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.

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.

DNA methylation near the INS gene is associated with INS genetic variation (rs689) and type 1 diabetes in the Diabetes Autoimmunity Study in the Young.

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.

Cepharanthine blocks TSH receptor peptide presentation by HLA-DR3: Therapeutic implications to Graves' disease.

We have previously identified a signature HLA-DR3 pocket variant, designated HLA-DRß1-Arg74 that confers a high risk for Graves' Disease (GD). In view of the key role of HLA-DRß1-Arg74 in triggering GD we hypothesized that thyroid-stimulating hormone receptor (TSHR) peptides that bind to the HLA-DRß1-Arg74 pocket with high affinity represent key pathogenic TSHR peptides triggering GD, and that blocking their presentation to CD4+ T-cells can be used as a novel therapeutic approach in GD. There were several previous attempts to identify the major pathogenic TSHR peptide utilizing different methodologies, however the results were inconsistent and inconclusive. Therefore, the aim of our study was to use TSHR peptide binding affinity to HLA-DRß1-Arg74 as a method to identify the key pathogenic TSHR peptides that trigger GD. Using virtual screening and ELISA and cellular binding assays we identified 2 TSHR peptides that bound with high affinity to HLA-DRß1-Arg74 - TSHR.132 and TSHR.197. Peptide immunization studies in humanized DR3 mice showed that only TSHR.132, but not TSHR.197, induced autoreactive T-cell proliferation and cytokine responses. Next, we induced experimental autoimmune Graves' disease (EAGD) in a novel BALB/c-DR3 humanized mouse model we created and confirmed TSHR.132 as a major DRß1-Arg74 binding peptide triggering GD in our mouse model. Furthermore, we demonstrated that Cepharanthine, a compound we have previously identified as DRß1-Arg74 blocker, could block the presentation and T-cell responses to TSHR.132 in the EAGD model.

Increasing Levels of Serum Heat Shock Protein 70 Precede the Development of AIDS-Defining Non-Hodgkin Lymphoma Among Carriers of HLA-B8-DR3.

BACKGROUND: We hypothesized that carriage of presumably high Hsp70-producing gene variants on a specific human major histocompatibility complex haplotype, the 8.1 ancestral haplotype (8.1AH), may predispose HIV-infected individuals to AIDS-non-Hodgkin lymphoma (NHL). SETTING: We compared serum Hsp70 levels in the years preceding the diagnosis of AIDS-NHL in a matched case-control study (n = 151 pairs) nested in the Multicenter AIDS Cohort Study. METHODS: We tested the impact of 8.1AH-specific single-nucleotide polymorphism (SNP) and joint SNP-human leukocyte antigen extended haplotypes previously associated with AIDS-NHL in the Multicenter AIDS Cohort Study on the circulating Hsp70 levels in mixed linear models. RESULTS: We report elevated serum levels of Hsp70 in the 4 years preceding the diagnosis of AIDS-NHL in cases that carry 8.1AH, but not in noncarrier cases and not in carrier- or non-carrier-matched controls. The strongest predictor of higher serum Hsp70 was the haplotype A-G-A-C formed by SNPs rs537160(A) and rs1270942(G) in the complement factor CFB gene cluster, and rs2072633(A) and rs6467(C) in nearby RDBP and CYP21A2 located 70 Kb apart from the Hsp70 gene cluster. The association with A-G-A-C haplotype (beta = 0.718; standard error = 0.182; P = 0.0002) and with other 8.1AH-specific haplotypes including the high-producing tumor necrosis factor-alpha haplotype rs909253(G)-rs1800629(A) (beta = 0.308; standard error = 0.140; P = 0.032) were observed only with NHL identified as an AIDS-defining condition, but not as a post-AIDS condition, nor in combined AIDS and post-AIDS cases. CONCLUSION: Our combined genetic and functional approach suggests that the altered level of Hsp70 is a correlate of 8.1AH-mediated AIDS-NHL. Further investigation of the Hsp70 gene cluster and nearby loci that are tagged by A-G-A-C could better elucidate the genetic determinants of the malignancy.

Immunotherapy With Apitopes Blocks the Immune Response to TSH Receptor in HLA-DR Transgenic Mice.

We have combined major histocompatibility complex-binding assays with immunization and tolerance induction experiments in HLA-DR3 transgenic mice to design apitopes (antigen-processing independent epitopes) derived from thyrotropin receptor (TSHR) for treatment of patients with Graves' disease (GD). A challenge model was created by using an adenovirus-expressing part of the extracellular domain of the thyrotropin receptor (TSHR289). This model was used to test whether current drug treatments for GD would have an impact on effective antigen-specific immunotherapy using the apitope approach. Furthermore, selected peptides were assessed for their antigenicity using peripheral blood mononuclear cell samples from patients with GD. A mixture of two immunodominant apitopes was sufficient to suppress both the T-cell and antibody response to TSHR when administered in soluble form to HLA-DR transgenic mice. Tolerance induction was not disrupted by current drug treatments. These results demonstrate that antigen-specific immunotherapy with apitopes from TSHR is a suitable approach for treatment of GD.

A quarter of patients with type 1 diabetes have co-existing non-islet autoimmunity: the findings of a UK population-based family study.

Individuals with type 1 diabetes (T1D) are at increased risk of coeliac disease (CD), autoimmune thyroiditis and autoimmune gastritis, but the absolute risks are unclear. The aim of this study was to investigate the prevalence of autoantibodies to tissue transglutaminase (TGA), thyroid peroxidase (TPOA) and gastric H+ /K+ -ATPase (ATPA) and their genetic associations in a well-characterized population-based cohort of individuals with T1D from the Bart's-Oxford family study for whom islet autoantibody prevalence data were already available. Autoantibodies in sera from 1072 patients (males/females 604/468; median age 11·8 years, median T1D duration 2·7 months) were measured by radioimmunoassays; HLA class II risk genotype was analysed in 973 (91%) using polymerase chain reaction with sequence specific primers (PCR-SSP). The prevalence of TGA (and/or history of CD), TPOA and ATPA in patients was 9·0, 9·6 and 8·2%, respectively; 3·1% had two or more autoantibodies. Females were at higher risk of multiple autoimmunity; TGA/CD were associated with younger age and TPOA with older age. ATPA were uncommon in patients under 5 years, and more common in older patients. Anti-glutamate decarboxylase autoantibodies were predictive of co-existing TPOA/ATPA. TGA/CD were associated with human leucocyte antigen (HLA) DR3-DQ2, with the DR3-DQ2/DR3-DQ2 genotype conferring the highest risk, followed by DR4-DQ8/DR4-DQ8. ATPA were associated with DR3-DQ2, DRB1*0404 (in males) and the DR3-DQ2/DR4-DQ8 genotype. TPOA were associated with the DR3-DQ2/DR3-DQ2 genotype. Almost one-quarter of patients diagnosed with T1D aged under 21 years have at least one other organ-specific autoantibody. HLA class II genetic profiling may be useful in identifying those at risk of multiple autoimmunity.

A case of fulminant Type 1 diabetes following anti-PD1 immunotherapy in a genetically susceptible patient.

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.

Of the multiple mechanisms leading to type 1 diabetes, T cell receptor revision may play a prominent role (is type 1 diabetes more than a single disease?).

A single determinant factor for autoimmunity does not exist; disease development probably involves contributions from genetics, the environment and immune dysfunction. Type 1 diabetes is no exception. Genomewide-associated studies (GWAS) analysis in T1D has proved disappointing in revealing contributors to disease prediction; the only reliable marker has been human leucocyte antigen (HLA). Specific HLAs include DR3/DR4/DQ2/DQ8, for example. Because HLA molecules present antigen to T cells, it is reasonable that certain HLA molecules have a higher affinity to present self-antigen. Recent studies have shown that additional polymorphisms in HLA that are restricted to autoimmune conditions are further contributory. A caveat is that not all individuals with the appropriate 'pro-autoimmune' HLA develop an autoimmune disease. Another crucial component is autoaggressive T cells. Finding a biomarker to discriminate autoaggressive T cells has been elusive. However, a subset of CD4 helper cells that express the CD40 receptor have been described as becoming pathogenic. An interesting function of CD40 on T cells is to induce the recombination-activating gene (RAG)1/RAG2 T cell receptor recombination machinery. This observation is contrary to immunology paradigms that changes in TCR molecules cannot take place outside the thymic microenvironment. Alteration in TCR, called TCR revision, not only occurs, but may help to account for the development of autoaggressive T cells. Another interesting facet is that type 1 diabetes (T1D) may be more than a single disease; that is, multiple cellular components contribute uniquely, but result ultimately in the same clinical outcome, T1D. This review considers the process of T cell maturation and how that could favor auto-aggressive T cell development in T1D. The potential contribution of TCR revision to autoimmunity is also considered.

HLA-DR3 antigen in the resistance to idiopathic dilated cardiomyopathy.

Idiopathic dilated cardiomyopathy (IDC) has been hypothesized as a multifactorial disorder initiated by an environment trigger in individuals with predisposing human leukocyte antigen (HLA) alleles. Published data on the association between HLA-DR3 antigen and IDC risk are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. Studies were identified by searching the PUBMED and Embase database (starting from June 2015). A total of 19 case-control studies including 1378 cases and 10383 controls provided data on the association between HLA-DR3 antigen and genetic susceptibility to IDC. Overall, significantly decreased frequency of HLA-DR3 allele (OR=0.72; 95%CI=0.58-0.90; P=0.004) was found in patients with IDC compared with controls. When stratified by myocardial biopsy or non-biopsy cases, statistically decreased risk was found for IDC in myocardial biopsy cases (OR=0.69; 95%CI=0.57-0.84; P=0.0003). In the subgroup analysis by ethnicity, borderline statistically significantly decreased risk was found among Europeans from 12 case-control studies (OR=0.76; 95%CI=0.58-1.00; P=0.05). In conclusion, our results suggest that individuals with HLA-DR3 antigen may have a protective effect against IDC.

HLA-DR3 antigen in the resistance to idiopathic dilated cardiomyopathy

Idiopathic dilated cardiomyopathy (IDC) has been hypothesized as a multifactorial disorder initiated by an environment trigger in individuals with predisposing human leukocyte antigen (HLA) alleles. Published data on the association between HLA-DR3 antigen and IDC risk are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. Studies were identified by searching the PUBMED and Embase database (starting from June 2015). A total of 19 case-control studies including 1378 cases and 10383 controls provided data on the association between HLA-DR3 antigen and genetic susceptibility to IDC. Overall, significantly decreased frequency of HLA-DR3 allele (OR=0.72; 95%CI=0.58-0.90; P=0.004) was found in patients with IDC compared with controls. When stratified by myocardial biopsy or non-biopsy cases, statistically decreased risk was found for IDC in myocardial biopsy cases (OR=0.69; 95%CI=0.57-0.84; P=0.0003). In the subgroup analysis by ethnicity, borderline statistically significantly decreased risk was found among Europeans from 12 case-control studies (OR=0.76; 95%CI=0.58-1.00; P=0.05). In conclusion, our results suggest that individuals with HLA-DR3 antigen may have a protective effect against IDC.

A Central Role for HLA-DR3 in Anti-Smith Antibody Responses and Glomerulonephritis in a Transgenic Mouse Model of Spontaneous Lupus.

MHC, especially HLA-DR3 and HLA-DR2, is one of the most important genetic susceptibility regions for systemic lupus erythematosus. Human studies to understand the role of specific HLA alleles in disease pathogenesis have been hampered by the presence of strong linkage disequilibrium in this region. To overcome this, we produced transgenic mice expressing HLA-DR3 (DRß1*0301) and devoid of endogenous class II (both I-A and I-E genes, AE(0)) on a lupus-prone NZM2328 background (NZM2328.DR3(+)AE(0)). Both NZM2328 and NZM2328.DR3(+)AE(0) mice developed anti-dsDNA and glomerulonephritis, but anti-dsDNA titers were higher in the latter. Although kidney histological scores were similar in NZM2328 and NZM2328.DR3(+)AE(0) mice (7.2 ± 4.3 and 8.6 ± 5.7, respectively, p = 0.48), the onset of severe proteinuria occurred earlier in NZM2328.DR3(+)AE(0) mice compared with NZM2328 mice (median, 5 and 9 mo respectively, p < 0.001). Periarterial lymphoid aggregates, classic wire loop lesions, and occasional crescents were seen only in kidneys from NZM2328.DR3(+)AE(0) mice. Interestingly, NZM2328.DR3(+)AE(0) mice, but not NZM2328 mice, spontaneously developed anti-Smith (Sm) Abs. The anti-Sm Abs were seen in NZM2328.DR3(+)AE(0) mice that were completely devoid of endogenous class II (AE(-/) (-)) but not in mice homozygous (AE(+/+)) or heterozygous (AE(+/-)) for endogenous MHC class II. It appears that only HLA-DR3 molecules can preferentially select SmD-reactive CD4(+) T cells for generation of the spontaneous anti-Sm immune response. Thus, our mouse model unravels a critical role for HLA-DR3 in generating an autoimmune response to SmD and lupus nephritis in the NZM2328 background.

Autoimmune hepatitis: East meets west.

Autoimmune hepatitis (AIH) is an inflammatory liver disease with diverse clinical spectrum, which predominantly affects females. This review provides detailed comparisons of epidemiology, genetic predispositions, clinical features, risk factors of hepatocellular carcinoma, and mortality in AIH patients between eastern and western countries. AIH prevalence and incidence are lower in Asia-Pacific area than in Europe and America. European and American patients seem to have more severe disease, characterized with human leukocyte antigen-DR3 haplotype, younger age, more AIH-induced "cirrhosis" at diagnosis, higher elevated serum immunoglobulin G levels, and positive rate of antisoluble liver antigen/liver pancreatitis. The overall AIH diagnostic accuracy of revised original criteria and simplified scoring system are similar in European/American populations and Asian. Cirrhosis at presentation and non-response to immunosuppressive therapy within 1 year are the most important predictors for poor prognosis of AIH patients.

Flow cytometric analysis of the graft-versus-Leukemia-effect after hematopoietic stem cell transplantation in mice.

Acute Graft-versus-Host-Disease (aGvHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Although rather helpful, the use of conventional immunosuppressive drugs leads to general immunosuppression and is toxic. The effects of CD4(+) T-cells, in respect to the development of aGvHD, can be altered by administration of antihuman CD4 monoclonal antibodies, here MAX.16H5 IgG1 . This approach must be tested for possible interference with the Graft-versus-Leukemia-Effect (GvL). Thus, in vitro experiments were conducted, exposing P815 leukemic cells to bone marrow and splenocytes from cd4(-/-) -C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice, [TTG]) as well as previously irradiated splenocytes from Balb/c(wt) mice. Using flow cytometry, the vitality of the various malignant and graft cells was analyzed over the course of 4 days. The survival rate of P815 cells did not change significantly when exposed to MAX.16H5 IgG1 , neither did the viability of the graft cells. This provides evidence that MAX.16H5 IgG1 does not impair the GvL effect in vitro. Additionally, P815-Balb/c(wt) leukemic mice were transplanted with P815(GFP) cells, bone marrow, and splenocytes from TTG mice with and without MAX.16H5 IgG1 . Without transplantation, P815(GFP) leukemic cells could be detected by flow cytometry in the liver, the bone marrow, and the spleen of recipients. The antibodies prevented aGvHD while leaving the GvL effect intact. These findings indicate no negative effect of MAX.16H5 IgG1 on the GvL effect in vitro and in vivo after HSCT in a murine model.

Systemic inflammatory response elicited by superantigen destabilizes T regulatory cells, rendering them ineffective during toxic shock syndrome.

Life-threatening infections caused by Staphylococcus aureus, particularly the community-acquired methicillin-resistant strains of S. aureus, continue to pose serious problems. Greater virulence and increased pathogenicity of certain S. aureus strains are attributed to higher prevalence of exotoxins. Of these exotoxins, the superantigens (SAg) are likely most pathogenic because of their ability to rapidly and robustly activate the T cells even in extremely small quantities. Therefore, countering SAg-mediated T cell activation using T regulatory cells (Tregs) might be beneficial in diseases such as toxic shock syndrome (TSS). As the normal numbers of endogenous Tregs in a typical host are insufficient, we hypothesized that increasing the Treg numbers by administration of IL-2/anti-IL-2 Ab immune complexes (IL2C) or by adoptive transfer of ex vivo expanded Tregs might be more effective in countering SAg-mediated immune activation. HLA-DR3 transgenic mice that closely recapitulate human TSS were treated with IL2C to increase endogenous Tregs or received ex vivo expanded Tregs. Subsequently, they were challenged with SAg to induce TSS. Analyses of various parameters reflective of TSS (serum cytokine/chemokine levels, multiple organ pathology, and SAg-induced peripheral T cell expansion) indicated that increasing the Tregs failed to mitigate TSS. On the contrary, serum IFN-γ levels were increased in IL2C-treated mice. Exploration into the reasons behind the lack of protective effect of Tregs revealed IL-17 and IFN-γ-dependent loss of Tregs during TSS. In addition, significant upregulation of glucocorticoid-induced TNFR family-related receptor on conventional T cells during TSS could render them resistant to Treg-mediated suppression, contributing to failure of Treg-mediated immune regulation.

Risk of pediatric celiac disease according to HLA haplotype and country.

BACKGROUND: The presence of HLA haplotype DR3-DQ2 or DR4-DQ8 is associated with an increased risk of celiac disease. In addition, nearly all children with celiac disease have serum antibodies against tissue transglutaminase (tTG). METHODS: We studied 6403 children with HLA haplotype DR3-DQ2 or DR4-DQ8 prospectively from birth in the United States, Finland, Germany, and Sweden. The primary end point was the development of celiac disease autoimmunity, which was defined as the presence of tTG antibodies on two consecutive tests at least 3 months apart. The secondary end point was the development of celiac disease, which was defined for the purpose of this study as either a diagnosis on biopsy or persistently high levels of tTG antibodies. RESULTS: The median follow-up was 60 months (interquartile range, 46 to 77). Celiac disease autoimmunity developed in 786 children (12%). Of the 350 children who underwent biopsy, 291 had confirmed celiac disease; an additional 21 children who did not undergo biopsy had persistently high levels of tTG antibodies. The risks of celiac disease autoimmunity and celiac disease by the age of 5 years were 11% and 3%, respectively, among children with a single DR3-DQ2 haplotype, and 26% and 11%, respectively, among those with two copies (DR3-DQ2 homozygosity). In the adjusted model, the hazard ratios for celiac disease autoimmunity were 2.09 (95% confidence interval [CI], 1.70 to 2.56) among heterozygotes and 5.70 (95% CI, 4.66 to 6.97) among homozygotes, as compared with children who had the lowest-risk genotypes (DR4-DQ8 heterozygotes or homozygotes). Residence in Sweden was also independently associated with an increased risk of celiac disease autoimmunity (hazard ratio, 1.90; 95% CI, 1.61 to 2.25). CONCLUSIONS: Children with the HLA haplotype DR3-DQ2, especially homozygotes, were found to be at high risk for celiac disease autoimmunity and celiac disease early in childhood. The higher risk in Sweden than in other countries highlights the importance of studying environmental factors associated with celiac disease. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and others.).

Autoantibody and human leukocyte antigen profiles in children with autoimmune liver disease and their first-degree relatives.

OBJECTIVE: Familial clustering of juvenile autoimmune liver disease (AILD), including autoimmune hepatitis and autoimmune sclerosing cholangitis (ASC), is rare, despite a high prevalence of autoimmune disorders in AILD families. METHODS: To investigate this discrepancy, we measured autoantibodies diagnostic for AILD, anti-nuclear, anti-smooth muscle, anti-liver kidney microsomal type 1, anti-liver cytosol type 1, and anti-soluble liver antigen antibodies, and human leukocyte antigen profiles in 31 patients and 65 of their first-degree relatives (FDR). The autoantibody profile was compared with that of 42 healthy subjects (HS). RESULTS: Autoantibodies were detected in 71% (22/31) patients. Anti-nuclear antibody or anti-smooth muscle antibody were present in 4/65 FDR (6.2%). HS were negative for all autoantibodies. The frequencies of homozygous HLA DRB1*0301 (DR3) genes and haplotype A1-B8-DR3 were higher in the patients (25% and 43%) than in FDR (9% and 27%) and HS (0% and 16%). The frequencies of disease-protective genes DR4 and/or DR15 were lower in the patients (25%) than in FDR (42%) and HS (42%). Only 1 family contained 2 patients with AILD, 1 with ASC and 1 with primary sclerosing cholangitis. Both patients possessed A1-B8-DR3 genes, the ASC being homozygous and the primary sclerosing cholangitis heterozygous. Six FDR had nonhepatic autoimmune disorders, none being autoantibody positive. CONCLUSIONS: Homozygosity for DR3 plays a major role in the predisposition to juvenile AILD. Diagnostic autoantibodies for AILD are rare among patients' FDR and not linked to clinical manifestation of AILD.

Genetic profiling in Graves' disease: further evidence for lack of a distinct genetic contribution to Graves' ophthalmopathy.

BACKGROUND: Graves' disease (GD), including Graves' ophthalmopathy or orbitopathy (GO), is an autoimmune disease with an environmental and genetic component to its etiology. The genetic contribution to the GO clinical phenotype remains unclear. Previous data from our laboratory and others have suggested that GO has no specific genetic component distinct from GD itself, while other reports have occasionally appeared suggesting that polymorphisms in genes such as CTLA4 and IL23R specifically increase the risk for GO. One of the criticisms of all these reports has been the clinical definition of the GO phenotype as distinct from hyperthyroid GD devoid of clinically significant eye involvement. The objective of this study was to take advantage of a phenotypically pure group of GD patients with GO and examine a series of genes associated with GD to determine if any were more definitively associated with GO rather than Graves' thyroid disease itself. METHODS: To further examine whether specific susceptibility genes are associated with GO, we have performed further genetic association studies using highly characterized GO patients, many of whom had undergone orbital decompression surgery for their exophthalmos. We genotyped HLA, CTLA4, IL23R, and TSHR genes in a group of 256 Caucasian patients with severe GO (n=199) and less severe GO (n=57), and 90 patients with GD but no clinically apparent GO. RESULTS: We found that the allele and genotype frequencies were not statistically different between GO and non-GO patients for any of the genes and gene combinations assessed. CONCLUSIONS: These results provide further evidence that patients with GO do not have a distinct genetic susceptibility to their eye disease and again suggest that environmental and/or epigenetic influences are at play.

Serum titres of anti-glutamic acid decarboxylase-65 and anti-IA-2 autoantibodies are associated with different immunoregulatory milieu in newly diagnosed type 1 diabetes patients.

Several studies correlated genetic background and pancreatic islet-cell autoantibody status (type and number) in type 1A diabetes mellitus (T1AD), but there are no data evaluating the relationship among these markers with serum cytokines, regulatory T cells and ß cell function. This characterization has a potential importance with regard to T1AD patients' stratification and follow-up in therapeutic prevention. In this study we showed that peripheral sera cytokines [interleukin (IL)-12, IL-6, II-1ß, tumour necrosis factor (TNF)-α, IL-10] and chemokines (CXCL10, CXCL8, CXCL9, CCL2) measured were significantly higher in newly diagnosed T1AD patients when compared to healthy controls (P < 0·001). Among T1AD, we found a positive correlation between CXCL10 and CCL-2 (r = 0·80; P = 0·000), IL-8 and TNF-α (r = 0·60; P = 0·000); IL-8 and IL-12 (r = 0·57; P = 0·001) and TNF-α and IL-12 (r = 0·93; P = 0·000). Glutamic acid decarboxylase-65 (GAD-65) autoantibodies (GADA) were associated negatively with CXCL10 (r = -0·45; P = 0·011) and CCL2 (r = -0·65; P = 0·000), while IA-2A showed a negative correlation with IL-10 (r = -0·38; P = 0·027). Human leucocyte antigen (HLA) DR3, DR4 or DR3/DR4 and PTPN22 polymorphism did not show any association with pancreatic islet cell antibodies or cytokines studied. In summary, our results revealed that T1AD have a proinflammatory cytokine profile compared to healthy controls and that IA-2A sera titres seem to be associated with a more inflammatory peripheral cytokine/chemokine profile than GADA. A confirmation of these data in the pre-T1AD phase could help to explain the mechanistic of the well-known role of IA-2A as a more specific marker of beta-cell damage than GADA during the natural history of T1AD.