Grete Sønderstrup

HLA class II transgenic mice: models of the human CD4+ T‐cell immune response

Summary: This review examines the field of current HLA class II transgenic mouse models and the individual approaches applied in production of these mice. The majority of these mice have been created with the objective of obtaining a disease model with clinical features mimicking human autoimmune disease. The development process of a different type of HLA class II transgenic mice, which are designed to function as a substitute for a normal human immune system in studies of human autoantigens, is described. Several HLA‐DR4 transgenic lines with normally expressed HLA‐DR4 molecules have been produced. To obtain adequate positive selection of the HLA‐DR4‐restricted CD4+ T‐cell repertoire in these mice it is essential both to introduce a human CD4 transgene. and to delete the murine major histocompatibility complex (MHC) class II molecules. These HLA‐DR4 transgenic mice have been used to determine the immunogenic CD4+ T‐cell epitopes of several human autoantigenic proteins.

Suppressive Effect of Glutamic Acid Decarboxylase 65-Specific Autoimmune B Lymphocytes on Processing of T Cell Determinants Located Within the Antibody Epitope

Type 1 diabetes is a T cell-mediated disease in which B cells serve critical Ag-presenting functions. In >95% of type 1 diabetic patients the B cell response to the glutamic acid decarboxylase 65 (GAD65) autoantigen is exclusively directed at conformational epitopes residing on the surface of the native molecule. We have examined how the epitope specificity of Ag-presenting autoimmune B cell lines, derived from a type 1 diabetic patient, affects the repertoire of peptides presented to DRB1*0401-restricted T cell hybridomas. The general effect of GAD65-specific B cells was to enhance Ag capture and therefore Ag presentation. The enhancing effect was, however, restricted to T cell determinants located outside the B cell epitope region, because processing/presentation of T cell epitopes located within the autoimmune B cell epitope were suppressed in a dominant fashion. A similar effect was observed when soluble Abs formed immune complexes with GAD65 before uptake and processing by splenocytes. Thus, GAD65-specific B cells and the Abs they secrete appear to modulate the autoimmune T cell repertoire by down-regulating T cell epitopes in an immunodominant area while boosting epitopes in distant or cryptic regions.

Allergen-Specific MHC Class II Tetramer+ Cells Are Detectable in Allergic, but Not in Nonallergic, Individuals

Allergen-specific cells are present in very low frequency in peripheral blood of humans, and differ in function in allergic and nonallergic individuals. We report in this study that soluble class II MHC tetramers can be used to directly identify and study such allergen epitope-specific CD4+ T cells in humans. We identified the major antigenic epitope of rye grass allergen Lol p 1 in HLA-DRB1*0401 individuals using HLA-DR*0401 transgenic mice and peripheral blood cells from HLA-DR*0401 individuals. Using DRB1*0401 tetramers loaded with this major epitope of Lol p 1, we detected allergen-specific CD4+ T cells in the peripheral blood of DRB1*0401 rye grass allergic individuals after ex vivo expansion with allergen. These tetramer-positive cells produced IL-4, but little IFN-γ. In contrast, we were unable to detect rye grass tetramer-positive cells in cultures from HLA-DR*0401 nonallergic individuals, even after expansion with IL-2. Thus, our results suggest that rye grass allergen-specific T cells in DR*0401 nonallergic subjects are present at very low levels (e.g., because of deletion or suppression), differ in a fundamental way in their requirement for ex vivo expansion (e.g., they may be anergic), or use TCRs distinct from those of allergic individuals. Thus, analysis using DRB1*0401 tetramers loaded with a major epitope of Lol p 1 indicates that allergen-specific CD4+ T cells in nonallergic individuals are distinct from those in allergic subjects.

Relationship between kinetic stability and immunogenicity of HLA-DR4/peptide complexes

Immunodominant T cell epitopes from the autoantigen human cartilage glycoprotein 39 have previously been mapped in the context of HLA‐DR*0401 and *0402, using mice expressing HLA‐DR4 transgenes. We measured the dissociation rates of these epitopes from soluble recombinant DR*0401 and DR*0402 to assess the relationship between peptide/HLA‐DR4 kinetic stability and immunogenicity. Experiments were performed at endosomal pH (5.5) and at cell surface pH (7), in the absence and presence of soluble recombinant HLA‐DM (sDM). All (4/4) immunodominant peptide/HLA‐DR complexes exhibit dissociation half‐times of 1 h to several days. In contrast, most (3/4) non‐immunodominant complexes dissociate with half‐times <30 min under at least one of these conditions. Interestingly, a complex which is stable except in the presence of HLA‐DM at pH 5.5 is immunogenic only following peptide immunization, while a complex which is stable at acidic but not at neutral pH, is non‐immunogenic following either whole protein or peptide immunization. These data indicate that kinetic stability of peptide/MHC complexes in vivo is a key determinant of immunogenicity.

DR, DQ, and you: MHC alleles and autoimmunity.

It has mystified immunologists for decades that antigen-specific T-cell clones obtained from human peripheral blood are, almost without exception, restricted by MHC class II molecules of the HLA-DR type, rather than by HLA-DQ molecules (1, 2). This is particularly surprising in that HLA-DQ alleles strongly influence susceptibility to many autoimmune diseases, including type 1 diabetes mellitus (T1DM) (3, 4). Thus, in humans, the HLA-DQB*0602 and HLA-DRB1*0403 alleles confer strong protection against T1DM (4, 5). The basis of this protective effect is undoubtedly complex and has proved difficult to study in humans. Unlike mice, where a number of inbred laboratory strains lack expression of H-2 IE (the murine equivalent to HLA-DR), humans always express both HLA-DR and HLA-DQ, usually a different set from each parent. In humans, it is therefore difficult to study the function of a single HLA class II allele in isolation, and in vivo experimentation with human subjects has many other constraints. These problems are further complicated by the strong linkage disequilibrium in the MHC class II region, which makes it difficult to distinguish the effects of individual HLA-DR alleles from the effects of linked HLA-DQ alleles, since particular DR/DQ allelic combinations tend to persist, with very little recombination. For these reasons, several laboratories have developed HLA class II transgenic mice to serve as an experimental surrogate for the CD4+ T-cell compartment of normal human subjects (6). David and his colleagues have produced HLA class II transgenic mice, which they and others have used to model human autoimmune diseases (7–9). By studying the influence of different combinations of coexpression of DR3, DQ8, DR2, and/or DQ6 alleles, and comparing these with HLA transgenic mice carrying only a single HLA allele, these authors found that coexpression of DR3 can modify the manifestations of an experimental arthritis associated with expression of DQ8 (7). DQ8 also increases the severity of other experimentally induced autoimmune diseases in this model system (8), whereas the DQ6 allele can prevent the spontaneous loss of tolerance to the pancreatic autoantigen GAD65 and the insulitis that are otherwise seen in HLA-DR3, DQ8 transgenic mice (9). In this issue of the JCI, Wen et al. wished to explore the influence of coexpression of HLA-DR alleles on the diabetes susceptibility conferred by the DQ8 allele (10). The HLA-DRB1*0401, DQ8 haplotype, the most common HLA haplotype in Caucasian T1DM patients, was therefore a logical candidate for this study (5). Because HLA-DQ8 transgenic mice do not develop diabetes spontaneously, the authors crossed them with a diabetes-prone transgenic strain, RIP-B7, which lacks endogenous MHC class II molecules and overexpresses the costimulatory molecule B7 specifically in the islet cells of the pancreas (11). When these RIP-B7 transgenic animals also carry the human HLA-DQ8 transgene, they develop spontaneous diabetes at a frequency of 73% (10, 11). Remarkably, a congenic RIP-B7 transgenic line carrying the DR4 (B1*0401) allele develops diabetes at a frequency of only 25%. To model the case of humans expressing the HLA-DR4, DQ8 haplotype, the authors then produced mice expressing both DR4 and DQ8 and found that the incidence of disease in these HLA double transgenic mice is almost identical to that of the DR4 transgenic mice (10). This suggests that DR4 can partly cancel the disease-promoting effect of DQ8. Following up on this finding, Wen and colleagues purified splenic CD4+ T cells from the diabetes-prone DQ8/RIP-B7 animals and from the other, relatively disease-insensitive strains (10). In vitro cytokine production by cells from DQ8/RIP-B7 mice produced mainly IFN-γ, compatible with a Th1 cytokine pattern. In contrast, cells from the DR4/RIP-B7 and the DQ8DR4/RIP-B7 mice produced IL-4 but very little IFN-γ, suggesting a Th2 cytokine pattern (10). However as the authors point out, the mechanisms of the HLA-DR4 effect are not clear (10). Similar HLA class II transgenic mice have been used to study the T-cell receptor (TCR) repertoire after immunization with recombinant GAD65 (12). These studies, using several DR/DR as well as DR/DQ combinations, have revealed that certain immunodominant GAD65-specific TCRs are all but absent in the repertoire of HLA class II double transgenic mice, if the diabetes-protective DRB1*0403 allele is present. This elimination of potentially pathogenic T cells occurs through still-unidentified mechanisms, operating at least in part at the level of intracellular antigen processing. Similarly, human antigen-presenting cells (APCs) carrying both the diabetes-protective DRB1*0403 allele and a second, diabetes-conducive DR4 allele (DRB1*0405) fail to present these processed immunodominant GAD65 epitopes to T cells (S. Parry and G. Sonderstrup, unpublished results), although they are perfectly capable of presenting these epitopes if they are provided as synthetic peptides. Further, these same human APCs can process and present other GAD65 epitopes normally to T cells that recognize these epitopes in the context of the DRB1*0405 molecule (S. Parry and G. Sonderstrup, unpublished results). This result indicates that this is a selective epitope-specific phenomenon. The ability to eliminate potentially pathogenic T cells presented by coexpressed HLA class II molecules, HLA-DR or DQ, may be responsible for the disease-protective effect of the HLA-DRB1*0403 allele and may therefore represent yet another mechanism for preventing autoimmunity in humans. The study of Wen et al. (10) and the results described above illustrate how HLA class II transgenic mice can provide a blueprint for unraveling DR/DQ allelic interactions in autoimmunity. This information should also provide new insight into pathogenesis and inspire novel strategies to prevent autoimmune disease.

Identification of autoantigen epitopes in MHC class II transgenic mice

Summary: MHC class II molecules function by selective binding of anti‐genic peptides, thereby both shaping the T‐cell receptor (TCR) repertoire in the thymus and influencing presentation of immunogenic peptides to CD4+ T cells in the periphery. The strong association between a number of human autoimmune diseases (type 1 diabetes, rheumatoid arthritis, and multiple sclerosis) and certain HLA‐DR/DQ alleles suggests that it may be possible to alter pathological autoimmune responses by deliberate introduction of autoantigenic peptides in a “tolerogenic” manner. Since there are likely to be differences in epitope selection and epitope spreading in different patients over time, this approach requires identification of all the immunogenic CD4‐ T‐cell epitopes (dominant, subdominant, or cryptic) of an autoantigen which elicit T‐cell responses restricted to the HLA‐DR/DQ alleles predisposing to these autoimmune diseases. This paper describes a new approach for the identification of immunogenic peptide epitopes of human autoantigenic proteins using HLA‐DR and DQ transgenic mice. These mice are engineered to select a full TCR repertoire which can identify immunogenic peptide epitopes similar or identical to human subjects of the same HLA‐DR/DQ genotype. This experimental system also allows comparison of autoantigenic immune responses restricted to disease‐susceptible and disease‐resistant HLA‐DR/DQ alleles.

Autoreactivity versus autoaggression: a different perspective on human autoantigens

Antigen-specific B and T cell responses against myelin basic protein, as well as responses against beta-islet-cells or joint tissue, are commonly found both in patients with autoimmune disease and in normal control subjects with disease-associated HLA-DR/DQ alleles. Thus, autoreactive immune responses are not disease-specific; however, the presence of certain autoantibodies may have prognostic value and may aid in disease management.

Autoantigenic HCgp39 Epitopes Are Presented by the HLA-DM-Dependent Presentation Pathway in Human B Cells

It is hypothesized that autoimmune diseases manifest when tolerance to self-Ags fails. One possible mechanism to break tolerance is presentation of self-Ag in an altered form. Most Ags are presented by APCs via the traditional presentation pathway that includes “epitope editing” by intracellular HLA-DM, a molecule that selects for stable MHC-peptide complexes. We were interested in testing the hypothesis that autoreactive MHC-peptide complexes may reach the cell surface by an alternate pathway without being edited by HLA-DM. We selected a cartilage autoantigen human cartilage glycoprotein 39 to which T cell responses are observed in rheumatoid arthritis (RA) patients and some DR*04 healthy subjects. RA is genetically associated with certain DRB1 alleles, including DRB1*0401 but closely related allele DRB1*0402 is either neutral or mildly protective with respect to RA. We generated human B lymphoblastoid cell line cells expressing DR*0401 or DR*0402 in the presence or absence of intracellular HLA-DM and assessed their ability to present a candidate autoantigen, human cartilage glycoprotein 39. Our results show that the presence of intracellular HLA-DM is critical for presentation of this autoantigen to CD4+ T cell hybridomas generated from DR*04-transgenic mice. Presentation of an autoantigen by the traditional HLA-DM-dependent pathway has implications for Ag presentation events in RA.

Aberrant MHC class II expression in mouse joints leads to arthritis with extraarticular manifestations similar to rheumatoid arthritis

Genetic susceptibility to rheumatoid arthritis (RA) is associated with certain MHC class II molecules. To clarify the role of these determinants in RA, we generated the D1CC transgenic mouse that expressed genes involved in antigen processing and presentation by the MHC class II pathway in joints. The class II transactivator, which was transcribed from the rat collagen type II promoter and enhancer, directed the expression of these genes. In D1CC mice congenic for the H-2q (DBA/1) background, small amounts of bovine collagen type II in adjuvant induced reproducibly an inflammatory arthritis resembling RA. Importantly, these stimuli had no effect in DBA/1 mice. Eighty-nine percent of D1CC mice developed chronic disease with joint swelling, redness, and heat in association with synovial proliferation as well as pannus formation and mononuclear infiltration of synovial membranes. Granulomatous lesions resembling rheumatoid nodules and interstitial pneumonitis also were observed. As in patients with RA, anticyclic citrullinated peptide antibodies were detected during the inflammatory stage. Finally, joints in D1CC mice displayed juxtaarticular demineralization, severe joint space narrowing, and erosions, which led to ankylosis, but without the appearance of osteophytes. Thus, aberrant expression of MHC class II in joints facilitates the development of severe erosive inflammatory polyarthritis, which is very similar to RA.

Human Risk Allele HLA-DRB1*0405 Predisposes Class II Transgenic Ab0 NOD Mice to Autoimmune Pancreatitis

BACKGROUND & AIMS Autoimmune pancreatitis (AIP) underlies 5%-11% of cases of chronic pancreatitis. An association between AIP and the human leukocyte antigen (HLA)-DRB1*0405/DQB1*0401 haplotype has been reported, but linkage disequilibrium has precluded the identification of predisposing HLA gene(s). We studied the role of single HLA genes in the development of AIP in transgenic mice. METHODS CD4(+) T-cell-negative I-Abeta chain(-/-) (Ab0) mice develop AIP spontaneously, likely due to dysregulation of CD8(+) T- cell responses. We generated Ab0 nonobese diabetic (NOD) mice transgenic for HLA-DR*0405, leading to rescue of CD4(+) T cells; we compared their susceptibility to AIP with HLA-DQ8 or HLA-DR*0401 (single) transgenic, or HLA-DR*0405/DQ8 (double) transgenic mice. RESULTS CD4(+) T-cell-competent HLA-DR*0405 transgenic Ab0 NOD mice develop AIP with high prevalence after sublethal irradiation and adoptive transfer of CD90(+) T cells, leading to complete pancreatic atrophy. HLA-DR*0405 transgenic mice can also develop unprovoked AIP, whereas HLA-DR*0401, HLA-DQ8, and HLA-DR*0405/DQ8 transgenic Ab0 NOD controls all remained normal, even after irradiation and adoptive transfer of CD90(+) T cells. Pancreas histology in HLA-DR*0405 transgenic mice was characterized by destructive infiltration of the exocrine tissue with CD4(+) and CD8(+) T cells, B cells, and macrophages. Mice with complete pancreatic atrophy lost weight, developed fat stools, and had reduced levels of serum lipase activity. CONCLUSIONS Because HLA-DR*0405 expression fails to protect mice from AIP, the HLA-DRB1*0405 allele appears to be an important risk factor for AIP on the HLA-DRB1*0405/DQB1*0401 haplotype. This humanized mouse model should be useful for studying immunopathogenesis, diagnostic markers, and therapy of human AIP.