Department of Microbiology and Immunology, Stanford, CA 94305, USA,
1 The Kennedy Institute of Rheumatology, 1, Aspenlea Road, Hammersmith, London W6 8LH, UK,
2 Cell Genesys Inc., Lakeside Drive, Foster City, CA 94404 and
3 Department of Microbiology and Immunology, Stanford, CA 94305, USA
Correspondence to:
G. Sønderstrup, D345 Fairchild Building, Department of Microbiology and Immunology, Stanford, CA 94305, USA.
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HLA association with disease: still a mechanistic mystery! |
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A variety of hypotheses have been advanced, based on current understanding of the biology of HLA molecules, to explain the HLA association with disease [6]. Crystallographic studies have demonstrated that MHC molecules bind peptide fragments in their binding cleft and present them to specific T-cell receptors (TCRs) [79]; in general, class I MHC molecules present intracellular peptides to CD8+ T cells, while class II molecules present peptides from extracellular proteins to CD4+ T cells. The remarkable polymorphism of MHC molecules dictates the specificity of peptide binding and, in turn, influences the processes of thymic selection and peripheral T-cell activation. The most popular hypothesis for HLA association with disease is that certain HLA molecules present a `pathogenic' peptide or peptides, and thereby elicit a deleterious immune response from specific T cells. Alternatively, disease susceptibility could be determined by variations in TCR repertoire. For example, during thymic selection, certain HLA molecules (or HLA-derived peptides presented by a second HLA molecule) may fail to delete potentially autoaggressive T cells from the repertoire. In the periphery, certain TCR specificities may be deleted by the presentation of superantigens on MHC class II molecules, further modifying the circulating TCR repertoire.
Despite a quarter of a century of investigation, mechanisms of HLA involvement in autoimmune disease remain speculative. This is largely due to the difficulty of isolating the effect of a particular HLA molecule on the disease studied. This problem has emerged in epidemiological, genetic and immunological approaches to the study of these diseases.
HLA is only one piece of the puzzle
HLA-associated autoimmune diseases are complex, arising as a result of the interaction of environmental influences with a polygenic background of susceptibility. In some cases, HLA appears to exert a large influence on the susceptibility to disease and in other diseases it is a relatively small component.
Neighbours tend to travel together
The HLA complex encodes multiple HLA molecules and a number of other immunologically active molecules [10]. Loci in close proximity exhibit linkage disequilibrium and HLA-DR and -DQ alleles, for example, are usually inherited together. The demonstration of an association between a disease and a particular locus may reflect an effect of either the locus studied, an adjacent genetic locus or a combination.
HLA functions in concert with the rest of the immunological orchestra
The influence of a particular HLA molecule on immune responses will be modified by the `landscape' of the other HLA molecules among which it is expressed. Each HLA molecule expressed modifies the TCR repertoire selected in the thymus and, in the periphery, may compete with other HLA molecules for peptide presentation. In addition, fragments of HLA molecules are themselves presented in the peptide-binding groove of other HLA molecules [11]. HLA molecules also interact with an array of immunological components involved in antigen processing, antigen recognition signalling and effector functions. For example, variations in degradative enzymes, proteasome components, the TCR repertoire, the signalling cascade or other co-stimulatory receptors, such as adhesion molecules, may all influence the immunogenicity of an epitope presented by a particular HLA molecule.
In addition, any study of human disease is hindered by the genetic variation between individuals and the obvious ethical constraints on immunizing individuals and analysing immune responses. Advances in molecular biology have provided a number of new technologies, including the development of HLA transgenic mice, with which to circumvent many of these problems. This enables the isolation, in vivo, of individual MHC molecules in order to assess their role in the regulation of immune responses and, ultimately, the development and resolution of disease.
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HLA transgenic mice: isolating the suspect |
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The process of generating a meaningful HLA transgenic model in which to study `humanized' immune responses is not trivial and extrapolating from murine to human disease poses an additional series of problems. The major issues can be summarized as follows:
Each of these will be discussed, along with strategies used by us and by others to address these problems.
Does the HLA transgene produce a physiological pattern of HLA expression?
The promoter regions of HLA class I and II genes determine the temporal pattern and tissue specificity of expression, as well as the modulation of expression during inflammation (reviewed in Ting and Baldwin [12] and Mach et al. [13]). In addition to differences in regulation between classes of HLA genes, the DRA and DRB loci may exhibit subtle regulatory differences and promoter polymorphisms may result in allelic differences in expression [14, 15]. Despite these complexities, it has proved possible to establish HLA transgenic lines by introducing HLA genes into mice under the control of their native human promoter. The tissue-specific expression of the transgenes has been variable. However, in some models, the HLA molecules produced are functional [1618]. An alternative approach is to introduce the HLA transgene under the control of a murine MHC class II promoter. There are subtle differences between mice and humans in the expression of MHC class II molecules, and the introduction of an HLA gene under the control of a murine promoter ensures more `physiological' patterns of expression in the mouse. The I-E promoter has been used to make HLA-DR4 transgenic mice [19]. The regulation of the transgenic HLA-DR appeared to be physiological, both in the thymus and the periphery (including the absence of MHC class II molecules on activated murine T cells), and the molecule was capable of functioning as a restriction element.
Even an optimal promoter does not guarantee physiological expression in transgenic models. The level and pattern of expression may also be influenced by the copy number and integration site of the transgene, and by the ability to associate with critical murine molecules (e.g. invariant chain and H-2M for class II molecules, and ß2m for class I molecules). The MHC class II overexpression syndrome, described in I-Aß transgenic mice, is often related to integration of over 50 copies of the transgene. It manifests as inflammatory disease with low levels of cell surface MHC class II molecules, progressive B-cell deficiency and abnormal extramedullary granulopoiesis with eosinophilia [2022]. This phenotype can easily be mistaken for an HLA-related model of human inflammatory disease, whereas it appears to be a non-specific effect resulting from the overexpression of any MHC class II allele. Overexpression of MHC class I molecules can also be associated with an inflammatory phenotype, as observed in the high-copy-number HLA-B27 mice [23], but it is unclear how this relates to the pathogenesis of HLA-B27-associated diseases in humans. The stoichiometry of transgenic products (class II and ß chains) may also influence expression and can be optimized by introducing the transgenes in tandem, within a single construct [19]. Given these various considerations, it is necessary to generate several founder lines, in order to obtain one with a physiological pattern of HLA expression.
Does the human MHC molecule interact normally with murine components of the antigen-processing and recognition pathways?
In general, it appears that trans-species interaction of HLA molecules with murine cellular and immunological machinery is sufficient to allow the transgenic molecules to function as restriction elements, at least under some circumstances. Both HLA-DR transgenic [24] and several DR
/DRß or DR
/DQß double transgenic mice exhibit reconstitution of HLA-restricted T-cell function [16, 18, 19, 2528].
Although transgenic HLA molecules can function in mice, it is possible that the nature of T cells activated by a given MHC/peptide stimulus may be different in HLA transgenic mice, compared with human subjects. T-cell activation is determined by the avidity of MHC/peptide/TCR interaction, which is influenced by the quality of co-receptor binding. For example, CD4, expressed on T cells, stabilizes the interaction of TCR with peptide/class II MHC by binding to the membrane-proximal domain of both MHC class II ß chains (2 and ß2 domains) and interacts with the src-family related tyrosine kinase, p56lck , intracellularly [29, 30]. CD8 performs a similar function for peptide/MHC class I interactions with TCR with binding sites on the
2 and ß3 domains of the MHC class I molecule [31]. Although murine CD4 and CD8 appear to perform adequately as HLA molecule co-receptors in some systems [16, 18], there are other examples where HLA-restricted immune responses have been rescued or augmented by the introduction of transgenic human CD4 [32, 33] or human CD8 [34]. The interaction of transgenic HLA molecules with CD4 or CD8 co-receptors can be optimized by the introduction of an additional transgene encoding either human CD4 or human CD8. An early model introduced the human CD4 transgene under the control of the murine CD3
promoter, resulting in human CD4 molecule expression on all CD3+ cells, both CD4+ and CD8+ subsets, and low-level expression on B cells [19]. Another approach has been to generate chimeric MHC molecules, in which the peptide-binding domains of the molecule were derived from HLA molecules, while the co-receptor binding site on the membrane-proximal ß chain was of murine origin [3537]. A modified HLA-DRB1*0401 transgenic line has also been developed, in which residues 110 and 139 of the DRß chain have been altered to resemble murine I-Aß, in order to improve interaction with murine CD4 [38]. The characterization of the murine CD4 transcriptional enhancer/silencer element by Littman and colleagues [39] enabled us to develop a new panel of human CD4, HLA-DR transgenic mice, in which human CD4 is appropriately expressed on CD4+, but not CD8+, cells and the unmodified HLA-DR molecule is expressed [28].
Does the presence of murine MHC molecules interfere with the study of human MHC molecules?
The presence of murine MHC molecules in an HLA transgenic model may confound attempts to study the function of the human MHC molecule. The murine counterparts will influence the selection of the TCR repertoire as well as competing with human MHC, in peripheral antigen-presenting cells, for binding to peptides or other components of the processing/presentation machinery. Mice lacking MHC molecules provide the means by which the transgenic HLA molecule can be isolated as the sole restriction element in a murine model. Mice deficient in ß2-microglobulin (ß2m°) usually lack surface expression of murine class I molecules, although certain class I heavy chains, such as HLA-B27, can be expressed in the absence of ß2m. These animals were developmentally normal, but lacked the CD8+CD4- T-cell compartment [40]. MHC class II-deficient mice (I-Aß°) have been generated in the 129 cell line, which carries a mutation in the I-E gene promoter, preventing expression of the I-E
ß heterodimer. Expression of the remaining class II molecule, I-A, was abolished by targeted disruption of the I-Aß gene [41]. The I-Aß° mice have very few CD4+ lymphocytes in the periphery and are unable to respond to T-cell-dependent antigens. Introduction of an HLA-DR transgene on the I-Aß° background partially restores the selection and function of CD4+ T cells (Table 1
). The size of the CD4+ compartment approaches that in wild-type mice in murine models which are homozygous for both HLA-DR4 and appropriately regulated human CD4 transgenes on a murine class II-deficient background [33]. In mice of this genotype, the avidity of TCR interaction with the HLA-DR/peptide should reflect most closely that observed in man and therefore provide the best model of HLA-DR-restricted immune responses.
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In murine models of autoimmune disease, how closely do murine autoantigens resemble their human counterparts, both structurally and functionally?
If HLA molecules present `pathogenic' peptides from autoantigens, then trans-species variations in the autoantigen may result in a different HLA-restricted response to the murine equivalent of a human autoantigen. The islet cell antigen, glutamic acid decarboxylase 65 (GAD65), is an autoantigen recognized early in the development of both human and murine IDDM. Published sequences reveal 95% amino acid sequence identity between mice and humans, and it can probably serve as an autoantigen on the relevant transgenic HLA class II molecules expressed in the mouse [42]. In contrast, the synovial autoantigen human cartilage glycoprotein-39 (HCgp-39) is only 74% identical with the murine equivalent BRP-39 [43, 44]. The evaluation of its role in murine models of inflammatory arthritis will probably necessitate the expression of human protein (HCgp-39) as a transgene, in the relevant model.
Concerns about the equivalence of human and murine disease pertain to all HLA transgenic murine models of autoimmune, allergic or infectious disease. In the non-obese diabetic (NOD) mouse model [45] and the NZB/NZW murine model of systemic lupus erythematosus [46], disease occurs spontaneously, as in the human patient. However, other models, including the collagen-induced arthritis (CIA) model [47] and the murine model of demyelinating disease, experimental allergic encephalitis [48], require the introduction of an antigenic trigger. All autoimmune diseases are multifactorial and develop from an interplay of environmental influences on a background of polygenic susceptibility. However, the genetic loci involved in humans are frequently different to those implicated in the murine model, as has been demonstrated through mapping the susceptibility loci for IDDM in humans and NOD mice [49, 50]. At best, murine models can be expected to reproduce selected features of human disease. However, a human disease susceptibility gene, expressed in mice, may have no demonstrable effect on the murine model of disease. This may occur either because the pathogenesis of the disease is different in humans and mice, or because the mouse lacks the `supporting' susceptibility genes, such as the critical human autoantigen(s) or the ability to produce a prominent interferon gamma (IFN-) response following antigen challenge.
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Compiling the evidence |
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Determination of immunodominant epitopes within a complex antigen
The association of particular HLA molecules with susceptibility to disease may be the result of a pathogenic immune response to the presentation of certain peptides. If this model is correct, then it is important to identify the autoantigens involved in pathogenesis and to define the T-cell epitopes presented by individual HLA molecules, which are associated with either disease susceptibility or resistance.
HCgp-39 is a synovial autoantigen, which is not constitutively expressed, but which is secreted by chondrocytes and synoviocytes during synovial inflammation. Peripheral blood T cells from >50% patients with RA, and from HLA-DR-matched controls, respond spontaneously to certain peptides from HCgp-39, which bear a DR*0401 motif [28, 51]. It is unclear whether HCgp-39 is directly involved in the pathogenesis of disease, but its expression in the inflamed joint and its autoantigenicity make it an attractive candidate for peptide-mediated immunotherapy. HLA-DR4 transgenic mice have been used to map the T-cell epitopes of HCgp-39 in the context of the RA-associated HLA-DR4 allele, DR*0401 and the closely related disease-neutral/resistant allele DR*0402 [28]. Mice transgenic for HLA-DR4 (either DR*0401 or DR*0402) and human CD4, but lacking murine class II molecules (I-Aß°), were immunized with HCgp-39 emulsified in incomplete Freund's adjuvant. A panel of T-cell hybridomas was raised to provide a `snapshot' of the primary TCR repertoire responding to HCgp-39 in the context of HLA-DR*0401 or -DR*0402. The specificity of the T-cell hybridomas was tested using HLA-DR4 positive antigen-presenting cells (APC), presenting whole HCgp-39, then pools of overlapping 16-mer peptides, which spanned the HCgp-39 molecule, by individual peptides from a stimulatory pool and, finally, truncated variants of an individual stimulatory peptide. This study revealed three immunodominant epitopes in the context of DR*0401 (100115, 262277, 322337) and two entirely distinct epitopes in the context of DR*0402 (2237, 298313) (Table 2). All could also be presented by human APC expressing the appropriate HLA-DR4 molecule, and each of the DR*0401 epitopes has been shown to elicit secondary T-cell proliferative responses in some RA patients. We are currently studying the kinetics of interaction of these peptides with soluble recombinant DR*0401 and DR*0402, and investigating the influence of physiological pH variation and of HLA-DM on the stability of the complexes.
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The DR*0401, hCD4, I-Aß° mice have been similarly used to map the epitopes of insulin and its prohormones, proinsulin and preproinsulin [52]. These results demonstrate that the prohormones, but not the mature insulin autoantigen, carry the immunodominant epitope in the context of DR*0401. The epitope, LALEGSLQK, was located at the junction of the C-peptide and A-chain, and it is proteolytically cleaved during maturation of the insulin molecule. This epitope can be processed and presented from prohormone by DR*0401-homozygous EpsteinBarr virus-transformed human B-cell lines and it is also recognized by T cells from the peripheral blood of HLA-DR4-positive patients with IDDM.
HLA transgenic mice have also been used to map T-cell epitopes from microbial antigens. For example, treatment-resistant Lyme arthritis is associated with HLA-DR4 and T-cell responses to the borrelial outer surface protein, OspA [53, 54]. Four immunodominant T-cell epitopes of OspA have been defined in the DR*0401/hCD4/I-Aß° mouse model, as described above [55].
The quality of the effector immune response
Defining T-cell epitopes in the context of disease-susceptible or disease-protective HLA molecules is a prelude to understanding how particular peptide/HLA partnerships might modify the nature of the immune response and hence the natural history of disease. In autoimmune and allergic disease, aggressive T-cell responses are stimulated by autoantigens and environmental agents, whereas, in healthy subjects, the behaviour of T cells specific for these antigens is benign. Many pathogens, including Mycobacterium tuberculosis, Borrelia burgdorferi, hepatitis C virus (HCV) and human immunodeficiency virus (HIV), elicit pathological rather than protective immune responses in the susceptible host, and the immune-mediated damage in chronic infectious disease and autoimmunity are often indistinguishable.
HLA transgenic murine models can be used to investigate the immune responses elicited by HLA-restricted T-cell epitopes from microbial antigens. The prominence of cytotoxic T cells, specific antibody or delayed-type hypersensitivity in the response to a panel of defined T-cell epitopes indicates which may be suitable for vaccine development. T-cell epitopes can also be modified in an attempt to improve the ability of the immune response to protect against, or eradicate, infection. For example, HLA-A2 transgenic mice were used to assess the immunogenicity of substituted peptides from an HLA-A2-restricted HCV epitope. One variant, 8A, proved to be a more potent stimulator of cytotoxic T-cell responses than the wild-type epitope [56].
Several autoimmune diseases are associated with apparent overproduction of certain cytokines. In both IDDM and RA, for example, prominent Th1-like responses are evident in T cells from the islets of Langerhans or the inflamed synovium, respectively [5759]. In murine models of diabetes, disease can be accelerated by the adoptive transfer of autoaggressive T-cell clones, which produce IFN- [60]. HLA-transgenic mice can be used to investigate the cytokine profiles elicited by the T-cell epitopes defined from autoantigens. For example, following immunization of DR*0401/hCD4/I-Aß° mice with HCgp-39, restimulation of the lymph node T cells in vitro with a pool of the DR*0401 immunodominant epitopes stimulated prominent IFN-
and tumour necrosis factor alpha (TNF-
) responses. Lymph node T cells from similarly immunized DR*0402/hCD4/I-Aß° mice produced small amounts of IFN-
and no TNF-
[28]. Lymph node T cells from DR*0402 transgenic mice immunized with the borrelial antigen, Osp A, elicited a substantial IFN-
response, indicating that DR*0401 and DR*0402 exert antigen-specific influences on the cytokine response. It is anticipated that the functional characterization of T-cell epitopes in association with particular HLA molecules will offer therapeutic strategies for autoimmune, allergic and certain infectious diseases based on the regulation of cytokine profiles.
Modification of disease
Murine disease models can be used to investigate the effect of single or multiple transgenic HLA molecules on disease expression. They also provide a method for testing novel immunomodulatory therapies in vivo, accepting the usual caveats associated with the extrapolation of results from murine to human disease.
The first HLA transgenic disease model was the HLA-B27 transgenic rat, which developed a spondyloarthropathy [61]. HLA-B27 transgenic mice also spontaneously develop inflammatory arthritis in transgenic mice which lack ß2-microglobulin [62]. This may reflect the unusual stability and surface expression of HLA-B27 heavy chain in the absence of ß2m [63].
CIA occurs only in the presence of susceptible murine MHC haplotypes, such as H-2q [47]. In I-Aß° mice, which lack murine class II molecules, CIA can also be induced in the presence of transgenic HLA-DR4 (DRA1*0401/DRB1*0401) back-crossed to the DBA/1J background [64] and HLA-DQ8 (DQA1*0301/DQB1*0302) [65]. Murine arthritis has also been reported following immunization of female BALB/c mice with the human synovial autoantigen HCgp-39 [51]. It remains to be seen whether spontaneous disease will arise in mice expressing HLA-DR4, human CD4 and the transgenic human molecule, HCgp-39.
In humans, the interaction between different HLA-DR molecules in the same individual, and between HLA-DR and -DQ molecules, modifies the susceptibility to RA. Two different RA-associated HLA-DR molecules may have a synergistic effect on the relative risk of disease [66, 67]. Both HLA-DQ7 and -DQ8 occur in linkage disequilibrium with DRB1*0401, but the DRB1*0401-DQB1*0301 (DQ7) haplotype appears to be associated with the production of rheumatoid factor [68] and Felty's syndrome [69]. The generation of mice expressing different combinations of HLA class II molecules may provide insight into the mechanisms underlying these interactions. Zanelli et al. [70] proposed that HLA-DQ molecules primarily determined susceptibility to RA by presenting `arthritogenic' peptides. Certain HLA-DR molecules could reduce this HLA-DQ associated susceptibility by providing peptides from the third hypervariable region which could compete successfully with the `arthritogenic' peptides. In this model, peptides carrying the consensus sequence, which is common to all RA-associated HLA-DR molecules, fail to protect against disease. In support of this model, transgenic HLA-DR2 has been reported to decrease the incidence of CIA in HLA-DQ8/I-Aß° transgenic mice, whereas HLA-DR3 was reported to have no modifying effect [71]. In contrast, the influence of HLA-DQ8 appeared negligible in the immunogenicity of the synovial autoantigen, HCgp-39, in mice expressing both HLA-DR*0401 and HLA-DQw8 transgenes. The majority (88%) of T-cell hybridomas raised from these mice following immunization with HCgp-39 were restricted by HLA-DR*0401 and the profile of DR*0401-restricted epitopes was almost identical to that previously observed in the HLA-DR*0401 single transgenic mice [28, 72].
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Conclusion |
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Acknowledgments |
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References |
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