Overrepresentation of HLA-DQ2 in small intestinal neuroendocrine tumor patients.

PURPOSE: To investigate whether celiac disease risk haplotypes HLA-DQ2 and DQ8 also increase the risk for developing small intestinal neuroendocrine tumor (SI-NET). METHODS: Thirty-five patients with serotonin-producing jejunal and ileal SI-NET were examined with HLA-DQ genotyping and serology for IgA anti-tissue transglutaminase (tTG) antibodies. RESULTS: Twenty-one patients (60 %) carried HLA-DQ2 or DQ8, twice the frequency of the general population (P < 0.001). In particular DQ2 was overrepresented (P = 0.013). Gender, age, disease stage, histopathological grade, or multifocality of primary tumor did not differ between patients with DQ2 or DQ8 and patients with other HLA-DQ haplotypes. No patient in the study was diagnosed with celiac disease (latent or symptomatic) as anti-tTG antibodies were negative in all 35. CONCLUSION: HLA-DQ haplotypes associated with celiac disease are overrepresented also in patients with SI-NET, in particular HLA-DQ2.

Pulse-chase analysis for studies of MHC class II biosynthesis, maturation, and peptide loading.

Pulse-chase analysis is a commonly used technique for studying the synthesis, processing and transport of proteins. Cultured cells expressing proteins of interest are allowed to take up radioactively labeled amino acids for a brief interval ("pulse"), during which all newly synthesized proteins incorporate the label. The cells are then returned to nonradioactive culture medium for various times ("chase"), during which proteins may undergo conformational changes, trafficking, or degradation. Proteins of interest are isolated (usually by immunoprecipitation) and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the fate of radiolabeled molecules is examined by autoradiography. This chapter describes a pulse-chase protocol suitable for studies of major histocompatibility complex (MHC) class II biosynthesis and maturation. We discuss how results are affected by the recognition by certain anti-class II antibodies of distinct class II conformations associated with particular biosynthetic states. Our protocol can be adapted to follow the fate of many other endogenously synthesized proteins, including viral or transfected gene products, in cultured cells.

A multiplex assay to rapidly exclude HLA-DQ2.5 and HLA-DQ8 expression in patients at risk for celiac disease.

BACKGROUND: Celiac disease (CD) is an inflammatory disorder of the small intestine induced by gluten ingestion. CD has a strong genetic association with human leukocyte antigen (HLA)-DQ2.5 and HLA-DQ8. The absence of HLA-DQ2.5 and HLA-DQ8 has a strong negative predictive value for CD. Genetic screening of HLA-DQ2.5 and HLA-DQ8 in patients at risk is of great value. METHODS: We designed, developed, and validated a multiplex assay based on multiplex ligation-dependent probe amplification (MLPA) technology, allowing the simultaneous detection of DQA1*05-DQB1*02, encoding HLA-DQ2.5, and DQA1*03-DQB1*03:02, encoding HLA-DQ8. The amplified products were separated and identified using capillary electrophoresis. RESULTS: When compared with a polymerase chain reaction followed by single-strand conformation polymorphism/ heteroduplex analysis, one discrepancy was found. Sequencing analysis showed that the developed MLPA assay result was correct. Furthermore, we demonstrated that the MLPA method is able to distinguish between the heterozygote and homozygote expression of HLA-DQ2.5 or HLA-DQ8. CONCLUSIONS: This study shows that it is possible to rapidly and accurately screen for the absence of HLA-DQ2.5 and HLA-DQ8 using MLPA, excluding patients at risk for CD for further serological or histological follow-up. In addition, MLPA might be an accurate tool to screen for other specific HLA types in the context of disease association in a diagnostic laboratory setting.

The relationship between human leukocyte antigens (HLA) and renal cell carcinoma.

Etiologies of Renal Cell Carcinoma (RCC) are not clear despite of the fact that many risk factors have been suggested. Especially in high stages RCC can affect the immune system in various ways. Human Leukocyte Antigens (HLA) may play a complementary role in the activation between the tumor and immunity. Our aim was to determine the existence of the relationship between HLA system and RCC. By using the standard microlymphocytotoxic method of Terasaki in our study, the HLA A, B, DR and DQ antigen types of 20 patients with RCC Stage T1 and T2 were compared with the control group consisting of healthy 30 people. In our RCC patient group, HLA-A23(9) and DQ7(3) antigens were significantly higher than the control group statistically (p=0.005, p=0.0028; respectively). HLA-A10, DQ1, DR10 and B44 antigens were significantly higher in the control group than the patient group (p=0.011; for all).The findings made us suggest that the people, carrying the antigens which were detected in the patient group, were at high risk for RCC and the people, carrying the protective antigens that were detected in the control group were at less risk for RCC. There may be a dramatic regression for the patients who underwent immunotherapy and HLA expression, which is known to play role in tumor biology, may direct the effects of immunotherapeutic agents. Immunologic description and destruction is avoided in case of change or disappearance of HLA expression by cancer cells. Further investigations which will be performed in our population in the future will be more illuminating to confirm those results. We have concluded that, HLA profiles may be evaluated for detection the people at risk of RCC, the prognosis of the patients and their treatments.

Alterations in CIITA constitute a common mechanism accounting for downregulation of MHC class II expression in diffuse large B-cell lymphoma (DLBCL).

OBJECTIVE: Significant decreases in patient survival are associated with downregulation of major histocompatibility complex class II (MHC-II) antigen expression in diffuse large B-cell lymphoma (DLBCL). However, the molecular mechanisms responsible for decreased MHC-II expression in DLBCL are poorly defined. We therefore examined these mechanisms in established DLBCL cell lines. MATERIALS AND METHODS: Human leukocyte antigen (HLA)-DR surface expression was examined by flow cytometry. Expression of the MHC-II genes and the MHC-II transcriptional activators class II transactivator (CIITA) and RFX was investigated by reverse transcriptase polymerase chain reaction. The integrity of the MHC-II genes was examined by polymerase chain reaction. Stable transfection assays were utilized to reconstitute CIITA expression. RESULTS: Dramatic variations in the levels of cell surface HLA-DR expression were observed on the DLBCL cell lines. OCI-Ly10 cells lack HLA-DR and HLA-DQ expression due to homozygous deletions within the MHC-II locus on chromosome 6. Dyscoordinate downregulation of MHC-II beta-chain expression in OCI-Ly3 cells mediates dramatic reductions of MHC-II surface expression. In SUDHL-4 and SUDHL-6 cells, expression of the MHC-II genes is coordinately reduced and quantitatively correlated with expression of the CIITA, the master regulator of MHC-II transcription. DB cells lack expression of CIITA and all of the MHC-II genes. Stable transfection of DB cells with CIITA expression vectors resulted in coordinate upregulation of MHC-II gene expression, which demonstrates the causal relationship between the lack of CIITA and MHC-II loss. CONCLUSIONS: These data demonstrate that downregulation of MHC-II expression occurs by multiple distinct mechanisms in DLBCL. However, decreases in CIITA expression appear to be the most prevalent mechanism.

T cell epitopes of the La/SSB autoantigen in humanized transgenic mice expressing the HLA class II haplotype DRB1*0301/DQB1*0201.

OBJECTIVE: T cells are implicated in the production of anti-La/SSB and anti-Ro/SSA autoantibodies commonly associated with the DR3/DQ2 haplotype in systemic lupus erythematosus and Sjögren's syndrome. This study was undertaken to investigate the DR3/DQ2-restricted T cell response to wild-type human La (hLa) and a truncated form of mutant La. METHODS: Humanized transgenic mice expressing HLA-DRB1*0301/DQB1*0201 (DR3/DQ2) were immunized with recombinant antigen and examined for development of autoantibodies and T cell proliferation against overlapping peptides spanning the La autoantigen. HLA restriction and peptide binding of identified T cell epitopes to DR3 or DQ2 were determined using blocking monoclonal antibodies and a direct binding assay. RESULTS: DR3/DQ2-transgenic mice generated an unusually rapid class-switched humoral response to hLa with characteristic spreading to Ro 52 and Ro 60 proteins following hLa protein immunization. Seven T cell determinants in hLa were restricted to the HLA-DR3/DQ2 haplotype. Six epitopes tested were restricted to HLA-DR and bound DR3 with semiconserved DR3 binding motifs. No DQ restriction of these epitopes was demonstrable despite efficient DQ binding activity in some cases. No neo-T cell epitopes were identified in mutant La; however, T cells primed with mutant La exhibited a striking increase in proliferation to the epitope hLa(151-168) compared with T cells primed with hLa. CONCLUSION: Multiple DR3-restricted epitopes of hLa have been identified. These findings suggest that truncation of La produced by somatic mutation or possibly granzyme B-mediated cleavage alters the immunodominance hierarchy of T cell responsiveness to hLa and may be a factor in the initiation or maintenance of anti-La autoimmunity.

Humanized transgenic mice expressing HLA DR4-DQ3 haplotype: reconstitution of phenotype and HLA-restricted T-cell responses.

Many autoimmune conditions have close genetic linkages to particular human histocompatibility leukocyte antigen (HLA) class II genes. With the aim of establishing a murine model of autoimmune disease, we have generated an HLA DR4-DQ3 haplotype transgenic (Tg) mouse that expresses a 440-kb yeast artificial chromosome harbouring DRA, DRB1*040101, DRB4*010301, DQA1*030101, DQB1*0302 and all the internal regulatory segments. This Tg mouse line was crossed to human CD4 (hCD4) Tg mice and endogenous class II knockout mice (I-A(o/o) and I-E(o/o)) lines to generate a DR4-DQ3.hCD4.IAE(o/o) Tg line. The Tg DR and DQ molecules are expressed on the physiological cell types in these animals, i.e. on most B cells (>85%), dendritic cells (DCs) and macrophages but not on T cells, with levels of expression comparable with those of human B cells (where DR > DQ expression). The DR4/DQ3 transgenes fully reconstituted the CD4 T-cell compartment, in both the thymus and the periphery, and the analysis of the T-cell receptor repertoire in the Tg mice confirmed that these class II molecules were able to mediate thymic selection of a broad range of Vbeta families. HLA DR4- and DQ3-restricted T-cell responses were elicited following immunization with known T-cell determinants presented by these molecules. Furthermore, the DR4-DQ3-restricted CD4(+) T cells conferred protective antibody-mediated immunity against an otherwise lethal infection with Salmonella enterica var. typhimurium. These new DR4-DQ3 Tg mice should prove to be valuable tools for dissecting the importance of this class II haplotype in autoimmune disorders like rheumatoid arthritis.

Conserved residues of the bare lymphocyte syndrome transcription factor RFXAP determine coordinate MHC class II expression.

RFXAP is required for the transcriptional regulation of MHC-II genes. Mutations in RFXAP are the genetic basis for complementation group D cases of the bare lymphocyte syndrome (BLS) immunodeficiency. Comparative genomic sequence analysis was conducted and found that only the C-terminal half of the protein is conserved among vertebrates. The C-terminal third of RFXAP, which contained an extensive glutamine-rich tract, could rescue HLA-DR, but not HLA-DQ or HLA-DP expression in a BLS cell line. To understand this phenomenon, a detailed analysis of the role of specific sequences in the C-terminal third of RFXAP with respect to MHC-II regulation was undertaken. Surprisingly, mutation of the conserved glutamine residues had no effect on activity, whereas mutation of hydrophobic and other conserved residues resulted in discoordinate MHC-II isotype expression. Moreover, mutation of potential phosphorylation sites abolished RFXAP activity. The ability of RFXAP mutants to rescue one isotype, but not another was investigated by their ability to form RFX complexes, bind DNA in vivo, recruit CIITA to promoters and to activate a series of chimeric reporter genes. The results suggest that certain RFXAP mutants exaggerate isotype promoter-specific differences and form transcriptionally inefficient activation complexes with factors at the neighboring cis-acting elements. These results show a distinction in factor recognition that is associated with specific MHC-II isotypes and may explain the basis of allele-specific expression differences.

HLA-DQ8 is a predisposing molecule for detergent enzyme subtilisin BPN'-induced hypersensitivity.

Several million individuals are exposed to agents in the workplace associated with atopy and asthma. Detergent enzymes have been implicated in occupationally induced hypersensitivity. However, the genetic susceptibility and T cell responses to detergent enzymes are undefined. We generated and used HLA-DQ6, -DQ8, -DR2, -DR3, and -DR4 transgenic mice to examine the immune and inflammatory components involved in the response to the detergent enzyme subtilisin BPN'. Based on in vitro and in vivo studies, for the first time, we present evidence that DQ8 is a strong susceptibility marker for BPN'-induced hypersensitivity. Only DQ8 mice showed consistent T cell responses to five immunodominant regions of BPN' comprising peptides #14 to 16, 36-37, 42-43, 62-63, and 80-81. The DQ8 mice also developed allergic eosinophilic inflammatory reactions in the airways following intranasal instillations of this enzyme. The DQ8 mice also responded to BPN' with a significant IgG1 and IgE production. We propose that the HLA Class II tg mice are useful for understanding allergenic responses to enzymes in humans, screening of allergenic and immunogenic properties of detergent enzymes, and for the development of modified enzymes to maintain efficient detergent qualities without allergic properties.

Cannabinoid CB2 receptors are expressed by perivascular microglial cells in the human brain: an immunohistochemical study.

Two types of cannabinoid receptors have been characterized so far, CB1 and CB2. While CB1 receptors are present both in the CNS and in the periphery, CB2 receptors showed an almost exclusive distribution within the immune system. We now report that CB2 receptors are present in a specific microglial cell type of the human cerebellum. Thus, we have performed immunohistochemical analysis of tissue sections of white matter areas of the human cerebellum and detected the presence of CB2 receptors in perivascular microglial cells. These findings match with the well-known immunomodulatory role of CB2 receptors and open new perspectives on the possible role that these receptors may play in pathophysiological events.

Allele-specific quantification of HLA-DQB1 gene expression by real-time reverse transcriptase-polymerase chain reaction.

In addition to coding region polymorphism, allele-specific variation in the upstream regulatory region of the HLA-DQB1 gene has been detected. Reporter gene assays and transfection studies have indicated that HLA-DQB1 promoter polymorphism may be of functional significance. The aim of this study was to utilize real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for allele-specific quantification of HLA-DQB1 expression and to analyze cell-specific HLA-DQB1 expression in vivo. For the allele-specific quantification of DQB1 gene products, a real-time RT-PCR set of primer pairs (n=27) and probes (n=5) targeting exon 2 variability was established. The robustness and integrity of the assay system were confirmed by using recombinant DQB1 exon 2 plasmid clones as active exogenous controls. Sensitivity and reproducibility were assessed by serial dilution and allelic mixing analyses. In application to the study of allele-specific expression of DQB1 gene products during cytokine-driven maturation of monocyte-derived dendritic cells, differential patterns of allelic expression in heterozygous individuals were observed for DQB1*0301, compared to DQB1*0501 and DQB1*0602. At maximum, 1.9-fold (*0301/*0501) and 2.5-fold (*0301/*0602) higher induction was seen for DQB*0301. In conclusion, HLA-DQB1 expression can be analyzed by real-time RT-PCR suitable for cell- and allele-specific detection of HLA-DQB1 transcripts in homo- and heterozygous combinations.

Humanized animal models for autoimmune diseases.

The development of transgenic mice expressing human DR and DQ major histocompatibility complex (MHC) class II molecules has been of value in studying the immunopathology of human MHC class II-associated autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes mellitus and celiac disease. Such mice have been used to identify the target antigens that are involved in the initiation of these diseases. Many of the mice develop aspects of the human diseases, either spontaneously or following immunization with the relevant antigen, thus providing an in vivo disease model, which may be used as a tool for further understanding the disease mechanisms and testing novel immunotherapies.

HLA-DQB1 alleles may influence the surface expression of DQ molecules in lymphomononuclear cells of type 1 diabetes mellitus patients.

To evaluate the expression of human leucocyte antigen (HLA) class II (DR and DQ) molecules on lymphomononuclear cells involved in the pathogenesis of type 1 diabetes, we studied 20 patients and 20 controls matched to patients for age, sex and HLA class II profile. The coexpression of HLA and CD3, CD4, CD8, CD19 and CD14 molecules was evaluated by flow cytometry. HLA-DRB1, -DQA1 and -DQB1 alleles were assigned using amplified DNA hybridized with sequence-specific primers. The fluorescence intensity of HLA-DR and -DQ molecules observed on the surface of the lymphomononuclear cells of patients did not differ significantly from controls. Patients presented decreased percentage of double-positive CD4(+)/DQ(+) cells and increased percentage of CD19(+)/DR(+) cells, irrespective of the HLA class II profile; however, the more dramatic alteration of the lymphomononuclear phenotype profile was observed for patients possessing the HLA-DQB1*0201 allele. These patients exhibited decreased percentage of CD3(+), CD4(+), CD8(+), CD19(+) and CD14(+) cells bearing HLA-DQ molecules and decreased fluorescence intensity for HLA-DQ molecules on CD19(+) cells compared to patients without the DQB1*0201 allele. Although type 1 diabetes patients shared CD4/DQ or CD19/DR phenotype abnormalities, patients typed as DQB1*0201 presented additional abnormalities in terms of DQ expression and cell phenotypes bearing DQ molecules.

A spontaneous model for autoimmune myocarditis using the human MHC molecule HLA-DQ8.

Genome-wide analyses have shown that the MHC class II region is the principal locus that confers susceptibility to a number of human autoimmune diseases. Due to the high degree of linkage disequilibrium across the MHC, it has been difficult to dissect the contribution of individual genes to disease susceptibility. As a result, intensive efforts have been made to generate mice transgenic for human class II molecules as models of autoimmune disease. However, in every case, additional manipulations-such as immunization with Ag in adjuvant, expression of immunostimulants on target tissues, or coexpression of TCR transgenes-have been required to induce disease. In this study, we show that expression of the human HLA-DQ8 (DQA1*0301/DQB1*0302) molecule alone in three lines of transgenic nonobese diabetic murine class II-deficient (mII(-/-)) mice results in the spontaneous development of autoimmune myocarditis. The disease shares key features of human myocarditis and was characterized by lymphocytic infiltrates in the myocardium and cardiac myocyte destruction, circulating IgG autoantibodies against cardiac myosin heavy chain, and premature death due to heart failure. We demonstrate that myocarditis could be transferred into healthy HLA-DQ8(+)RAG-1(-/-)mII(-/-) nonobese diabetic recipients with lymphocytes, but not sera. It has been widely thought that autoimmune myocarditis is of infectious etiology, with the immune responses arising secondary to cardiac damage from pathogens. These studies provide direct experimental evidence that spontaneous autoimmune myocarditis can occur in the absence of infection and that expression of HLA-DQ8 confers susceptibility to this organ-specific autoimmune disease.

Fine-scale mapping at IGAD1 and genome-wide genetic linkage analysis implicate HLA-DQ/DR as a major susceptibility locus in selective IgA deficiency and common variable immunodeficiency.

Selective IgA deficiency (IgAD) and common variable immunodeficiency (CVID) are the most common primary immunodeficiencies in humans. A high degree of familial clustering, marked differences in the population prevalence among ethnic groups, association of IgAD and CVID in families, and a predominant inheritance pattern in multiple-case pedigrees have suggested a strong, shared genetic predisposition. Previous genetic linkage, case-control, and family-based association studies mapped an IgAD/CVID susceptibility locus, designated IGAD1, to the MHC, but its precise location within the MHC has been controversial. We have analyzed a sample of 101 multiple- and 110 single-case families using 36 markers at the IGAD1 candidate region and mapped homozygous stretches across the MHC shared by affected family members. Haplotype analysis, linkage disequilibrium, and homozygosity mapping indicated that HLA-DQ/DR is the major IGAD1 locus, strongly suggesting the autoimmune pathogenesis of IgAD/CVID. This is supported by the highest excess of allelic sharing at 6p in the genome-wide linkage analysis of 101 IgAD/CVID families using 383 marker loci, by previously reported restrictions of the T cell repertoires in CVID, the presence of autoantibodies, impaired T cell activation, and a dysregulation of a number of genes in the targeted immune system. IgAD/CVID may thus provide a useful model for the study of pathogenesis and novel therapeutic strategies in autoimmune diseases.

The bacterial superantigen streptococcal mitogenic exotoxin Z is the major immunoactive agent of Streptococcus pyogenes.

The gene encoding streptococcal mitogenic exotoxin Z (SMEZ) was disrupted in Streptococcus pyogenes. Despite the presence of other superantigen genes, mitogenic responses in human and murine HLA-DQ transgenic cells were abrogated when cells were stimulated with supernatant from the smez(-) mutant compared with the parent strain. Remarkably, disruption of smez led to a complete inability to elicit cytokine production (TNF-alpha, lymphotoxin-alpha, IFN-gamma, IL-1 and -8) from human cells, when cocultured with streptococcal supernatants. The potent effects of SMEZ were apparent even though transcription and expression of SMEZ were barely detectable. Human Vbeta8(+) T cell proliferation in response to S. pyogenes was SMEZ-dependent. Cells from HLA-DQ8 transgenic mice were 3 logs more sensitive to SMEZ-13 than cells from HLA-DR1 transgenic or wild-type mice. In the mouse, SMEZ targeted the human Vbeta8(+) TCR homologue, murine Vbeta11, at the expense of other TCR T cell subsets. Expression of SMEZ did not affect bacterial clearance or survival from peritoneal streptococcal infection in HLA-DQ8 mice, though effects of SMEZ on pharyngeal infection are unknown. Infection did lead to a rise in Vbeta11(+) T cells in the spleen which was partly reversed by disruption of the smez gene. Most strikingly, a clear rise in murine Vbeta4(+) cells was seen in mice infected with the smez(-) mutant S. pyogenes strain, indicating a potential role for SMEZ as a repressor of cognate anti-streptococcal responses.

Expression of activation markers on alveolar macrophages in allergic asthmatics after endobronchial or whole-lung allergen challenge.

We examined the effect of endobronchial (EB) or whole-lung (WL) challenge with ragweed or Timothy grass extract on alveolar macrophage (AM) activation. Expression of 17 constitutive activation markers on AM was examined by flow cytometry. Late-phase bronchial obstruction was greater after WL challenge, while changes in bronchoalveolar lavage cytology (eosinophil accumulation) were greater after EB challenge. After EB challenge, levels of 10 of 17 markers (CD11a, CD11b, CD14, CD18, CD23, CD32, CD63, CD64, HLA-class I, and HLA-DR) were significantly increased (by 33-234%, P < 0.05). Six markers (CD16, CD29, CD33, CD35, CD44, CD71, and HLA-DQ) remained unchanged. Levels of seven markers following EB challenge (CD14, CD16, CD18, CD29, CD32, HLA-class I, and HLA DQ) correlated with airway sensitivity to methacholine. WL challenge only increased expression of HLA-class I. The different results obtained with the two challenge methods probably depend on higher local concentrations of allergen in the EB challenge. We suggest that activation of AM occurs following EB challenge with antigen in asthmatics.

Long-term expression of an HLA-DQ molecule in the EBV-transformed bare lymphocyte cell line, BLS-1, using a plasmid vector.

The HLA class II molecule, DQ6, confers strong natural protection against the development of type 1 diabetes. The mechanism of disease protection is unknown, but is likely to be related to the function of the molecule in antigen presentation. In order to investigate this function, we have created an in vitro model which expresses DQ6 in isolation by introducing the relevant DQ alleles into an Epstein-Barr virus (EBV)-transformed, human leucocyte antigen (HLA) class II-deficient B cell line, bare lymphocyte syndrome (BLS)-1. A recent report suggested that the expression of transferred genes in human EBV-transformed B cells might be limited in duration. We present a plasmid-based transfection method that allows long-term stable expression of the DQ molecule. The DQA1*0102 and DQB1*0602 alleles were cloned into the pCIneo expression vector and the constructs were introduced into BLS-1 by electroporation. Stable transfectants were selected using magnetic sorting and cloned by limiting dilution. Two clones were shown to express functionally active DQ6 molecules even after 14 months of continuous culture. These clones will be used in functional studies to investigate the antigen binding and T-cell activation properties of the DQ6 molecule.

Gene expression profiling in DQA1*0501+ children with untreated dermatomyositis: a novel model of pathogenesis.

Juvenile dermatomyositis (JDM), the most common pediatric inflammatory myopathy, is a systemic vasculopathy affecting young children. Epidemiology studies documenting an antecedent illness in the 3 mo before the first definite symptom (rash and/or weakness) of JDM are supported by immunologic data that suggest that the disease pathophysiology is Ag driven. The purpose of this study was to compare the gene expression profiles in muscle biopsies of four untreated DQA1*0501(+) JDM children with profiles from children with a known necrotizing myopathy (Duchenne muscular dystrophy), as well as an in vitro antiviral model (NF90), and healthy pediatric controls. Nearly half (47%) of the dysregulated genes in JDM were associated with the immune response. In particular, increased expression of IFN-alphabeta-inducible genes 6-16, myxovirus resistance protein p78, latent cytosolic transcription factor, LMP2, and TAP1 was observed. This profile is consistent with an IFN-alphabeta transcription cascade seen in the in vitro viral resistance model. The IFN-alphabeta-inducible profile was superimposed on transcription profiles reflective of myofiber necrosis and regeneration shared with Duchenne muscular dystrophy. Expressed genes were confirmed by quantitative real-time PCR (6-16), immunofluorescence (thrombospondin 4), and immunolocalization (IFN-gamma, p21). We hypothesize that these data support a model of Ag (?viral) induction of an apparent autoimmune disease based on dynamic interaction between the muscle, vascular, and immune systems in the genetically susceptible (DQA1*0501(+)) child.

A 320-kilobase artificial chromosome encoding the human HLA DR3-DQ2 MHC haplotype confers HLA restriction in transgenic mice.

MHC class II haplotypes control the specificity of Th immune responses and susceptibility to many autoimmune diseases. Understanding the role of HLA class II haplotypes in immunity is hampered by the lack of animal models expressing these genes as authentic cis-haplotypes. In this study we describe transgenic expression of the autoimmune prone HLA DR3-DQ2 haplotype from a yeast artificial chromosome (YAC) containing an intact similar320-kb region from HLA DRA to DQB2. In YAC-transgenic mice HLA DR and DQ gene products were expressed on B cells, macrophages, and dendritic cells, but not on T cells indicating cell-specific regulation. Positive selection of the CD4 compartment by human class II molecules was 67% efficient in YAC-homozygous mice lacking endogenous class II molecules (Abeta(null/null)) and expressing only murine CD4. A broad range of TCR Vbeta families was used in the peripheral T cell repertoire, which was also purged of Vbeta5-, Vbeta11-, and Vbeta12-bearing T cells by endogenous mouse mammary tumor virus-encoded superantigens. Expression of the HLA DR3-DQ2 haplotype on the Abeta(null/null) background was associated with normal CD8-dependent clearance of virus from influenza-infected mice and development of CD4-dependent protection from otherwise lethal infection with Salmonella typhimurium. HLA DR- and DQ-restricted T cell responses were also elicited following immunization with known T cell determinants presented by these molecules. These findings demonstrate the potential for human MHC class II haplotypes to function efficiently in transgenic mice and should provide valuable tools for developing humanized models of MHC-associated autoimmune diseases.