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The Journal of Immunology, 1998, 160: 4169-4174.
Copyright © 1998 by The American Association of Immunologists

Polymorphism at the HLA-DQ Locus Determines Susceptibility to Experimental Autoimmune Myasthenia Gravis1

Raghavan Raju*, Wen-Zhi Zhan{dagger}, Peter Karachunski{ddagger}, Bianca Conti-Fine{ddagger}, Gary C. Sieck{dagger} and Chella David2,*

Departments of * Immunology and {dagger} Anesthesiology Research, Mayo Clinic, Rochester, MN 55905; and {ddagger} Department of Biochemistry and Pharmacology, University of Minnesota, St. Paul, MN 55108


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-Aß0 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-Aß0) 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Myasthenia gravis (MG)3 is an important model for the study of molecular mechanisms of autoimmunity, since the target autoantigen is known (nicotinic acetylcholine receptor (AChR)), sequenced, and well characterized (1). Muscle AChR is formed by four homologous subunits in a stoichiometry of {alpha}2ß{gamma}(or {epsilon}){delta} (1, 2). Anti-AChR CD4+ T cells from MG patients are known to recognize several epitopes on all muscle AChR subunits (3, 4, 5, 6, 7, 8, 9, 10). MG is an Ab-mediated disease, as Abs have been shown to reduce the number of available muscle AChRs (Ref. 11 and references therein). CD4+ T cells are required for generation of the high affinity IgG Abs that bind muscle AChR (12, 13, 14).

MHC class II molecules serve the dual function of selection of specific peptides to be bound and presented to the TCR and of regulation of TCR specificities during the process of T cell differentiation and maturation in the thymus. Identification of class II molecules associated with autoimmune diseases helps formulate specific models of pathogenesis of autoimmune diseases. Specific class II susceptibility genes have been described in different autoimmune diseases, such as type I diabetes (HLA DR4, DR3, DQ8), rheumatoid arthritis (HLA DR4), pemphigus vulgaris (HLA DR4, DR6), and other diseases (as reviewed in 15 .

HLA linkage in MG as reported in literature varies with ethnic groups, age, and sex. However, some of the initial epidemiologic studies demonstrate that HLA B8 and HLA DR3 are linked to MG in Caucasians (16, 17, 18, 19, 20, 21, 22). One of the first reports that established the significance of HLA-DQ ß gene polymorphism in MG pathogenesis was that of Bell et al. (16). However, the specific role of any particular HLA-DQ gene in the etiopathogenesis of MG is not as clear as in other autoimmune diseases such as type I diabetes or rheumatoid arthritis.

In young Caucasian MG patients, mainly women without thymoma and with high levels of AChR Abs have a high frequency of the B8 and/or DR3 haplotypes (119–22). MG was also found to be positively associated with the DQB1*0604 allele, particularly in patients with thymoma (23). Horiki et al. (24) reported that combinations of HLA-DPB1 and HLA-DQB1 alleles determine the susceptibility to early onset MG in Japan. In Jamaicans, MG is most strongly associated with HLA-B8, HLA B13, and DQ4 and is negatively associated with HLA-A2. Female MG patients under 30 yr of age at the onset of disease had a significantly higher frequency of DQB1*03, which includes *0301, *0302, and *0303, compared with healthy controls (25). In a Swedish study, it was reported that two different DQ2 haplotypes (DQA1*0501/DQB1*0201 and DQA1*0201/DQB1*0201) were positively associated with MG (26). Carlsson et al. (27) observed a strong association in Caucasian MG patients of a DR-DQ haplotype (DR3DR52DQ6) with MG in young females and an association with DR4-DQ8 in elderly non-DR3 males. A recent study of 79 Swedish patients and 155 unrelated, population-based controls, found that polymorphic domains on the HLA-DQ molecule are associated with disease heterogeneity in MG (28). By analyzing polymorphic domains on HLA-DQ molecules contributing to positive and negative association with MG, they found that a domain with residues common to DQB1*05 and DQB1*06 alleles is negatively associated with the disease in patients with thymic hyperplasia or an early disease onset.

In mice the H2b haplotype is strongly associated with susceptibility to experimental autoimmune myasthenia gravis (EAMG) (17, 29). Other susceptible haplotypes are H2q, H2r, and H2i. The resistant strains include the H2d, H2k, and H2p haplotypes. In C57BL6 (H2b) mice, the susceptibility to EAMG was mapped to the H2-Ab genes (30, 31, 32). The role of H2-Ab in the development of EAMG is further supported by the finding that a mutation in Aßb at positions 67, 70, and 71 generates the H2bm12 strain that is not susceptible to EAMG (30, 31).

In this study we investigated the roles of HLA-DQA1*0301/DQB1*0302 (DQ8) and HLA-DQA1* 0103/DQB1*0601 (DQ6) in the pathogenesis of MG by using mice transgenic to the respective HLA class II molecule in the absence of endogenous mouse class II molecules. The mice were immunized with Torpedo acetylcholine receptor (TAChR) to examine the effect of polymorphism at the HLA DQ locus on their susceptibility for EAMG.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Transgenic mice expressing functional HLA-DQ8 (HLA-DQA1*0301/DQB1*0302) and DQ6 (HLA-DQA1* 0103/DQB1*0601) genes were generated as described previously (33, 34, 35, 36, 37). Cosmid clones H11A and X10A containing the DQA1*0301 and DQB1*0302 genes were provided by Dr. J. Strominger, while cosmids pAKQ 4116 and pAKQ 056 containing DQA1*0103 and DQB1*0601 genes, respectively, were gifts from Dr. H. Inoko. Cosmids pAKQ 4116 and pAKQ 056 were derived from the AKIBA cell line, linearized by restriction enzyme digestion, and microinjected into (CBA/J x B10.M)F2 or (SJL x SWR)F2 embryos (34). HLA-DQ8 and DQ6 transgenic mice were bred into H2b mice lacking Aßb expression (the latter hereafter referred to as Aß0 mice) to obtain DQ8.Aß0or DQ6.Aß0 mice. The mice were all homozygous for the transgene, as demonstrated by the absence of nontransgenic offsprings in the generations previous to that studied.

Purification of TAChR

TAChR was purified from Torpedo californica (Aquatic Research Consultants, San Pedro, CA) electric organ as postsynaptic membrane fragments that were further enriched for TAChR by alkaline pH treatment (38, 39). The purity of the isolated TAChR was ascertained by SDS-PAGE (40). TAChR preparations were characterized using 125I-labeled {alpha}-bungarotoxin; the purified receptor preparation contained 4 to 6 nmol of {alpha}-bungarotoxin binding sites/mg of protein (41).

Induction of the disease

Mice were given three injections (s.c.) of TAChR (20 µg/mice) at 4-wk intervals. The first injection was given in CFA, and boosters were given in IFA in a 1:1 ratio. After 12 wk, mice were sacrificed.

Assay of serum anti-AChR Abs

Anti-AChR Abs in the sera were assayed by a radioimmunoprecipitation assay using 125I-labeled {alpha}-bungarotoxin (38).

Disease assessment

Clinical assessment. Muscle weakness was assessed every week in a blind study (42). Briefly, mice were allowed to grip their paws on cage top grids, were pulled off the grid by tail consecutively for 25 times for forced exercise, and were scored as follows: grade 0, there was no weakness at rest or after exercise; grade 1, normal strength at rest, but weak with chin on the floor and inability to raise the head after exercise; grade 2, the mice exhibit grade 1 weakness at rest; and grade 3, moribundity or quadriplegia. The methods used for the clinical assessment of EAMG are subjective, and various factors may interfere with them. To confirm that the mouse weakness was of a myasthenic nature, muscle weakness of grade 1 was verified by pancuronium-sensitized forced exercise tests, which exacerbate myasthenic weakness. Grade 2 weakness was confirmed by the use of tensilon (edrophonium chloride).

Electrophysiology. Since neuromuscular junction failure is the hallmark of myasthenia, we measured the neuromuscular transmission failure in a few DQ8 transgenic mice that showed muscle weakness and in mice that were normal. Due to practical reasons we were unable to perform this test in all the mice we studied. Although use of pharmacologic agents such as pancuronium or edrophonium chloride was sufficient to establish the myasthenic nature of muscle weakness, we believed that it would be more informative if electrophysiologic experiments were also performed (43, 44). Briefly, the right midcostal diaphragm muscle was excised together with the phrenic nerve and mounted vertically in a glass tissue chamber containing Ringer’s solution. The solution was aerated with 95% O2/5% CO2 and maintained at 26°C. The central tendon was attached in series to a calibrated force transducer, while the rib insertion was clamped to a micromanipulator. The muscle was stimulated directly by 0.5-ms pulses using a pair of platinum electrode. Muscle fiber length was adjusted until maximum isometric twitch force responses were obtained. The phrenic nerve was stimulated using 0.2-ms duration pulses delivered via a suction electrode. Neuromuscular transmission failure was assessed at 40 Hz. Nerve stimulation was presented at 40 Hz in 330-ms duration trains repeated one train per second for a period of 2 min. Every 15 s, direct muscle stimulation at 40 Hz was superimposed. The force decline during direct muscle stimulation reflects only the contribution of muscle fatigue, while the force loss during nerve stimulation reflects the contributions of both muscle fatigue and neuromuscular transmission failure.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rationale

The susceptibility of different strains of mice to EAMG is well characterized. The H2-A molecule is important for the induction of the disease, and polymorphism at this locus influences the susceptibility to EAMG. MG is an Ab-mediated disease, and Th cells are important in providing help for the generation of high affinity Abs by B cells. The presentation of pathogenic epitopes to CD4+ T cells is important in the manifestation and perpetuation of MG through generation of high affinity Abs. The selection of CD4+ T cells in the thymus is regulated by MHC class II molecules. MHC class II molecules can present pathogenic epitopes to CD4+ T cells, and polymorphism at this locus should influence such peptide binding and presentation, leading to susceptibility or protection to disease. In humans, the study of the significance of HLA in autoimmune diseases is limited by the fact that every APC would express at least three different (DR, DQ, and DP) class II isotypes along with different class I molecules, making interpretations of the role of any single molecule difficult. Mice transgenic for the MHC genes are a useful model to study the role of HLA molecules in the pathogenesis of MG and other autoimmune diseases. To understand the influence of polymorphism at human class II locus in the pathogenesis of MG and EAMG, we used two different HLA transgenic mice in this study. We used three groups of mice, Aß0. DQ8 (DQA1*0301/DQB1*0302)., Aß0.DQ6 (DQA1*0103/DQB1* 0601), and Aß0. These mice do not express endogenous class II molecules, since they contain the disrupted H2-Aß gene. Previous studies have shown that no hybrid molecules (DQßA{alpha} or DQ{alpha}Eß) are generated (37). The DQ8 and DQ6 mice differ only in the DQ genes. Since mice that are deficient in endogenous class II molecules are resistant to EAMG, the susceptibility of HLA transgenic mice underscores the significance of HLA single class II molecules in the initiation and perpetuation of MG.

EAMG in HLA-DQ8.Aß0 mice

HLA-DQ8 transgenic mice were highly susceptible to EAMG as demonstrated in Figure 1GoA. Seven of the 10 mice studied developed EAMG; in six of them the disease score was 2 or more, demonstrating increased severity of disease in these mice. Clinical symptoms of EAMG were transient in the mice that had grade 1 disease. EAMG onset in all the mice except one occurred immediately after the second immunization. One mouse developed EAMG before the third immunization. By the time of the third immunization four mice (one of them had had transient grade 1 disease earlier) had no disease, and none of these mice showed symptoms of EAMG even after the third immunization. This is consistent with the Ab titers in these mice, which remained practically unchanged after the second immunization (Fig. 2Go). Figure 3Go, A and B, shows the repetitive nerve stimulation test of a normal HLA-DQ8 transgenic mouse compared with that of a mouse that showed grade 3 clinical symptoms of EAMG. Figure 3Go, A and B, clearly demonstrates marked differences in the forces evoked by muscle stimulation vs nerve stimulation, reflecting extensive neuromuscular transmission failure.



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  		FIGURE 1. EAMG in mice immunized with TAChR. Arrows on the x-axis show the days immunized. Score: grade 0, no weakness at rest or after exercise (25 consecutive paw grips on cage top steel grids); grade 1, normal strength at rest, but weak with chin on the floor and inability to raise the head after exercise; grade 2, grade 1 weakness at rest; grade 3, moribundity, dehydration, or quadriplegia. * indicates that the mouse was killed or died. A horizontal line with a blunt end denotes death of the mouse due to reasons other than EAMG. A, HLA DQ8 transgenic mice; B, HLA DQ6 transgenic mice; C, Aß0 (MHC class II-deficient) mice.

 


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  		FIGURE 2. TAChR Ab levels. TAChR Ab levels were estimated in five mice in each group at three different time points (days 0, 20, and day 45) and expressed as micromolar concentrations of BGTx binding sites.

 


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  		FIGURE 3. Repetitive nerve stimulation test in HLA DQ8 transgenic mice. A, Control mice not immunized with TAChR; B, mice immunized with TAChR and reached grade 3 weakness. For experimental details, see Materials and Methods.

 
EAMG in HLA-DQ6.Aß0 mice

HLA-DQ6 transgenic mice showed only a moderate susceptibility to EAMG (Fig. 1GoB). Six of the nine mice in this group developed EAMG, but five of these six mice had very modest symptoms (grade 1), and the other one had grade 2 symptoms. Most of the mice that had grade 1 EAMG did not show persistence of the clinical symptoms, which were transient. The anti-TAChR Ab titer was lower in this group than in the DQ8 transgenic mice (Fig. 2Go). The titer of the anti-TAChR Abs remained same at 3 and 7 wk. In this group we did not observe any clinical signs of muscle weakness before the second immunization. However, after the second immunization, transient symptoms continued to occur until termination of the experiment at 12 wk.

Absence of EAMG in class II knockout mice

The Aß0 mice did not show any clinical signs of muscle weakness. Among the 10 mice in this group, one developed weakness in one of the rear limbs. The clinical symptom in this mice was not changed after administration of either pancuronium or edrophonium chloride. We concluded that this could not be due to EAMG and hence deleted this mouse from the study.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The clinical symptoms in HLA-DQ8 transgenic mice immunized with TAChR were very severe compared with those in HLA-DQ6 transgenic mice. None of the Aß0 mice developed clinical EAMG. This strongly points out the influence of polymorphism at the human class II locus in the development of MG. The influence of H2-A gene polymorphism on susceptibility to EAMG in the mice is well studied (29, 30, 31, 32). However, our knowledge of the effect of HLA gene polymorphism in humans is limited, probably due to the low incidence of MG as well as the MHC heterogeneity among individuals. Our model, in which a human class II gene is introduced in isolation in the absence of the endogenous mouse class II genes, is a valuable tool in addressing the immunogenetics of human MG.

The only MHC class II genes functional in these mice are the human DQ8 and DQ6 molecules, respectively. The H2-Aßb knockout mice express H2-A{alpha}b and H2-Eßb in the cytoplasm, but these two chains do not combine to form functional heterodimers expressed on the cell surface (36). Also, surface expression of heterodimers formed by H2-A{alpha}b and DQ ß-chains was not observed in either DQ8.Aß0 or DQ6.Aß0 transgenic mice (34, 37). Previous reports from our laboratory have demonstrated an HLA-DQ8-restricted T cell response in DQ8.Aß0 mice and an HLA-DQ6-restricted T cell response in DQ6.Aß0 mice (37, 45, 46). The functional significance of HLA-DQ8 molecules in DQ8.Aß0 mice is underscored by the finding that these mice were highly susceptible to collagen-induced arthritis (37).

The present study compares the susceptibility of mice transgenic to HLA-DQ8 and HLA-DQ6 molecules to assess the differential susceptibility to EAMG due to polymorphism at the DQ locus in an experimental system. Our observation of zero incidence of EAMG in class II-deficient mice is similar to that reported in a previous study that concluded that functional class II molecules and CD4+ T cells are essential for the development of EAMG and rule out any pathogenic effector role for MHC class I-restricted CD8+ T cells, {gamma}{delta} TCR-bearing cells, or NK cells, which are intact in the MHC class II mutant mice (32).

Previous studies using bm12 mutant mice concluded that polymorphism at the H2-A locus (homologous to HLA-DQ) strongly influences EAMG susceptibility (31, 47, 48, 49). C57BL6 mice were susceptible to TAChR-induced EAMG, while C57BL6bm12 mice were resistant to the disease (31, 47, 48, 49). The Abm12 mutation drastically affected the epitope repertoire of murine CD4+ T cells sensitized to Torpedo AChR (30). Such changes in the epitope repertoire and the lack of recognition of the otherwise immunodominant CD4+ T cell epitope were proposed as likely reasons for the disease resistance of bm12 mutant mice (30). In another study it was observed that the expression of the Aßb:A{alpha}k transpair in C57BL10 transgenic mice suppressed the cellular and humoral autoimmune responses to AChR and reduced the incidence of muscle weakness and its associated abnormal electrophysiologic response (50).

However, such stringent genetic studies with single variant genetic elements are impossible to perform with human subjects due to the considerable polymorphism at the HLA locus and the presence of multiple class II and class I molecules on the cell surface. This had been a major limitation for the study of HLA restriction of different autoimmune diseases in which in vitro experiments using blocking Abs and HLA-identical APCs were the only limited options to understand the roles of individual HLA genes in the disease pathogenesis. Such limitations are overcome by transgenic technology, by the introduction of a specific HLA transgene into a mouse enabling study of the function of these molecules in isolation. The obvious drawback is the fact that these human MHC molecules select mouse T cells, and hence the pathogenic epitopes are recognized in the context of the mouse T cells. Also, in the presence of endogenous mouse class II molecules, there is a potential problem of preferential interaction of these molecules with mouse CD4 molecules, leading to the selection of predominantly mouse class II-restricted T cells. This was overcome by crossing the HLA transgenic mice to class II knockout mice. In our HLA class II transgenic mice that do not express the endogenous mouse class II molecules, we found that normal levels of CD4+ T cells are restored, confirming that HLA class II molecules can efficiently interact with mouse CD4 molecule and TCRs (37).

EAMG was severe in HLA-DQ8 transgenic mice and very moderate in HLA-DQ6 transgenic mice (Table IGo). This shows that the HLA-DQ8 haplotype might be contributing to a serious predisposition to MG, whereas the role of HLA-DQ6 may be limited. The manifestation of the disease is underscored by the drastic reduction in nerve conduction, as shown by the repetitive nerve stimulation experiment presented in Figure 3Go. We conducted this experiment in four normal DQ8 mice and did not observe more than a 10% decline in the initial force ratio between tetanic nerve stimulation vs direct muscle stimulation (data not shown). The weakness observed in the TAChR-immunized HLA transgenic mice should therefore be due to severe neuromuscular junction failure correlating with the clinical score.


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  		Table I. Summary of EAMG scores of mice studied in each group

 
The necessity of class II molecules and hence the CD4+ T cells in MG has been consistently demonstrated by different laboratories (32, 51). This finding corroborates the evidence that MG is an Ab-mediated disease, and the help of CD4+ T cells is required for the production of high affinity pathogenic Abs. The role of CD8+ T cells in EAMG is less clear. One study used ß2m-deficient mice, which do not express functional class I molecules on the cell surface and are deficient in CD8+ T cells, and found that the mice were susceptible to EAMG. This suggested that CD8+ T cells are not necessary for the development of EAMG. However, another study found that CD8+ T cell deficiency caused resistance to EAMG and proposed that both CD4+ and CD8+ T cells are needed for the development of EAMG, perhaps due to a CD4+-CD8+ T cell collaboration in the pathogenesis of the disease (51). Nevertheless, both studies confirmed the important role of class II molecules in the disease process. The present study verifies the importance of class II molecules in human MG and demonstrates that HLA-DQ8 could be one of the predisposing haplotypes for the development of MG. As the role of class I and hence CD8+ T cells in EAMG is a matter of controversy, it is essential to study its role in HLA class II transgenic mice either using class I-deficient mice or using strains in which different class I transgenes are introduced into the HLA class II transgenic animals.

This is the first report to directly demonstrate the role of any particular HLA molecule in isolation in the modulation of MG in vivo in an experimental system. This humanized mouse model is an excellent resource toward better understanding the immunogenetic basis of MG as well and to develop treatment and prophylactic strategies for MG.


   Acknowledgments
 
We thank Drs. Jack Strominger and Hidetoshi Inoko for providing the HLA-DQ8 and DQ6 cosmids, Drs. Christophe Benoist and Diane Mathis for the Aß0 mice, and Drs. Paul Zhou, Shen Cheng, and Jeanine Baisch for generating the transgenic mice used in this study. The assistance of Michelle Smart in screening the mice is appreciated. Thanks are also due to Julie Hanson and her crew for the breeding and maintenance of the mice used in this study.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI14764 (to C.S.D), NS23919 (to B.M.C.-F.), HL34817 (to G.C.S.), and HL37680 (to G.C.S.); and an Osserman Postdoctoral Fellowship of the Myasthenia Gravis Foundation of America (to R.R.). Back

2 Address correspondence and reprint requests to Dr. Chella David, Department of Immunology, Guggenheim-310, Mayo Clinic, Rochester, MN 55905. E-mail address: Back

3 Abbreviations used in this paper: MG, myasthenia gravis; AChR, acetylcholine receptor; EAMG, experimental autoimmune myasthenia gravis; TAChR, Torpedo acetylcholine receptor. Back

Received for publication October 17, 1997. Accepted for publication December 22, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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