HLA class II disease associations: Molecular basis

Journal of Autoimmunity (1992) 5 (Supplement A), 45-53

H L A C l a s s II D i s e a s e A s s o c i a t i o n s : M o l e c u l a r B a s i s

Dominique Charron

Laboratoire d'Immunog~n~tique Mol~culaire, Institut Biomedical des Cordeliers, Universit~ Paris VI, Paris, France

Introduction T h e initial observation concerning H L A and disease associations has been an increase in the frequency of certain serologically defined alleles of the H L A complex in several autoimmune diseases [ 1]. T h e most recent data assign disease susceptibility (D S) to common amino-acid sequences present on one or several chains of an H L A class II molecule within the 'active' site [2]. It can be postulated that a specific antigen (or set of antigens) presumably in the form of short peptides will bind with high affinity to the precise structure of the class I I molecules associated with a particular disease. This would lead to inappropriate antigen presentation and T cell activation resulting in an impaired immune response. Because of the strong linkage disequilibrium between loci and alleles the description of the genetic associations will be restricted to the most recent epidemiological data. I will discuss the central contribution of three-dimensional conformational structures of the H L A class II molecules and emphasize the possibilities generated by hybrid H L A class II molecules. Such genetic and molecular approaches have implications in predictive medicine for identifying individuals at risk for a disease and may provide new rationales for therapy.

HLA class II genes and m o l e c u l e s Class II molecules have a central immunoregulatory role. T h e y are cell surface glycoproteins expressed principally on macrophages, monocytes and B lymphocytes, which present processed antigens (in the form of peptides) to antigen-specific T lymphocytes leading to their specific activation and proliferation. Such T cell features are implicated in the development of autoimmunity. A description of the class II genes and molecules is presented in Figure 1. 45 0896-8411/92/0A0045÷ 09 $03.00/0

© 1992AcademicPress Limited

46

D. Charron

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HLA Class II disease associations

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Most class I I p o l y m o r p h i s m is located at the first domain of the molecule and clustered into two, three or four hypervariable regions. T h e s e structural variations between the different class I I gene p r o d u c t s are the fundamental differences permitting i m m u n e recognition and are therefore likely to be critical in disease susceptibility. An antigen recognition site can be p r o p o s e d which is c o m p o s e d of a cavity lined by two a-helical structures and closed at the b o t t o m by a p l a t f o r m of eight [3-pleated sheet structures [3].

H L A class II p o l y m o r p h i s m and t y p i n g Class I I serology is presently being replaced by two new techniques for H L A typing. T h e s e are P C R - A S O and P C R - R F L P [4-6]. Both rely on the known nucleotide sequences of the alleles which allow their detection either using allele specific oligonucleotides ( P C R - A S O ) hybridization or enzymatic cleavage which generates allele specific polymorphic fragments ( P C R - R F L P ) . T h e first step in either technique consists of an amplification by P C R of the exon I I of the class I I (~ or [3 genes.

H L A class II h y b r i d m o l e c u l e s (Figure 2) Cell surface expression of the class I I molecule as a stable heterodimer appears to be a logical requirement for immunological function and is therefore likely to play a major role in disease association. T h e expressed repertoire of the class I I molecules is the result of allelic and isotypic variations. I n addition to the n u m b e r of class I I (z-13loci combined 2 x 2 within an haplotype to f o r m isotypes, a powerful way to increase class I I antigen diversity would be the association of the a and [3 chains of the two haplotypes by trans complementation within an isotype. T h e s e molecules will be found

48

D. Charron RA

IDDM

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Figure 3. Molecular localization of the HLA class II epitopes involved in disease susceptibility. ?, Amino acids not precisely mapped; + , heterozygous effect (trans-complementation). RA, Rheumatoid arthritis; IDDM, insulin-dependent diabetes mellitus.

only in heterozygous individuals. An even more efficient way of generating additional diversity would be to pair ~ and [3 chains from different isotypes either in cis and/or t r a n s [7]. T h e H L A - D Q products have the potential of forming hybrid molecules which are dimers created by a-[3 chain pairing resulting from gene transcomplementation. Such molecules include chains belonging to both the paternal and the maternal haplotypes [8]. Indeed both the D Q a and the D Q 13 chains are highly polymorphic. Many, but not all, of the possible combinations of ~ and 13chains are found [9]. Whether the absence of a given a-[3 combination is due to the inability to associate (forbidden a-[3 pairing) or whether the population studies are, as yet, insufficiently extensive to include all possible combinations is unresolved. In numerous epidemiological studies of insulin-dependent diabetes mellitus ( I D D M ) , juvenile rheumatoid arthritis (JRA) and coeliac disease (CD) an unexpectedly high incidence of the disease has been observed in particular combinations of D R haplotypes (heterozygous effect) [10]. As a consequence, the relative risk (RR) is dramatically higher in some heterozygous situations than for any unique allele, even when present in a homozygous state. Such data cannot be explained by the model of monoallelic association. If H L A class II molecules are involved it becomes logical to propose that the particular determinant derives from a combination of products, one from the first haplotype and the other from the second haplotype. T h e D Q loci fulfill the two requirements of being in strong linkage disequilibrium with D R alleles and of having structurally polymorphic subunits. T r a n s association of D Q (t-DQ [3 chains creates hybrid molecules which may be consistent with the observed heterozygous effect since only these H L A - D Q hybrid molecules will bear conformational epitopes unique to the combination of paternal and maternal haplotypes. Altogether the suggestion that specific conformation within H L A class II dimers may represent a critical element for susceptibility and that hybrid class II molecules occur either by cis or t r a n s complementation within an isotype or isotypes will be discussed in the context of two well characterized diseases RA and I D D M (Figure 3).

HLA Class II disease associations

49

R h e u m a t o i d arthritis (RA) Although likely to be multifactorial, the search for an aetiology of rheumatoid arthritis has mostly focused only on a few immunological aspects; rheumatoid factors and H L A studies. Approximately 70% of RA are DR4 versus 28% in controls [11]. Shortly after its serological definition the DR4 haplotypes were subdivided into five (or six) cellular specificities defined by homozygous typing cells: w4, w 10, w 13, w 14, w15, K T 2 . Among these subtypes only two, Dw4 and D w l 4 , are prevalent in RA and account for the association with RA found in caucasian studies, while D wl 0 appears neutral. T h e position of the molecules which differ in amino acid composition between D w l 0 (not associated or protective) and D w 4 - D w l 4 (both associated to D S in RA) can be considered as the most likely to be critical in determining the impaired immune response which underlies the susceptibility to RA. These include primarily amino acids 67, 70, 71, and additionally 86. Moreover DR1 is over-represented in the group of DR4 negative RA patients. Overall the data localize the functional RA associated epitope (shared epitope) to an area of the DR[31 molecule common to DR1, DR4w4 and D R 4 w l 4 (also D w l 5 and D w l 6 ) [12]. However, it was impossible to strictly correlate the recognized epitope with this sequence. This suggests that the D S factor is not a common continuous linear sequence between all the recognized epitopes but is likely to be a three-dimensional structure of a conformational epitope which may be of importance independently of the fine structure of the molecule on which it is found. Apart from the overwhelming evidence for a role for DR[31 gene in RA susceptibility, involvement of other loci products remains unestablished. Indeed, DR4 haplotypes include polymorphic DQ]3 genes. However individuals with D R 4 associated RA carry either the DQw7 or the DQw8 species and the DQw7 and DQw8 specificities are equally distributed in RA patients and in normal controls. However D Q may influence the phenotypic expression of RA and superimpose some feature on the main genetic susceptibility due to the DR[31 gene association. In this respect an increase of the DQw3.1 (DQwT) specificity has been found in severe RA (seropositive RA) and in Felty's syndrome or RA associated with nodules and/or erosions [13, 14].

Insulin-dependent diabetes mellitus (Table 1) I D D M has been the most investigated pathology with regard to the H L A class II system association and autoimmunity. This is quite remarkable for a disease which was not considered to involve the immune system less than two decades ago. T h e first association was described in 1973 with the specificity B15 reported, followed in 1974 by a report of a B8 association. T h e most exquisite recent association is with the absence of aspartic acid at position 57 in the DQ[3 chain [15]. This illustrates the development of H L A typing procedures from serology to molecular biology and the subsequent subdivisions of loci and alleles. When inspecting the individual class I I amino acid substitutions T o d d et al. noticed that all class II haplotypes which were not positively associated but neutral or negatively associated with I D D M possessed in common an aspartic acid at position 57 in the DQ[3 chain. In contrast there is no common polymorphic determinant (or structural stretch of amino acids) in the I D D M positively-associated haplotypes (noticeably DR4, DR3 and DR1) and the

50

D. C h a r r o n

T a b l e 1. H L A factors contributing to I D D M susceptibility or resistance* Susceptibility D R4 DR3 DR1 DRwl6 DRw13 DQw8 DQw2 DQw5 Dwl9 DQI357: Ala-Val-Ser

Resistance D Rw 15 DRwl5 DR4 DR5 DRw13 DQw6 DQw6 DQw7 Dwl8 DQI357: Asp

*Heterozygous effect: hybrid HLA class II molecules. DR4, DQw8/DR3, DQw2 and DR4, DQw8/DRw8, DQw4: synergistic effect.DR7, DQw2 in blacks (DQw213+ DR4 DQ~); DRg, DQw2 in blacks (DQw213+ DR4 DQct).

amino acid 57 of DQ]3 is either an alanine, a valine or a serine. Interestingly ASP57 is also found in every mouse IA]3 gene so far sequenced with the exception of N O D mice, which represent the mouse model for spontaneous I D D M , where it is a serine. T h e emerging picture suggests that it is the charge of the polymorphic residue at position 57 of DQ[3 that is associated with I D D M susceptibility. T h u s the presence at position 57 of the DQ[3 chain of an amino acid with a non-polar h y d r o p h o b i c R group (alanine, valine) or an amino acid with a polar but uncharged R group (serine) is preferentially associated with an a u t o i m m u n e response to an as yet unknown diabetes-related antigen. T h i s contrasts with the positively charged R of aspartic acid found in the I D D M negatively-associated haplotypes. Several important exceptions exist to the absence of aspartic acid DQ[357 as a requirement for I D D M susceptibility. T h e D R w 9 haplotypes associated with I D D M in the Japanese and Chinese have aspartic acid at position 57 of DQ[3 [16]. M o r e o v e r the presence of a nonaspartic acid residue at this position may not be sufficient to confer the highest susceptibility to I D D M . In fact a heterozygous effect was reported very early in I D D M studies. Early studies in which B8 and B15 were found associated with I D D M suggested that both haplotypes could contribute to I D D M . A dramatically increased R R was further demonstrated for individuals possessing a particular heterozygous combination of H L A class I I antigens namely, D R 3 / D R 4 . T h i s p r o m p t e d a search for the class I I molecule inducing most susceptibility. Since the D R a chain is m o n o m o r p h i c it cannot therefore contribute to the heterozygous effect. T h e discovery that both a and [3 chains of the D Q molecules were polymorphic suggested that formation of hybrid class I I molecules obtained by trans complementations could occur and that those molecules only present in the heterozygous individuals will fulfill the requirement imposed to explain the heterozygous effect (similar to an F1 effect in animal

HLA Class II disease associations

51

genetics). Ultimately it supports the concept that it is more the structural threedimensional conformation than an individual sequence which is the likely genuine susceptibility element and that several distinct amino acids can contribute to its formation. Indeed this would agree with our present understanding of the structural model for antigen presentation which implies a series of dynamic interactions between the antigenic peptide, the M H C and the T cell receptor (TCR). H y b r i d class II molecules provide the best model to explain the heterozygous effect observed for I D D M . T h e data are consistent with the highest susceptibility being mediated by a conformational structure present in a hybrid molecule formed from a DQw3.2 [3 chain (DQwS) of the DR4 haplotype and the DQw2 a chain of the DR3 haplotype. T h e minimal requirement at the DQ]3 chain level would be the absence of aspartic acid at position 57. Indeed the heterozygous effect was not only observed in D R 3 / D R 4 individuals but also in DR4/DR1 individuals. This could be interpreted in that the conformation of the D Q hybrid molecule formed in DR4/DR1 is very close to that formed in DR4/3 (functionally and/or for peptide binding) and thus confers a similar level of susceptibility. When other DQt~ ]3 combinations are present the level of susceptibility appears lower, which may reflect a less functional efficiency of the hybrid molecule created in these cases. Overall this may mean that the structural requirement may be less stringent for D Q u than for DQ]3. This would explain that the heterogenous effect can still be accounted for by an identical capacity of association of the DQw3.213 chain with any type of DQct chain found in DR3 haplotypes. Alternatively the heterozygous effect may not concern a D Q u ]3 dimer but other type of class II molecules. In this respect it is noteworthy that in Chinese and Japanese IDDM-susceptible haplotypes D R 4 DQ4 and DR9 DQ9, the DQ]3 chain has aspartic acid at position 57 but that it is the DR]3 chains which may contribute to the D S (absence of aspartic acid/presence of serine in I D D M DR]3 chains from Asians). In this population a characteristic DR]3 D Q a dimer could be equivalent to the DQ]3 D Q ~ dimer found in Caucasians.

Concluding remarks and perspectives

T h e enigma of H L A and disease association has been clarified by the present knowledge of the structure of the class II molecules involved in disease susceptibility. Clearly the H L A class I I molecules which are involved in disease susceptibility have a normal structure and are found in the normal population, although at a lower frequency. T h e data reviewed here support the concept that the disease susceptibility element is composed of amino acids (contiguous and/or distantly spaced on the same chain or on different chains of the same or of a distinct class II isotype) which delineate a specific three-dimensional conformation. It is suggested that it is more the conformational epitope created than the linear stretch of amino acids which is important. Clearly even when a portion of one class I I chains appears predominant in DS, additive and often synergistic contributions are found for a second chain. In conjunction with the fact that H L A class II molecules are u ]3 dimers capable of forming hybrid molecules (intra, inter isotypic) by cis and/or trans complementation this provides an ideal structural framework for the localization of the conformational D S epitope(s). T h e capacity of the conformational epitope to bind with high

52

D. C h a r r o n

affinity to a set of defined peptides varies, presumably according to the polym o r p h i s m of the epitope. Future key studies include the definition of antigenic peptides whether they are environmental (foreign) or autologous (self). Candidate peptides are presently being tested for their ability to bind class I I D S elements. T h e T C R represents the third partner of the functional trimolecular complex involved in the control of the i m m u n e response. Cell surface expression of the H L A class I I molecules is likely to elicit the pathogenic effect of the D S gene. Indeed n u m e r o u s abnormalities of H L A expression have been observed in a u t o i m m u n e diseases. In particular, an aberrantly high expression of class I I molecules on the diseased tissue such as in Langherhans [3 islet cells in I D D M and synovial lining cells in RA. T h e genetic control of such hyperexpression may involve a p o l y m o r p h i s m within the class I I regulatory regions. Ultimately the H L A class I I molecules are the potential target of therapeutic strategies. T h i s possibility was suggested when anti-class I I monoclonal antibodies were shown to prevent or reverse induced as well as spontaneous a u t o i m m u n e diseases in animal models. T h i s concept suggests that either blocking or competing antibodies or peptides may be tailored towards the D S element and will therefore display high specificity and predictably high efficacy against the disease associated i m m u n e response. Alternatively anti-lymphokine reagents (antibodies, antagonists, drugs) which could downregulate H L A class I I expression may also be considered. H o w e v e r the exact mechanisms of such treatments are not known. T h e y m a y block i m m u n e responses at the effector level but m a y also solicit induction of tolerance and/ or stimulation of suppression. In this perspective adverse potential affects mediated by immunological and also by non-immunological pathways should not be ignored [ 17].

References

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9. Bontrop, R. E., B. M. Baas, N. Otting, G. M. Schreuder, and M. J. Giphart. 1987. Immunogenetics 25; 305-312 10. Charron, D. 1986. U n mod61e et trois exemples pour comprendre H L A et maladies: cis et trans associations mol~culaires de classe II du C M H dans le diab~te juvenile insulinodependant, l'arthrite rhumatoide de l'enfant et la maladie coeliaque. Path. Biol. 34: 795-800 11. Stasny, P. 1978. N. Engl.J. Med. 298:869 12. Gregersen, P., M. Shen, Q. Song, P. Merryman, S. Degar, T. Seki, J. Maccari, D. Goldberg, H. Murphy, J. Schenzer, C. Wang, R. Winchester, G. Nepom, and J. Silber. 1986. Proc. Natl. Acad. Sci. U S A 83:2642-2645 13. Sansom, D. M., J. L. Bidwell, P. J. Maddison, G. Campion, P. T. Klouda, and B. A. Bradley. 1987. Hum. Immunol. 19:269 14. Sansom, D. M., J. L. Bidwell, P. T. Klouda, and S. N. Amin. 1988. Lancet i: 58 15. T o d d , J. A., J. L. Bell, and H. O. McDevitt. 1987. HLA-DQ[3 gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Nature 329:599-605 16. Ju, L. Y., Y. P. Sun, G. Semana, X. F. Gu, R. Krishnamoorthy, R. Fauchet, and D. Charron. 1991. Aspartic acid at position 57 of the H L A DQ[3 chain in insulin dependent diabetes mellitus: an association with one D R w g - D Q w 9 subtype in the chinese population. Tissues Antigens 37:218-223 17. Mooney, N., C. Grillot-Courvalin, C. Hivroz, L. Y. Ju, and D. Charron. 1990. Early biochemical events following M H C class I I mediated signaling on human B lymphocytes. J. Immunol. 145:2070-2076