- and postimmune TCRs showed that the SE of HLA-DR4 represented a main TCR contact zone. Immunization with a double citrullinated epitope (Fibβ-72,74cit69-81) altered the responding HLA-DR4 tetramer+ T cell repertoire, which was due to the P2-citrulline residue interacting with the TCR itself. We show that the SE of HLA-DR4 has dual functionality, namely, presentation and a direct TCR recognition determinant. Analogous biased TCR β chain usage toward the Fibβ-74cit69-81 peptide was observed in healthy HLA-DR4+ individuals and patients with HLA-DR4+ RA, thereby suggesting a link to human RA.

CD4+ T cells orchestrate adaptive immune responses via binding of antigens to their receptors through specific peptide/MHC-II complexes. To study these responses, it is essential to identify protein-derived MHC-II peptide ligands that constitute epitopes for T cell recognition. However, generating cells expressing single MHC-II alleles and isolating these proteins for use in peptide elution or binding studies is time consuming. Here, we express human MHC alleles (HLA-DR4 and HLA-DQ6) as native, noncovalent αβ dimers on yeast cells for direct flow cytometry-based screening of peptide ligands from selected antigens. We demonstrate rapid, accurate identification of DQ6 ligands from pre-pro-hypocretin, a narcolepsy-related immunogenic target. We also identify 20 DR4-binding SARS-CoV-2 spike peptides homologous to SARS-CoV-1 epitopes, and one spike peptide overlapping with the reported SARS-CoV-2 epitope recognized by CD4+ T cells from unexposed individuals carrying DR4 subtypes. Our method is optimized for immediate application upon the emergence of novel pathogens.

The importance of the human leukocyte antigen (HLA) system in kidney transplantation is well-known, but it remains unexplored if patient HLA antigens constitute independent risk factors in complications after transplantation. We hypothesized that specific HLA class II phenotypes associated with immune-mediated disease (HLA-IMD) predispose to immunological activity and/or complications after kidney transplantation. Based on the literature we defined HLA-DR2-DQ6; -DR3-DQ2 and -DR4-DQ8 as HLA-IMD phenotypes. We investigated associations between HLA-IMD phenotypes in patients, biomarkers of systemic chronic inflammation at the time of transplantation, and the outcome after kidney transplantation in a retrospective cohort study of 611 kidney transplanted patients. The HLA-IMD phenotypes were associated with higher levels of biomarkers of systemic inflammation. The HLA-DR4-DQ8 phenotype was associated with mortality after transplantation in Cox analyses with adjustments for confounders. Data support the hypothesis that specific HLA class II phenotypes affects immunological pathways that determine the midterm clinical outcome of kidney transplantation.

The bioactive sphingolipid ceramide affects immune responses although its effect on antigen (Ag) processing and delivery by HLA class II to CD4+ T-cells remains unclear. Therefore, we examined the actions of a novel cell-permeable acid ceramidase (AC) inhibitor [(1R,2R) N myristoylamino-(4′-nitrophenyl)-propandiol-1,3] on antigen presentation and inflammatory cytokine production by Ag-presenting cells (APCs) such as B-cells, macrophages, and dendritic cells. We found that AC inhibition in APCs perturbed Ag-processing and presentation via HLA-DR4 (MHC class II) proteins as measured by coculture assay and T-cell production of IL-2. Mass spectral analyses showed that B13 treatment significantly raised levels of four types of ceramides in human B-cells. B13 treatment did not alter Ag internalization and class II protein expression, but significantly inhibited lysosomal cysteinyl cathepsins (B, S and L) and thiol-reductase (GILT), HLA class II Ag-processing, and generation of functional class II-peptide complexes. Ex vivo Ag presentation assays showed that inhibition of AC impaired primary and recall CD4+ T-cell responses and cytokine production in response against type II collagen. Further, B13 delayed onset and reduced severity of inflamed joints and cytokine production in the collagen-induced arthritis mouse model in vivo. These findings suggest that inhibition of AC in APCs may dysregulate endolysosomal proteases and HLA class II-associated self-antigen presentation to CD4+ T-cells, attenuating inflammatory cytokine production and suppressing host autoimmune responses.

T cells are vital for adaptive immune responses that protect against pathogens and cancers. The T cell receptor (TCR)-CD3 complex comprises a diverse αβ TCR heterodimer in noncovalent association with three invariant CD3 dimers. The TCR is responsible for recognizing antigenic peptides bound to MHC molecules (pMHC), while the CD3 dimers relay activation signals to the T cell. However, the mechanisms by which TCR engagement by pMHC is transmitted to CD3 remain mysterious, although there is growing evidence that mechanosensing and allostery both play a role. Here, we carried out NMR analysis of a human autoimmune TCR (MS2-3C8) that recognizes a self-peptide from myelin basic protein presented by the MHC class II molecule HLA-DR4. We observed pMHC-induced NMR signal perturbations in MS2-3C8 that indicate long-range effects on TCR β chain conformation and dynamics. Our results demonstrate that, in addition to expected changes in the NMR resonances of pMHC-contacting residues, perturbations extend to the Vβ/Vα, Vβ/Cβ, and Cβ/Cα interfacial regions. Moreover, the pattern of long-range perturbations is similar to that detected previously in the β chains of two MHC class I-restricted TCRs, thereby revealing a common allosteric pathway among three unrelated TCRs. Molecular dynamics (MD) simulations predict similar pMHC-induced effects. Taken together, our results demonstrate that pMHC binding induces long-range allosteric changes in the TCR β chain at conserved sites in both representative MHC class I- and class II-restricted TCRs, and that these sites may play a role in the transmission of signaling information.

We have engineered a Human Immune System (HIS)-reconstituted mouse strain (DRAGA mouse: HLA-A2. HLA-DR4. Rag1 KO. IL-2Rγc KO. NOD) in which the murine immune system has been replaced by a long-term, functional HIS via infusion of CD34(+) hematopoietic stem cells (HSC) from cord blood. Herein, we report that the DRAGA mice can sustain inducible and transmissible H1N1 and H3N2 influenza A viral (IAV) infections. DRAGA female mice were significantly more resilient than the males to the H3N2/Aichi infection, but not to H3N2/Hong Kong, H3N2/Victoria, or H1N1/PR8 sub-lethal infections. Consistently associated with large pulmonary hemorrhagic areas, both human and murine Factor 8 mRNA transcripts were undetectable in the damaged lung tissues but not in livers of DRAGA mice advancing to severe H1N1/PR8 infection. Infected DRAGA mice mounted a neutralizing anti-viral antibody response and developed lung-resident CD103 T cells. These results indicate that the DRAGA mouse model for IAV infections can more closely approximate the human lung pathology and anti-viral immune responses compared to non-HIS mice. This mouse model may also allow further investigations into gender-based resilience to IAV infections, and may potentially be used to evaluate the efficacy of IAV vaccine regimens for humans.

OBJECTIVE: Mechanisms underlying the role of non-human leukocyte antigen (HLA) genetic risk variants in type 1 diabetes (T1D) are poorly understood. We aimed to test the association between methylation and non-HLA genetic risk. METHODS: We conducted a methylation quantitative trait loci (mQTL) analysis in a nested case-control study from the Dietary Autoimmunity Study in the Young. Controls (n = 83) were frequency-matched to T1D cases (n = 83) based on age, race/ethnicity, and sample availability. We evaluated 13 non-HLA genetic markers known be associated with T1D. Genome-wide methylation profiling was performed on peripheral blood samples collected prior to T1D using the Illumina 450 K (discovery set) and infinium methylation EPIC beadchip (EPIC validation) platforms. Linear regression models, adjusting for age and sex, were used to test to each single nucleotide polymorphism (SNP) -probe combination. Logistic regression models were used to test the association between T1D and methylation levels among probes with a significant mQTL. A meta-analysis was used to combine odds ratios from the two platforms. RESULTS: We identified 10 SNP-methylation probe pairs (false discovery rate (FDR) adjusted P < .05 and validation P < .05). Probes were associated with the GSDMB, C1QTNF6, IL27, and INS genes. The cg03366382 (OR: 1.9, meta-P = .0495), cg21574853 (OR: 2.5, meta-P = .0232), and cg25336198 (odds ratio: 6.6, meta-P = .0081) probes were significantly associated with T1D. The three probes were located upstream from the INS transcription start site. CONCLUSIONS: We confirmed an association between DNA methylation and rs689 that has been identified in related studies. Measurements in our study preceded the onset of T1D suggesting methylation may have a role in the relationship between INS variation and T1D development.

Vogt-Koyanagi-Harada (VKH) disease is a systemic inflammatory disorder that affects pigment cell-containing organs such as the eye (e.g., chronic and/or recurrent granulomatous panuveitis). While the exact etiology and pathogenic mechanism of VKH disease are unclear, HLA-DR4 alleles have been documented to be strongly associated with VKH disease in various ethnic groups. Recently, a genome-wide association study (GWAS) found two new genetic risk factors (IL23R-C1orf141 and ADO-ZNF365-EGR2) in a non-HLA region from a Han Chinese population. In this study, we replicated these GWAS findings in a Japanese population. A total of 1,643 Japanese samples (380 cases with VKH disease and 1,263 healthy controls) were recruited. We assessed four single nucleotide polymorphisms (SNPs) shown in previous GWAS: rs78377598 and rs117633859 in IL23R-C1orf141, and rs442309 and rs224058 in ADO-ZNF365-EGR2. A significant allelic association with VKH disease was observed for all of the four SNPs (rs78377598: pc = 0.0057; rs117633859: pc = 0.0017; rs442309: pc = 0.021; rs224058: pc = 0.035). In genotypic association analysis, the minor alleles of IL23R-C1orf141 rs78377598 and rs117633859 had the strongest association with disease susceptibility under the additive model (pc = 0.0075 and pc = 0.0026, respectively). The minor alleles of ADO-ZNF365-EGR2 rs442309 and rs224058 were most strongly associated with disease susceptibility under the dominant model (pc = 0.00099 and pc = 0.0023, respectively). The meta-analysis of the current and previous studies found that all of the four SNPs exhibited a significantly strong association with VKH disease (meta-p < 0.00001: rs78377598, meta-odds ratio (OR) = 1.69; rs1176338, meta-OR = 1.82; rs442309, meta-OR = 1.34; rs224058, meta-OR = 1.33). In summary, our study replicated significant associations with VKH disease susceptibility reported in a previous GWAS. Thus, the IL23R-C1orf141 and ADO-ZNF365-EGR2 loci may play important roles in the development of VKH disease through genetic polymorphisms.

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.

Typhoid fever is a severe illness in humans, caused by Salmonella typhi, a Gram-negative bacterium. Membrane proteins of S. typhi have strong potential for its use in development of subunit vaccine against typhoid. In current study, peptide-based subunit vaccine constructed from AI-2 import ATP-binding cassette transporter protein (LsrA) against S. typhi. B-cell and T-cell epitopes were identified at fold level with validated 3-D theoretical modelled structure. T-cell epitope from LsrA (LELPGSRPQ) has binds to maximum number (82.93%) of MHC class I and class II alleles. LsrA epitope was docked with HLA-DR4 and contact map were constructed to analyze molecular interaction (docking) studies. Simulation search for the binding site for full flexibility of the peptide from CABS-dock shows the stable interactions. MD simulation analysis reveals that LsrA epitope was binding and interacting firmly with the HLA-DR4. Hence, we are proposing that LsrA epitope would be a prominent epitope vaccine for human specific pathogen of S. typhi, which requires further steps to be elevated as a vaccine drug in near future.

Antigen-specific immunotherapy of autoimmune disease currently remains the only potentially curative approach. However, translation of promising pre-clinical results into successful clinical application has proven challenging. In part, this is because pre-clinical findings in mouse models have to be redesigned for human application due to differences in MHC II. To reduce the gap between pre-clinical and clinical studies, we have created a novel mouse model that expresses human HLA-DR4, but no endogenous MHC on antigen-presenting cells. Moreover, human B7.1 (CD80) is expressed in the pancreatic islets under the control of the rat insulin promoter. Although this model does not develop diabetes spontaneously, it is susceptible to the induction of type 1 diabetes by challenging mice with overlapping peptides derived from murine proinsulin-2 in adjuvant. Unlike the NOD model of spontaneous type 1 diabetes, but akin to the human condition, this model does not have a gender bias. Furthermore, similar to the human condition, the disease is characterised by a diverse leucocyte infiltration of the pancreatic islets and the formation of anti-proinsulin auto-antibodies. The model that we report here offers detailed insights into type-1 diabetes and is expected to prove instrumental when studying the mechanism of action in translational, antigen-specific immunotherapy.

Aim: To investigate the clinical significance of detecting several biomarkers collectively in the diagnosis of rheumatoid arthritis (RA). Methods: 128 RA patients, 174 non-RA patients and 80 healthy controls were enrolled. HLA-DR4 and HLA-DR53 were detected by the PCR-SSP method, 14-3-3η protein, anti-CCP and anti-Sa were detected by ELISA and DD was detected by latex immunoturbidimetric assay. Results: The positive rates of HLA-DR4, HLA-DR53, 14-3-3η protein, anti-CCP and anti-Sa were obviously higher in the RA group (43.8, 38.3, 51.6, 80 and 40.6%, respectively); anti-CCP was of highest sensitivity (79.68%), highest specificity (97.5%) and Youden index (0.77). The AUC of 14-3-3η protein, DD, anti-CCP, anti-Sa were 0.813, 0.859, 0.930, 0.861, respectively. Conclusion: All biomarkers were strongly correlated risk factors for RA; the combination of multiple biomarkers might be of help for diagnostic and therapeutic strategies in RA of recent onset.

The mucosal tissues of the gut and female reproductive tract (FRT) are susceptible to pathogen infections including bacteria, viruses, and parasites, and are also the targets for immune disorders such as Crohn's disease, inflammatory bowel disease (IBD), and many types of cancers. However, the role of the mucosal immune cells to control these diseases is largely unknown. The limited availability of human mucosal biopsy tissue and the low number of cells that can be isolated from these tissues hampers the characterization of the phenotype and function of human mucosal immune cell subsets. Therefore, human-immune-system humanized mice are surrogate models to investigate the human mucosal immune cell responses during the course of the disease. The current protocols used to harvest the immune cells from the mucosal tissues, however, result in low recovery of cells with poor viability. We have established a novel protocol, which results in a high yield of human lymphocytes with high viability to overcome this issue. The immune cells obtained from a single DRAG mouse by our protocol were sufficient for conducting functional assays and for flow cytometry analyses including phenotypic, exhaustion, and functional panels.

Objective:Both environmental and genetic factors are important in antiphospholipid antibody(aPL) development in antiphospholipid syndrome(APS). Currently, the only available data on predisposing genetic factors have been obtained from epidemiological studies without mechanistic evidence. Therefore, we studied the influence of Major Histocompatibility Complex Class II(MHC-II) alleles on the production of aPL in an APS mouse model. Methods:Three groups of mice: MHC-II deficient (MHC-II-/-) mice, MHC-II-/- mice transgenic for human DQ6, DQ8 or DR4 alleles and their corresponding wild-type(WT) strains were each immunized, half with human β2glycoprotein-I(β2GPI) and the other half with control ovalbumin(OA) protein. Thrombus formation in-vivo, tissue factor(TF) activity in carotids and peritoneal macrophages(PMs) and serum levels of tumor necrosis factor-α(TNFα), IgG anticardiolipin(aCL), anti-β2GPI and anti-OA were determined. Results:β2GPI immunization induced significant aCL and anti-β2GPI production in WT compared to control mice immunized with OA(p

OBJECTIVE We tested the associations between genetic background and selected environmental exposures with respect to islet autoantibodies and type 1 diabetes. RESEARCH DESIGN AND METHODS Infants with HLA-DR high-risk genotypes were prospectively followed for diabetes-related autoantibodies. Single nucleotide polymorphisms (SNPs) came from the Illumina ImmunoChip and environmental exposure data were by parental report. Children were followed to age 6 years. RESULTS Insulin autoantibodies occurred earlier than GAD antibody (GADA) and then declined, while GADA incidence rose and remained constant (significant in HLA-DR4 but not in the DR3/3 children). The presence of SNPs rs2476601 (PTPN22) and rs2292239 (ERBB3) demonstrated increased risk of both autoantibodies to insulin (IAA) only and GADA only. SNP rs689 (INS) was protective of IAA only, but not of GADA only. The rs3757247 (BACH2) SNP demonstrated increased risk of GADA only. Male sex, father or sibling as the diabetic proband, introduction of probiotics under 28 days of age, and weight at age 12 months were associated with IAA only, but only father as the diabetic proband and weight at age 12 months were associated with GADA only. Mother as the diabetic proband was not a significant risk factor. CONCLUSIONS These results show clear differences in the initiation of autoimmunity according to genetic factors and environmental exposures that give rise to IAA or GADA as the first appearing indication of autoimmunity.

Myocarditis, the principal cause of dilated cardiomyopathy and heart failure in young adults, is associated with autoimmunity to human cardiac α-myosin (hCAM) and the DR4 allele of human major histocompatibility II (MHCII). We developed an hCAM-induced myocarditis model in human HLA-DR4 transgenic mice that lack all mouse MHCII genes, demonstrating that immunization for 3 weeks significantly increased splenic T-cell proliferative responses and titres of IgG1 and IgG2c antibodies, abolished weight gain, provoked cardiac inflammation and significantly impaired cardiac output and fractional shortening, by echocardiography, compared to adjuvant-injected mice. Neither cardiac dilatation nor fibrosis occurred at this time point but prolonging the experiment was associated with mortality. Treatment with mixtures of hCAM derived peptides predicted to have high affinity for DR4 significantly preserved ejection fraction and fractional shortening. Our new humanized mouse model of autoimmune cardiomyopathy should be useful to refine hCAM-derived peptide treatment., Highlights • We developed a novel fully-humanized mouse model of autoimmune myocarditis. • Human leukocyte antigen DR4 transgenic mice were immunized with human cardiac myosin. • After 3 weeks, mice developed myocarditis and impaired myocardial function. • A pilot study established the feasibility of peptide immunotherapy in this model.

Programmed cell death-1 protein (PD-1) is an immune checkpoint that has gained popularity in the treatment of several advanced cancers. Inhibiting this checkpoint is known to enhance immune response, but is also known to diminish immune tolerance and to increase autoimmune toxicity. We discuss a case of rapid onset fulminant Type 1 diabetes induced by treatment with anti-programmed cell death-1 monoclonal antibody, nivolumab, in a patient with late-stage non-small-cell lung adenocarcinoma. The patient had no history of previous diabetes but did reveal a high-risk genotype for Type 1 diabetes development (DR3-DQ2; DR4-DQ8). This finding supports that acute Type 1 diabetes can be an important adverse effect of immunotherapies targeting T-cell activation regulation. Because of the severity of this adverse effect, physicians should be aware of it, and studies directed to the detection of new biomarkers for early risk stratification (e.g., HLA) should be sought.

International audience; Vogt-Koyanagi-Harada (VKH) syndrome is a rare condition implicating systemic immune reaction against melanocytes. The pathophysiology is unclear. A genetic predisposition has been suggested as HLA-DR4/DRB1*04 is more common among VKH patients. Drug induced VKH syndrome has been reported in advanced melanoma patients receiving immunotherapy, including ipilimumab and adoptive cell transfer of Tumor-Infiltrating Lymphocyte associated with IL-2. To date, no case of anti PD-1 -induced VKH syndrome has been described. We report here the case of a HLA-DR4/DRB1*04 patient successfully treated with anti PD-1 for advanced melanoma who developed a systemic immune reaction against melanocytes for whom we discuss a VKH-like syndrome diagnosis in a potentially genetically predisposed patient

Background and aims Type 1 diabetes (T1D) is a multifactorial autoimmune disease that combines genetics and environmental factors. The aim of this study is to determine the environmental risk factors and to investigate how virals infections are risks factors for type 1 diabetics whom have HLA DR3/DR4 predisposition in our population. Methods This study includes 233 subjects, 145 diabetics and 88 controls from regions of the extreme western of Algeria. All the informations related to the disease were collected using predesigned questionnaire. Using in silico approach, we attempt to improve the understanding of this analytical result by molecular mimicry, which is associated with the breakdown of several autoimmune pathologies. Results The statistical study showed that history of varicella and measles infection and T1D related inheritance and type 2 diabetes are risk factors for T1D in the population of Tlemcen. We have determined the homologous antigenic regions between the glycoprotein “gE” of the varicella virus, the “hemagglutinin” of measles and the human protein “HSP60” at the level of their sequence and 3D structure. These cross-reactive epitopes bind to MHC class II molecules (HLA DR3/DR4) that predispose to T1D but not to MHC class II molecules (HLA DR2) that protect against T1D. This epitopes induce Th2 cells but only “hemagglutinin” and “Hsp60” can activate Th1 differentiation. This indicates their potential to destroy pancreatic cells β. Conclusion Our study can allow us to adapt biological markers to genetically predisposed T1D and to establish a preventive strategy for healthy genetic predisposed individuals in Tlemcen population.