The influence of the BoLA-A locus on reproductive traits in cattle

Associations between the major histocompatibility complex (MHC) and reproductive performance have been reported in humans, mice, rats, pigs and chickens. Only the A locus of the bovine major histocompatibility complex (BoLA-A) has been well characterized, and 42 alleles of this locus have been identified in American cattle. Four studies were conducted to examine the association between alleles of the BoLA-A locus and reproductive performance. Testis size, which is an indicator of early puberty and increased fertility in young bulls, was examined in 440 yearling bulls from nine breeds with a gene substitution model that included the effects of breed, sire, age of dam and age or weight of the bull. Estimated breeding value for twinning was examined with a gene substitution model with 204 cattle from a herd with a high frequency of twinning. Fertility of potential partners having BoLA-A locus alleles in common was examined in a prospective study involving 101 pure-bred Hereford cows mated by artificial insemination to four pure-bred Hereford bulls. The effect of homozygosity on birth weight, preweaning weight gain and post-weaning weight gain was estimated in a sample of 683 calves from nine breeds; 22% of the calves were apparently homozygous and 78% were heterozygous at the BoLA-A locus. There were significant and large effects of some BoLA-A locus alleles on paired testicular volume, but the analyses on the other traits did not show significant associations. Substitution of the W6.1 allele for the W9A allele reduced paired testicular volume by 150 +/- 44 cm3. The W6.1 allele has now been shown to influence a reproductive trait, a production trait and susceptibility to an economically important disease. Selection for these traits may influence the frequency of the large number of alleles at the BoLA-A locus.     

Transplantation in Miniature Swine. III: Effects of MSLA and A-O Blood Group Matching on Skin Allograft Survival

The fate of split thickness skin allografts between and among major histocompatibility complex (MSLA) homozygous herds of miniature swine has been examined. Among animals matched for all known blood groups, MSLA-identical skin grafts survived 11.8 ± 0.89 days, while skin grafts from animals differing for one or two haplotypes survived 7.0 ± 0.36 days. Cytotoxic antibodies invariably appeared in the serum of animals rejecting MSLA-nonidentical grafts, and were never detected among animals rejecting MSLA-identical grafts.
Among the homozygous DD herd, it was possible to assess the effect on skin allograft survival of a defined difference at the A-O blood group locus. Skin grafts between DD animals identical at the A-O blood group locus. Skin grafts between DD animals differing at this locus survived 7.66 ± 0.54 days. Blood group O animals rejecting skin from blood group A animals produced a low liter of antibodies which were cytotoxic for donor lymphocytes. These antibodies were at least partially absorbable by donor red cells. An absorption analysis using other blood group-defined animals of these herds showed that ability to absorb activity segregated with the A blood group.
These studies confirm the expectation that MSLA is a major histocompatibility complex of miniature swine, having important implications for the outcome of allografts. In addition, they show that the A-O blood group, or a locus closely linked to it, is also a histocompatibility locus in pigs, and must be respected in allografting studies.     

Lymphocyte alloantigens of the horse

A genetic system controlling lymphocyte alloantigens of the horse is described. Alloantisera to paternal histocompatibility antigens induced as a result of pregnancy in mares were used in an antibody-mediated complement-dependent microcytotoxicity assay to define 15 Equine Leukocyte Antigen (ELA) specificities using cluster analysis. In this study 369 sera were screened for alloantibody using lymphocytes from 10 randomly selected, unrelated horses. A high proportion (83%) of these sera were found to be positive for antibody to lymphocyte alloantigens. After initial cluster analysis, 120 of the most discriminating sera were tested against a further 400 horses. The phenotypic distribution of the ELA antigens in 304 randomly selected horses and their segregation behavior in a family study of 161 offspring and their sires and dams indicated that 13 of the alloantigens behaved as members of a single allelic series, provisionally named locus ELA-A. These 13 alloantigens accounted for approximately 90% of the genes at this locus. Large differences in the frequencies of some of the ELA-A specificities were found between the Standardbred and Thoroughbred breeds of horses. Rare 'blank' alleles which were predicted by the estimated cumulative gene frequency of known alleles at ELA-A were identified in informative families. The ELA system is probably the Major Histocompatibility Complex of the horse, although the evidence for this is not yet conclusive. The high incidence of sensitization to ELA antigens in pregnant mares suggests that the horse may be an interesting model for investigations of the maternal immunological response to fetal histocompatibility antigens.     

Characterization of bovine MHC class II polymorphism using three typing methods: serology, RFLP and IEF.

Various methods, with different strengths and weaknesses, are currently used to define polymorphism of the bovine major histocompatibility complex (MHC) class II genes. A more complete characterization of bovine lymphocyte antigen (BoLA) haplotypes can be achieved by combining several of these methods. In this study BoLA class II polymorphism was characterized using three typing methods: serology, restriction fragment length polymorphism (RFLP), and isoelectric focusing (IEF). Twenty six Holstein-Friesian and 15 Angus cattle that carried an array of serologically defined BoLA haplotypes were selected for the study. The panel included 12 BoLA complex homozygotes. The three class II typing methods recognized polymorphism associated with the same or very tightly linked genes in the DQ-DR class II subregion. In total 25 BoLA-A locus (class I)--DQ-DR subregion (class II) haplotypes were defined. Three of the serological class II specificities, Dx1, Dx3, and Dx4, were associated with more than one RFLP defined DQ-DR haplotype. The other 4 class II specificities behaved as private specificities. One BoLA haplotype was found in both Holstein and Angus cattle. Two other BoLA haplotypes defined here have previously been described in other breeds. This suggests that these haplotypes exist in strong linkage disequilibrium.     

Bovine alloreactive cytotoxic cells generated in vitro: target specificity in relation to BoLA phenotype.

Cytotoxic cells of bovine origin were generated in primary MLC using stimulator cells of BoLA w8/w11 phenotype. Bovine lymphoblasts transformed by the protozoan parasite Theileria parva parva acted as target cells in studies of the specificity of cytotoxicity. When responder cells in MLC did not share w8 or w11 with stimulator cells, cytotoxicity was evident with all targets bearing w8 or w11, or both, and was almost entirely restricted to these products of the BoLA-A locus. When responder and stimulator cells shared both w8 and w11, cytotoxicity was also generated. Whether this was specific for the products of other putative Class I loci in cattle, or for the products of a Class II region, remains to be determined. These results suggest that the determinants recognized by appropriately generated bovine alloreactive cytotoxic cells are identical with, or closely related to, determinants characterized by BoLA w8 and w11 defining alloantisera.     

The major histocompatibility complex of rhesus monkeys. VI. Serology and genetics of Ia-like antigens.

The serology and genetics of11 new cell surface alloantigens of rhesus monkeys are described: They are controlled by the mamor histocompatibility complex but are distinct from the conventional serologically defined (SD) antigens ofRhL-A. The new specifications are termed "Ia-like" because ofserological, immunocytological and other characteristics reminiscent of Ia-antigens of the mouse. Population and family analyses led to the postulation of two segregant series controlling eight of the 11 Ia-like specificities of the monkey. Strong linkage disequilibria with SD2 antigens and genetic mapping on the basis of segregation studies in recombinant offspring in the monkey families, places at least one of the two loci in the vicinity of the SD2 locus of RhL-A, not in the region of the major MLC or LD1 locus. For this and other reasons, the new B-cell alloantigens of rhesus monkeys are not believed to be similar to or associated with the stimulator antigens of LD1. The biological function(s) of the Ia-like antigens of primates are as yet unknown.     

Genetic analysis of the antigens defined at the third international BoLA workshop

A comparison of lymphocyte antigens showed that 32 of the 33 BoLA antigens defined at the third international BoLA workshop (Bull et al., 1989) corresponded to previously defined local antigens (Stear et al., 1988). The third workshop antigen w18 had no locally defined equivalent. All 32 antigens were shown in family studies to be expressed by autosomal co-dominant genes, and all 32 workshop antigens were shown to be products of the BoLA system. After excluding the supertypic antigens, nearly all animals tested possessed only one or two antigens and there were no observed recombinants in family studies. These results do not exclude the possibility that the 32 workshop antigens are the products of one locus (BoLA-A).     

The Histocompatibility Complex of Rhesus Monkeys. II. A Major Locus Controlling Reactivity in Mixed Lymphocyte Cultures

Rhesus monkeys have a genetic locus which has a major influence on reactivity in mixed lymphocyte cultures (MLR). This MLR locus is closely linked to the RhL–A system², which controls the serologically defined histocompatibility antigens of this species. Three cases of recombination between RhL–A and the MLR region are described. Consequently, the MLR locus is located outside the RhL–A region proper, possibly adjacent to the locus defining the antigens of the 1st series. The recombination frequency between the MLR and RhL–A regions of rhesus monkeys is similar to that found for the analogous systems of man.
Inhibition of MLR can be achieved by pre-incubation of responder or stimulator cells with cytotoxic anti-RhL–A sera. Such inhibition is highly specific and probably caused by an interaction of the sera with RhL–A surface antigens, not with MLR determinants. Yet, there is circumstantial evidence that non-cytotoxic sera can be produced which inhibit MLR, presumably by affecting MLR determinants on the cell surface.
The relevance of MLR to histocompatibility and the genetic control of the immune response of rhesus monkeys to synthetic antigens, are the subjects of current studies which are briefly discussed.     

The mixed leukocyte reaction in chickens. Evidence for control by the major histocompatibility complex

One-way and two-way mixed leukocyte reactions (MLR) have been performed using cells isolated from peripheral blood of chickens from four different inbred lines (CA, CB, WA and WB). MLR between chickens from the same line, or from lines CA and CB (which have the same major histocompatibility antigens (B antigens)) were always negative; MLR between chickens from lines differing at the B locus were always positive, even when the lines compared (WA, WB) had otherwise identical or very similar genetic backgrounds. This evidence that a locus or loci controlling the MLR is linked to the major histocompatibility locus in the chicken was confirmed by the results of MLR performed with leukocytes from the F2 progeny of a WA × CB cross. These experiments also exclude (at least for the WA/CB combination) the role in the reaction of other, non-B-linked MLR loci. The evolution of the vertebrate major histocompatibility complex is discussed in the light of these findings.     

The dominant role of la antibodies in the passive enhancement of H-2 incompatible skin grafts

The ability of H-2 antisera and their constituent K and la antibodies to enhance the survival of skin allografts was investigated. Ia sera were prepared from H-2 alloantisera by exhaustive absorption with donor-strain RBC and the absorbed K antibodies were also recovered by acid elution of the RBC. The removal of conventional K/D antibody in no way diminished the activity of enhancing sera over wide dose ranges in two different incompatibility systems. The recovered K/D antibodies in the doses used had at best a trivial enhancing effect. The dominant role of Ia antibodies in enhancement was confirmed by showing significant prolongation of graft survival in third-party systems where the sera covered only some of the Ia antigens in incompatibilities involving K,D and Ia differences and in homologous systems using Ia sera fractionated into their constituent mono-specificities. It is concluded that enhancement is a function of antibodies directed against Ia antigens (I region products) and that antibody against conventional histocompatibility antigens such as H-2.K, D, and by homology HL-A and Ag-B has only a minor role in passive enhancement.     

Antibody response to antigens distinct from smooth lipopolysaccharide complex in Brucella infection.

The smooth lipopolysaccharide complex of the outer surface of smooth Brucella abortus cells is believed to be the antigenic component involved in serological tests routinely used for the diagnosis of brucellosis. Sera from cattle vaccinated or infected with B. abortus generally contain antibody directed toward the smooth lipopolysaccharide complex. The brucella organism contains a large number of other antigenically distinct components. The biological significance of some of these antigens has been demonstrated by showing that sera from infected cattle have precipitins to these components. These sera revealed up to seven distinct lines in immunoelectrophoresis with a protein-rich antigen mixture prepared from rough strain B. abortus 45/20, whereas sera from strain 19-vaccinated cattle did not reveal these lines at 4 or more months after vaccination. Monospecific antisera were prepared against six antigens in this mixture, and the purification of two of them by antibody affinity chromatography is described.     

The major histocompatibility complex of rhesus monkeys, RhL--A. VII. Identification of five new serologically defined antigens.

Five new serologically defined (SD) tissue antigens of rhesus monkeys are described. Results of a population study and a segregation analysis in families were consistent with their control by the major histocompatibility complex (MHC), as alleles of the two previously established SD loci of RhL--A. The number of identifiable SD specificities of the rhesus monkeys' MHC is now twenty-five, thirteen controlled by the SD1 locus and twelve by SD2. The recombination frequency between SD1 and SD2 is estimated to be 0.3%. No evidence of a third SD series, as analogue of the human HLA--C locus, has yet been found.     

Breed differences in the frequency of bovine lymphocyte antigens

Lymphocytes from 1,564 cattle of 18 breeds and cross-bred groups in Australia were tested for major histocompatibility system class 1 antigens. Gene frequencies were calculated for the Angus, Belmont Red, Brahman, Hereford and Holstein-Friesian breeds. There were substantial differences among these breeds in antigen and gene frequency. There were striking differences among all 18 breeds in the presence or absence of certain antigens. Two antigens, CA13 and CA36, were strongly associated in Hereford cattle but occurred independently of each other in the other breeds.     

Immunologic enhancement of rat renal allografts. III. Immunopathologic lesions and rejection in long-surviving passively enhanced grafts.

Immunologic enhancement of renal allografts from (Lewis times Brown Norway) F1 to Lewis rats was achieved by administering a single dose of antidonor serum at the time of transplantation. A series of grafts functioning for 1 to 4 months after transplantation were examined by light and immunofluorescence microscopy to evaluate the long-term protective effects of the enhancing serum and to determine if previously unobserved lesions appeared in long survivors. Despite the absence of detectable circulating cytotoxic alloantibody, long-term allografts showed necrotizing glomerular and arterial lesions which resembled those seen in acutely rejecting grafts and were compatible with humoral rejection. Thus, in this model, there is a late decline in the ability of passive enhancement to inhibit humoral rejection. Long-term grafts also developed tubular lesions with deposition of immunoglobulin and complement on the tubular basement membranes (TBM). Anti-TBM antibodies were demonstrated in recipients' sera and found to be organ specific but not major histocompatibility antigen or species specific. This tubular lesion is therefore a unique form of allograft injury in which the immune response is directed against tissue antigen(s) which are distinct from the major histocompatibility antigens that induce rejection.     

IMMUNOGENETIC PROPERTIES OF SL-A INHIBITOR SUBSTANCES IN SERUM OF PIGS*

One hundred and twelve pig sera were screened for their inhibitory properties to cytolytic reactions of eighteen reagents obtained after skin grafting. All of the tested pig sera discriminately inhibited the cytotoxicity of various reagents, which clearly demonstrated that the inhibitions are of defined specificities. This could be confirmed in pig families. In a number of instances serum contained inhibitors of such specificities that were not detectable on the lymphocyte membrane of the animal the serum was taken from. The aim of this study was not to identify the specificities involved. Nevertheless the SL-A3 specificity was observed in serum of eighty-four out of 112 animals, whereby the SL-A3 antigen was detectable on the lymphocyte membrane of only fourteen of those animals. The occurrence of histocompatibility inhibitors in serum of pigs is of importance in experimental organ grafting and in immunizations of animals when reagents of certain specificities are attempted to be produced. Granulocytes appear also to be furnished with cell bound histocompatibility antigens.     

The Mo91 Antigen, an Allele at the First HL-A Locus

Mo91 is an antigen "intermediate" between HL-A10 and HL-A11 which is defined by the coexisting positivities of several apparently monospecific anti-HL-A10 and anti-HL-A11 sera. Mo91 cells completely absorb the antibodies contained in these anti-HL-A10 and anti-HL-A11 sera. Family investigations show that Mo91 is controlled by an allele at the first HL-A locus.
There are probably other intermediate antigens in the HL-A system. Their constitution is discussed. Are they distinct from two classic antigens, and linked by a strong cross reaction with these two antigens? Are they double antigens governed by single genes, that may be called "bispecific alleles"     

Cloned cellular reagents to define antigens encoded between HLA-DR and glyoxylase

Primed lymphocyte typing reagents have been used to define antigens encoded by genes of a locus (loci) mapping between HLA-DR and glyoxalase I. This locus, which we shall refer to as the third locus of the HLA-D region, has been variously referred to as D beta, PL beta, PL3, and SB. Generating discriminatory primed lymphocyte typing reagents which can be used to define these antigens, however, has been extremely difficult. Donors of responding and stimulating cells for the priming combinations have usually been matched not only for the DR, D, and MB/MT antigens but also for the HLA-A, -B, and -C antigens. Even under these very restricted conditions, not all bulk primed lymphocyte typing reagents that are generated are discriminatory enough to be useful for antigen definition. We have derived "clones" from bulk priming combinations in which stimulator and responder differed for known antigens of this third locus. Even though the bulk reagents that were prepared did not provide discriminatory results, approximately 7-12% of the clones derived from the bulk priming combination proved to be highly discriminatory. We have been able to obtain these results with regard to all three antigens of the third locus so far evaluated. The very ease of screening clones and deriving discriminatory reagents, as compared with screening responder-stimulator combinations, allows the ready derivation of cellular reagents that define the antigens of this third locus.     

Rhesus lymphocyte alloantigens. III. Identification of new antigens.

Rhesus monkey lymphocytotoxic alloantisera were tested in 437 random unrelated monkeys and in members of 19 pedigreed families. Groups of sera or individual sera identified 11 specificities. Genetic analysis of the associations of these antigens revealed behavior consistent with the current concept of the major histocompatibility complex of several mammalian species including man in that the antigens could be grouped into two series of closely linked co-dominantly expressed alleles.     

Heterogeneity of Trichomonas vaginalis and discrimination among trichomonal isolates and subpopulations with sera of patients and experimentally infected mice

The antibody response in trichomoniasis patients was examined with a variety of methodologies including enzyme-linked immunosorbent assays, indirect immunofluorescence, immunoblotting, and radioimmunoprecipitation-electrophoresis-autoradiography. Based on enzyme-linked immunosorbent assay recognition of trichomonal isolates, sera from patients with trichomoniasis were categorized into reactive class I (IA, IB, and IC) and nonreactive class II sera. A diminished ability to precipitate antibody-binding trichomonad membrane proteins by the whole cell radioimmunoprecipitation assay was noted from class IA to class II sera. The antigenic distinctions among various Trichomonas vaginalis isolates appeared due to high-molecular-weight protein antigens detected by class IA sera in a whole cell radioimmunoprecipitation assay. The heterogeneity in antigenic patterns was confirmed among the isolates with sera from experimental animals. Also, live T. vaginalis cells appear to have only a few of the entire repertoire of major immunogenic surface proteins accessible to antibody binding. Immunoblotting demonstrated that the high-molecular-weight proteins responsible for trichomonal isolate heterogeneity are present in all isolates. The data suggest that trichomonads of a given isolate express only a subset of internally synthesized protein antigens on their surface. Importantly, the presence of these protein antigens on T. vaginalis membranes correlated with antibody production in subcutaneously challenged mice. Finally, indirect immunofluorescence studies with highly reactive, pooled sera from either patients or mice revealed a subpopulation of nonstaining trichomonads. These data support the view that heterogeneity among T. vaginalis is dependent upon the surface disposition of highly immunogenic protein antigens. Strategies may now be developed not only for studying potential vaccine reagents, but also for examining possible antigenic phenotypic variations in this experimental model.     

Detection by membrane immunofluorescence of isoantibodies during rejection of skin allografts

Isoantibodies which react strongly and specifically with donor lymphocytes have been demonstrated by membrane immunofluorescence in the sera of mice and rabbits bearing first and second set allografts of skin. Changes in the titre of these antibodies closely paralleled the course of the rejection reaction. With other types of cells the immunofluorescent staining was less intense and the reactions were less consistent. Staining was completely inhibited by serum absorption with spleen, lymph node or thymus, but myocardium, aorta or tendon had no specific absorbing effect. The antibodies detected are probably directed against histocompatibility antigens.