Genetics and Classification of Major Histocompatibility Complex Antigens of the Chicken

Genetics and Classification of Major Histocompatibility Complex Antigens of the Chicken W. E. BRILES and RUTH W. BRILES Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115 (Received for publication January 7, 1987)

1987 Poultry Science 66:776-781 INTRODUCTION

Antigenic differences between erythrocytes of individual members of a species are most effectively detected by antisera produced by injecting the blood of one animal into another of the same species-a procedure known as alloimmunization, resulting in the production of alloantisera. This procedure results in the production of antibodies reactive with antigenic determinants present only in some members of the species, while heteroimmunization (immunization across species) produces primarily antibodies reactive with determinants common to all members of the donor species. For example, rabbit anti-chicken serum must be absorbed by erythrocytes of individual chickens in order to obtain antibodies specific for some antigen types within the species (Landsteiner and Miller, 1924). Todd (1930, 1931), combining the techniques of alloimmunization and differential absorption, demonstrated that the cells of every chicken-with the exception of close relativescould be differentiated from the cells of every other chicken. Further, he was able to show that any cellular antigen possessed by an individual was present in the cells of one or both of its parents. In an effort to account for such a multitude of antigenic differences, Weiner (1934) hypothesized the existence of three or more alleles at some antigen-determining locus. POLYMORPHISM OF THE MAJOR HISTOCOMPATIBILITY COMPLEX

The major histocompatibility complex (MHC) of the chicken was originally identified

as an erythrocyte antigen or blood group system in early studies at the University of Wisconsin (Briles et al., 1950). Twelve antigenic traits were shown by genetic segregation to be controlled by genes at either of two independent loci-seven segregated as a series of alleomorphs and were assigned to locus A and five segregated as if they were alleomorphs belonging to a second locus, which was designated B and later recognized as the chicken MHC (Schierman and Nordskog, 1961). The finding of extensive multiple alleles at each of the two loci was suggestive of a selective advantage of heterozygous birds over those homozygous for genes of these blood group systems. If such were generally true for poultry populations maintained as closed flocks or even under moderate inbreeding, two or more alleles should be detectable at such loci. In 1948, two moderately inbred White Leghorn lines (inbreeding coefficients of at least .50) developed by the Regional Poultry Research Laboratory at East Lansing, Michigan, were tested with A and B reagents; both of the lines were found to be segregating for two or more alleles at each of the loci, in agreement with the hypothesis of natural selection favoring heterozygotes (Briles and McGibbon, 1948). An opportunity to test further the validity of the observation that the A and B loci normally exhibit multiple allelism came with the initiation of my research at Texas A and M University in 1948. I elected to begin anew by investigating alloantigens in local stocks without the use of reference reagents from the University of Wisconsin. By the spring of 1949 three loci had

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ABSTRACT The chicken MHC consists of a gene complex divisable into three chromosomal regions, each producing a series of genetically antithetical molecules with regionally specific functions. Most of the genes (alleles) of each region are present in unique haplotype combinations with genes (alleles) from the other two regions; relatively few haplotypes appear to share identical regional genes. Even with the high degree of polymorphism existing within and between regions, general typing of erythrocytes for MHC haplotypes can still be performed as economically as ever with alloimmune reagents of known specificity. (Key words: alloantigen, blood group, major histocompatibility complex antigens, alleles, polymorphism)

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TABLE 1. B Allele frequencies in three related Texas lines1 Alleles 2

Lines

B

T22 T23 T24

.28 .53 .26

1

B

6

B1

B"

.65 .12 .66

.35

B' .07

.08

Data from Briles et al. (1957).

TABLE 2. Cross-reactivity of a sample of B reagents prepared in Line T23* Reagents

of of test cell

test cell

B2-168

B2-199

B6-156

B6-157

B6-209

T22, T23, andT24 T23 T22, T23, and T24 T24 T22

2/2 6/6 7/7 8/8 9/9

+2 0 0 + 0

+ 0 0 0 +

0 + 0 0 +

0 + 0 + +

0 + 0 + +

1 2

Data from Briles et al. (1950). + = Agglutination; 0 = no agglutination.

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been identified and genetically proven to be segregating independently (Briles, 1949, 1950); testing segregating families with reference reagents previously used in the Wisconsin study clearly showed that the A and B loci were among the three recently identified in the Texas material. The results of the work during the next few years (Briles et al., 1957) contributed substantially toward an understanding of the B system. The primary genetic material used in initiating the work consisted of three moderately inbred lines (inbreeding coefficients of .52 to .59) derived from a single open-bred flock. The gene frequencies of each of five alleles-B2, B 6 , B7, B 8 , and B9-over the three lines are depicted in Table 1. The B2 and B1 alleles were present in all three of the derived lines while the other alleles were each present exclusively in only one of the lines-B6 in Line T23, B 8 in T24, and B9 in T22. Therefore, a minimum of five alleles must have been segregating in the original stock at the time the three lines were initiated. Antisera produced against particular B antigens exhibit distinct cross-reactivity patterns against other antigens of the series. This crossreactivity serves as a powerful tool for the detection and genetic analysis of the array of B alleles in other populations of chickens. The use of

antisera of confirmed specificity in one population or line as serological reagents in identifying antigens produced by alleles segregating in a second population is exemplified by data presented in Table 2. Reagents B2-168 and B2-199 were produced in Line T23 by injecting cells containing the B 2 antigen into recipients of the genotype B6/B1. Reagents B6-156, B6-157, and B6-209 were produced by injecting red blood cells containing the B 6 antigen into B2/B7 recipients. In order to perform agglutination tests, each reagent was diluted with physiological saline according to titer to give satisfactory agglutination of cells possessing the homologous (donor) antigen. The initial search for B antigen types in an unexplored population is greatly facilitated by testing parents and their respective progeny in family groups as exemplified by Line H10 birds in Table 3. The results of testing with reagents B2-199 and B6-157 proved that two B alleles were segregating in the line. Agglutination of the cells of the sire and dam number 1 with both reagents and the simultaneous or alternate agglutination of the cells of the progeny showed that the two reagents were detecting antithetical antigen types, designatedB13 andB . The progeny from dam number 2 (B14/B14) mated to the same sire (B,3/B14) were either B l3 /B 14 , orB 14 / B 14 , in conformity with the interpretation of the results from the first family. Having arrived at a genetic interpretation of the agglutination data from the line H10 families (Table 3), it is instructive to note the cross-reactivity of the five Line T23 reagents used in the tests on H10 with the other two Texas lines, T22 and T24 (lower portion of Table 2). The B2-168 cross-reacted with antigen B 8 while B2199 cross-reacted with B9 of Line T22, even though both reagents appeared identical within Line T23. Similiarly, the three B6 reagents

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TABLE 3. Cross-reactivity of the red blood cells of members of Sire Family H10 with T23 reagents'

Assigned B genotype

Sire Dam 1 Dam 1 progeny

13/14 13/14 13/13 13/14 14/14 14/14 13/14 14/14

Dam 2 Dam 2 progeny

10 21 11 13 12

1

Data from Briles et al. (1957).

2

Test dilution for respective reagents.

reacted alike in T23 but exhibited two different cross-reactive patterns in the other Texas lines. The B6-156 reacted with B 9 , whereas the other two B6 reagents, B6-157 and B6-209, reacted with both B g and B 9 . Considering the tests on the chickens from Line H10 along with those for the Texas lines, each of the five reagents displayed a different total cross-reactivity pattern. Using the basic discrimination afforded by serological cross-reactions as presented in Tables 2 and 3, Texas reagents were used to test numerous populations of chickens from other laboratories. Alloantigen genotypes identified by these reagents were used as the basis for alloimmunizations within lines, resulting in antisera useful as typing reagents. Reagents produced within inbred lines were used to analyze the specificity of B alleles in 12 commercial inbred lines. Sixteen reagents identified, by cross-reactivity patterns, a total of 21 distinctly different antigens, each of which had already been designated as the product of a B allele segregating in one or more of the lines (Briles etal., 1957). The occurrence of B system heterogeneity within closed populations of chickens is essentially universal. Of 73 populations (including the 12 lines noted previously) sampled by nine or more birds per line, 71 clearly possessed two or more B alleles each; even the eleven most highly inbred of the lines tested, with inbreeding coefficients of .66 to .86, possessed two alleles each (Briles et al., 1957). In a separate investigation of four highly inbred White Leghorn lines, Gilmour (1959) found that two or three of nine distinctly different B alleles were seg-

Reagents B2-168 1:82

B2-199 1:8

B6-156 1:32

B6-1S7 1:48

B2-209 1:16

0 0 0 0 0 0 0 0

+ + + + 0 0 + 0

0 0 0 0 0 0 0 0

+ + 0 + + + + +

+ + + + + + + +

regating in each line, with no common alleles in any of the lines. The laboratory at Northern Illinois University has devoted considerable research effort to establishing identities or differences or both in B types in chicken populations used for immunogenetic studies in various laboratories in the United States and Europe. Numerous B system reagents were used in these studies, 15 of which were included in testing all 32 populations under investigation. These 15 reagents, covering a broad spectrum of specificity, differentiated a total of 27 haplotypes (combinations of very closely linked genes of a genetic complex). The segregating units during the early years of the specificity investigations were viewed as alleles of a single genetic locus. However, before the study was completed, the three locus complexity of the B system had become apparent. Therefore, the term haplotype is appropriate for the 27 composite specificities in the experimental lines which have been examined. Judging from the presence of 27 haplotypes disclosed in this limited sample of chicken lines (some of which were of common origin), the number of haplotypes demonstrable in the chicken populations of the world would probably extend well above a hundred. MULTIPLE LOCUS STRUCTURE OF THE AVIAN MAJOR HISTOCOMPATIBILITY COMPLEX

The first indication of recombination of antigenic determinants of the B system was an unexpected skin-graft rejection between a pair of half-sibs who should have been compatible according to results of blood typing (Schierman

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Identity of cell

Number of progeny of each genotype

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patterns should aid the eventual establishment of the structure of this series of very polymorphic antigens. Biochemical and functional evidence indicate the existence in the B complex of a third region, B-L, which produces a molecule of an apparent molecular weight of 30,000 and which corresponds to the la or Class II antigen of the mammalian MHC (Ziegler and Pink, 1976; Pink et al., 1977; Ewert and Cooper, 1978; Ewert et al., (1980). The B-L antigens are restricted to certain leukocytes, primarily B lymphocytes and cells of the monocyte-macrophage series; the molecules function primarily as recognition molecules during immune response involving certain types of T cells (Benacerraf, 1981). Although the separate existence of the B-L system within the MHC is clearly evident from experimental data, crossing over between B-L and B-F has not been reported. Analysis of seven recombinants has revealed consistent transmission of B-L and B-F from the same parental chromosome (Briles et al., 1982b). Thus, a reasonable working model for the arrangement of the three regions is B-L, B-F and B-G-with the tightest linkage between B-L and B-F. Of course, present data do not exclude the possibility that B-L lies between the two other regions though more closely linked to B-F than to B-G. The multiplicity of loci in functional regions of the H-2 complex of the mouse (Klein, 1975) suggests the possibility of multiple genes within the B-L, B-F, and B-G regions of the chicken MHC. Biochemical analysis of B-L antigens by Crone et al. (1981) demonstrated two distinct populations of molecules, indicating the likely presence of two genetic loci in the B-L region. The existence of two loci in the B-G region is indicated by a recombinant possessing B-G determinants derived from each of the B haplotypes present in the heterozygous parent (Briles et al., 1982a). The point of crossing over in one of the parental chromosomes divides the B-G region into two genetic subregions, each of which produces a different set of B-G determinants (Briles, unpublished data). Although there are presently no genetic data indicating more than one locus in the B-F region, addition F loci may well be disclosed by future investigations. POLYMORPHISM OF CHICKEN MAJOR HISTOCOMPATIBILITY COMPLEX REVISITED

Present data show that the B-F and B-G are very polymorphic. Fifteen B-F antigens pro-

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and McBride, 1969). On investigation, it was found that the abberrant chicken had received only part of the antigenic determinants characterizing each of the two B alleles present in the heterozygous sire. The data regarding this apparent recombinant are fully compatible with the subsequent conclusions of Hala et al., (1976) regarding a recombinant, BR{, recovered among 1,206 progeny from a special mating designed to detect recombination within the B chromosomal region. Analysis of the recombinant revealed that antigenic specificity derived from one of the parental chromosomes also controlled histocompatibility associated with the B system, whereas that originating from the other parental chromosome was not related to histocompatibility. Thus, investigation of the BR] recombinant revealed that erythrocyte antigen specificity associated with the B system was determined by genes in at least two separate but closely linked chromosomal regions; B-F was assigned to the region also determining histocompatibility and B-G to the region not associated with histocompatibility. Subsequent investigation of the BRX recombinant and Z?13 haplotype, with which it shares the B-G segment, led Pink et al., (1977) to propose a three-locus arrangement for the production of three corresponding species of glycoprotein molecules characterizing the B complex. Antigens produced by the gene (or genes) in the B-F region are present on both erythrocytes and leukocytes and act as primary histocompatibility antigens corresponding to the class I molecules of the mammalian MHC. The molecule consists of a heavy chain with an apparent molecular weight of 40,000 to 43,000; it is associated noncovalently with a beta-2 microglobulin molecule with a weight of 11,000 to 12,000. Antigens produced by the B-G region are present on erythrocytes only and may play a role in erythroid differentiation (Simonsen, 1981), though this has not been firmly established; B-G antigens do not appear to have a known counterpart in mammalian cells. Pink et al. (1977) reported that B-G antigens consist of two chains of apparent molecular weights of 40,000 and 31,000 though the smaller chain may represent a protolytic breakdown product of the larger. Using monoclonal antibodies against a widely shared B-G determinant in high resolution two-dimensional gel electrophoresis, Miller etal.(\984) obtained distinct polypeptide patterns for individual B-G antigens previously identified by alloantisera; these characteristic

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BRILES AND BRILES

of the same relative magnitude as that of regular B-F/B-G reagents. Thus, there is little advantage of using F-specific or G-specific reagents in routine typing, but for further investigation of the basic structure and function of the B complex such reagents offer distinct advantages. Monoclonal antibodies against antigens of each of the three regions have been produced by mouse hybridomas. Ewert et al. (1984) reported a monoclonal antibody reactive with B cells of all B system haplotypes tested. Reactivity is assumed to be due to one or more determinants common to all B-L antigens, this reagent is widely used in biochemical and immunopathological studies. A monoclonal anti-F antibody reactive with four of seven B haplotypes tested has recently been reported by Pink et al. (1985). Mice show a strong preferential response to B-G antigens of the chicken MHC complex and monoclonal antibodies against these antigens are thus readily formed by mouse hybridomas (Longenecker et al., 1979; Longenecker and Mosmann, 1981). In workshop comparisons in Innsbruck, Austria, in 1981, some monoclonals were very specific for particular B-G antigens and others exhibited various degrees of crossreactivity with a large panel of haplotypes (Briles et al., 1982b). ACKNOWLEDGMENTS

Research reported from the Northern Illinois University was supported by Public Health Service Grant CA-12796 and AI-22957, awarded by the National Cancer Institute and the National Institute of Allergy and Infectious Diseases, respectively, Department of Health and Human Services. REFERENCES Benacerraf, B., 1981. Role of MHC gene products in immune regulation. Science 212:1229—1237. Briles, W. E., 1949. Heterozygosity of inbred lines of chickens at two loci effecting cellular antigens in the chicken. Genetics 33:97. (Abstr.) Briles, W. E., 1950. Antigenic differences between inbred lines of chickens. Genetics 35:656-657. Briles, W. E., C. P. Allen and T. W. Millin, 1957. The B blood group system in chickens. I. Heterozygosity in closed populations. Genetics 42:631-648. Briles W. E., and R. W. Briles, 1982. Identification of haplotypes of the chicken major histocompatibility complex (B). Immunogenetics 15:449^159. Briles, W. E., R. W. Briles, and R. E. Taffs, 1982a. An apparent recombinant within the B-G region of the B complex. Poultry Sci. 61:1425-1426. Briles, W. E., N. Bumstead, D. L. Ewert, D. G. Gilmour,

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duced by different haplotypes were tested with 14 anti-F reagents (Simonsen et al., 1982); only one case of identity was found-that of haplotypes B4 and B 13 , already studied by Pink et al. (1977). In a similar test using B-G reagents on the 15 haplotypes, B2 and B were found to share identical determinants and B6 and a haplotype designated B 187 were identified as probably also possessing identical B-G antigens. In a similar test with nine B-G reagents on 23 haplotypes, two likely homologies were identified; B2 and B'2 and B3 and B'4 (Briles, unpublished). Data regarding the polymorphism of the B-L system are presently more limited than of the B-F and B-G systems. The arduousness of reagent preparation and indirect immunofluorescent staining method of typing B-L antigens on peripheral lymphocytes are obstacles to rapid progress on this system. Simonsen et al. (1982) produced alloantisera by immunization with leukocytes, absorption with donor erythrocytes to remove F-specific antibodies, and finally absorption with spleen cells of selected B-L genotypes to narrow B-L specificity. Eleven qualitatively distinct B-L reagents prepared in this manner were tested on peripheral blood lymphocytes from known genotypes representing nine B-L antigens. The L antigens in B4 and B 13 were identical, as already reported by Pink et al. (1977), and the L antigens exhibited by B12 and B 19 showed parallel reactivity and thus are probably also identical. The latter identity suggests that the B19 haplotype probably resulted from recombination of B12 with some haplotype possessing F 19 and G19 in which the chromosome break occurred between the B-F and B-L regions. This certainly indicates that crossing over between these two regions has occurred and can be expected in future searches for this type of recombination. Practical erythrocyte typing of lines or strains of chickens for B standardized reference types B1 through B 29 (Briles and Briles, 1982) and characterization of populations with unknown haplotypes are relatively uninfluenced by the separate existence of B-F and B-G regions. Many, and perhaps most, of the alloimmune reagents prepared in populations of known B genotypes consist of mixtures of antibodies formed against F and G antigens. When these reagents are compared with reagents specific for F or G antigens (prepared by immunizations between recombinant and parental haplotypes), the occurrence of cross-reactivity of the latter with other haplotypes is, in general, essentially

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polymorphic antigenic determinants of the chicken MHC analyzed with mouse hybridoma (monoclonal) antibodies. Immunogenetics 9:137-147. Miller, M. M., R. Goto, andH. Abplanalp, 1984. Analysis of the B-G antigens of the chicken MHC by two-dimensional gel electrophoresis. Immunogenetics 20:373385. Pink, J.R.L., W. Droege, K. Hala, V. C. Miggiano, and A. Ziegler, 1977. A three-locus model of the chicken major histocompatibility complex. Immunogenetics 5:203-216. Pink, J.R.L., M. W. Kieran, A. M. Rijnbeek, and B. M. Longenecker, 1985. A monoclonal antibody against chicken MHC class I (B-F) antigens. Immunogenetics 21:293-297. Schierman, L. W., and R. A. McBride, 1969. Evidence for mutational event at the B blood group histocompatibility locus in chickens. Transplantation 8:515-516. Schierman, L. W., and A. W. Nordskog, 1961. Relationship of blood type to histocompatibility in chickens. Science 134:1008-1009. Simonsen, M., 1981. The major histocompatibility complex in a bird's eye view. Pages 192-201 in: Immunobiology of the major histocompatibility complex. 7th Convoc. Immunol., Niagara Falls, NY 1, 1980. Karger, Basel, Switzerland. Simonsen, M., M. Crone, C. Koch, and K. Hala, 1982. The MHC haplotypes of the chicken. Immunogenetics 16:513-532. Todd, C , 1930. Cellular individuality in higher animals, with special reference to the individuality of the red blood corpuscle. Proc. R. Soc. Lond. B. Biol. Sci. 106:20-44. Todd, C , 1931. Cellular individuality in higher animals, with special reference to the individuality of the red blood corpuscle. II. Proc. R. Soc. Lond. B Biol. Sci. 107:197-205. Weiner, A. S., 1934. Individuality of the blood of higher animals. III. J. Genet. 29:1-8. Ziegler, A., and R. Pink, 1976. Chemical properties of two antigens controlled by the major histocompatibility complex of the chicken. J. Biol. Chem. 251:53915396.

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J. Gogusev, K. Hala, C. Kock, B. M. Longenecker, A. W. Nordskog, J.R.L. Pink, L. W. Schierman, M. Simonsen, A. Toivanen, P. Toivanen, O. Vainio, and G. Wick, 1982b. Nomenclature for chicken major histocompatibility (B) complex. Immunogenetics 15:441^147. Briles W. E., W. H. McGibbon, and M. R. Irwin, 1950. On multiple alleles effecting cellular antigens in the chicken. Genetics 35:633-652. Briles, W. E., andW. H. McGibbon, 1948. Heterozygosity of inbred lines of chickens at two loci effecting cellular antigens. Genetics 33:605. (Abstr.) Crone, M. J. Jensenius, and C. Kock, 1981. Evidence for two populations of B-L (la-like) molecules encoded by the chicken MHC. Immunogenetics 13:381-391. Ewert, D. L., and M. D. Cooper, 1978. Ia-like alloantigens in the chicken: serologic characterization and ontogeny of cellular expression. Immunogenetics 7:521-535. Ewert, D. L., D. G. Gilmour, W. E. Briles, and M. D. Cooper, 1980. Genetics of Ia-likc alloantigens in chickens and linkage with the B major histocompatibility complex. Immunogenetics 10:169-174. Ewert, D. L., M. S. Munchus, C. H. Chen, and M. D. Cooper, 1984. Analysis of structural properties and cellular distribution of avian la antigen by using monoclonal antibody to monomorphic determinants. J. Immunol. 132:2524-2530. Gilmour, D. G., 1959. Segregation of genes determining red cell antigens at high levels of inbreeding in chickens. Genetics 44:14-33. Hala, K., M. Vilhelmova, and J. Hartmanova, 1976. Probable crossing-over in the B blood group system of the chicken. Immunogenetics 3:97-103. Klein, J., 1975. Biology of the Mouse Histocompatibility Complex. Springer-Verlag, New York, NY. Landsteimer, K., and C. P. Miller, 1924. On individual differences in chicken blood. Proc. Soc. Exper. Biol. Med. 22:100-102. Longenecker, B. M., and T. R. Mosmann, 1981. Nomenclature of chicken MHC (B) antigens defined by monoclonal antibodies. Immunogenetics 13:25-28. Longenecker, B. M., T. R. Mosmann, and C. Shiozawa, 1979. A strong preferential response of mice to

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