Polymorphisms of the HLA-DQ subregion

Immunology Today, vol. 7, No. 10, 1986

ros/rl mPolymorphismsof the HLA-DQ subregion

The HLA-D region (analogous to the I region of the mouse H-2 complex gene) is now known to contain three subregions, one of which is HLA-DQ. The products of the HLA-DQ subregion are important in T-cell recognition. Three DQ specificities have been recognized by the International Histocompatibility Workshop but additional polymorphisms of DQ gene products have also been described. In this article, Massimo Trucco and Ren~ Duquesnoy discuss the relationship between serologically, cellularly and molecularly defined polymorphisms of HLA-DQ. During recent years our knowledge of the human major histocompatibility complex (MHC) and in particular its gene structure, has dramatically expanded. Serological and cellular studies have helped to unravel many of the complexities of the human leukocyte antigen (HLA) system. Within the HLA gene complex the HLA-D region, the human equivalent of the I region of the murine H-2 complex, is now known to contain multiple genes organized into three subregions: HI_A-DR, HLA-DQ (formerly MB or DC), and HLA-DP (formerly SB). Each subregion encodes one or more distinct class II MHC molecules. HI_A-DR consists of the'genes for one e~ chain and three 13 chains. The DRc~ gene appears rather nonpolymorphic, whereas the three DR~3 genes show different degrees of polymorphism ~,2. The DR subregion expresses at least two types of class II molecules, comprised of dimers of e~and 13chains. One e~-I~dimer is the classical DR molecule, the human homologue of the murine I-E molecule. The second c~-~ dimer is considered to carry the specificities DRw52 and DRw53 (formerly called MT2 and MT3) recognized by alloantisera. These specificities are strongly associated with groups of DR antigens, namely DRw52 with DR3, DR5, DRw6, and DRw8; and DRw53 with DR4, DR7, and DRw9. The third /3 gene is considered to be a pseudogene 3'4. The HLA-DQ subregion contains two a and two /3 genes. One pair of a and /3 genes encodes the DQ molecule, the human homologue of the murine I-A molecule. The second pair of HLA-DQ subregion genes have been referred to as DXc~ and DX/3s,6. The HLA-DP subregion also expressestwo ~ and two/3. genes. One ~-13 polypeptide pair encodes class II molecules which carry the DPwl-6 (SB1-6) antigens, whereas the other pair of genes does not seem to be expressed7.

Polymorphismsof DQ In this paper, we focus on the polymorphisms of the HLA-DQ subregion. Since the Ninth International Histocompatibility Workshop (1984), three DQ specificities have officially been recognized, and each of them correlates with groups of DR antigens. In caucasians, DQwl (MB1 or DC1) associates with DR1, DR2, and DRw6; DQw2 (MB2) associates with DR3 and most DR7; and DQw3 (MB3) associates with DR4, DR5, and sometimes DR7. These DQw antigens can be serologically defined by

TPittsburghCancerlnstitute, Pittsburgh,PA 15213;and 2the Divisionof Clinical Immunopathology, Department of Pathology, University of PittsburghSchoolof Medicine,Pittsburgh,PA 15261, USA ~) 1986, ElsevierScience Publishers B.V. Amsterdarn 0167-4919/86/$02.00

Massimo TruccoI and Ren J. Duquesnoy2 alloantisera and murine monoclonal antibodies 8-1°. Although three DQw specificities have been recognized, additional polymorphisms of DQ gene products have been described 1°'31. HLA-DQ subregion products are important in T-cell recognition: DQw antigens can serve as stimulators of secondary proliferation and as targets of cell-mediated cytotoxicity of alloreactive. T-cell clones12-14; they can also function as MHC restriction determinants for antigen-specific T cellsls'16. Although the currently defined DQw polymorphism has been attributed to the 13 chain of the dimer, both DQc~ and DQ/3 genes are polymorphic 17. This has become apparent particularly from the analysis of restriction fragment length polymorphism (RFLP) in endonuclease-digested genomic DNA hybridized with DNA probes for DQ~ and DQ/3 genes ~8'~9.Little is known about the polymorphisms of the corresponding gene products. Here we describe studies directed toward finding what we called "le trait d'union ' between RFLPand cell surface polymorphisms determined by serological and cellular assays. The findings indicate that DQ subregion polymorphisms have to be defined at the individual c~and chain levelsz°. In addition, we have obtained evidence that specific DQ~-I~ chain combinations are recognized by alloreactive T cells21,31. RFLP is generally seen when there are particular nucleotide sequences located outside of the exons which encode the gene products of interest. Considering this, it is remarkable how well the two gene stretches sometimes correspond: the one, present in the exon, encodes the allelic differences of the gene products, and the other, frequently present in the introns, is the target of the restriction enzyme 22. Unequal intrachromosomal gene conversion has been proposed to explain this strong correspondence 23,24. Perhaps the same process, occurring between different class II genes, underlies the cross-reaction found between different but related gene products 17. We thought it necessary to narrow the scope of our study by identifying an RFLP, generated by a specific restriction enzyme, that correlated closely with a polymorphic serological profile. The RFLPpatterns recognized by DQ~ and DQ/3 probes also had to be simple enough to allow direct analysis of their segregation in families. AIIoreactive T-cell clones could then be tested against the same family members in a primed lymphocyte test (PLT) to see whether the RFLP of the stimulators correlated with the T-cell response. Among a large number of restriction enzymes tested, we observed that Pstl generated RFLP which appeared to meet our criteria 2°. Pstl digestion of genomic DNA from HLA homozygous cell lines and informative family members produces, in each haplotype, only two bands revealed by the DQ~ or the DQ/3 probe: one band corresponds to a DQ allelic form, and the other repre-

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Immunology Today, voL 7, No. 10, 1986

Table 1. Pstl-DQa and Pstl-DQt3 RFLPs Probe Fragment Allelic Cell surface size in kb pattern determinant DQa 15.2 apl AQ1 4.1 ap2 AQ2 2.2 ap3 AQ3

DR associations DR1,DR2,DRw6 DR3,DR5,DRw8 DR4,DR7,DRw9

4.5 bpl BQ1 DRI,(DR2),DRw6 5.5 bp2 BQ2 DR3,DR7a 5.0 bp3 BQ3 DR4,DR5,DRw8,DRw9 13.0 bp4 BQ4 DR2,DRw6 aDR7-positive haplotypes which type DQw2. DR7 haplotypes which type DQw3 expressthe bp3 allelic pattern. DQ13

sents the cross-hybridizing DX form 32. The three different positions of the DQa bands correspond to the three DQ~ allelelic forms, which have been called DQapl, DQap2 and DQap3 (in this notation 'DQ' refers to the subregion, 'a' means ~ gene, and "p' refers to the restriction enzyme used, Pstl). Each band associates with a different group of DR antigens: DQapI associates with DR1, DR2, and DRw6; DQap2 with DR3, DR5, and DRw8; and DQap3 with DR4, DR7, and DRw9 (Table 1). Pstl-RFLP has been useful in determining whether the DQ polymorphic markers reside on the ~ or 13chains 2°. To distinguish the products of these two ~ or/3 genes, we have begun to use a terminology whereby AQ1, AQ2, etc. represent a-chain markers, and BQ1, BQ2, etc. represent 13-chain polymorphic gene products. Interestingly, the DQapl form, but none of the DQI3 forms, correlates with the serologically defined DQwl specificity. This suggests that DQwl is an a-chain allelic marker and not a 13-chain marker. We refer to this s-encoded gene product as AQ1. The DQap2 allelic form corresponds to a serological determinant which we call AQ2, and which associates with DR3 and DR5, but not with DRw6. The AQ2 determinant may be equivalent to the serologically defined DCa3 specificity described by Tosi eta/. 2s. The determinant present on the product coded by the DQap3 gene is called AQ3. The strong associations of AQ3 with DR4 and DR7 are similar to the DR association of DRw53, which is encoded by a DR~ gene26. The serological distinction between AQ3 and DRw53 may be difficult because of similarities in DR associations due to strong linkage disequilibrium. However, previous cellular studies have demonstrated the detection of AQ3 by two alloreactive T-cell clones (HJ1 and HJ39) which specifically react with DR4- and DR7-positive cells12. These clones were inhibited by DQ-specific, but not by DR-specific, monoclonal anti-

DR3, DQw2

298

DR5, OQw;5

DR4, DQw3

DR7, OQw2

Fig. 1. Hypothetica/modelfor chain-specificdeterminantson HLA-DQ molecules.Fourhaplotypesdefinedby classicalserology,aregivenas examples.

bodies, suggesting that they recognize a DQ-encoded determinant 12. These findings indicate that each of three allelic forms of the Pstl-RFLP of DQa corresponds to a distinct alloantigenic determinant which can be defined by serological and/or cellular methods. Similar observations have been made about the correlation between Pstl-RFLP of the DQ~ gene and serologically defined determinants, which we now refer to as BQ1, BQ2, BQ3, and BQ4. Two DQ~ bands, DQbp2 and DQbp3, correlate with DR3 and DR7 and with DR4 and DR5, respectively. The DQbp2 gene product expressed the BQ2 (DQw2) alloantigenic determinant, whereas the DQbp3 gene encodes the BQ3 (DQw3) determinant. The other two allelic forms of DQ~ (DQbpl and DQpb4) correlate with two different groups of DR1-, DR2-, and DRw6-positive haplotypes. The DQbpl allelic form is found in most DR1, some DR2 and about half of DRw6-positive cells. DQbp4 is found in most DR2, and the other half of DRw6 cells. The DQbpl and DQbp4 genes appear to have two corresponding serological determinants: BQ1 can be defined by antisera specific for DR1(+2)+6, and BQ4 can be defined by DR2+6specific antisera. Both types of these so-called 'DQwlincluded' specificities have been described a, and their respective specific antibodies appear to recognize two different allelic gene products.

DQ subregionterminology These observations support the concept that the DQ polymorphism can be defined at the level of individual a and 13 chains. The currently used DQw nomenclature lacks this consideration and is also somewhat misleading, particularly because DQwl does not seem to be allelic to DQw2 and DQw3. The proposed terminology, which differentiates between antigenic specificities on a and 13 chains (AQ versus BQ), stresses the need to learn more about individual DQ gene product polymorphisms, and also reflects our increased understanding of the complexity of DQ molecules expressed on the cell surface (Fig. 1). For instance, DQw2 is strongly associated with DR3 and DR7. Although the DQ molecules of DR3,DQw2 and DR7,DQw2 haplotypes appear to have similar 13 chains expressing BQ2, they have different a chains expressing AQ2 and AQ3, respectively. Similarly, DR4,DQw3- and DR5,DQw3-positive haplotypes express DQ molecules with similar 13-chain but different a-chain determinants. On the other hand, it can also be predicted that many DR3,DQw2- and DR5,DQw3-positive haplotypes express DQ molecules with a chains expressing the same AQ2 determinant, even if their 13chains are different. In the case of DQwl, we have observed that the DQapl gene product associates with either DQbpl or DQbp4 gene products. In other words, DR1, DR2, and DRw6 cells will generally type as either AQ1,BQ1 or AQ1 ,BQ4. Antibody specificity studies are not the only source of evidence that the DQa and DQ~ allelic forms recognized at the DNA level are consistently expressed in their products at the cell surface. We have also observed that alloreactive T-cell clones can discriminate among different Pstl-defined allelic forms of DQ. An example was provided by a pair of clones (DS6 and DS9) that defined a DR2-associated determinant. These clones were inhi-

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ImmunologyToday,voL7, No. 10, 1986

bited by monoclonal antibodies directed against DQ molecules, suggesting that they recognized a DQ gene product ~3. Pstl-RFLP analysis of our cell panel showed that those DR2 cells which stimulated these clones were positive for DQapl and DQbpl. No stimulation was observed with those DR2 cells that were DQapl,bp4 or DR1 and DRw6 cells that were DQapl,bpl. These findings suggested additional DNA polymorphisms of DQ genes which have not been defined by Pstl-RFLP. Among numerous restriction enzymes tested, only Taql generated RFLPpatterns which could differentiate between the DQapl and DQbpl genes of the DR1- and DR2-positive haplotypes. Only one association of specific and 13 patterns corresponded to the DR2-associated DQ molecule recognized by our T-cell clones21. The Taql analysis also provided us with the opportunity to identify additional allelic forms of DQa and DQ~ genes (Table 2). DQapl is always found to be associated with one of three different Taql band patterns, namely DQapl.tl in DRl-positive cells; DQapl.t2 in DR2 cells; DQapl.t6 in DRw6-positive cells. We have also found some DRw6positive cells which express DQapl.tl or DQapl.t2. DQbpl can also be 'subdivided' by Taql into DQbpl.tl associated with DR1, and DQbpl.t2 associated with DR2 (Ref. 32). Only those panel cells which exhibited both the DQapl.t2 and DQbpl.t2 allelic forms together can stimulate the two alloreactive T-cell clones with their, DR2-associated DQ specificity 21. Recently we have generated another T-cell clone (DB29) which specifically recognizes the DR2-associated DQ molecule encoded by DQapl.t2 and DQbp4.tl allelic forms 31. These data demonstrate that the genetic basis of cellular determinants recognized by T-cell clones can be established at the DNA level21. The data in Table 2 also show that the Taql-RFLP analysis yields two allelic forms associated with DQbp2 and DQbp3. DQbp2 is subdivided into DQbp2.tl and DQbp2.t2, while DQbp3 is split into DQbp3.tl and DQbp3.t2. The remaining Pstl-defined allelic forms, DQap2, DQap3 and DQbp4, correspond to single Taql allelic patterns which are called DQap2.tl, DQap3.tl and DQbp4.tl, respectively32. We may hypothesize that cell surface determinants corresponding to all the different Taql-defined allelic forms of DQa and DQ~ genes exist, but we do not have, at the moment, the serological evidence that can confirm this hypothesis. Taql splits of Pstl-defined DQa or DQ~ allelic forms, however, are informative regarding genetic differences in DQ genes of DRw6 homozygous typing cells with different HLA-Dw specificities as defined by mixed leukocyte culture (MLC). DRw6-associated Dw splits, in fact, show a strong association with different DQ~-13 combinations2~: DQapl.tl,bpl.tl with Dw9; DQapl.t6,bp4.tl with Dw18; and DQapl.t2,bpl.t2 with Dw19. DQapl.t6,bp4.tllDQapl.t2,bpl.t2 heterozygotes, such as the DRw6-homozygous Daudi cell line, expresses both Dw18 and Dw19 specificities and type Dw6 in MLC (Dw18 and Dw19 were first defined as Dw6 splits)27'28. The Dw splits associated with DRw6 haplotypes, then, quite likely reflect DQ specificities. It is possible that the same relationship exists between the DR7-positive haplotypes and the DR7-associated Dw splits. Dw7 associates, in fact, with DQap3.tl,bp2.t2 while Dw11 associates with DQap3.tl,bp3.tl (Table 3). These findings suggest that genetic analysis of the

Table 2. Taq 1-DQa and Taq 1-DQI3 RFLPs

Probe

Fragment size in kb 2.6

DQ~

5.8 6.2 4.6 5.3

Allelic pattern

apl.tl ap 1.t2 ap I. t6 ap2.tl ap3.tl

DR associations DR1 DR2 DRw6a DR3,DR5,DRw8 DR4,DR7,DRw9

5.3/6.0 bpl.tl DR1 6.0 bpl.t2 DR2 2.8/5.3 bp2.tl DR3 2.8/5.3/7.6 bp2. t2 DR7b 1.9/5.3 bp3. t l DR4,DRw9 2.5/5.3 bp3.t2 DR5,DRw8 3.0/5.3 bp4.tl DR2 aDRw6-positive haplotypescan also expresseither the apl .tl or the apl .t2 DQc~allelic patterns, as well as bpl .tl, bpl .t2, and bp4.tl DQI3 allelic ~Datterns. R7-positive haplotypes which type DQw2. DR7 haplotypes which type DQw3 expressthe bp3.tl allelic pattern. DQ13

HLA-DQ subregion should consider individual oL and 13 polymorphisms. This type of analysis will also increase our understanding of the existing relationship between serological and cellular antigenic systems encoded by the HLA-DQ subregion and DNA polymorphisms of the DQa and DQ~ genes. Such studies will also provide information about the primordial significance of various RFLP in the HLA-D region. A better definition of the polymorphisms of individual DQa and DQI3gene products may also provide opportunities to detect combinatorial determinants on DQ molecules. By definition these determinants are epitopes formed as a result of dimerization between ~ and 13 Table3. DR-associated DNA polymorphisms of DQa and DQ~ and cellular recognition of the corresponding cell surface markers Serology

RFLP

DR1

DQa apl.tl

DQI3 bpl.tl

DR2 DR2

apl.t2 apl.t2

bpl.t2 bp4.tl

DRw6 DRw6 DRw6

apl.tl apl.t2 apl.t6

bpl.tl bpl.t2 bp4.tl

DR3

ap2.tl

bp2.tl

DR5 DR5

ap2.tl apl.t6

bp3.t2 bp4.tl

DRw8

ap2.tl

bp3.t2

Cellular recognition MLC

PLTa

DS6(+)DB29(-) DS6(-)DB29(+) Dw9 Dw19 Dw18

Ref.

31,21 31,21 21,27 21,27 21,27

HJ1,HJ39(+) 21 DR4 ap3.tl bp3.tl HJ1,HJ39(+) 21,27 DR7 ap3.tl bp2.t2 Dw7 21,27 DR7 ap3.tl bp3.tl Dwl 1 HJ1,HJ39(+) HJ1,HJ39 nd DRw9 ap3.tl bp3.tl apositive (+) and negative (-) PLT experiments were performed with DS6,DB29,HJ1,and HJ39 T-cell clones as responders(nd = not done).

-ros/rum chains and are not found on the separate chains. In principle, there are two types of combinatorial determinants: one pertaining to products of genes in cis position and the other resulting from trans gene complementation ('hybrid' molecules). Although combinatorial determinants have been well documented on mouse class II molecules, there is only biochemical evidence for the existence of human class II hybrid molecules 29'3°. No specific antisera or cellular reagents that define determinants created by dimerization have been described. The best opportunity to define combinatorial determinants would be with those class II molecules in which both and 13 chains are polymorphic. Since the DR~ gene is relatively non-polymorphic, it would be difficult to detect combinatorial determinants on DR molecules other than those controlled genetically only by the DR~ gene. On the other hand, DQ molecules may express combinatorial determinants because both DQ~ and DQ~8 are polymorphic. Our observations suggest that the cellular approach which uses T-cell clones could be useful towards defining the different types of combinatorial determinants. We take this opportunity to thank I. Cascino, M. Marrari, S. Rosenshine, E. Turco, and A. Zeevi for their excellent collaboration. This research was supported by research grants CA10815, CA-37932, and AI-21410 from the National Institutes of Health. References

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