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The human MHC : evidence for multiple HLA-D-region genes
Immunology Today, vol. 4, No. 8, 1983
219
The human MHC: evidence for multiple HLA-D-regaongenes Carolyn Katovich Hurley, Robert C. Giles and J. Donald Capra The human major histocompatibility complex regulates immune responsiveness through a complex series of H L A - D region-controlled molecules. The use of monoclonal antibodies to dissect this expanding array of molecules has allowed the structural characterization of several of these molecular species. Here biochemical studies are discussed which, coupled with classic serological techniques, have been used to construct a model of the genetic organization of the human H L A - D region. The h u m a n major histocompatibility complex ( M H C ) or human leukocyte antigen (HLA) complex, a linked array of genes on chromosome 6, plays a major role in many phases of the immune response. It controls the recognition of foreign substances or antigens and is important in disease susceptibility and tissue-graft rejection. At least three classes of molecules are controlled by the M H C , each functioning in a distinct way to mediate the response to a foreign antigen. The class-I molecules, H L A - A , -B and -C, the classic transplantation antigens, are responsible for graft rejection and regulate the killing of virusinfected cells. The class-II molecules, the HLA-D-region antigens, mediate mixed lymphocyte stimulation and are involved in communication between lymphoid cells. Both class-I and class-II molecules exhibit a high degree of polymorphism. Their diversity is thought to enhance the capacity of the immune system to respond to the diverse antigenic onslaught of the environment. In contrast, the polymorphism of the M H G class-III molecules, the complement components, C2, C4 and Bf, is only moderate. These serum proteins participate in cell lysis and mediate inflammatory responses. Class-I molecules are expressed by practically all cells~ but class-II molecules are expressed most abundantly by B lymphocytes, activated T lymphocytes and antigenpresenting cells including peripheral blood monocytes, macrophages, Langerhans' cells, and dendritic cells of the lymphoid organs, i.e. on immunologically active cells. These HLA-D-region antigens have been less readily characterized than the class-I molecules, primarily because no well-defined, high-titer alloantisera were available with which to isolate the antigens. The advent of highly discriminatory monoclonal antibodies has now simplified the isolation of the class-II molecules and the description of several HLA-D-region-encoded molecules has followed. This review explores the evidence for the existence of these multiple species. T h e m u r i n e I region serves as a m o d e l for the H L A - D region
The murine homologue of the human H L A - D region is the I region. The I region has been divided into five subDepartment of Microbiology, The University of Texas Health Science Center at Dallas, Dallas, TX 75235, USA.
regions: l-A, I-B, I-J, I-E and I-C, based on a number of traits including immune responsiveness, mixed lymphocyte reactions, and serologically detectable antigens 2. The /-region antigens (Ia antigens) encoded by the I-A a n d / - E subregions consist of heterodimers containing an a chain of approximately 32 000 molecular weight and a fl chain of approximately 280003 . These chains may also be associated with an invariant chain 4. The I-A and I-E antigens are structurally distinct from one another ~'5. These structural differences correlate with functional differences as demonstrated by the ability of the I-A or I-E antigens to control the immune response to particular simple antigens 2. The I-A and I-E antigens are polymorphic. The fl chains exhibit more or most of the polymorphism, although the a chains, in particular the I-A a chain, are also polymorphic3. Additional polymorphism is generated by the presence of unexpressed alleles of the I-E a or/3 chains and combinatorial association of I-A or I-E allelic products 6. In a heterozygous animal, therefore, the I-A chain of one haplotype has been observed in combination with both the I-A/3 chain of the same haplotype and the I-A /3 chain of the alternative haplotype. Association between I-A and I-E polypeptide chains, however, has not been detected. I-A a-chains have not been observed complexed with I-E/3 chains and vice versa. The extent of polymorphism generated by combinatorial association is modulated by preferential association between the Ia products of particular H-2 haplotypes 7. A further level of polymorphism may derive from the presence of multiple I-E and/or I-A gene products 8-H. The Ia a and/3 polypeptide chains are encoded within the murine M H C : the I-A a and/3 chains and the I-E/3 chain are encoded in the I-A subregion while the I-E a chain is encoded in the I-E Subregion (Fig. 1)3'12'13. The linkage of these genes encoding the Ia heterodimers is unusual for molecules that are not synthesized as polyproteins. In addition, the genetic organization of the I-E a- and/3-chain genes into separate subregions appears to explain the I gene complementation observed in the response of some recombinant strains to several simple antigens 3. Complementation requires the presence of responder alleles at two distinct/-region loci, I-A a n d / - E , in order to generate an immune response. The products of the /-B, I-J and I-C subregions have not been © Elsevier Biomedical Press 1983 0167-4919/83/0000-00(}0/$1.00
Immunology Today, vol. 4, No. 8, 1983
220
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At the time this figure was published3, the order of the genes was not known.
B. The genetic organization of the murine I region based on recent DNA data~. characterized 2. Although there is m o u n t i n g evidence that these regions do not exist within the M H C 2'12, D N A studies have demonstrated the presence of a d d i t i o n a l / region related genes 12. At present, it is not clear whether these genes represent pseudogenes or expressed genes perhaps encoding other/-region molecules. Elucidation of the genetic organization of the m u r i n e I region has prompted several laboratories to extend their studies into the homologous h u m a n H L A - D region, especially since this region appears to be involved in susceptibility to a large n u m b e r of h u m a n diseases. This vast array of studies, which have included genetic, serological, cellular and biochemical analyses, have already begun to demonstrate that the h u m a n H L A - D region appears to possess a higher degree of complexity than does the I region of the mouse.
T h e t t L A - D r e g i o n is genetically c o m p l e x Historically, the H L A - D region was localized within a region encompassing approximately four centimorgans, between the H L A - B locus a n d a n e n z y m e locus, glyoxylase (GLO), located on the centromeric side of the H L A complex (Fig. 2). W i t h i n this chromosomal region, several serological a n d cellular specificities have been mapped: D, D R , MB, M T a n d SB. (In addition to these associated HLA-D-region specificities, a n u m b e r of complement components m a p within this region of the h u m a n MHC14). T h e primary specificities traditionally mapped to this region are H L A - D and H L A - D R . At present, twelve alleles of H L A - D have been identified by mixed lymphocyte reactivity ( M L R ) . Serological typing has identified ten alleles of HI_,A-DR. W i t h i n the Caucasian population, there has been a high correlation between D a n d D R typing, suggesting that mixed lymphocyte reactivity a n d serology m a y be identifying the same molecules. A n u m b e r of additional specificities m a p p i n g to the H L A - D region have been defined serologically. These include the MB, M T , DC, B R a n d T e series 15-18.Each of these specificities is found in linkage disequilibrium or associated strongly with a n u m b e r of D R specificities a n d is called a 'supertypic' specificity. For example, in the MB series, MB1 is associated with D R 1 , 2, w6, w8 a n d wl0; MB2 with D R 3 and 7; a n d MB3 with DR4, 5, a n d w9. M e m b e r s of a given supertypic series are thought to represent allelic products since they segregate from one another in family studies and, particularly in the case of the M B series, are in H a r d y - W e i n b e r g equilibrium. T h e relationship between these independently defined supertypic series has not been clear. For example, on the one hand, m a n y MB, D C and T e specificities appear to have similar, if not identical, DR-associated distributions suggesting that they may represent the same segregant series (Table I). O n the other hand, the MB and M T series (with the exception of MB1 a n d M T 1 ) have very different DR-associated distributions and probably represent different series. A simplified scheme relating the supertypic specificities to each other is presented in T a b l e I. For the purpose of simplicity a n d in order to develop a unified construct, specificities which have similar distributions are assumed to be identical; slight differences observed by different laboratories in the association of the supertypic specificities with D R haplotypes and the degree of linkage disequilibrium are assumed to be differences in
TABLE I. Supertypic series MB series MT series Associated Equivalent Associated Equivalent DR specificities specificities DR specificities specificities MBI DR1, 2, w6, w8a, wl0a DC1, Te21, MT1 b MT1 DR1, 2, w6, wl0 a DC1, Te21, MB1b MB2 DR3,7 DC3,Te24 MT2 DR3, 5, w6, w8" BR3 MB3 DR4, 5, w9a DC4,Te22,MT4 MT3 DR4, 7, w9, wl0 a BR4 aAssignments tentative. bSince MB1 and MT1 apparendy recognize the same population of DR molecules, they may represent equivalentspecificities.However, ifMB and MT are two different series of determinants, then there may be a split in the sera defining MB1/MTI/DC1/Te21, one set ofsera defining MB1 determinants and another set defining MT1 determinants. A second possibility is that the MT determinants do not represent an allelic series but simply public determinants shared by several DR specificities. In that case, MT2 and MT3 have no molecular relationship to each other and the need for an MT1 allelic specificity is negated.
Immunology Today, voL 4, No. 8, 1983
I
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like t~ chains in a single D R homozygous cell line2s,29. It has been difficult, however, to determine if D R determinants are, indeed, expressed by each of these multiple I-E-like species. The convention in biochemical studies has been to call any molecule with an I-E-like sequence, DR. For the purpose of this review, we will use this broad definition unless indicated.
MB
Fig. 2. Region of the human chromosome 6 containing the HLA-D region 8°. In map units, the region between the GLO and HLA-B loci is approximately 30 times longer than the mouse I region.
alloantisera used by these groups. In this way, the multiple supertypic series can be condensed into two series of aUoantigenic specificities, MB and M T (see also Ref. 15). The DC and Te series appear to be identical to the MB series, and the BR series appears to be identical to the M T series. A n additional specificity, SB, has been also mapped to the HLA-D region using primed lymphocyte typing. This specificity is independent of D R specificity, suggesting that SB and DR are distinct loci19.
Molecules bearing HLA-D and HLA-DR determinants are polymorphic Almost perfect correlation has been observed between some D and D R specificities, suggesting that D and D R typing are identifying the same molecules2°. Recent studies, however, have split certain D R specificities into multiple subsets based on H L A - D typing2°'21. For example, DR4 has been shown to be associated with H L A - D specificities Dw4, Dwl0, DB3, D Y T and LD4021. Discrepancies in correlations between H L A - D and H I , A - D R typing have also appeared in non-Caucasian populations 2°a2. In these studies, the H L A - D determinant normally associated with a particular D R specificity was absent and a different specificity was sometimes observed. These data suggest that H L A - D and H L A - D R typing do not define the same determinants; however, these data do not distinguish whether the D and D R determinants reside on the same or different molecules 23. Structural studies have characterized the molecules bearing D R determinants. H L A - D R is the predominant class-II molecule. Like the murine Ia antigens, D R is a heterodimer consisting of two non-covalently linked glycoproteins (Fig. 3). These chains may also be associated with an invariant chain 24. Both a and fl chains are integral membrane proteins 2~. Amino acid sequences derived from molecules isolated by xenoantisera indicate that the human molecules are homologues of the murine I-E antigens 25'26. A comparison of D R antigens isolated from cell lines bearing different D R specificities shows that the molecules possess relatively invariant a chains but polymorphic/3 chains 24. The molecules controlled by a single D R haplotype may, however, vary from one another at the primary structural level23'2730. U p to seven DR-like/3 chains have been detected as well as two DR-
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Some HLA-D-region antigens are homologues of the murine I-A antigens While the D R antigens are the equivalent of the murine I-E antigens, the DS antigens are the equivalent of the I-A antigens 31. Six DS haplotypes have been described, each haplotype in linkage disequilibrium with a particular D R haplotype ~2. Both the t~ and fl chains of the DS molecules are polymorphic, in contrast to the D R antigens in which only the fl chain exhibits noticeable polymorphism.
The supertypic specificity MB resides on DS molecules As mentioned above, two series of supertypic specificities linked to H L A - D R have been defined: MB (DC, Te) and M T (BR). While molecules bearing these specificities resemble D R by having a similar two-chain structure, the molecular bases of the two supertypic series differ. By several criteria33-37, the serologically defined MB (DC, Te) specificities and the D R specificities reside on separate molecules. However, this distinction is not complete as certain MB specificities have also been detected on D R molecules by the same criteria~5'38.(This discrepancy is discussed in the section describing M T . ) Partial amino acid sequences of molecules carrying the supertypic specificities DC 1 (MB 1)s9'~°and MB3 41clearly
Immunology Today, vol. 4, No. 8, 1983
222 TABLE III. Amino acid sequencesof DR, SB and DS (MB) moleculesfrom the cellline PRIESS Positionno. DR4(IIIE3) SB3 (I-LR1) DS (MB3) (IVD12) DR4 (IIIE3) SB3 (I-LR1) DS(MB3)(IVD12)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 a chains F F F I V I I F Y L F F F Y F L Y Y Y F I V V Y V fl chains F F Y Y F L Y F F Y Y Y Y Y F V V Y Y F V Y F
demonstrate that these molecules are distinct from D R molecules. The sequence homology detected between the DC1 (MB1) and MB3 molecules suggests that the two molecules are allelic forms of the molecule bearing the MB specificity4~and provides a biochemical basis for the MB series presented in Table I. Furthermore, the amino acid sequence data indicate that the DC1 (MB1) and MB3 molecules are homologous to the murine I-A antigens and, therefore, can be classified as DS molecules. Two molecules that bear the same supertypic specificity can be structurally distinct from one another. Molecules bearing the DC 1 (MB 1) specificity but derived from cell lines bearing different D R specificities can diffe?3'3L This difference may correspond to the different DS haplotypes observed by Goyert and Silv& 2. An additional complexity is the incomplete linkage disequilibrium of the supertypic specificities. For example, the MB3 determinant in Caucasians, as detected by the monoclonal antibody IVD 12, has been found on 100 % of donors bearing the DR4 allele but on only approximately 90% of DR5 individuals4~. Alloantisera give similar indications. It is not known if the DR5 individuals that are negative for IVD12 binding bear an MB3 determinant not detected by the monoclonal antibody. Alternatively, these individuals could bear another untyped aUelic product or a null allele of MB. Nevertheless, molecules bearing MB determinants have been described which are distinct from D R molecules and can be classified by amino acid sequence as DS molecules. The possibility also exists that, in some cell lines, the MB specificity may also reside on D R molecules. T h e supertypic specificity M T resides on several
molecular species In contrast to the MB specificities examined in the sequence analysis, the M T (or BR) specificities appear to reside on several different molecules. Some M T determinants appear on molecules distinct from those molecules bearing D R determinants31'~5'36'~2.These may include molecules bearing MB determinants31'35'~8 or entirely new molecules representing an additional set of HLA-D-region molecules36. Other studies have localized M T specificities on molecules bearing D R determinants35.3s. Clearly, as above with MB, there is some confusion as to which molecules bear the M T supertypic specificities. While some of the confusion may be the result of using complex alloantisera for analysis of MB and M T determinants, it is possible that the molecules I~earing the supertypic specificities could vary from cell line to cell
line. Based on DNA data discussed below, there may be several molecules related to D R controlled by the HLA-D region. Exchange of genetic segments between these related molecules by gene conversion, as suggested in the H-2 system43'", could spread supertypic determinants throughout the HLA-D protein family. This conversion, however, must take place primarily in a cis fashion as conversion between homologous chromosomes would cause linkage breakdown between D R and the supertypic specificities. This has not been commonly observed. Some interesting observations concerning the sharing of determinants between HLA-D-region molecules can be made using monoclonal antibodies directed against HLA-D-region specificities. Table II lists some of the available monoclonal antibodies and their characteristics. The majority of monoclonal antibodies directed against D R molecules recognize determinants present on essentially all h u m a n B cells tested. In contrast, the majority of monoclonal antibodies directed against the MB and M T supertypic specificities or SB recognize previously defined polymorphic specificities. Unusual binding patterns, however, have been observed with some of these antibodies. For example, I-LR1, in addition to detecting certain SB allelic products, also appears to recognize DR5 molecules19. The monoclonal antibody SG171 appears to detect D R molecules in all cell lines; however, in a DR7 line, this monoclonal antibody also detects the DS molecule31. The sharing of determinants between different molecules as observed with these monoclonal antibodies could be producing some of the variability in the alloantisera studies. TABLE II. Monodonal antibodies directed against H/A-D-region molecules Monoclonal antibody 203 243 227 IIIE3 SG171 Genox IVD12 I-LR2 109d6 I-LR1
Predominant specificity DR DR DR DR DR,DS MB MB MT MT SB,DR
Monomorphic/ polymorphic Monomorphic Monomorphic Monomorphic Monomorphic MonomorphicDR, DS7 DC1 (MB1) MB3 MT2 MT3 SB2,3,DR5
Ref. 27 27 27 27 31 33 41 45 46 19
The use of monoclonal antibodies rather than alloantisera directed against the supertypic specificities may provide a more fruitful means of analysing the molecular bases for these determinants. Two monoclonal antibodies
Immunology Today, vol. 4, No. 8, 1983 DRa
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rare ~. One monoclonal antibody, I-LR1, has been identified that reacts with certain SB allelic produc& 9. Although similar to H L A - D R in its two-chain structure, both a and fl chains of the SB antigens are structurally distinct from the D R a and fl chains by amino acid sequence analysis. Based on these limited sequence data, the SB a chain, like its D R counterpart, is homologous to the murine I-E a chain 49. A B cell expresses at least three HLA-D-region molecules The studies detailed above describe several HI,A-Dregion specificities. Some of these specificities appear to be on structurally distinct molecules; alternatively, others may reside on the same molecule. Most of these data describing multiple HLA-D-region specificities are
Fig. 4. Model of the genetic organization of the HLA-D region. The order of the genes is not known.
have been used to characterize molecules carrying M T like determinants (unpublished observations). The antibody I-LR2 binds to cells in a pattern similar to that found with MT2 alloantisera45 and 109d6, in a pattern similar to an M T 3 alloantisera~. Preliminary amino • acid sequence characterization of MT2-1ike reactive molecules from two different cell lines suggests that the M T 2 molecule is D R or DR-like. Tyrosine is observed at position 13 in the a chain and at positions 10, 26 and 30 in the 13chain of an MT2-1ike molecule isolated from a DR3 line, positions identical to those observed when an antiD R monoclonal antibody (L203) is used. These data suggest that one of the M T 2 determinants is borne solely on D R or DR-like molecules but do not preclude the occurrence of additional MT2 determinants on other HLA-D-region molecules. The MT3-1ike determinant, as detected with the monoclonal antibody, 109d6, in two homozygous DR4 cell lines, appears to be present on two populations of molecules, D R and DS (see below and Table III). Partial amino-terminal amino acid sequences of the 109d6reactive molecule shows tyrosines at positions characteristic of D R in both the a and/3 chains. In addition, a DSlike sequence is also observed. The approximate ratio of D R to DS in these preparations varies, probably reflecting the amounts of D R and DS in the cell. By these criteria, the MT3 determinant appears to be present on both D R - and DS-like molecules. At this time, these data provide no evidence for M T as a molecule different from D R or DS and support the notion that M T 2 and M T 3 determinants are found on molecules closely resembling D R and/or DS molecules. The SB antigens represent an additional set o f H L A - D region molecules Cellular typing of cells matched for known HLA-Dregion antigens has been a fruitful means of identifying additional HLA-D-region loci19'47. Allogeneic mixed lymphocyte responses have been used to define an additional series of polymorphic HLA-D-region antigens, S B I9. SB is a poor immunogen compared to the D R , MB and M T determinants and alloantisera defining SB are
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FRACTION Fig. 5. Isolation of antigens from radiolabelled cell lysates by chromatography on lentil lectin-'Sepharose', immunoprecipitation or affmity chromatography with monoclonal antibodies and SDS polyacrylamide gel electrophoresis. The figures show the results of SDS gel electrophoresis of molecules isolated from the cell line PRIESS using monoclonal antibodies: (A) IIIE3 to isolate a D R molecule; (B) IVDI2 to isolate an MB3-bearing molecule; (C) I-LR1 to isolate an SB3 molecule; and (D) 109d6 to isolate an MT3-bearing molecule. The 109d6 immunoprecipitates contain a higher molecular weight molecule which we are in the process of characterizing. The approximate positions of immunoglobulin heavy and light chains are indicated by the arrows. Migration was from left to right.
224 consistent with the construction of a model (Fig. 4) depicting three distinct subregions within the H L A - D region: DR, D S and SB. Each of these subregions controls a distinct a and 0 chain. It should be emphasized that the gene order has not been established. Most of the genes encoding HLA-D-region molecules have been mapped to the M H C on human chromosome 62~'5°'51. This model can be further supported by the recent isolation and characterization of three structurally distinct molecules from a single cell line. Using monoclonal antibodies directed against antigens bearing D R 27, MB 41, M T (unpublished observations), and SB .9 specificities, three structurally distinct molecules have been isolated from the cell line PRIESS 52, a homozygous DR4 cell line which bears the MB3 and MT3 supertypic speeificities. The line is heterozygous for SB3/4; however, only the SB3 allelic product is reactive with the anti-SB monoclonal antibody 19. The four monoclonal antibodies used in the study, IIIE3, IVD12, 109d6 and I-LR1, are listed in Table II. All of the HLA-D-region molecules isolated were heterodimers of approximately the same molecular weight (Fig. 5). The molecules bearing the DR, MB and SB specificities were characterized by amino acid sequencing and the results are shown in Table III. Both a and O chains of the DR, DS (MB) and SB molecules are structurally distinct from one another, implying the presence of at least six distinct structural genes. In preliminary studies of the cell line PRIESS, the anti-MT3 monoclonal antibody recognized two populations of molecules, D R and DS, as described above. By comparison to murine sequences, the SB and D R a chains and the D R 0 chain are homologues of the murine I-E antigens; the DS (MB) a and 0 chains are homologues of the I-A antigens; and the SB O chain appears to be distinct from both I-E and I-A. Evidence from our laboratory and several others suggests that the D R subregion encodes multiple D R molecules 27-~°.Although the multiplicity of the D R population in PRIESS has not been examined by amino acid sequencing, sequential immunodepletion experiments suggest that these populations exist27. Multiple D R 0chain loci have, therefore, been included in the model. Since previous studies had detected at least three structurally distinct D R 0 chains in other cell lines, three E-chain genes have been included in the model. This model predicts only one D R a chain although multiple a chains have been suggested by other studies ~.
The DNA data suggest multiple HLA-D region loci Many of the HLA-D-region genes have been isolated by D N A cloning techniques and correlations can be made between the D N A data and the protein studies described above. The DNA encoding the D R and the DC 1 (MB 1) ot chains have been isolated by several investigators 51'53-5~. Portions of the a chains were found to exhibit homology to the HLA-D-region 0 chains, H L A class-I molecules, immunoglobulin and/3-2 microglobulin. Hybridization with the D R a-chain probe to genomic DNA under stringent conditions detects only one D R a-chain gene 5°'56'57 although eleven bands hybridize under less
Immunology Today, vol. 4, No. 8, 1983
stringent conditions ~. This suggests that there are multiple related a-chain genes in the genome. One of the additional hybridizing bands observed with the D R achain probe could be the gene encoding SB a, a protein which appears to be structurally similar to D R a ~. cDNA clones containing the D R and DC1 (MB1) 0chain genes have been isolated and sequenced 4°'59. Like the other HLA-D-region molecules that have been characterized, the E-chain sequences share homology with elass-I antigens and immunoglobulin. The homology between mouse and human gene products observed in the protein sequence comparisons was also observed at the D N A level with cross-hybridization of human HLA-D-region cDNA probes to murine cosmids containing/-region genes 12. Examination of D R and DC (MB) E-chain hybridization patterns from individuals with different DR specificities indicated polymorphism of the majority of hybridizing bands 6°. This finding complements the protein data which suggest that the 0 chains carry the allelic polymorphism24. Hybridization of D R and DC1 (MB1) E-chain gene probes to genomie D N A produced a complex pattern suggesting that multiple E-chain genes encoding the two subsets of molecules are present within the cell°°. The DNA data support the protein chemistry data which demonstrate multiple HLA-D-region a- and E-chain gene products and suggest that as yet unidentified HLA-Dregion-related E-chain genes may exist.
The functional roles of HLA-D-region antigens appear to vary HLA-D-region antigens act as primary and secondary stimulators in mixed lymphocyte reactions, as targets for cytotoxic T cells, and as controlling elements in antigen presentation. In the mouse, the I-A and I-E molecules control the immune response to different simple antigens 2. Similar delineations of function may exist among the multiple human HLA-D-region antigens as well. The functional roles of the HLA-D-region molecules are summarized in Table IV. While functional studies of the HLA-D-region molecules are still in their infancy, the role of the H L A - D region in susceptibility to disease has been studied extensively. Each HLA-D subregion is associated with distinct human diseases The varied functional roles played by the HLA-Dregion molecules in the control of immune responsiveness may be important in determining susceptibility to particular diseases. Correlations can be found between particular alleles at H L A - D and a variety of human diseases. Early studies of disease association showed association of certain diseases to certain alleles of the HLA-A and HLAB loci. Upon discovery of the H L A - D region, many of these diseases were shown to be more closely associated with H L A - D R alleles. Now that additional HLA-Dregion specificities have been identified and reagents directed against these molecules have become available, some of the disease associations have shifted from D R to MB, M T and SB specificities. While SB6ahas not yet been
225
Immunology Today, ool. 4, No. 8, 1983 T A B L E IV. T h e functional roles of HLA-D-region antigens Function Stimulation in Specificity MLR
Target for CTL
Antigen presentation
Disease association
DR a
+
+
+
MB b
?e
?
?
M'Ix
+
ND f
+
SBd
+
+
+
Multiple sclerosis (DR2) Type-I diabetes (DR3,4) Rheumatoid arthritis (DR4) Asthma (MB3) Chronic lymphatic leukemia (MB3) Hairy-cell leukemia (MB3) Primary sicca syndrome (MT2) Juvenile-onset diabetes (MT3) Dermatitis herpetiformis (SB1)
aData on D R was generated from Refs 20, 66-68, 71-76. hData on MB was generated from Refs 67, 68, 70, 78. CData on M T was generated from Refs 68, 69, 74, 78. dData on SB was generated from Refs 19, 77. ~The data are conflicting. fNot determined.
widely studied, certain alleles or specificities of D R 62, MB 46'63and M q "~'65 have all been strongly associated with particular disease states. Examples of such associations are shown in Table IV. Recent studies have demonstrated the association of certain malignancies to the MB3 specificity using the monoclonal antibody described above, IVD12 ~. These studies were the first to show association of the HLA-D region and malignancies. While these malignancies show weak association with particular D R alleles, the association with MB3 is striking. Table V illustrates this for the case of chronic lymphocytic leukemia where the relative risk of individuals bearing DR4 or DR5 is 1.4 or 6.0 while the relative risk of MB3-bearing individuals is 13.5. These observations suggest that the products of HLA-D subregions play important and varied roles in disease susceptibility. The use of monoclonal antibodies detecting H/M-D-region specificities should aid in elucidating the association of additional diseases with particular HLA-D-region molecules. Such antibodies will be TABLE V. Contrasting frequencies of certain HLA-D-region alloantigenicdeterminants between individualswith chronic lymphocyticleukemia and a control population~ HLA-D-re#on alloantigen specificity
Individuals with leukemia Normal control (n = 29) (n = 28) Relative (% positive) (% positive) risk
DR1 DR2 DR3 DR4 DR5 DRw6 DRw7 MB3(IVD12)
10.3 6.9 20.7 31.0 62.1 13.8 20.7 93.1
21.4 35.7 25.0 25.0 21.4 17.9 21.4 50.0
- 2.4 - 7.5 - 1.3 + 1.4 + 6.0 - 1.4 - 1.0 + 13.5
valuable in future studies to determine how these molecules function in immune responsiveness. Conclusion The human HLA-D region controls the expression of cell-surface antigens involved in communication between lymphoid cells. This communication appears to be critically important in immune responsiveness as suggested by the linkage of disease susceptibility in humans to particular H/M-D-region alleles or specificities. In order to understand how these H/M-D-region molecules function in cellular collaboration and antigen presentation, it is important to elucidate the assortment of H/M-D-region molecules found on the surface of immune response related cells. Using monoclonal antibodies, three structurally distinct H/A-D-region molecules, DR, DS and SB have been isolated from a single cell line. This effectively divides the HLA-D region into at least three subregions encoding a minimum of six protein chains. These studies have also attempted to define the biochemical basis for the serologically and functionally defined antigens bearing D R , MB, M T and SB specificities. Based on structural homology with the murine/-region antigens, the D R and SB antigens appear to be related to the murine I-E antigens while the DS (MB) antigens are similar to the murine I-A antigens. Thus, in the human, it appears that there has been a duplication of at least the loci encoding I-E to produce multiple gene products. The number of loci already described in the HLA-D region provide a large repertoire of cell-surface molecules which can be used in communication between immune response related cells. The murine data suggest that in the HLA-D region too, transassociation of chains within a subregion will generate additional molecules. Since some of the subregions such as DR appear to encode multiple fl chains, the number of possible chain associations is greatly increased. Additional mechanisms which generate polymorphism, as found in mice, probably exist. The association between susceptibility to particular diseases and certain allelic products ofHLA-D subregions suggests that roles played by the multiple H/A-D-region molecules in cellular collaboration and antigen presentation are varied. The development of carefully characterized monoclonal antibodies directed against subsets of these H/A-D-region molecules will be crucial in understanding the function of these molecules in immune responsiveness.
References 1 Ploegh, H. L., Orr, H. T. and Strominger, J. L. (1981) Cd124, 287-299 2 Klein, J., Juretic, A., Baxevanis, C. N. and Nag),, Z. A. (1981) Nature
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3 Uhr, J. W., Capra, J. D., Vitetta, E. S. and Cook, R. G. (1979)Science 206, 292-297 4 Jones, P. P., Murphy, D. B., Hewgill, D. and McDevitt, H. O. (1979) MoL Immunol. 16, 51-60 5 Cook, R. G., Vitetta, E. S., Uhr, J. W. and Capra, J. D. (1980)J~ Immunol. 124, 1594-1598 6 Jones, P. P., Murphy, D. B. and McDevitt, H. O. (1981) Immunogenetics 12, 321-337 7 McNicholas, J. M., Murphy, D. B., Matis, L. A. et al. (1982),/ Exp. Med. 155, 490-507 8 Delovitch, T. L. and Barber, B. H. (1979)J. Exp. Med. 150, 100-107
Immunology Today, vol. 4, No. 8, 1983
226 9 Huber, B. T., Jones, P. P. and Thorley-Lawson, D. (1981) Proc. Natl Acad. Sci. USA 78, 4525-4529 10 Lafuse, W. P., Corser, P. S. and David, C. S. (1982) Immunogenetics 15, 365-375 11 Ozato, K. and Sachs, D. H. (1982)J. Immunol. 128, 807-810 12 Steinmetz, M., Minard, K., Horvath, S. et al. (1982)Nature (London)300, 35-42 13 Benoist, C. O., Mathis, D.J., Kanter, M. R. etal. (1983) Proc. NatlAcnd. Sci. USA 80, 534-538 14 Shreffier, D. C. (1982) in Histocompatibility Antigens." Structure and Function (Parham, P. and Strominger, J., eds), pp. 187-219, Chapman and Hall, New York 15 Duquesnoy, R. J., Marrari, M. and Annen, K. (1979) Transplant. Proc. 11, 1757-1760 16 Park, M. S., Terasaki, P. I. and Bernoco, D. (1980) in Histocompatibility Testing (Terasaki, P. I., ed.), pp. 572-573, UCLA Tissue Typing Laboratory, Los Angeles 17 Park, M. S., Terasaki, P. I., Bernoco, D. and Iwaki, Y. (1978) Transplant. Proc. 10, 823-828 18 Tanigaki, N. and Tosi, R. (1982) lmmunol. Rev. 66, 5-37 19 Shaw, S., DeMars, R., Schlossman, S. F. etal. (1982),]. Exp. Med. 156, 731-743 20 Termijtelen, A., van Leeuwen, A. and van Rood, J. J. (1982) Immunol. Rto. 66, 79-101 21 Reinsmoen, N. L. and Bach, F. H. (1982) Hum. Immunol. 4, 249-258 22 Troup, G. M.,Jameson, J., Thomsen, M. teal. (1978) TissueAntigtns 12, 44-57 23 Groner, J. P., Watson, A. J. and Bach, F. H. (1983).]. Exp. Med. 157, 1687-1691 24 Shackelford, D. A., Kaufman, J. F., Korman, A. J. and Strominger, J. L. (1982) Immunol. Rev. 66, 133-187 25 Allison, J. P., Walker, L. E., Russell, W. A. et al. (1978)Proc. NatlAcod. Sci. USA 75, 3953-3956 26 Springer, T. A., Kaufman, J. F., Terhorst, C. and Strominger, J. L. (1977) Nature (London) 268, 213-218 27 Hurley, C. K., Nunez, G., Winchester, R. etal. (1982).]. Immunol. 129, 2103-2108 28 Kratzin, H., Yang, C., Gotz, H. et al. (1981) Hoppt-Sqltr's Z PhysioL Chern. 362, 1665-1669 29 Yang, C., Kratzin, H., Gotz, H. tt al. (1982) Hoppe-Styler's Z Physiol. Chem. 363, 671-676 30 Andrews, D. W., Bono, M. R. and Strominger, J. L. (1982) Biochemistry 21, 6625-6628 31 Goyert, S. M., Shively, J. E. and Silver, J. (1982)`]. Exp. Med. 156, 550-566 32 Goyert, S. M. and Silver, J. (1983) Fed. Proc. Fed. Am. Soc. Exp. Biol. 42, 836 33 Shackelford, D. A., Mann, D. L., van Rood, J. J. etal. (1981)Proc. Natl Acad. Sci. USA 78, 4566-4570 34 De Kretser, T. A., Crumpton, M.J., Bodmer, J. G. and Bodmer, W. F. (1982) Eur. J. Immunol. 12, 600-606 35 Karr, R. W., Kannapell, C. C., Stein, J. A. teal. (1982)`]. Immunol. 128, 1809-1818 36 Tanigaki, N., Tosi, R., Duquesnoy, R. J. and Ferrara, G. B. (1983)J. Exp. Med. 157, 231-247 37 Corte, G., Calabi, F., Damiani, G. et al. (1981) Nature (London) 292, 357-360 38 Markert, M. L. and Cresswell, P. (1982)J. ImmunoL 128, 2004-2008 . 39 Bono, M. R. and Strominger, J. L. (1982) Nature (London) 299, 836-838 40 Larhammar, D., Schenning, L., Gustafsson, K. et al. (1982) Proc. Natl Acad. Sci. USA 79, 3687-3691 41 Giles, R. C., Nunez, G., Hurley, C. K. et al. (1983)J. Exp. Med. 157, 1461--1470 42 Katagiri, M., Ikeda, H., Maruyama, N. et al. (1979) Immunogenetics 9, 335-351 43 Pease, L. R., Schulze, D. H., Pfaffenbach, G. M. and Nathenson, S. G. (1983) Proc. Natl Acad. Sci. USA 80, 242-246 44 Weiss, E. H., Mellor, A., Golden, L. et al. (1983) Nature (London) 301, 671-674 45 Nadler, L. M., Stashenko, P., Hardy, R. etaL (1981)Hum. ImmunoL 1, 77-90 46 Winchester, R., Toguchi, T., Szer, I. et aL (1983) Immunol. Rev. 70, 155-166
47 SegaU, M., Reinsmoen, N. L., Iwaki, J. and Bach, F. H. (1982) Hum. ImmunoL 4, 239-248 48 van Leeuwen, A., Termijtelen, A., Shaw, S. and van Rood, J. J. (1982) Nature (London) 298, 565-567 49 Hurley, C. K., Shaw, S., Nadler, L. et aL (1982)`] Exp. Med. 156, 1557-1562 50 Lee, J. S., Trowsdale, J. and Bodmer, W. F. (1982) Proc. NatlAcad. Sd. USA 79, 545-549 51 Trowsdale, J., Lee, J., Carey, J. et al. (1983) Abstract from The Second Meeting on Cloning of the H L A and H-2 Regions, Airlie, Virginia 52 Thomsen, M., Jacobsen, B., Platz, P. et al. (1975) in Histocompatibility Testing 1975 (KissmeyeroNielsen, F., ed.), pp. 509-518, Munksgaard, Copenhagen 53 Lee, J. S., Trowsdale, J., Travers, P.J. et al. (1982) Nature (London) 299, 750 752 54 Korman, A. J., Auffray, C., Schamboeck, A. and Strominger, J. L. (1982) Pr0c. NatlAcad. Sci. USA 79, 6013-6017 55 Larhammar, D., Gustafsson, K., Claesson, L. et al. (1982) Cell 30, 153-161 56 Stetler, D., Das, H., Nunberg, J. H. etal. (1982) Proc. NatlAcad. Sd. USA 79, 5966-5970 57 Wake, C. T., Long, E. O., Strubin, M. etaL (1982) Pr0c. NatlAcad. Sci. USA 79, 6979-6983 58 Auffray, C., Korman, A. J., Roux-Dosseto, M. et al. (1982) Proc. Natl Acad. SoL USA 79. 6337-6341 59 Long, E. O., Wake, C. T., Gorski, J. and Mach, B. EMBOJ. (in press) 60 Wake, C. T., Long, E. O. and Mach, B. (1982) Nature (London) 300, 372-374 61 Shaw, S., Hall, R. P., Duquesnoy, R.J. and Katz, S. I. (1981) Abstract from the American Association for Clinical Histocompatibility Testing 62 Winchester, R. J. and Kunkel, H. G. (1979) in Adv. lmmunol. (Dixon, F.J. and Kunkel, H. G., eds), Vol. 28, pp. 221-292, Academic Press, New York 63 Nakai, Y., Kano, T., Moriuchi,J. etaL (1982) Hum. lmmunol. 4, 265-270 64 Moutsopoulos, H. M., Mann, D. L.,Johnson, A. H. and Chused, T. M. (1979) N. Engl. `] Med. 301, 761-763 65 Moriuchi, J., Katagiri, M., Wakisaka, A. et al. (1980)Hum. ImmunoL 4, 357-362 66 Accolla, R. S., Moretta, A. and Cerottini, J. C. (1981)~ Immunol. 127, 2438-2442 67 Koide, Y., Awashima, F., Yoshida, T. O. et al. (1982)`]. Immunol. 129, 1061-1069 68 Zeevi, A., Scheffel, C., Annen, K. etal. (1982) Immunogenotics 16, 209-218 69 Zeevi, A., Annen, K., Scheffel, C. and Duquesnoy, R. J. (1983) Human Immunology 6, 97-109 70 Corte, G., Moretta, A., Cosulich, M. E., Ramarli, D. and Bargellesi, A. (1982)J. Exp. Med. 156, 1539-1544 71 Feighery, C. F. and Stastny, P. (1980) Immunogenetics 10, 31-38 72 Krensky, A. M., Reiss, C. S., Mier, J. W., Strominger, J. L. and Burakoff, S. J. (1982) Proc. NatlAcad. Sci. uSA 79, 2365-2369 73 Burakoff, S. J., Engelhard, V. H., Kanfman, J. and Strominger, J. L. (1980) Nature (London) 283, 495-497 74 Thorsby, E., Berle, E. and Nousiainen, H. (1982) Immunol. Rev. 66, 39-56 75 Eckels, D. D., Lamb, J. R., Lake, P. et al. (1982) Hum. Immunol. 4, 313-324 76 Hirschberg, H., Braathen, L. R. and Thorsby, E. (1982) ImmunoL Rev. 66, 57-77 77 Eckels, D. D., Lake, P., Lamb, J. R. et al. (1983) Nature (London) 301, 716-718 78 Gonwa, T. A., Picker, L. J., Raft, H. V. et al. (1983)`]. Immunol. 130, 706-711 79 Hood, L., Steinmetz, M. and Malissen, B. Annu. Rev. Immunol. (in press) 80 Weitkamp, L. and Francke, U. (1978) Cell Genet. 22, 92-105
Acknowledgements The authors would like to thank M. Firra, J. Mills, P. Presley, A. GarciaSpain and K. van Egmond for their assistance and the Capra laboratory, G. Nunez, P. Stastny, S. Shaw, L. Nadler, and R. Winchester for their helpful discussions. This work is supported by NIH grant AI18922 to J. Donald Capra, a UTHSC grant and ACS grant IN-142 to Carolyn Katovich Hurley and NIH grant CA-09082 to Robert C. Giles.
219
The human MHC: evidence for multiple HLA-D-regaongenes Carolyn Katovich Hurley, Robert C. Giles and J. Donald Capra The human major histocompatibility complex regulates immune responsiveness through a complex series of H L A - D region-controlled molecules. The use of monoclonal antibodies to dissect this expanding array of molecules has allowed the structural characterization of several of these molecular species. Here biochemical studies are discussed which, coupled with classic serological techniques, have been used to construct a model of the genetic organization of the human H L A - D region. The h u m a n major histocompatibility complex ( M H C ) or human leukocyte antigen (HLA) complex, a linked array of genes on chromosome 6, plays a major role in many phases of the immune response. It controls the recognition of foreign substances or antigens and is important in disease susceptibility and tissue-graft rejection. At least three classes of molecules are controlled by the M H C , each functioning in a distinct way to mediate the response to a foreign antigen. The class-I molecules, H L A - A , -B and -C, the classic transplantation antigens, are responsible for graft rejection and regulate the killing of virusinfected cells. The class-II molecules, the HLA-D-region antigens, mediate mixed lymphocyte stimulation and are involved in communication between lymphoid cells. Both class-I and class-II molecules exhibit a high degree of polymorphism. Their diversity is thought to enhance the capacity of the immune system to respond to the diverse antigenic onslaught of the environment. In contrast, the polymorphism of the M H G class-III molecules, the complement components, C2, C4 and Bf, is only moderate. These serum proteins participate in cell lysis and mediate inflammatory responses. Class-I molecules are expressed by practically all cells~ but class-II molecules are expressed most abundantly by B lymphocytes, activated T lymphocytes and antigenpresenting cells including peripheral blood monocytes, macrophages, Langerhans' cells, and dendritic cells of the lymphoid organs, i.e. on immunologically active cells. These HLA-D-region antigens have been less readily characterized than the class-I molecules, primarily because no well-defined, high-titer alloantisera were available with which to isolate the antigens. The advent of highly discriminatory monoclonal antibodies has now simplified the isolation of the class-II molecules and the description of several HLA-D-region-encoded molecules has followed. This review explores the evidence for the existence of these multiple species. T h e m u r i n e I region serves as a m o d e l for the H L A - D region
The murine homologue of the human H L A - D region is the I region. The I region has been divided into five subDepartment of Microbiology, The University of Texas Health Science Center at Dallas, Dallas, TX 75235, USA.
regions: l-A, I-B, I-J, I-E and I-C, based on a number of traits including immune responsiveness, mixed lymphocyte reactions, and serologically detectable antigens 2. The /-region antigens (Ia antigens) encoded by the I-A a n d / - E subregions consist of heterodimers containing an a chain of approximately 32 000 molecular weight and a fl chain of approximately 280003 . These chains may also be associated with an invariant chain 4. The I-A and I-E antigens are structurally distinct from one another ~'5. These structural differences correlate with functional differences as demonstrated by the ability of the I-A or I-E antigens to control the immune response to particular simple antigens 2. The I-A and I-E antigens are polymorphic. The fl chains exhibit more or most of the polymorphism, although the a chains, in particular the I-A a chain, are also polymorphic3. Additional polymorphism is generated by the presence of unexpressed alleles of the I-E a or/3 chains and combinatorial association of I-A or I-E allelic products 6. In a heterozygous animal, therefore, the I-A chain of one haplotype has been observed in combination with both the I-A/3 chain of the same haplotype and the I-A /3 chain of the alternative haplotype. Association between I-A and I-E polypeptide chains, however, has not been detected. I-A a-chains have not been observed complexed with I-E/3 chains and vice versa. The extent of polymorphism generated by combinatorial association is modulated by preferential association between the Ia products of particular H-2 haplotypes 7. A further level of polymorphism may derive from the presence of multiple I-E and/or I-A gene products 8-H. The Ia a and/3 polypeptide chains are encoded within the murine M H C : the I-A a and/3 chains and the I-E/3 chain are encoded in the I-A subregion while the I-E a chain is encoded in the I-E Subregion (Fig. 1)3'12'13. The linkage of these genes encoding the Ia heterodimers is unusual for molecules that are not synthesized as polyproteins. In addition, the genetic organization of the I-E a- and/3-chain genes into separate subregions appears to explain the I gene complementation observed in the response of some recombinant strains to several simple antigens 3. Complementation requires the presence of responder alleles at two distinct/-region loci, I-A a n d / - E , in order to generate an immune response. The products of the /-B, I-J and I-C subregions have not been © Elsevier Biomedical Press 1983 0167-4919/83/0000-00(}0/$1.00
Immunology Today, vol. 4, No. 8, 1983
220
A.
l REGION A Aa
~
i~/./~/J//'/A/
i
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A~ SCALE[kI~/ I. - J 0
I 20
Aa I 40
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E0 E~ Eo I 60
I 80
I 100
I 120
I 140
I 160
Fig. 1. A. Model of genetic organization and expression of the I-A and I-E alloantigens.
At the time this figure was published3, the order of the genes was not known.
B. The genetic organization of the murine I region based on recent DNA data~. characterized 2. Although there is m o u n t i n g evidence that these regions do not exist within the M H C 2'12, D N A studies have demonstrated the presence of a d d i t i o n a l / region related genes 12. At present, it is not clear whether these genes represent pseudogenes or expressed genes perhaps encoding other/-region molecules. Elucidation of the genetic organization of the m u r i n e I region has prompted several laboratories to extend their studies into the homologous h u m a n H L A - D region, especially since this region appears to be involved in susceptibility to a large n u m b e r of h u m a n diseases. This vast array of studies, which have included genetic, serological, cellular and biochemical analyses, have already begun to demonstrate that the h u m a n H L A - D region appears to possess a higher degree of complexity than does the I region of the mouse.
T h e t t L A - D r e g i o n is genetically c o m p l e x Historically, the H L A - D region was localized within a region encompassing approximately four centimorgans, between the H L A - B locus a n d a n e n z y m e locus, glyoxylase (GLO), located on the centromeric side of the H L A complex (Fig. 2). W i t h i n this chromosomal region, several serological a n d cellular specificities have been mapped: D, D R , MB, M T a n d SB. (In addition to these associated HLA-D-region specificities, a n u m b e r of complement components m a p within this region of the h u m a n MHC14). T h e primary specificities traditionally mapped to this region are H L A - D and H L A - D R . At present, twelve alleles of H L A - D have been identified by mixed lymphocyte reactivity ( M L R ) . Serological typing has identified ten alleles of HI_,A-DR. W i t h i n the Caucasian population, there has been a high correlation between D a n d D R typing, suggesting that mixed lymphocyte reactivity a n d serology m a y be identifying the same molecules. A n u m b e r of additional specificities m a p p i n g to the H L A - D region have been defined serologically. These include the MB, M T , DC, B R a n d T e series 15-18.Each of these specificities is found in linkage disequilibrium or associated strongly with a n u m b e r of D R specificities a n d is called a 'supertypic' specificity. For example, in the MB series, MB1 is associated with D R 1 , 2, w6, w8 a n d wl0; MB2 with D R 3 and 7; a n d MB3 with DR4, 5, a n d w9. M e m b e r s of a given supertypic series are thought to represent allelic products since they segregate from one another in family studies and, particularly in the case of the M B series, are in H a r d y - W e i n b e r g equilibrium. T h e relationship between these independently defined supertypic series has not been clear. For example, on the one hand, m a n y MB, D C and T e specificities appear to have similar, if not identical, DR-associated distributions suggesting that they may represent the same segregant series (Table I). O n the other hand, the MB and M T series (with the exception of MB1 a n d M T 1 ) have very different DR-associated distributions and probably represent different series. A simplified scheme relating the supertypic specificities to each other is presented in T a b l e I. For the purpose of simplicity a n d in order to develop a unified construct, specificities which have similar distributions are assumed to be identical; slight differences observed by different laboratories in the association of the supertypic specificities with D R haplotypes and the degree of linkage disequilibrium are assumed to be differences in
TABLE I. Supertypic series MB series MT series Associated Equivalent Associated Equivalent DR specificities specificities DR specificities specificities MBI DR1, 2, w6, w8a, wl0a DC1, Te21, MT1 b MT1 DR1, 2, w6, wl0 a DC1, Te21, MB1b MB2 DR3,7 DC3,Te24 MT2 DR3, 5, w6, w8" BR3 MB3 DR4, 5, w9a DC4,Te22,MT4 MT3 DR4, 7, w9, wl0 a BR4 aAssignments tentative. bSince MB1 and MT1 apparendy recognize the same population of DR molecules, they may represent equivalentspecificities.However, ifMB and MT are two different series of determinants, then there may be a split in the sera defining MB1/MTI/DC1/Te21, one set ofsera defining MB1 determinants and another set defining MT1 determinants. A second possibility is that the MT determinants do not represent an allelic series but simply public determinants shared by several DR specificities. In that case, MT2 and MT3 have no molecular relationship to each other and the need for an MT1 allelic specificity is negated.
Immunology Today, voL 4, No. 8, 1983
I
15
I 3 GLO
221
I 0.8 I D B
35
I
Cenlromere SB
DR
MT
like t~ chains in a single D R homozygous cell line2s,29. It has been difficult, however, to determine if D R determinants are, indeed, expressed by each of these multiple I-E-like species. The convention in biochemical studies has been to call any molecule with an I-E-like sequence, DR. For the purpose of this review, we will use this broad definition unless indicated.
MB
Fig. 2. Region of the human chromosome 6 containing the HLA-D region 8°. In map units, the region between the GLO and HLA-B loci is approximately 30 times longer than the mouse I region.
alloantisera used by these groups. In this way, the multiple supertypic series can be condensed into two series of aUoantigenic specificities, MB and M T (see also Ref. 15). The DC and Te series appear to be identical to the MB series, and the BR series appears to be identical to the M T series. A n additional specificity, SB, has been also mapped to the HLA-D region using primed lymphocyte typing. This specificity is independent of D R specificity, suggesting that SB and DR are distinct loci19.
Molecules bearing HLA-D and HLA-DR determinants are polymorphic Almost perfect correlation has been observed between some D and D R specificities, suggesting that D and D R typing are identifying the same molecules2°. Recent studies, however, have split certain D R specificities into multiple subsets based on H L A - D typing2°'21. For example, DR4 has been shown to be associated with H L A - D specificities Dw4, Dwl0, DB3, D Y T and LD4021. Discrepancies in correlations between H L A - D and H I , A - D R typing have also appeared in non-Caucasian populations 2°a2. In these studies, the H L A - D determinant normally associated with a particular D R specificity was absent and a different specificity was sometimes observed. These data suggest that H L A - D and H L A - D R typing do not define the same determinants; however, these data do not distinguish whether the D and D R determinants reside on the same or different molecules 23. Structural studies have characterized the molecules bearing D R determinants. H L A - D R is the predominant class-II molecule. Like the murine Ia antigens, D R is a heterodimer consisting of two non-covalently linked glycoproteins (Fig. 3). These chains may also be associated with an invariant chain 24. Both a and fl chains are integral membrane proteins 2~. Amino acid sequences derived from molecules isolated by xenoantisera indicate that the human molecules are homologues of the murine I-E antigens 25'26. A comparison of D R antigens isolated from cell lines bearing different D R specificities shows that the molecules possess relatively invariant a chains but polymorphic/3 chains 24. The molecules controlled by a single D R haplotype may, however, vary from one another at the primary structural level23'2730. U p to seven DR-like/3 chains have been detected as well as two DR-
u
i
-t--"
-+--
S-
SS-
??gYY? J AAA*AA [
?yy?y. A&AAA
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9
Fig. 3. Schematic representation of the DR molecule showing the two-chain structure, carbohydrate moieties, the disulfide bonds, and its relationship to the plasma mmbrane and cytoplasm. Adapted from Ref. 24.
Some HLA-D-region antigens are homologues of the murine I-A antigens While the D R antigens are the equivalent of the murine I-E antigens, the DS antigens are the equivalent of the I-A antigens 31. Six DS haplotypes have been described, each haplotype in linkage disequilibrium with a particular D R haplotype ~2. Both the t~ and fl chains of the DS molecules are polymorphic, in contrast to the D R antigens in which only the fl chain exhibits noticeable polymorphism.
The supertypic specificity MB resides on DS molecules As mentioned above, two series of supertypic specificities linked to H L A - D R have been defined: MB (DC, Te) and M T (BR). While molecules bearing these specificities resemble D R by having a similar two-chain structure, the molecular bases of the two supertypic series differ. By several criteria33-37, the serologically defined MB (DC, Te) specificities and the D R specificities reside on separate molecules. However, this distinction is not complete as certain MB specificities have also been detected on D R molecules by the same criteria~5'38.(This discrepancy is discussed in the section describing M T . ) Partial amino acid sequences of molecules carrying the supertypic specificities DC 1 (MB 1)s9'~°and MB3 41clearly
Immunology Today, vol. 4, No. 8, 1983
222 TABLE III. Amino acid sequencesof DR, SB and DS (MB) moleculesfrom the cellline PRIESS Positionno. DR4(IIIE3) SB3 (I-LR1) DS (MB3) (IVD12) DR4 (IIIE3) SB3 (I-LR1) DS(MB3)(IVD12)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 a chains F F F I V I I F Y L F F F Y F L Y Y Y F I V V Y V fl chains F F Y Y F L Y F F Y Y Y Y Y F V V Y Y F V Y F
demonstrate that these molecules are distinct from D R molecules. The sequence homology detected between the DC1 (MB1) and MB3 molecules suggests that the two molecules are allelic forms of the molecule bearing the MB specificity4~and provides a biochemical basis for the MB series presented in Table I. Furthermore, the amino acid sequence data indicate that the DC1 (MB1) and MB3 molecules are homologous to the murine I-A antigens and, therefore, can be classified as DS molecules. Two molecules that bear the same supertypic specificity can be structurally distinct from one another. Molecules bearing the DC 1 (MB 1) specificity but derived from cell lines bearing different D R specificities can diffe?3'3L This difference may correspond to the different DS haplotypes observed by Goyert and Silv& 2. An additional complexity is the incomplete linkage disequilibrium of the supertypic specificities. For example, the MB3 determinant in Caucasians, as detected by the monoclonal antibody IVD 12, has been found on 100 % of donors bearing the DR4 allele but on only approximately 90% of DR5 individuals4~. Alloantisera give similar indications. It is not known if the DR5 individuals that are negative for IVD12 binding bear an MB3 determinant not detected by the monoclonal antibody. Alternatively, these individuals could bear another untyped aUelic product or a null allele of MB. Nevertheless, molecules bearing MB determinants have been described which are distinct from D R molecules and can be classified by amino acid sequence as DS molecules. The possibility also exists that, in some cell lines, the MB specificity may also reside on D R molecules. T h e supertypic specificity M T resides on several
molecular species In contrast to the MB specificities examined in the sequence analysis, the M T (or BR) specificities appear to reside on several different molecules. Some M T determinants appear on molecules distinct from those molecules bearing D R determinants31'~5'36'~2.These may include molecules bearing MB determinants31'35'~8 or entirely new molecules representing an additional set of HLA-D-region molecules36. Other studies have localized M T specificities on molecules bearing D R determinants35.3s. Clearly, as above with MB, there is some confusion as to which molecules bear the M T supertypic specificities. While some of the confusion may be the result of using complex alloantisera for analysis of MB and M T determinants, it is possible that the molecules I~earing the supertypic specificities could vary from cell line to cell
line. Based on DNA data discussed below, there may be several molecules related to D R controlled by the HLA-D region. Exchange of genetic segments between these related molecules by gene conversion, as suggested in the H-2 system43'", could spread supertypic determinants throughout the HLA-D protein family. This conversion, however, must take place primarily in a cis fashion as conversion between homologous chromosomes would cause linkage breakdown between D R and the supertypic specificities. This has not been commonly observed. Some interesting observations concerning the sharing of determinants between HLA-D-region molecules can be made using monoclonal antibodies directed against HLA-D-region specificities. Table II lists some of the available monoclonal antibodies and their characteristics. The majority of monoclonal antibodies directed against D R molecules recognize determinants present on essentially all h u m a n B cells tested. In contrast, the majority of monoclonal antibodies directed against the MB and M T supertypic specificities or SB recognize previously defined polymorphic specificities. Unusual binding patterns, however, have been observed with some of these antibodies. For example, I-LR1, in addition to detecting certain SB allelic products, also appears to recognize DR5 molecules19. The monoclonal antibody SG171 appears to detect D R molecules in all cell lines; however, in a DR7 line, this monoclonal antibody also detects the DS molecule31. The sharing of determinants between different molecules as observed with these monoclonal antibodies could be producing some of the variability in the alloantisera studies. TABLE II. Monodonal antibodies directed against H/A-D-region molecules Monoclonal antibody 203 243 227 IIIE3 SG171 Genox IVD12 I-LR2 109d6 I-LR1
Predominant specificity DR DR DR DR DR,DS MB MB MT MT SB,DR
Monomorphic/ polymorphic Monomorphic Monomorphic Monomorphic Monomorphic MonomorphicDR, DS7 DC1 (MB1) MB3 MT2 MT3 SB2,3,DR5
Ref. 27 27 27 27 31 33 41 45 46 19
The use of monoclonal antibodies rather than alloantisera directed against the supertypic specificities may provide a more fruitful means of analysing the molecular bases for these determinants. Two monoclonal antibodies
Immunology Today, vol. 4, No. 8, 1983 DRa
GLO
DR ~"1
223 DR ~l'~
:;'---.....0,....---"i2 /,,,,, /,, /
DR ~'~
.
,
.
•
•
\
\
/
-
\
•
SB=
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\
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rare ~. One monoclonal antibody, I-LR1, has been identified that reacts with certain SB allelic produc& 9. Although similar to H L A - D R in its two-chain structure, both a and fl chains of the SB antigens are structurally distinct from the D R a and fl chains by amino acid sequence analysis. Based on these limited sequence data, the SB a chain, like its D R counterpart, is homologous to the murine I-E a chain 49. A B cell expresses at least three HLA-D-region molecules The studies detailed above describe several HI,A-Dregion specificities. Some of these specificities appear to be on structurally distinct molecules; alternatively, others may reside on the same molecule. Most of these data describing multiple HLA-D-region specificities are
Fig. 4. Model of the genetic organization of the HLA-D region. The order of the genes is not known.
have been used to characterize molecules carrying M T like determinants (unpublished observations). The antibody I-LR2 binds to cells in a pattern similar to that found with MT2 alloantisera45 and 109d6, in a pattern similar to an M T 3 alloantisera~. Preliminary amino • acid sequence characterization of MT2-1ike reactive molecules from two different cell lines suggests that the M T 2 molecule is D R or DR-like. Tyrosine is observed at position 13 in the a chain and at positions 10, 26 and 30 in the 13chain of an MT2-1ike molecule isolated from a DR3 line, positions identical to those observed when an antiD R monoclonal antibody (L203) is used. These data suggest that one of the M T 2 determinants is borne solely on D R or DR-like molecules but do not preclude the occurrence of additional MT2 determinants on other HLA-D-region molecules. The MT3-1ike determinant, as detected with the monoclonal antibody, 109d6, in two homozygous DR4 cell lines, appears to be present on two populations of molecules, D R and DS (see below and Table III). Partial amino-terminal amino acid sequences of the 109d6reactive molecule shows tyrosines at positions characteristic of D R in both the a and/3 chains. In addition, a DSlike sequence is also observed. The approximate ratio of D R to DS in these preparations varies, probably reflecting the amounts of D R and DS in the cell. By these criteria, the MT3 determinant appears to be present on both D R - and DS-like molecules. At this time, these data provide no evidence for M T as a molecule different from D R or DS and support the notion that M T 2 and M T 3 determinants are found on molecules closely resembling D R and/or DS molecules. The SB antigens represent an additional set o f H L A - D region molecules Cellular typing of cells matched for known HLA-Dregion antigens has been a fruitful means of identifying additional HLA-D-region loci19'47. Allogeneic mixed lymphocyte responses have been used to define an additional series of polymorphic HLA-D-region antigens, S B I9. SB is a poor immunogen compared to the D R , MB and M T determinants and alloantisera defining SB are
A.
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I
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FRACTION Fig. 5. Isolation of antigens from radiolabelled cell lysates by chromatography on lentil lectin-'Sepharose', immunoprecipitation or affmity chromatography with monoclonal antibodies and SDS polyacrylamide gel electrophoresis. The figures show the results of SDS gel electrophoresis of molecules isolated from the cell line PRIESS using monoclonal antibodies: (A) IIIE3 to isolate a D R molecule; (B) IVDI2 to isolate an MB3-bearing molecule; (C) I-LR1 to isolate an SB3 molecule; and (D) 109d6 to isolate an MT3-bearing molecule. The 109d6 immunoprecipitates contain a higher molecular weight molecule which we are in the process of characterizing. The approximate positions of immunoglobulin heavy and light chains are indicated by the arrows. Migration was from left to right.
224 consistent with the construction of a model (Fig. 4) depicting three distinct subregions within the H L A - D region: DR, D S and SB. Each of these subregions controls a distinct a and 0 chain. It should be emphasized that the gene order has not been established. Most of the genes encoding HLA-D-region molecules have been mapped to the M H C on human chromosome 62~'5°'51. This model can be further supported by the recent isolation and characterization of three structurally distinct molecules from a single cell line. Using monoclonal antibodies directed against antigens bearing D R 27, MB 41, M T (unpublished observations), and SB .9 specificities, three structurally distinct molecules have been isolated from the cell line PRIESS 52, a homozygous DR4 cell line which bears the MB3 and MT3 supertypic speeificities. The line is heterozygous for SB3/4; however, only the SB3 allelic product is reactive with the anti-SB monoclonal antibody 19. The four monoclonal antibodies used in the study, IIIE3, IVD12, 109d6 and I-LR1, are listed in Table II. All of the HLA-D-region molecules isolated were heterodimers of approximately the same molecular weight (Fig. 5). The molecules bearing the DR, MB and SB specificities were characterized by amino acid sequencing and the results are shown in Table III. Both a and O chains of the DR, DS (MB) and SB molecules are structurally distinct from one another, implying the presence of at least six distinct structural genes. In preliminary studies of the cell line PRIESS, the anti-MT3 monoclonal antibody recognized two populations of molecules, D R and DS, as described above. By comparison to murine sequences, the SB and D R a chains and the D R 0 chain are homologues of the murine I-E antigens; the DS (MB) a and 0 chains are homologues of the I-A antigens; and the SB O chain appears to be distinct from both I-E and I-A. Evidence from our laboratory and several others suggests that the D R subregion encodes multiple D R molecules 27-~°.Although the multiplicity of the D R population in PRIESS has not been examined by amino acid sequencing, sequential immunodepletion experiments suggest that these populations exist27. Multiple D R 0chain loci have, therefore, been included in the model. Since previous studies had detected at least three structurally distinct D R 0 chains in other cell lines, three E-chain genes have been included in the model. This model predicts only one D R a chain although multiple a chains have been suggested by other studies ~.
The DNA data suggest multiple HLA-D region loci Many of the HLA-D-region genes have been isolated by D N A cloning techniques and correlations can be made between the D N A data and the protein studies described above. The DNA encoding the D R and the DC 1 (MB 1) ot chains have been isolated by several investigators 51'53-5~. Portions of the a chains were found to exhibit homology to the HLA-D-region 0 chains, H L A class-I molecules, immunoglobulin and/3-2 microglobulin. Hybridization with the D R a-chain probe to genomic DNA under stringent conditions detects only one D R a-chain gene 5°'56'57 although eleven bands hybridize under less
Immunology Today, vol. 4, No. 8, 1983
stringent conditions ~. This suggests that there are multiple related a-chain genes in the genome. One of the additional hybridizing bands observed with the D R achain probe could be the gene encoding SB a, a protein which appears to be structurally similar to D R a ~. cDNA clones containing the D R and DC1 (MB1) 0chain genes have been isolated and sequenced 4°'59. Like the other HLA-D-region molecules that have been characterized, the E-chain sequences share homology with elass-I antigens and immunoglobulin. The homology between mouse and human gene products observed in the protein sequence comparisons was also observed at the D N A level with cross-hybridization of human HLA-D-region cDNA probes to murine cosmids containing/-region genes 12. Examination of D R and DC (MB) E-chain hybridization patterns from individuals with different DR specificities indicated polymorphism of the majority of hybridizing bands 6°. This finding complements the protein data which suggest that the 0 chains carry the allelic polymorphism24. Hybridization of D R and DC1 (MB1) E-chain gene probes to genomie D N A produced a complex pattern suggesting that multiple E-chain genes encoding the two subsets of molecules are present within the cell°°. The DNA data support the protein chemistry data which demonstrate multiple HLA-D-region a- and E-chain gene products and suggest that as yet unidentified HLA-Dregion-related E-chain genes may exist.
The functional roles of HLA-D-region antigens appear to vary HLA-D-region antigens act as primary and secondary stimulators in mixed lymphocyte reactions, as targets for cytotoxic T cells, and as controlling elements in antigen presentation. In the mouse, the I-A and I-E molecules control the immune response to different simple antigens 2. Similar delineations of function may exist among the multiple human HLA-D-region antigens as well. The functional roles of the HLA-D-region molecules are summarized in Table IV. While functional studies of the HLA-D-region molecules are still in their infancy, the role of the H L A - D region in susceptibility to disease has been studied extensively. Each HLA-D subregion is associated with distinct human diseases The varied functional roles played by the HLA-Dregion molecules in the control of immune responsiveness may be important in determining susceptibility to particular diseases. Correlations can be found between particular alleles at H L A - D and a variety of human diseases. Early studies of disease association showed association of certain diseases to certain alleles of the HLA-A and HLAB loci. Upon discovery of the H L A - D region, many of these diseases were shown to be more closely associated with H L A - D R alleles. Now that additional HLA-Dregion specificities have been identified and reagents directed against these molecules have become available, some of the disease associations have shifted from D R to MB, M T and SB specificities. While SB6ahas not yet been
225
Immunology Today, ool. 4, No. 8, 1983 T A B L E IV. T h e functional roles of HLA-D-region antigens Function Stimulation in Specificity MLR
Target for CTL
Antigen presentation
Disease association
DR a
+
+
+
MB b
?e
?
?
M'Ix
+
ND f
+
SBd
+
+
+
Multiple sclerosis (DR2) Type-I diabetes (DR3,4) Rheumatoid arthritis (DR4) Asthma (MB3) Chronic lymphatic leukemia (MB3) Hairy-cell leukemia (MB3) Primary sicca syndrome (MT2) Juvenile-onset diabetes (MT3) Dermatitis herpetiformis (SB1)
aData on D R was generated from Refs 20, 66-68, 71-76. hData on MB was generated from Refs 67, 68, 70, 78. CData on M T was generated from Refs 68, 69, 74, 78. dData on SB was generated from Refs 19, 77. ~The data are conflicting. fNot determined.
widely studied, certain alleles or specificities of D R 62, MB 46'63and M q "~'65 have all been strongly associated with particular disease states. Examples of such associations are shown in Table IV. Recent studies have demonstrated the association of certain malignancies to the MB3 specificity using the monoclonal antibody described above, IVD12 ~. These studies were the first to show association of the HLA-D region and malignancies. While these malignancies show weak association with particular D R alleles, the association with MB3 is striking. Table V illustrates this for the case of chronic lymphocytic leukemia where the relative risk of individuals bearing DR4 or DR5 is 1.4 or 6.0 while the relative risk of MB3-bearing individuals is 13.5. These observations suggest that the products of HLA-D subregions play important and varied roles in disease susceptibility. The use of monoclonal antibodies detecting H/M-D-region specificities should aid in elucidating the association of additional diseases with particular HLA-D-region molecules. Such antibodies will be TABLE V. Contrasting frequencies of certain HLA-D-region alloantigenicdeterminants between individualswith chronic lymphocyticleukemia and a control population~ HLA-D-re#on alloantigen specificity
Individuals with leukemia Normal control (n = 29) (n = 28) Relative (% positive) (% positive) risk
DR1 DR2 DR3 DR4 DR5 DRw6 DRw7 MB3(IVD12)
10.3 6.9 20.7 31.0 62.1 13.8 20.7 93.1
21.4 35.7 25.0 25.0 21.4 17.9 21.4 50.0
- 2.4 - 7.5 - 1.3 + 1.4 + 6.0 - 1.4 - 1.0 + 13.5
valuable in future studies to determine how these molecules function in immune responsiveness. Conclusion The human HLA-D region controls the expression of cell-surface antigens involved in communication between lymphoid cells. This communication appears to be critically important in immune responsiveness as suggested by the linkage of disease susceptibility in humans to particular H/M-D-region alleles or specificities. In order to understand how these H/M-D-region molecules function in cellular collaboration and antigen presentation, it is important to elucidate the assortment of H/M-D-region molecules found on the surface of immune response related cells. Using monoclonal antibodies, three structurally distinct H/A-D-region molecules, DR, DS and SB have been isolated from a single cell line. This effectively divides the HLA-D region into at least three subregions encoding a minimum of six protein chains. These studies have also attempted to define the biochemical basis for the serologically and functionally defined antigens bearing D R , MB, M T and SB specificities. Based on structural homology with the murine/-region antigens, the D R and SB antigens appear to be related to the murine I-E antigens while the DS (MB) antigens are similar to the murine I-A antigens. Thus, in the human, it appears that there has been a duplication of at least the loci encoding I-E to produce multiple gene products. The number of loci already described in the HLA-D region provide a large repertoire of cell-surface molecules which can be used in communication between immune response related cells. The murine data suggest that in the HLA-D region too, transassociation of chains within a subregion will generate additional molecules. Since some of the subregions such as DR appear to encode multiple fl chains, the number of possible chain associations is greatly increased. Additional mechanisms which generate polymorphism, as found in mice, probably exist. The association between susceptibility to particular diseases and certain allelic products ofHLA-D subregions suggests that roles played by the multiple H/A-D-region molecules in cellular collaboration and antigen presentation are varied. The development of carefully characterized monoclonal antibodies directed against subsets of these H/A-D-region molecules will be crucial in understanding the function of these molecules in immune responsiveness.
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Acknowledgements The authors would like to thank M. Firra, J. Mills, P. Presley, A. GarciaSpain and K. van Egmond for their assistance and the Capra laboratory, G. Nunez, P. Stastny, S. Shaw, L. Nadler, and R. Winchester for their helpful discussions. This work is supported by NIH grant AI18922 to J. Donald Capra, a UTHSC grant and ACS grant IN-142 to Carolyn Katovich Hurley and NIH grant CA-09082 to Robert C. Giles.