David, Chella S.

Abstract HLA-DQB1*0302/DQA1*0301 renders humans and transgenic mice susceptible to develop arthritis while HLA-DQB1*0601/DQA1*0103 has been associated with resistance to arthritis in both. However, some patients of rheumatoid arthritis carry the non-associated alleles. Most of the studies do not take in to account the role of DQA1 alleles. DQB1*0604 generally occurs in linkage with DQA1*0102. To understand the role of trans-heterodimer, we generated DQB1*0604/ DQA1* 0103 transgenic mice lacking endogenous class II molecules. Previous studies have shown that DQB1*0601/DQA1*0103 transgenic mice are resistant to develop collagen-induced arthritis. The DQB1*0604/A1*0103 and DQA1*0103 mice developed severe arthritis and generated antigen-specific response in vitro. The DQB1*0604/ DQA1*0103 could present CII-derived peptides that are not presented by RA resistant DQ6 allele suggesting that trans-heterodimer molecules between two DQB1 and DQA1 molecules may result in presentation of unique antigens and susceptibility to develop arthritis. The data provides a possible explanation for the presence of HLA alleles not associated with arthritis in some patients.

Abstract Autoimmune uveitis affects 150,000 Americans annually and is a leading cause of blindness. Pathogenesis is thought to involve T cell mediated responses to retinal proteins, such as retinal arrestin (S-Ag). Susceptibility to autoimmune uveitis is strongly associated with HLA class I and class II, but relatively little is known about the nature of HLA class II alleles and about the epitopes that might be pathogenic in the context of different HLA class II molecules. Using a ‘humanized’ mouse model for experimental autoimmune uveitis (EAU) in HLA transgenic mice, we have identified permissive and non-permissive alleles of the HLA-DR and -DQ genes and characterized allele-specific uveitogenic epitopes. The sequences of these epitopes overlap with some of the previously identified peptides of S-Ag (M and N), which elicit memory responses in lymphocytes of uveitis patients. HLA-DR restricted S-Ag specific CD4+ T cells could be detected in blood and draining lymph nodes of immunized mice using HLA class II tetramers, demonstrating the potential use of this reagent to detect and isolate antigen specific T cells in uveitis patients. Our data provide new insights into the pathogenesis of human uveitis, and may help in the development of antigen specific therapies.

Abstract MS is an inflammatory demyelinating disease of CNS with a presumed autoimmune origin. Prevalent data from animal model of disease, experimental autoimmune encephalomyelitis (EAE) suggests that myelin-specific CD4 T-cells play a major role in disease. Role of CD8 T-cells in MS/EAE has been enigmatic as it has been assigned either regulatory or pathogenic role in disease. Previously we have shown that PLPp91-110 peptide can induce inflammatory neurological disease in HLA-DR3 transgenic (Tg) mice. In order to understand the role of CD8 T cells in this humanized model, we generated HLA-DR3 Tg mice lacking CD8 T cells (DR3.CD8-/-). These mice showed earlier onset and more severe disease when compared to CD8 sufficient HLA-DR3 Tg mice indicating that CD8 T-cells plays a regulatory role. However, pathological analysis of DR3.CD8-/- mice with EAE showed less inflammation and demyelination in CNS compared to DR3.CD8 sufficient mice suggesting a second pathogenic role of CD8 T cells. Thus, our data suggests both a regulatory as well as a pathogenic role of CD8 T cells in EAE and possibly in MS. We have characterized both subsets in detail and show that CD8+CD122- T cells are regulatory subset of CD8 T cells and suppress CD4 T cells by inhibiting antigen presentation pathway, while CD8+CD122- T cells are pathogenic subset. Thus ours is the first study to show a dual role of CD8 T cells in the onset, incidence, progression, severity and pathogenesis of EAE/MS.

Abstract Studies in myasthenia gravis (MG) patients demonstrate that polymorphism at the HLA-DQ locus influences the development of MG. Several studies using the mouse models also demonstrate the influence of class II molecules, especially the H2-A, which is the mouse homologue of HLA-DQ, in experimental autoimmune myasthenia gravis (EAMG). We used transgenic mice expressing two different DQ molecules, DQ8 (DQA1*0301/B1*0302) and DQ6 (DQA1*0103/B1*0601), to evaluate the role of HLA-DQ genes in MG. These mice do not express endogenous mouse class II molecules since they contain the mutant H2-Aβ0 gene. The mice were immunized with Torpedo acetylcholine receptor, and EAMG was assessed by clinical evaluation and was confirmed by electrophysiology. Clinical scores for EAMG were highest in HLA-DQ8 transgenic mice, whereas the scores of HLA-DQ6 mice rarely exceeded grade 1. There was no incidence of EAMG in class II-deficient (H2-Aβ0) mice. These results demonstrate that polymorphism at the HLA-DQ locus affects the incidence and the severity of EAMG. The manifestation of susceptibility to EAMG in the context of human class II molecules underscores the important roles of these molecules in the initiation and perpetuation of EAMG.

Abstract Hybrids of New Zealand Black (NZB) and New Zealand White (NZW) mice spontaneously develop a disease similar to human systemic lupus erythematosus. MHC and non-MHC genes contribute to disease susceptibility in this murine model. Multiple studies have shown that the NZW H2z locus is strongly associated with the development of lupus-like disease in these mice. The susceptibility gene(s) within H2z is not known, but different lines of evidence have pointed to class II MHC genes, either H2-E or H2-A (Ez or Az in NZW). Recent studies from our laboratory showed that Ez does not supplant H2z in the contribution to lupus-like disease. In the present work we generated C57BL/10 (B10) mice transgenic for Aaz and Abz genes (designated B10.Az mice) and used a (B10.Az × NZB)F1 × NZB backcross to assess the contributions of Az genes to disease. A subset of backcross mice produced high levels of IgG autoantibodies and developed severe nephritis. However, no autoimmune phenotype was linked to the Az transgenes. Surprisingly, in the same backcross mice, inheritance of H2b from the nonautoimmune B10 strain was strongly linked with both autoantibody production and nephritis. Taken together with our previous Ez studies, the present work calls into question the importance of class II MHC genes for lupus susceptibility in this model and provides new insight into the role of MHC in lupus-like autoimmunity.

Abstract We have generated transgenic (tg) mice expressing HLA-DQ8αβ (DQA1*0301/DQB*0302) or HLA-DQ6αβ (DQA1*0103/DQB1*0601) molecules lacking endogenous murine class II expression (Aβ0) to investigate the ability of these HLA class II to present type II collagen (CII) and induce collagen-induced arthritis. The DQ8αβ tg mice responded strongly to CII, developing severe arthritis, while DQ6αβ tg mice were nonresponsive to CII. The addition of the mixed haplotype DQ8α6β molecule did not significantly influence CII reactivity. To examine the interaction of DQ6αβ and DQ8αβ molecules in vivo, we generated double tg DQ6αβ/8αβ (Aβ0) mice expressing both the α- and β-chains of DQ6 and DQ8 molecules by mating DQ6αβ (Aβ0) and DQ8αβ (Aβ0) tg mice. CII-immunized DQ6αβ/8αβ tg mice developed severe experimental polychondritis, exhibiting both polyarthritis and auricular chondritis. The clinical, serologic, and histologic manifestations of experimental polychondritis are similar to those symptoms in human relapsing polychondritis. The susceptibility of DQ6αβ/8αβ tg mice compared with resistance in the parental strains suggests that expression of both the DQ6αβ and DQ8αβ tgs, unique to the DQ6αβ8αβ tg strain, is important in susceptibility to experimental polychondritis. The DQ6αβ/8αβ tg mice provide a model to investigate putative autoantigens and the mechanisms of pathogenesis involved in relapsing polychondritis as well as the influence of the expression of multiple HLA class II molecules on the disease process.

Abstract We have investigated the genetic and molecular basis of immune responsiveness to short ragweed (SRW) (Ambrosia artemisiifolia) extract, and group 5 allergens from short and giant (Ambrosia trifida) ragweed using transgenic mice expressing DQ6 (HLA-DQA1*0103, HLA-DQB1*0601) and DQ8 (HLA-DQA1*0301, HLA-DQB1*0302) genes in class II knockout (Aβ0) mice. Panels of overlapping peptides spanning the Amb a 5 and Amb t 5 Ags were synthesized. Mice were immunized with whole SRW extract or individual peptides s.c. and lymph node cells (LNC) were challenged in vitro. Strong T cell responses to SRW extract were measured in both HLA-DQ transgenic mice, while control, HLA-DQ6−/DQ8−/H-2Aβ0, mice were unresponsive. IL-5 and IL-10 were the primary cytokines produced by in vitro challenged LNC of SRW-primed transgenic mice. HLA-DQ6-restricted T cell responses were detected to all three peptides of Amb t 5 and two determinants (residues 1–20 and 11–30) on Amb a 5. In contrast, LNC of HLA-DQ8 mice did not recognize peptide 11–30 of Amb t 5 Ag, but recognized several Amb a 5 determinants. The immune response in transgenic mice was dependent upon CD4+ T cells and was HLA-DQ restricted. Primed with purified Amb t 5, both transgenics recognized peptide 21–40, and an additional DQ6-restricted epitope was found within residue 1–20. SRW-immunized HLA-DQ6 mice respond to peptide 11–30 of Amb a 5, while HLA-DQ8 mice strongly recognize peptide 1–20. These results demonstrate the specificity of HLA class II polymorphism in allergen sensitivity and pave the way for developing antagonistic peptides for desensitization.

Abstract HLA-DQA1*0301 and HLA-DQB1*0302 genes encoding the HLA-DQ8 molecule and HLA-DQA1*0103 and HLA-DQB1*0601 genes encoding the HLA-DQ6 molecule were introduced into H-2Aβo knockout mice. Three lines of transgenic mice were established: HLA-DQ8, HLA-DQ6, and HLA-DQ8β6α. HLA-DQ8 mice are susceptible to collagen-induced arthritis, while HLA-DQ6 mice are resistant. HLA-DQ8β6α mice develop polychrondritis in addition to arthritis. Transgenic mice were primed and challenged with individual synthetic peptides representing human type II collagen. A total of 101 synthetic peptides were tested in each transgenic line of mice. HLA-DQ8 mice responded to 15 synthetic peptides representing all cyanogen bromide fragments. In contrast, HLA-DQ6 mice responded to a subset of the peptides recognized by HLA-DQ8 T cells. HLA-DQ8β6α mice, although exhibiting diminished responses to the majority of HLA-DQ8-restricted determinants, elicited enhanced responses to two peptides. In addition, HLA-DQ8β6α mice respond to two unique peptide determinants contained within cyanogen bromide fragments CB10 and CB11 showing the significance of mixed isotype dimers in the immune response. The determinants recognized by the HLA-DQ transgenic mice are distinct from those previously identified using conventional laboratory mice. These results suggest that human class II transgenic mice offer a means of identifying human class II-restricted epitopes associated with potential human autoantigens.

Abstract MHC class I genes are important in the regulation of cyst number in mice perorally infected with Toxoplasma gondii. Since human MHC class I molecules may be capable of functioning as restriction elements in mice, to determine whether class I genes also control outcome of T. gondii infection for humans, a transgenic mouse model was developed using human class I genes in mice. Human HLA-B27 and Cw3 transgenic mice, with the cyst-susceptible B10 mouse background, were tested for resistance to cyst formation after peroral infection with the Me49 strain of T. gondii. Introduction of the B27 gene into susceptible mice made them even more susceptible to T. gondii brain cyst formation. In contrast, HLA-DB27 transgenic mice that have a substitution of the alpha 3 portion of the human class I molecule with the alpha 3 portion of the mouse Kd molecule had the same number of brain cysts as susceptible B10 control mice when perorally infected. Similarly, HLA-Cw3 transgenic mice had the same number of brain cysts as the susceptible B10 controls. These results indicate that the interaction between CD8 on mouse T cells and the human class I molecule in transgenic mice does not always occur, and sometimes may hinder a normal response. Furthermore, B27 and Cw3 transgenes did not generate resistance to cyst formation in infection by T. gondii.

Abstract Infection of certain strains of mice with Theiler’s murine encephalomyelitis virus results in persistence of virus and an immune-mediated primary demyelination in the central nervous system that resembles multiple sclerosis. Because susceptibility/resistance to demyelination in B10 congeneic mice maps strongly to class I MHC genes (D region) we tested whether expression of a human class I MHC gene (HLA-B27) would alter susceptibility to Theiler’s murine encephalomyelitis virus-induced demyelination. Transgenic HLA-B27 mice were found to co-express human and endogenous mouse class I MHC genes by flow microfluorimetry analysis of PBL. In the absence of the human transgene, H-2stf, or v mice but not H-2b mice had chronic demyelination and persistence of virus at 45 days after infection. No difference in degree of demyelination, meningeal inflammation, or virus persistence was seen between transgenic HLA-B27 and nontransgenic littermate mice of H-2f or H-2v haplotype. In contrast, H-2s (HLA-B27+) mice showed a dramatic decrease in extent of demyelination and number of virus-Ag+ cells in the spinal cord compared with H-2s (HLA-B27-) littermate mice. In addition, none of the eight H-2s mice homozygous for HLA-B27 gene had spinal cord lesions even though infectious virus was isolated chronically from their central nervous system. Expression of HLA-B27 transgene did not interfere with the resistance to demyelination normally observed in B10 (H-2b) mice. These experiments demonstrate that expression of a human class I MHC gene can modulate a virus-induced demyelinating disease process in the mouse.