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Minor Histocompatibility Genes and Their Antigens 1

Ralph J. Graff

Waldheim Department of Surgery
Jewish Hospital of St. Louis
Washington University School of Medicine
St. Louis, Missouri

Progress in transplantation immunogenetics has depended heavily on the development of inbred mouse strains. The earliest observations indicating that acceptance of allografts was at least partially heritable were made in closed mouse colonies. With the production of inbred mouse strains, the basic principles of transplantation immunogenetics developed more readily: outstanding examples were the finding that the acceptance of allografts is a polygenic trait, and that histocompatibility genes can be isolated in congenic strain pairs.

Since the present report will deal with histocompatibility genes and their antigens, particularly the minor histocompatibility genes and antigens, it would be appropriate to define these terms. Histocompatibility antigens (H-antigens) are found on a broad distribution of tissues and immunological reaction against them results in the rejection of antigen-bearing tissues. This definition excludes tissue-specific antigens such as the blood group antigens (the Ea series in the mouse) and the lymphocyte antigens (the Ly series in the mouse). In addition, it excludes modulating antigens -- antigens that evoke immunological reactions not resulting in tissue rejection (TL in the mouse). The term minor H-antigen is complementary to the term major H-antigen. The major H-antigens are under the genetic control of the H-2 complex, a group of closely linked genes on chromosome 17. The minor H-antigens are controlled by genes scattered throughout the remainder of the chromosomes in addition to four genes located on chromosome 17.

Estimates of the Number of H-Loci

The role of genetics in transplantation was first suggested by Loeb ( 1) and Tyzzer ( 2), but it remained for Little ( 3) in his classical publication, "A Possible Mendelian Explanation for a Type of Inheritance Apparently Non-Mendelian in Nature," to hypothesize that the susceptibility or resistance to the growth of an allograft was under the control of multiple genes, subsequently named histocompatibility genes by Snell ( 4). In a later publication, Little and Tyzzer ( 5) produced F2 mice from the initial cross of a Japanese waltzing mouse and a common house mouse. They used the acceptance or rejection of a Japanese waltzing mouse tumor by the F2 mice to estimate that the Japanese waltzing mouse and the common house mouse differed by 14-15 H-genes. The technique suffered from two shortcomings: 1) because of the large number of H-genes by which the parental strains differed, very few grafts were accepted and the resultant error was great, and 2) the technique allowed only one opportunity for crossover; therefore multiple closely linked H-genes behaved as one gene. Bailey and Mobraaten ( 6) minimized these two shortcomings by basing their estimates on the survival of skin grafted between partially inbred mice and from multiple backcrossed mice to parental strain mice. In both cases the number of surviving skin grafts and the opportunities for crossovers are increased manyfold. Using these techniques Bailey and Mobraaten estimated that strains BALB/cBy and C57BL/6By differ by 28 or 29 H-genes.

To determine if Bailey's estimate, which was significantly larger than previous estimates, was typical of other strain combinations, we have tested additional strain pairs. We used the multiple backcross technique to estimate the number of genes by which DBA/2J and B10.D2 differ (these two strains both possess the H-2d haplotype). These strains were crossed and female offspring backcrossed to B10.D2 males for multiple generations. Skin was grafted to B10.D2 males from females of backcross generations N4 through N8. If the skin grafts were not rejected within 100 days, whenever possible a second B10.D2 male was immunized with 5 x 106 spleen cells from the female donor, skin grafted, and scored for 250 days. The formula L = sn-1 was used to estimate the number of histocompatibility differences between DBA/2J and B10.D2 (L = number of H-genes, n = the generation in which all grafts of a given line were accepted). Based on the responses of unimmunized hosts an H-gene difference of 28 genes was estimated. Based on the responses of immunized hosts an H-gene difference of 63 genes was estimated. In order to estimate the number of H-differences by which C57BL/6J and DBA/2J differ, orthotopic tail skin grafts were exchanged between unimmunized, sex-matched, full siblings of 16 sublines derived from a C57BL/6J-DBA/2J cross. These sublines had been through 13 to 17 sibling matings. The grafts were scored for 100 days. Using the formula S = (3/4)rL (5/8)vL (S = surviving fraction, r and v are constants dependent on the generation number), it was estimated that these strains differed by 34 H-genes (28 in males, 39 in females) ( 7). Similarly the number of H-genes by which C57BL/6J and C3H/HeJ differ was estimated to be 32 (37 in males, 27 in females) ( 7). These results for strain combinations B10.D2 - DBA/2J, C57BL/6J - DBA/2J, and C57BL/6J - C3H/HeJ are of the same order of magnitude as Bailey's estimate for C57BL/6By - BALB/cBy. It is likely that the counts of 28 to 34 for histocompatibility differences between unrelated strains are representative.

Estimates of the total number of H-genes also can be made from the frequency of H-gene mutations, Based on the ratio of H-gene mutation (as determined by rejection of skin grafts exchanged between F1's) to the mutation rate of other genes, Bailey ( 8) estimated a total of 720 H-genes. By another method, based on lost type mutations and the number of H-gene differences between the strains being tested, he estimated 430 H-genes. Bailey explained extremely large estimates by the facts that they reflect non-polymorphic (silent) as well as polymorphic H-loci, that they are not dependent on gene segregation and thereby not affected by linkage, and that there may be more than one mutation site at each locus.

Isolation, Mapping, and Allelism of Weak Loci

It was possible to identify and study the H-2 locus in inbred mouse strains which differed by many non-H-2 loci as well as by the H-2 locus because of the strength of the H-2 antigens and because specific antibodies could be produced against H-2 antigens which could then be used for identification. The relative weakness of the non-H-2 antigens and their failure to stimulate good antisera has necessitated the isolation of non- H-2 genes in congenic mouse strains for identification. Through the use of congenic strains 35 minor histocompatibility loci have been reported in addition to loci on the X and Y chromosomes (the latter two not requiring congenic strains). H-1, H-3, H-4, H-7, H-8, H-9, H-10, H-11, H-12, and H-13 were described by Snell and co-workers ( 9, 10, 11), H-15, H-16, H-17, H-18, H-19, H-20, H-21, H-22, H-23, H-24, H-25, H-26, H-27, H-28, H-29, H-30, H-34, H-35, H-36, H-37, H-38, and H-X by Bailey ( 12, 13), H-31, H-32, and H-33 by Flaherty and co-workers ( 14, 15), H-39 by Artzt ( 16) and H-Y by Eichwald ( 17) ( Table 1). Sixteen of these loci have been mapped on eight chromosomes ( Figure 1). As many as eight additional H-genes have been demonstrated by the rejection of skin grafts exchanged between non-histocompatibility congenic strains ( 18). The temporary designations and the congenic strains are indicated in Table 2. H-(JS), H-(Lt), and H-(tn) have not been proven to be distinct from one another; H-(go) and H-(pi) have not been proven to be distinct from one another; and none of these potential loci have been proven to be distinct from the previously described unmapped H-genes.

Although many congenic strains have been produced for the purpose of defining new H-loci, relatively few have been produced to define additional alleles of the known loci. Six alleles of the H-3 locus, four alleles each of the H-1 and H-13 loci, three alleles each of H-8 and H-11, and two alleles of each of the remaining H-loci have been isolated in congenic strain pairs ( 9, 10, 11, 12, 13, 14, 15, 19, 20, 21, 22, 23, 24, 25, 26; Graff, unpublished data).

The Cellular Immune Response to Non-H-2 Antigens

The most frequently used technique to study the cellular immune response to non-H-2 antigens has been allograft rejection. The first allografts to be used were tumor, which proved to be a technically easy but insensitive test. Skin grafting techniques were found to be more sensitive because the hosts could be observed for rejection for prolonged periods, whereas tumor recipients would have succumbed. Two major techniques of skin grafting have been used, the grafting of skin onto the flank ( 27) and the grafting of tail skin orthotopically ( 28). Median survival times (MST's) tend to be shorter with the orthotopic tail skin technique, probably because the grafts are smaller.

The magnitude of the immune response by a particular host to a given antigen depends on the degree of "differentness" of the antigen (immunogenicity) and the immune responsiveness of the host. Data are being accumulated documenting variation in host responsiveness to a given antigen. In 1969 Graff and Snell ( 26) noted a variation in the immune capability of a group of F1 mice as indicated by their ability to reject non-H-2 disparate skin grafts. Subsequent studies have indicated that genes in the H-2 complex control the speeds of rejection of non-H-2 disparate skin grafts. The immune response to H-Y by female mice has been shown to be under the control of an H-2-linked gene ( 29, 30). To study the H-2-linked genetic control of immune responsiveness to non-H-2 antigens, Wettstein and Haughton are producing groups of "double congenic" strains. These strains are developed in groups of at least four strains. All of the strains are on a C57BL/10 background, two of the strains share one H-2 haplotype and differ at a given non-H-2 locus, and the other two share a second H-2 haplotype and possess the same non-H-2 difference. With these groups of double congenic mice, Wettstein and Haughton ( 31, 32) have demonstrated that the magnitude of the allograft rejection reaction against the H-4.2 and H-7.2 antigens is effected by the H-2 haplotype of the host, indicating the existence of immune response genes in the H-2 complex. Through the use of crossover strains it appears that the immune response genes for different non-H-2 antigens may be located in different places in the H-2 complex.

To properly study the immunogenicity of histocompatibility antigens, variations in host immune responsiveness must be minimized. This can most effectively be accomplished by using only one host strain in a given experiment. If this is not possible, one should use host strains with a common background and preferably the same H-2 haplotype. The subsequently described studies have been carried out with these guidelines in mind. The total immunogenicity (as indicated by skin allograft rejection) of the antigens of the H-2 complex plus adjacent histocompatibility genes has been shown to be about equal to the cumulative non-H-2 immunogenicity ( Table 3). The individual non-H-2 antigens are weaker, falling on a continuum from the strong H-4b antigen (which is slightly weaker than H-2D) to the extremely weak H-9 antigens. Great variations exist in the strengths of the antigens controlled by the alleles of many of the loci ( Table 3) ( 20, 21). As data accumulate on the H-2 complex, it is apparent that the "H-2 immunogenicity" is really the cumulative immunogenicity of multiple histocompatibility antigens. In addition to the immunogenicity of H-2K, H-2A, H-2C, H-2D, there are now added H-31, H-32, H-33, and H-39. It would appear that when the appropriate crossover and congenic strains are produced the immunogenicity of the individual antigens of the H-2 complex and adjacent genes may prove to be comparable to that of the individual non-H-2 antigens.

To quantitate more accurately the cellular immune response to histocompatibility antigens of varying strengths, B10.A mice were injected with varying numbers of lymphocytes differing at the H-2 locus, and C57BL/10Sn mice were injected with varying numbers of lymphocytes differing at the H-3, H-4, H-7, and H-13 loci. The B10.A and C57BL/10Sn mice were then grafted with skin from the lymphocyte donors at varying intervals thereafter. The speed of rejection of the skin grafts was taken as an indication of the level of immunity. A first set rejection was taken to indicate that the antigen dose was too small to be recognized (a subthreshold dose); a second set rejection was taken to indicate that an immunizing dose had been given; prolonged survival was taken to indicate that the immune system had been overwhelmed (I avoid the word "tolerance" because I wish to make no implications concerning mechanism). The responses of mice tested one week after antigen challenge indicated that the ability to immunize and overwhelm relates directly to immunogenicity: the weaker the antigen, the larger the amount of antigen needed to immunize and the smaller the amount of antigen needed to overwhelm. With H-2 antigens and the strongest non-H-2 antigens, small doses of allogenic lymphocytes sensitized (as indicated by accelerated rejection of subsequent skin grafts) and the largest doses used did not produce non-reactivity. With weaker antigens the threshold immunizing dose increased and the dose producing non-reactivity decreased until, with extremely weak antigens, no single dose sensitized and relatively small numbers of cells overwhelmed ( 33, 34).

Following the injection of H-2 disparate lymphocytes the immune response to the H-2 antigen was apparent one day later, peaked three days later, and waned 14 days later, persisting in a low grade manner for 98 days. Following the injection of H-4 disparate lymphocytes the immune response to the H-4 antigens appeared two days later, peaked four days later, and waned at 14 days, persisting at a moderate level until 98 days. Following the injection of H-7 disparate lymphocytes the immune response to the H-7 antigen first appeared seven days later and reached a persistent maximum at 28 days, persisting at that high level for 98 days. Following the injection of H-13 disparate lymphocytes the immune response to the H-13 antigen was suppressed for 14 days. Immunity first appeared 35 days after injection and disappeared by day 56 ( Figure 2). This assay has the limitation of being very slow (the length of the assay is the duration of the graft survival time) and relatively inaccurate in estimating latency. Nevertheless, comparisons between the patterns of the responses to antigens of different strengths can be made. Clearly, the weaker the antigen the longer the latent period. As in previous studies of Snell et al. ( 35) using Winn's assay, strong cellular immunity to H-2 antigens is short lived. If cellular immunity persists at a high level only as long as antigen is present, the short duration of strong cellular immunity against H-2 antigens and the long duration of cellular immunity against H-4 and H-7 antigens may reflect the rate of clearance of the antigen. The fluctuating response against the weak H-13 antigen probably reflects the small size of the clone capable of responding.

The immunogenicity of transplantation antigens can be cumulative under some circumstances and not cumulative under others. The immunogenic relationship between multiple transplantation antigens on a single cell has been studied using congenic strains differing at one or more H-loci ( 36). The immune response against a cell containing two antigens theoretically can be directed at either or both antigens; if the magnitude of the immune response against both antigens is no stronger than the magnitude of the response to the stronger antigen alone, then it can be said that the weaker antigen has made no contribution to total immunogenicity and there is no cumulative effect. if, however, the magnitude of the response to the double difference is stronger than the response to the stronger antigen alone, then it can be stated that there is a cumulative effect. When grafts were exchanged between strains differing by two loci, the greatest cumulative effects were noted when the rejection rates were of similar magnitudes. If the immunogenicities of the individual antigens were different, no cumulative effect was noted. If a third antigen was added to two others, its immunogenicity contributed only if there was a similarity in the rates of rejection of the single and double differences. It would appear that if one continues to add weaker antigens, they would be cumulative only as long as the rate of rejection of the multiple differences was sufficiently similar to the rate of rejection of the last antigen to be added. Although B10.D2 and DBA/2 differ by about 34 minor H-antigens, it is probable that only the two or three of the strongest antigens affect the magnitude of the immune response. These observations held true whether H-2 antigens were involved or not. The mechanism of the cumulative effect is not known. It has been hypothesized ( 36) that if two antigens of disparate immunogenicity existed on the same cell, the stronger antigen would lead to sensitization and cell destruction before the weak antigen could trigger an immune response in its clone. If the immunogenicities were similar, both clones would be triggered. Nothing is known about the mechanisms of immunologic destruction of cells bearing single antigenic differences versus cells bearing multiple differences. The observed results can be explained on the basis of independent action on each antigen. This does not mean that an effect beyond this independent action does not exist ( 37).

The H-2 antigens elicit near-maximal responses which obliterate many characteristics of allograft rejection that are apparent with the weaker responses to non-H-2 antigens. Although the size of grafts exchanged across H-2 barriers does not affect graft rejection, the size of grafts exchanged across non-H-2 barriers does. Both the ability to sensitize and the time required for graft destruction are size dependent with larger grafts sensitizing more rapidly and taking longer to reject ( 38, 39). Rejection of grafts from hemi-allogeneic donors occurs significantly more slowly that rejection of grafts from totally allogeneic donors ( 40). These observations tend to indicate that both the affector and the effector mechanisms are dose dependent. The female immune response is often stronger than the male response to the same antigen (reviewed in 41). This is quite apparent in the immune response to many non-H-2 antigens as indicated by shorter MST's in females than in males. This difference is obliterated following gonadectomy and adrenalectomy. In fact, the strength of male rejection and to a lesser extent the strength of female rejection were greater than the strength of unaltered mice. It has been hypothesized that the strengthened immune response was due to the removal of endogenous immunosuppressive adrenal and gonadal steroids and that the difference between male and female rejection times was due to quantitative and qualitative differences in the endogenous male and female adrenal and gonadal steroids.

In Vitro Assays for Non-H-2 Antigens

Until recently, in vitro assays generally have been unsuccessful in demonstrating non-H-2 antigens. Perhaps as a result of improved technology, positive results have been obtained. Tests using humoral immune techniques to demonstrate non-H-2 transplantation antigens have rarely been successful. Winn, Stevens, and Snell ( 42) produced hemolytic antibodies directed against H-1 and H-3 antigens. Snell and Graff (unpublished data) produced monospecific hemagglutinating antibodies directed against H-1 and H-7 by exchanging skin grafts between congenic strains differing by these genes and, following rejection, injecting donor strain lymphocytes. The hemagglutinating antibodies produced had extremely high titers but lost activity rapidly following freezing. Subsequent attempts to produce these antisera were unsuccessful. Zink and Heyner ( 43) produced polyspecific non-H-2 antisera by injecting B10.D2/n spleen and lymph node cells into (BALB/cJxDBA/2J)F1 mice. Anti H-3 and H-8 activity was demonstrated using hemagglutination, immunofluorescence, and mixed hemabsorption techniques with B10.LP-a and B10.D2(57N) target cells, respectively. It is of particular note that none of these studies have been able to demonstrate lymphocytotoxic activity. The indirect radioimmunoassay has been used successfully to demonstrate TL antigen ( 44) and tumor-specific antigens ( 45). In pilot studies in our laboratory target cells were reacted with putative antisera and either 125I-labeled goat-anti-mouse globulin or 125I-labeled staphylococcal protein A as binding agents. Although early studies were encouraging, we have not yet succeeded in applying this technique to non-H-2 antigens.

Although Mangi and Mardiney ( 46) have reported weak mixed lymphocyte reaction to individual non-H-2 antigens, most investigators have been unsuccessful in doing so. Recent personal communications have indicated success (Wettstein personal communication) and failure (Dunlop, personal communication) with this technique. The cell-mediated lysis (CML) assay has proven more effective, Gordon et al. ( 47) using CML against the H-Y antigen and Bevan ( 48) using CML against H-3, H-13, H-7, and H-8 antigens.


In summary, many polymorphic cell membrane structures under the control of multiple genes scattered throughout the genome are specifically recognizable by the immune system. Although these structures are collectively called by the name histocompatibility antigens, and the controlling genes are collectively called by the name histocompatibility genes, they may have no more in common than being membrane structures that are immunologically recognizable. Actually we know nothing about the functions of these structures, which may be different from one another and different from the functioning of the antigens of the H-2 complex. Although these studies were stimulated by a need to know about allograft rejection and histoincompatibility, the knowledge gained may have greater implications for cell biology in general and the function of the cell membrane specifically.

1Supported by U.S. Public Health Service Research Grant AI 07437 from the National Institute of Allergy and Infectious Diseases and Veterans Administration Grant MRIS #3177.


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