- Email: [email protected]
HLA-DR Region Proteins and Genes*
HLA-DR Region Proteins and Genes*
J.L. Strominger, D.W. Andrews, C A . Auffray, M.R. Bono, J.F. Kaufman, P.J. Knudsen, A.J. Korman, M. Roux-Dosseto, A. Schamboeck, and D. Shackelford
Department of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts 02138
The major histocompatibility complex (MHC) of the mouse is located on chromosome 17 and that of man on chromosome 6. In the mouse immunogenetic experiments have divided the I region, which encodes the class II antigens, into a number of subregions (I-A, I-E, etc.). In man, where planned inbreeding is much more difficult, no comparable subdivision of the HLA-DR region had been accomplished. However, as I will show you, the DR region in man is very complex, and as far as we see it now, more complex than the picture so far seen in the mouse. One of the most interesting features of the system of histocompatibility antigens is their extraordinary polymorphism. A large numbers of alleles are known to occur at many of the MHC loci. The polymorphism must have arisen from some evolutionary pressure related to the function of these molecules. To a biochemist, one of the most interesting questions is: where in these molecules are the polymorphic regions and how do they reflect the functions of these molecules? *
This research has been supported by NIH grants AI 13230, AI 10736, and AI 30241.
H U M O RLA F A C T OSR IN H O ST D E F E NES
343
C o p y r i gth © 1983 by the T a k ead S c i e nec F o u n d a tni o All right s of reproductio n in any form r e s e r v e. d I S BN 0 - 1 2 - 7 6 8 2 21 0 -
344
J. L. STROMINGER ET AL.
Structure of Class I and Class II Antigens (2,9,12) The heavy chains of the class I antigens (HLA-A,B,C in man or H-2K, D, L in the mouse) consist of three extracellular domains called α 1, α 2 and α 3, plus transmembrane and m c riois o the nonintracytoplasmic regions. The fourth domain covalently associated light chain, j32"" ^ S bulin. Two of these domains, a. 3 and /32, are highly conserved, Ig-like domains. One of the other domains (al) is not apparently Ig-like, although it has a disulfide loop of the same size as an Ig domain. The fourth region (al) does not contain a disulfide loop. Polymorphic segments occur in both al and 02. The class II antigens are remarkably similar in structure, although as you know class I and class II antigens have very different functions. Class II antigens were first observed in 1975 because they co-purified with class I antigens; they were separated only at the last step of purification. The sum of the molecular weights of the two chains of the class I antigens and that of the two chains of the class II antigens are very similar. The meaning of that became clear later when it was discovered that the class II antigens have a very similar structure to the class I antigens and also contain 4 domains. However, the domains are organized so that two occur in each chain (al and α2, β 1 and
Fig. 1
Various molecules containing Ig-like domains. Modified from a figure of Alan William (University of Oxford) originally published in Biosci. Rep. 2: 277 (1982) with his permission.
IgM V-domains MHC A n t i g e n s
N
Class I
Thy-1
A V ^ L
p<
F
^ y^ y n
n
J t
J^O^
Λ
c_domains
^ΠΪΪΠΜΠΪΪ
MEMBRANE
Cytoplasm
346
J. L. STROMINGER
ETAL.
|32). Again, in the class II antigens there are two highly conserved, Ig-like domains (a 2 and j32), a polymorphic third domain which contains a disulfide loop which is not apparently Ig-like (ßl) and a fourth domain which is not defined by a disulfide loop (al). The class II antigens differ from the class I antigens in two respects: 1. the way in which domains are linked to each other to form the two chains and 2. in that both chains of the class II antigens span the membrane and both have small intracytoplasmic regions. The relationship of various molecules built up of Ig-like domains is illustrated in Fig.l. IgM has 14 such domains. Thy-1 is a molecule which has a single such domain. The al domain of the heavy chain of the class I antigen is shown bent over above β 2~microglobulin. This representation accommodates the fact that the study of the crystal structure of a class I antigen (HLA-A2) is now sufficiently advanced so that the two-fold rotational symmetry of the molecule is apparent (P. Β jorkmann and D. Wiley). This representation illustrates the striking similarity in structure of class I and class II antigens. The Polymorphic Region of Class II Antigens (9,10,12) A single chymotryptic cleavage site which occurs in the light chain of the class II antigens was very important in elucidating the structural organization of the light chain. It also permitted the separation of the N-terminal region of the light chain from the C-terminal region. Separation of the fragments SDS gels followed by isoelectric focussing of the N-terminal and C-terminal regions from eight homozygous human cell lines (DRI to DR ) showed that the C-terminal regions (the Ig-like domains) are highly conserved, and the N-terminal regions are polymorphic. Moreover, 8-12 different 15 kilodalton fragments were seen from the N-terminal region of a single homozygous human cell line. They must represent at least 3 and probably more expressed light chains in the cell. The number of C-terminal fragments also suggests some complexity. Separation of Subsets of HLA-DR Antigens (8,11,12,13) Subsets of the DR antigens could also be separated by immunoaffinity chromatography using monoclonal antibodies. First a monoclonal antibody specific for the DC-1 subset
HLA-DR REGION PROTEINS A N D GENES
was used and then another antibody which is specific for all the DR subsets. The DC-1 subset is the human homologue of the mouse I-A antigen and the DR subsets are the human homologues of the mouse I-E antigens. The DR subset was further fractionated by monoclonal antibodies to give two subsets, each of which appeared to have a complex light chain pattern. The amino acid sequence of the light chains of these two subsets, i.e. the dominant sequence, indicated clearly that they represent different gene products. Cloning of the HLA-DR Antigen Heavy Chain Genes (1-4) First, a cDNA clone was obtained after purification of polysomes, using an anti-DR heavy chain monoclonal antibody. About a 3,000 fold purification of the polysomes was obtained in one step. The mRNA was recovered from these polysomes. Only a single protein, the DR heavy chain precursor, was translated from that purified mRNA preparation. With mRNA of that degree of purity it was relatively easy to make a cDNA transcript and clone it. The cDNA clone was used to probe a human placental phage genomic library. A phage containing the genomic clone for the DRa chain was identified. It consists of 5 exons. The 5' exon, containing the leader sequence is located about 2 kb upstream from the main coding sequences. The latter, all contained in a 3.2 kb EcoRI fragment, include four exons which encode: a. the al domain, b. the a2 domain, c. the connecting peptide, transmembrane region, intracytoplasmic region and a small portion of the 3' untranslated sequence and d. the main part of the 3' untranslated sequence. The entire sequence of the DR heavy chain was obtained. The sequence of the α 2 region showed conclusively that it is an Ig-like domain. The gene for the DRa chain has been localized to 6p21.1 band on the short arm of human chromosome 6 by in situ hybridization. cDNA Clones for the DC-1 Heavy Chain A differential screening procedure was then employed to identify additional clones: a cDNA library from a human Β cell line was screened with cDNA probes made from Β cell and Τ cell mRNA (the Τ cell line does not express class II antigens). cDNA clones hybridizing only with the Β cell cDNA were selected and analyzed with the DC ά chain gene. First, several cDNA clones were identified which hybridized
347
J. L. STROMINGER ET AL.
348
1 with fragments derived from the 5 and 3' end of the DRa chain gene. Two kinds of clones were identified. They both hybridized very strongly to the 3' probe, but one of them hybridized weakly to the 5' probe. When this clone was sequenced, it turned out to be a cDNA clone for the chain of the DC-1 antigen, as demonstrated by comparison to the protein sequence which had been obtained from the isolated protein. Its complete sequence revealed that the DR and DC-1 heavy chains (i.e. the human proteins equivalent to murine I-E and I-A) are in fact remarkably similar. They differ most at the N-terminus where there is almost no homology. Of course, all the amino acid sequencing done previously to compare these chains has been N-terminal sequencing. They also differ in several other regions, but are otherwise quite homologous. Remarkably, in the transmembrane region there is almost complete conservation of 1 of almost complete conservation of sequence. Another area sequence is at the 3 end of the 0.2 exon. The DC-1 heavy chain cDNA clone has been used for a number of purposes. 1.
A human DNA library has been probed to isolate genomic clones.
2.
Southern blots have been carried human typing cell lines.
3.
γ ray induced deletion mutants of hemizygous cell lines have also been examined by Southern blots in collaboration with Robert DeMars (University of Wisconsin) .
4.
The murine homologue of the DC-1 heavy chain gene has been mapped to the mouse I-A subregion using mouse recombinant strains in collaboration with J.G. Seidman (Harvard Medical School).
out on homozygous
Two DC-Like Heavy Chain Genes (5) When human genomic library was probed, two classes of genomic clones were obtained and differentiated by restriction maps. One, of course, represented the DC chain gene. The second, however, was a third heavy chain gene present in the human genomic library. It was provisionally called DX because it was not known whether it will be an
HLA-DR REGION PROTEINS A N D GENES
349
expressed gene or a pseudogene. A partial sequence has revealed no unusual feature to prevent its expression, and evidence that the DX gene is transcribed has been obtained recently through the isolation of a cDNA clone corresponding to it. Polymorphism of the DC
Chain Genes (5)
The DR α chain appears to be virtually invariant, and only a small number of nucleotide changes have been found when the DR α chain gene has been sequenced by several groups. In contrast, the DC α and DX α genes appear to be polymorphic genes. The DX gene is very homologous to the DC a chain gene. The largest region of divergence is found at residues 60 to 75 in the amino acid sequence. That position is also the position of greatest sequence diversity in the al a2 domains of the class I antigens. It now appears generally as a region of amino acid diversity in a domain of a class II antigen. The region of amino acids 60 to 80 in these domains appears to be a very important sequence in terms of the polymorphism of these proteins. A remarkable restriction enzyme polymorphism of the DCa chain gene was found by Southern blot analysis of Hindlll digested DNA of HLA-DR homozygous human Β cell lines, which appears to correspond to the MT alloantiserum classification system of Terasaki. The DX gene also hybridized with the DC-1 probe in these blots. It also appeared to have a more limited polymorphism. All three of the light chains are also polymorphic, i.e. the DC-1 light chain and the light chains of the two DR subsets. At least four extensively polymorphic chains occur, therefore, in the DR antigens, viz.: the three light chains of the three major subsets and the heavy chain of one of the subsets. Study of Deletion Mutants (6) Much information can be gained from study of deletion mutants. First hemizygous mutants are obtained, i.e. mutants in which one of the HLA regions is completely deleted by X-irradiation. The other haplotype is then subjected to further irradiation to obtain DR-null cells which do not react with anti-DR sera. Some of these are also DC-null. The DC-null mutants are all missing the same Hindlll restriction enzyme band, thus identifying it as the restriction fragment containing the DCa gene. The DX gene is split by Hindlll into two fragments. In addition, at least one
J. L. STROMINGER ET AL.
350
other weakly hybridizing gene is detected in this experiment, suggesting that a minimum of four heavy chain genes occur in the human genome. We have just begun to examine the 3 chain genes. An ΙΑ 3 cDNA clone (obtained from J.G. Seidman) was used to probe the human Β cell cDNA library. The first cDNA clone obtained encoded neither a DR nor a DC Β chain, but a related β chain. Other laboratories have been working on DR ß chain genes (principally the laboratories of Per Peterson in Uppsala and Bernard Mach in Geneva). They have obtained cDNA clones for both DR ß and DC β chain genes. The clone we isolated had equal homology to each, but considerable differences from each. DR β chain genomic clones are also being studied and, from the complexity of the proteins so far observed, even greater complexity may be observed than in the case of the α chain genes. The DR region in man may be much more complex than has so far been seen in the I region of the mouse. Questions Q.
Do you know how many specificities there are which are so far identifiable by serological studies in that region?
A.
Serological studies are very complicated. There are various classification systems. First of all there is that provided by the classical DR alloantiserum. I'm not sure that they define a single locus. Even these DR alloantisera may be defining several different segregating loci (for example, the light chains of the two DR subsets). Secondly, there are the MT, MB, DC and TE systems. At least 5 different sets of serological reagents have been used in man. It is not absolutely clear what those define at the present time. References
This article is an edited transcript of the Symposium presentation of work from our laboratory. It has been published in the following papers. A complete list of reference, including contributions from other laboratories, can be found in these papers.
HLA-DR REGION PROTEINS A N D GENES
351
1.
Korman, A.J., Ploegh, H.L., Kaufman, J.F., Owen, M.J.and Strominger, J.L. Cell free synthesis and processing of the heavy and light chains of HLA-DR antigens. J. Exp. Med. 152: 65s-82s (1980)
2.
Korman, A.J., Knudsén, P.J., Kaufman, J.F. minger, J.L. cDNA Clones for the Heavy Chain Antigens Obtained after Immunopurification somes by Monoclonal Antibody. PNAS USA 79: (1982)
3.
Korman, A.J., Auffray, C , Schamboeck, A. and Strominger, J.L. The sequence and gene organization of the HLA-DR heavy chain: homology to immunoglobulins. PNAS USA 79: 6013-6017 (1982)
4.
Auffray, C.A., Korman, A.J., Roux-Dosseto, Μ., Bono, R. and Strominger, J.L. cDNA Clone for the Heavy Chain of the Human Β Cell Alloantigen DC1: Strong Sequence Homology to the HLA-DR Heavy Chain. PNAS USA 79: 6337-6341 (1982)
5.
Auffray, C.A., Ben-Nun, Α., Roux-Dosseto, Μ., Germain, R.N., Seidman, J.G. and Strominger, J.L. Polymorphism and Complexity of the Human DC and Murine I-Αα Chain Genes. Eur. Mol. Biol. Organiz. J., in press
6.
Auffray, C.A., Kuo, J., DeMars, R. and Strominger, J.L. Mapping of the Human DCa Chain Gene to Chromosome 6 Using Deletion Mutants. Nature, submitted for publication
7.
Kirsch, I.R., Morton, C.C., Nance, W.E., Evans, G.A., Korman, A.J. and Strominger, J.L. Orientation of loci within the human major histocompatibility complex by chromosome in situ hybridization. Nature, submitted for publication
8.
Andrews, D.W. and Strominger, J.L. A homozygous human cell line contains three subsets of HLA-DR-like antigens distinguishable by amino acid sequencing. Biochemistry 21: 6625-6628 (1982)
and Stroof HLA-DR of Poly1844-1848
J. L. STROMINGER
ETAL.
Kaufman, J.F. and Strominger, J.L. HLA-DR light chain has a polymorphic N-terminal region and a conserved Ig-like C-terminal region. Nature 297: 694-696 (1982) Kaufman, J.F. and Strominger, J.L. The extracellular region of light chains from human and murine NHC class II antigens consists of 2 domains. Immunol. 130: 808 -817 (1983) Bono, R. and Strominger, J.L. Direct evidence of homology between human DC-1 antigen and murine I-A molecules. NH2-terminal amino acid sequence analysis of human class II molecules carrying DC-1 and DR determinants. Nature 299: 836-838 (1982) Schackelford, D.A., Kaufman, J.F., Korman, A.J. and Strominger, J.L. HLA-DR antigens: Structure, separation of subpopulations, gene cloning and function. Immunol. Rev. 66: 133-187 (1982) Shackelford, D.A., Lampson, L.A. and Strominger, J.L. Separation of three class II antigens from a homozygous human Β cell line. J. Immunol. 130: 289-296 (1983)
J.L. Strominger, D.W. Andrews, C A . Auffray, M.R. Bono, J.F. Kaufman, P.J. Knudsen, A.J. Korman, M. Roux-Dosseto, A. Schamboeck, and D. Shackelford
Department of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts 02138
The major histocompatibility complex (MHC) of the mouse is located on chromosome 17 and that of man on chromosome 6. In the mouse immunogenetic experiments have divided the I region, which encodes the class II antigens, into a number of subregions (I-A, I-E, etc.). In man, where planned inbreeding is much more difficult, no comparable subdivision of the HLA-DR region had been accomplished. However, as I will show you, the DR region in man is very complex, and as far as we see it now, more complex than the picture so far seen in the mouse. One of the most interesting features of the system of histocompatibility antigens is their extraordinary polymorphism. A large numbers of alleles are known to occur at many of the MHC loci. The polymorphism must have arisen from some evolutionary pressure related to the function of these molecules. To a biochemist, one of the most interesting questions is: where in these molecules are the polymorphic regions and how do they reflect the functions of these molecules? *
This research has been supported by NIH grants AI 13230, AI 10736, and AI 30241.
H U M O RLA F A C T OSR IN H O ST D E F E NES
343
C o p y r i gth © 1983 by the T a k ead S c i e nec F o u n d a tni o All right s of reproductio n in any form r e s e r v e. d I S BN 0 - 1 2 - 7 6 8 2 21 0 -
344
J. L. STROMINGER ET AL.
Structure of Class I and Class II Antigens (2,9,12) The heavy chains of the class I antigens (HLA-A,B,C in man or H-2K, D, L in the mouse) consist of three extracellular domains called α 1, α 2 and α 3, plus transmembrane and m c riois o the nonintracytoplasmic regions. The fourth domain covalently associated light chain, j32"" ^ S bulin. Two of these domains, a. 3 and /32, are highly conserved, Ig-like domains. One of the other domains (al) is not apparently Ig-like, although it has a disulfide loop of the same size as an Ig domain. The fourth region (al) does not contain a disulfide loop. Polymorphic segments occur in both al and 02. The class II antigens are remarkably similar in structure, although as you know class I and class II antigens have very different functions. Class II antigens were first observed in 1975 because they co-purified with class I antigens; they were separated only at the last step of purification. The sum of the molecular weights of the two chains of the class I antigens and that of the two chains of the class II antigens are very similar. The meaning of that became clear later when it was discovered that the class II antigens have a very similar structure to the class I antigens and also contain 4 domains. However, the domains are organized so that two occur in each chain (al and α2, β 1 and
Fig. 1
Various molecules containing Ig-like domains. Modified from a figure of Alan William (University of Oxford) originally published in Biosci. Rep. 2: 277 (1982) with his permission.
IgM V-domains MHC A n t i g e n s
N
Class I
Thy-1
A V ^ L
p<
F
^ y^ y n
n
J t
J^O^
Λ
c_domains
^ΠΪΪΠΜΠΪΪ
MEMBRANE
Cytoplasm
346
J. L. STROMINGER
ETAL.
|32). Again, in the class II antigens there are two highly conserved, Ig-like domains (a 2 and j32), a polymorphic third domain which contains a disulfide loop which is not apparently Ig-like (ßl) and a fourth domain which is not defined by a disulfide loop (al). The class II antigens differ from the class I antigens in two respects: 1. the way in which domains are linked to each other to form the two chains and 2. in that both chains of the class II antigens span the membrane and both have small intracytoplasmic regions. The relationship of various molecules built up of Ig-like domains is illustrated in Fig.l. IgM has 14 such domains. Thy-1 is a molecule which has a single such domain. The al domain of the heavy chain of the class I antigen is shown bent over above β 2~microglobulin. This representation accommodates the fact that the study of the crystal structure of a class I antigen (HLA-A2) is now sufficiently advanced so that the two-fold rotational symmetry of the molecule is apparent (P. Β jorkmann and D. Wiley). This representation illustrates the striking similarity in structure of class I and class II antigens. The Polymorphic Region of Class II Antigens (9,10,12) A single chymotryptic cleavage site which occurs in the light chain of the class II antigens was very important in elucidating the structural organization of the light chain. It also permitted the separation of the N-terminal region of the light chain from the C-terminal region. Separation of the fragments SDS gels followed by isoelectric focussing of the N-terminal and C-terminal regions from eight homozygous human cell lines (DRI to DR ) showed that the C-terminal regions (the Ig-like domains) are highly conserved, and the N-terminal regions are polymorphic. Moreover, 8-12 different 15 kilodalton fragments were seen from the N-terminal region of a single homozygous human cell line. They must represent at least 3 and probably more expressed light chains in the cell. The number of C-terminal fragments also suggests some complexity. Separation of Subsets of HLA-DR Antigens (8,11,12,13) Subsets of the DR antigens could also be separated by immunoaffinity chromatography using monoclonal antibodies. First a monoclonal antibody specific for the DC-1 subset
HLA-DR REGION PROTEINS A N D GENES
was used and then another antibody which is specific for all the DR subsets. The DC-1 subset is the human homologue of the mouse I-A antigen and the DR subsets are the human homologues of the mouse I-E antigens. The DR subset was further fractionated by monoclonal antibodies to give two subsets, each of which appeared to have a complex light chain pattern. The amino acid sequence of the light chains of these two subsets, i.e. the dominant sequence, indicated clearly that they represent different gene products. Cloning of the HLA-DR Antigen Heavy Chain Genes (1-4) First, a cDNA clone was obtained after purification of polysomes, using an anti-DR heavy chain monoclonal antibody. About a 3,000 fold purification of the polysomes was obtained in one step. The mRNA was recovered from these polysomes. Only a single protein, the DR heavy chain precursor, was translated from that purified mRNA preparation. With mRNA of that degree of purity it was relatively easy to make a cDNA transcript and clone it. The cDNA clone was used to probe a human placental phage genomic library. A phage containing the genomic clone for the DRa chain was identified. It consists of 5 exons. The 5' exon, containing the leader sequence is located about 2 kb upstream from the main coding sequences. The latter, all contained in a 3.2 kb EcoRI fragment, include four exons which encode: a. the al domain, b. the a2 domain, c. the connecting peptide, transmembrane region, intracytoplasmic region and a small portion of the 3' untranslated sequence and d. the main part of the 3' untranslated sequence. The entire sequence of the DR heavy chain was obtained. The sequence of the α 2 region showed conclusively that it is an Ig-like domain. The gene for the DRa chain has been localized to 6p21.1 band on the short arm of human chromosome 6 by in situ hybridization. cDNA Clones for the DC-1 Heavy Chain A differential screening procedure was then employed to identify additional clones: a cDNA library from a human Β cell line was screened with cDNA probes made from Β cell and Τ cell mRNA (the Τ cell line does not express class II antigens). cDNA clones hybridizing only with the Β cell cDNA were selected and analyzed with the DC ά chain gene. First, several cDNA clones were identified which hybridized
347
J. L. STROMINGER ET AL.
348
1 with fragments derived from the 5 and 3' end of the DRa chain gene. Two kinds of clones were identified. They both hybridized very strongly to the 3' probe, but one of them hybridized weakly to the 5' probe. When this clone was sequenced, it turned out to be a cDNA clone for the chain of the DC-1 antigen, as demonstrated by comparison to the protein sequence which had been obtained from the isolated protein. Its complete sequence revealed that the DR and DC-1 heavy chains (i.e. the human proteins equivalent to murine I-E and I-A) are in fact remarkably similar. They differ most at the N-terminus where there is almost no homology. Of course, all the amino acid sequencing done previously to compare these chains has been N-terminal sequencing. They also differ in several other regions, but are otherwise quite homologous. Remarkably, in the transmembrane region there is almost complete conservation of 1 of almost complete conservation of sequence. Another area sequence is at the 3 end of the 0.2 exon. The DC-1 heavy chain cDNA clone has been used for a number of purposes. 1.
A human DNA library has been probed to isolate genomic clones.
2.
Southern blots have been carried human typing cell lines.
3.
γ ray induced deletion mutants of hemizygous cell lines have also been examined by Southern blots in collaboration with Robert DeMars (University of Wisconsin) .
4.
The murine homologue of the DC-1 heavy chain gene has been mapped to the mouse I-A subregion using mouse recombinant strains in collaboration with J.G. Seidman (Harvard Medical School).
out on homozygous
Two DC-Like Heavy Chain Genes (5) When human genomic library was probed, two classes of genomic clones were obtained and differentiated by restriction maps. One, of course, represented the DC chain gene. The second, however, was a third heavy chain gene present in the human genomic library. It was provisionally called DX because it was not known whether it will be an
HLA-DR REGION PROTEINS A N D GENES
349
expressed gene or a pseudogene. A partial sequence has revealed no unusual feature to prevent its expression, and evidence that the DX gene is transcribed has been obtained recently through the isolation of a cDNA clone corresponding to it. Polymorphism of the DC
Chain Genes (5)
The DR α chain appears to be virtually invariant, and only a small number of nucleotide changes have been found when the DR α chain gene has been sequenced by several groups. In contrast, the DC α and DX α genes appear to be polymorphic genes. The DX gene is very homologous to the DC a chain gene. The largest region of divergence is found at residues 60 to 75 in the amino acid sequence. That position is also the position of greatest sequence diversity in the al a2 domains of the class I antigens. It now appears generally as a region of amino acid diversity in a domain of a class II antigen. The region of amino acids 60 to 80 in these domains appears to be a very important sequence in terms of the polymorphism of these proteins. A remarkable restriction enzyme polymorphism of the DCa chain gene was found by Southern blot analysis of Hindlll digested DNA of HLA-DR homozygous human Β cell lines, which appears to correspond to the MT alloantiserum classification system of Terasaki. The DX gene also hybridized with the DC-1 probe in these blots. It also appeared to have a more limited polymorphism. All three of the light chains are also polymorphic, i.e. the DC-1 light chain and the light chains of the two DR subsets. At least four extensively polymorphic chains occur, therefore, in the DR antigens, viz.: the three light chains of the three major subsets and the heavy chain of one of the subsets. Study of Deletion Mutants (6) Much information can be gained from study of deletion mutants. First hemizygous mutants are obtained, i.e. mutants in which one of the HLA regions is completely deleted by X-irradiation. The other haplotype is then subjected to further irradiation to obtain DR-null cells which do not react with anti-DR sera. Some of these are also DC-null. The DC-null mutants are all missing the same Hindlll restriction enzyme band, thus identifying it as the restriction fragment containing the DCa gene. The DX gene is split by Hindlll into two fragments. In addition, at least one
J. L. STROMINGER ET AL.
350
other weakly hybridizing gene is detected in this experiment, suggesting that a minimum of four heavy chain genes occur in the human genome. We have just begun to examine the 3 chain genes. An ΙΑ 3 cDNA clone (obtained from J.G. Seidman) was used to probe the human Β cell cDNA library. The first cDNA clone obtained encoded neither a DR nor a DC Β chain, but a related β chain. Other laboratories have been working on DR ß chain genes (principally the laboratories of Per Peterson in Uppsala and Bernard Mach in Geneva). They have obtained cDNA clones for both DR ß and DC β chain genes. The clone we isolated had equal homology to each, but considerable differences from each. DR β chain genomic clones are also being studied and, from the complexity of the proteins so far observed, even greater complexity may be observed than in the case of the α chain genes. The DR region in man may be much more complex than has so far been seen in the I region of the mouse. Questions Q.
Do you know how many specificities there are which are so far identifiable by serological studies in that region?
A.
Serological studies are very complicated. There are various classification systems. First of all there is that provided by the classical DR alloantiserum. I'm not sure that they define a single locus. Even these DR alloantisera may be defining several different segregating loci (for example, the light chains of the two DR subsets). Secondly, there are the MT, MB, DC and TE systems. At least 5 different sets of serological reagents have been used in man. It is not absolutely clear what those define at the present time. References
This article is an edited transcript of the Symposium presentation of work from our laboratory. It has been published in the following papers. A complete list of reference, including contributions from other laboratories, can be found in these papers.
HLA-DR REGION PROTEINS A N D GENES
351
1.
Korman, A.J., Ploegh, H.L., Kaufman, J.F., Owen, M.J.and Strominger, J.L. Cell free synthesis and processing of the heavy and light chains of HLA-DR antigens. J. Exp. Med. 152: 65s-82s (1980)
2.
Korman, A.J., Knudsén, P.J., Kaufman, J.F. minger, J.L. cDNA Clones for the Heavy Chain Antigens Obtained after Immunopurification somes by Monoclonal Antibody. PNAS USA 79: (1982)
3.
Korman, A.J., Auffray, C , Schamboeck, A. and Strominger, J.L. The sequence and gene organization of the HLA-DR heavy chain: homology to immunoglobulins. PNAS USA 79: 6013-6017 (1982)
4.
Auffray, C.A., Korman, A.J., Roux-Dosseto, Μ., Bono, R. and Strominger, J.L. cDNA Clone for the Heavy Chain of the Human Β Cell Alloantigen DC1: Strong Sequence Homology to the HLA-DR Heavy Chain. PNAS USA 79: 6337-6341 (1982)
5.
Auffray, C.A., Ben-Nun, Α., Roux-Dosseto, Μ., Germain, R.N., Seidman, J.G. and Strominger, J.L. Polymorphism and Complexity of the Human DC and Murine I-Αα Chain Genes. Eur. Mol. Biol. Organiz. J., in press
6.
Auffray, C.A., Kuo, J., DeMars, R. and Strominger, J.L. Mapping of the Human DCa Chain Gene to Chromosome 6 Using Deletion Mutants. Nature, submitted for publication
7.
Kirsch, I.R., Morton, C.C., Nance, W.E., Evans, G.A., Korman, A.J. and Strominger, J.L. Orientation of loci within the human major histocompatibility complex by chromosome in situ hybridization. Nature, submitted for publication
8.
Andrews, D.W. and Strominger, J.L. A homozygous human cell line contains three subsets of HLA-DR-like antigens distinguishable by amino acid sequencing. Biochemistry 21: 6625-6628 (1982)
and Stroof HLA-DR of Poly1844-1848
J. L. STROMINGER
ETAL.
Kaufman, J.F. and Strominger, J.L. HLA-DR light chain has a polymorphic N-terminal region and a conserved Ig-like C-terminal region. Nature 297: 694-696 (1982) Kaufman, J.F. and Strominger, J.L. The extracellular region of light chains from human and murine NHC class II antigens consists of 2 domains. Immunol. 130: 808 -817 (1983) Bono, R. and Strominger, J.L. Direct evidence of homology between human DC-1 antigen and murine I-A molecules. NH2-terminal amino acid sequence analysis of human class II molecules carrying DC-1 and DR determinants. Nature 299: 836-838 (1982) Schackelford, D.A., Kaufman, J.F., Korman, A.J. and Strominger, J.L. HLA-DR antigens: Structure, separation of subpopulations, gene cloning and function. Immunol. Rev. 66: 133-187 (1982) Shackelford, D.A., Lampson, L.A. and Strominger, J.L. Separation of three class II antigens from a homozygous human Β cell line. J. Immunol. 130: 289-296 (1983)