Structure of the HLA-D Region. (Received for publication, February 19, 1979)

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1 Microbial. Immunol. Vol. 23 (11), , 1979 Structure of the HLA-D Region Katsuaki ITAKURA* *Department of Pathology, Asahikawa Medical College, Asahikawa (Received for publication, February 19, 1979) The major histocompatibility region of man (HLA) codes for two groups of polymorphic cell surface membrane antigens (4). One group comprises the products of the A, B, and C loci. The second group represents the human analogues of the mouse Ia antigens, which are coded by a series of alleles in the HLA-D region. The HLA-D locus was originally defined as a locus whose alleles code for HLA-D antigens defined by the mixed lymphocyte culture (MLC) test (5). The discovery of the I region gene products by the serological method in the mouse was a great stimulus for HLA-ologists, and the study of the human homologues of the murine Ia antigens, i.e. HLA-linked B cell alloantigens detected serologically, has attracted many investigators in the field of HLA and has been one of the most important topics in this field for the last four years (1). HLA-DR antigens. In the 7th international histocompatibility workshop held in Oxford in 1977, 180 antisera, which had been shown locally or in regional workshops to react with B lymphocytes or with chronic lymphatic leukemia (CLL) cells or B cell lymphoid lines, were selected from those submitted from all over the world. These typing sera were distributed to the participating laboratories, where the experiments were carried out. The data were collected by the organizers of each region, on punched cards in the standard format, and sent on magnetic tape to Oxford, where they were analyzed on Oxford University's computer (3). In the workshop, seven antigen specificities (DRw 1 DRw7), predominantly expressed on B lymphocytes and supposed to be coded by a series of alleles of one locus (HLA-DR), were established. These HLA-DR antigen specificities defined serologically showed a high correlation with HLA-D antigen specificities assigned on the basis of the MLC test (1) (Table 1). Thus DRw 1 is correlated with Dw1, DRw2 with Dw2, and so on. A good Hardy-Weinberg fit was observed for these seven specificities in the population study, suggesting that a set of these specificities is coded for by allelic genes at the same locus. In further consideration of the problem of cross-reaction between the DRw antigens, only one locus (HLA-DR) was proposed to elucidate the genetic structure of the HLA-linked B cell alloantigen system (3). In the Oxford workshop a cluster of antisera was noticed which reacted with both DRw4 and DRw7 antigens. One representative serum in this group, serum P3796, reacted almost exclusively with cells that have either DRw4 or DRw7 antigen. Bodmer et al used this serum in an absorption experiment (4). After absorption with 1133

2 1134 K. ITAKURA Table 1. r Values of HLA-DRw and -Dw factors in 189 haplotypes from healthy Caucasoids (Data from Lamm et al 1978) DRw4 homozygote-positive cells, the serum reacted with only DRw7. Absorption with DRw7 homozygote cells, however, removed both the DRw4 and DRw7 activity. Bodmer et al interpreted this experimental result as indicating that in this serum the DRw4 activity must be the result of cross-reaction. The Ia antigens of the mouse, in contrast, are coded by the alleles of more than two loci in the H-2I region (8). Recent biochemical study on the HLA-linked B cell alloantigens by Katagiri et al presented firm evidence opposing this widely accepted view of one DR locus in the HLA-D region (10, detailed later). HLA-linked B cell alloantigens detected by radioimmune inhibition assay. Katagiri et al developed a radioimmune inhibition method for the detection of HLA-linked B cell alloantigens. The details of the assay system have been described elsewhere (10), and the outlines of the method are shown in Fig. 1. Briefly, an HLA-D homozygous B cell line (EBV-Wa) was solubilized with Brij 58 (a nonionic detergent) and was partially purified by gel filtration with Bio-Gel A 1.5 M, Con A-coupled Sepharose 4B affinity chromatography and column electrophoresis on Bio-Gel P-2. The preparation was trace-labeled with 1251 by the chloramine T method (125I-EBV-Wa) (7). Two kinds of antisera were used for the detection of B cell alloantigens. Hon 7 serum was originally obtained from a renal allograft recipient, whose graft came from his HLA-A and B identical but MLC-antigen nonidentical mother. The antiserum was mixed with LG 10 (Dw7 homozygous) cells and incubated for 2 hr at 4 C. Antibodies bound with LG 10 cells during this procedure were eluted by acid treatment. The dissociated antibody (Hon 7 antibody) reacted well with the 125I- EBV-Wa antigen preparation. The antigen responsible for this binding reaction was tentatively designated as Hon 7 antigen. The other antiserum, H 2075, was obtained from a multipara and was shown not to contain anti HLA-A, B, or C antibodies by the standard microcytotoxicity test. This serum also reacted well with the 1251-EBV-Wa preparation. The antigen detected by this reaction was named 2075 antigen. The antigenic activities of Hon 7 and 2075 were determined by the radioimmune inhibition test, in which the direct binding of the radiolabeled antigen preparation with the corresponding antiserum was inhibited by the antigen to be assayed.

3 NOTES 1135 Fig. 1. Principle of radioimmune inhibition test. EBV-Wa cells, which have both Hon 7 and 2075 antigens, proved to be DRw4 x 7 and DRw4 positive serologically. These two antigens, 2075 and Hon 7, were shown to be predominantly expressed on B cells. Family studies showed that both the antigens segregated concordantly with their respective HLA haplotypes, indicating a close linkage between HLA-A and B antigens and these antigens. A sequential coprecipitation test showed that 2075 and Hon 7 antigens are on different molecules. In this test, Hon 7 antigen molecule was removed from 125I-EBV-Wa by coprecipitation with an excess of Hon 7 serum. The pretreated 125I-EBV-Wa still retained the binding reactivity with H 2075, indicating that the antigen (2075) left in the supernatant after this procedure is distinct from Hon 7 antigen and that the antigens are on different molecules. Relationship between B cell alloantigens detected serologically ( DRw4, DRw4 x 7) and those detected by radioimmune inhibition test ( 2075, Hon 7).. B cell alloantigens of 26 Japanese who had been assigned to the HLA-DR group in the Oxford workshop were typed for Hon 7 and 2075 antigens by the radioimmune inhibition test. The results were surprising. As shown in Table 2, all the 2075 antigen positives were DRw4 positive and all the 2075 antigen negatives were DRw4 negative. No discrepant case was found. In the same manner, all the Hon 7 antigen positives were

4 1136 K. ITAKURA Table 2. Correlation of 2075 and Hon 7 antigens with HLA-DR specificity Table 3. Phenotype frequencies of Hon 7 and 2075 antigens and their linkage disequilibrium DRw4 x 7 positive, and all those who were Hon 7 antigen negative had no DRw4 x 7 antigen. It is, therefore, highly probable that the antigenic determinants (DRw4 and DRw4 x 7) detected serologically are the same as those (2075 and Hon 7) detected by the radioimmune inhibition method. Furthermore, none of the Hon 7 antigen negatives proved to be positive for 2075 antigen. The reverse, however, did not hold; i.e. those who were 2075 antigen negative but Hon 7 antigen positive were often found (Table 3). These results indicate that two genes encoding Hon 7 and 2075 antigens are in strong linkage disequilibrium. New aspects of the structure of the HLA-D region,clarified by the application of radio - immune inhibition assay. As a method for the detection of HLA-linked B cell alloantigens, the radioimmune inhibition assay has at least three advantages over the usual complement dependent cytotoxicity test. First, the amount of antiserum and the number of cells used for typing in this method are much less than are necessary for the conventional cytotoxicity test. Second, separation of B cells from T cells is not necessary for this test. The procedure of T cell-b cell separation is one of the most important factors that make the typing results of a cytotoxicity test ambiguous. Finally, this method is highly reproducible and a quantitative assay of the B cell alloantigen is also possible. The important contribution of the development of this assay method is the discov-

5 NOTES 1137 ery of the existence of at least two DR loci in the HLA-D region. The existence of two human Ia loci has been suggested by Ting et al (13), Mann et al (10), and van Rood et al (15), based on both population and recombination family data. These results need further confirmation by a method other than the cytotoxicity test. As stated earlier, the final concensus of the participants at the 7th international workshop on the HLA-linked B cell alloantigen system was that all the results of B cell alloantigen specificities analyzed in the workshop could be regarded as indicating that all of the antigens are coded by the alleles of one locus in the HLA-D region (2). The experimental results obtained by the use of radioimmunoassay definitely oppose this view. A cluster of antisera named antidrw4 x 7 and supposed to detect es - sentially DRw7 antigen but also to cross-react with DRw4, consists, in fact, of a single type of antiserum directed against the independent specificity "DRw4 x 7." The result of the sequential coprecipitation test clearly showed that the genes which code for DRw4 and DRw4 x 7 are not the same. Sasazuki et al observed that both the HLA-D homozygous cells, HLA-Dw2 and HLA-DHO, were typed as HLA-DRw2, but they were mutually strongly stimulatory in the MLC test (12). Based on these observations, Sasazuki considered the possi - bility that the HLA-D "locus" in man may, like the I "region" of the mouse H-2 complex, be divisible into several subregions. He proposed four models to explain the correlation between HLA-D and DR specificities (12). Model 1 assumes two distinct loci which code HLA-DR antigen and HLA-D antigen respectively. Model 2 proposes one HLA-D "region" and HLA-DR is considered to be a part of the HLA- D "region." According to Model 3, HLA-DR is HLA-D itself but there is another locus comparable to the mouse "M-locus." In Model 4, HLA-DR is a part of the several antigenic determinants on a single molecule coded for by one gene on the HLA-D "locus." The results obtained by Katagiri et al favor Model 1. Also the results seem rather to favor the view suggested by Bodmer in 1973 (2). He suggested that serologically detected allelic differences may actually be products of different, closely linked genes, and that the genetic polymorphism is for the control of which - ever of these genes is expressed. Immunochemical analysis of Hon 7 and 2075 antigens clearly demonstrated that these two antigens are on different molecules and, therefore, are coded by different genes in the HLA-D region, although the genes coding both antigens are closely linked and are in extremely strong linkage disequilibrium. Very recently, Tosi et al made a similar immunochemical analysis of Daudi Ia molecules, and they recognized at least three molecular species carrying different antigenic determinants (14). The last but not the least contribution of the study of HLA-linked B cell alloantigens by the immunochemical method is the discovery of the extremely close association between some organ specific autoimmune diseases and gene(s) in the newly found locus in the HLA-D region. Moriuchi et al (11) used this radioimmune inhibition technique to investigate the relationship of the HLA-linked B cell alloantigens to juvenile onset diabetes mellitus ( JOD) and to Vogt-Koyanagi - Harada's syndrome. As Tables 4 and 5 show, out of 50 JOD patients, 48 (phenotype

6 1138 K. ITAKURA Table 4. Association of Hon 7 antigen with juvenile onset diabetes mellitus Table 5. Association of Hon 7 antigens with Vogt-Koyanagi- Harada syndrome frequency 96.0%, relative risk 21.5) had Hon 7 (DRw4 ~ 7) antigen. All the 41 patients with Vogt-Koyanagi-Harada's syndrome had this Hon 7 antigen (41/ 41 = 100%, relative risk 74.6). The phenotype frequency of Hon 7 among healthy Japanese is 52.7% (39/ 74). The gene that codes Hon 7 (DRw4 ~ 7) antigen, in contrast to the 2075 antigen encoding gene, is distinct from the set of alleles of the HLA-DR locus thus far defined. This newly discovered gene in the HLA-D region seems to have a much more important role for the pathogenesis of disease of autoimmune etiology. Recently Gibofsky et al presented a similar report suggesting the special value of Ia-like B cell alloantisera for demonstrating disease associations with histocompatibility antigens (6). The characteristics of one of the sera they have used in their experiment are quite similar to those of the Hon 7 serum, thus emphasizing the importance of the further analysis of such sera. The immunogenetic factors relevant to the development of autoimmune diseases will be made clearer in the future by the application of immunochemical analysis to the HLA-D region antigens. REFERENCES 1) Bodmer, J.G Ia antigens. Brit. Med. Bull. 34: ) Bodmer, W.F A new genetic model for allelism at histocompatibility and other complex loci: Polymorphism for control of gene expression. Transplant. Proc. 5: ) Bodmer W.F Report of the Seventh International Histocompatibility Workshop and Conference, Oxford, p In Bodmer, W.F., Batchelor, J.R., Bodmer, J.G., Festenstein, H., and Morris, P. J. (eds), Histocompatibility testing 1977, Munksgaard, Copenhagen. 4) Bodmer, W.F., Jones, E.A., Barnstable, C. J., and Bodmer, J.G Genetics of HLA: the major histocompatibility system. Proc. R. Soc. Lond. B. 202: ) Bradley, B.A., and Festenstein, H Cellular typing. Brit. Med. Bull. 34: ) Gibofsky, A., Winchester, R. J., Patarryo, M., Fotino, M., and Kunkel, H.G Disease associations of the Ia-like human alloantigens. Contrasting patterns in rheumatoid arthritis and systemic lupus erythematosus. J. Exp. Med. 148: ) Greenwood, F.C., Hunter, W.M., and Gloner, J.S The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89:

7 NOTES ) Ivanyi, P Some aspects of the H-2 system, major histocompatibility system in the mouse. Proc. R. Soc. Lond. B. 202: ) Katagiri, M., Ikeda, H., Maruyama, N., Moriuchi, J., Wakisaka, A., Aizawa M., and Itakura, K Evidence for two B cell alloantigen loci in HLA-D region. Immunogenetics (in press). 10) Mann, D.L., Abelson, L., Harris, S., and Amos, D.B Second genetic locus in the HLA region for human B-cell alloantigens. Nature, Lond. 259: ) Moriuchi, J., Katagiri, M., Wakisaka, A., Matsuura, N., Aizawa, M., and Itakura, K B cell alloantigens and juvenile diabetes mellitus in the Japanese (submitted to Tissue Antigens). 12) Sasazuki, T., Kohno, Y., Iwamoto, I., Tanimura, M., Naito, S., Kashiwagi, N., Itakura, K., Aizawa, M., Hasegawa, T., Miyajima, T., Akiyama, N., Juji, T., Tsuji, K., Sekiguchi, S., Yoshida, T.O., Akaza, T., Matsuyama, M., and Toyoda, K The relationship between HLA-D and WIA specificities in Japanese population, Oxford, p In Bodmer, W.F., Batchelor, J.R., Bodmer, J.G., Festenstein, H. and Morris, P. J. (eds), Histocompatibility testing 1977, Munksgaard, Copenhagen. 13) Ting, A., Mickey, M.R., and Terasaki, P B lymphocyte alloantigens in Caucasians. J. Exp. Med. 143 : ) Tosi, R., Tanigaki, N., Centis, D., Ferrara, G.B., and Pressman, D Immunological dissection of human Ia molecules. J. Exp. Med. 148: ) van Rood, J. J., van Leewen, A., Jonker, M., Termijtelen, A., and Bradley, B.A Polymorphic B-cell determinants in man. Cold Spring Harb. Symp. Quant. Biol. 41: