Identification and genetics of horse lymphocyte alloantigens

Six hundred horses were tested with lymphocytotoxic antisera derived from 550 parous mares and 58 antisera produced by alloimmunization with horse blood cells. Seven equine lymphocyte specificities were identified using correlation analysis of the test data, absorption analysis and lysostripping. These specificities are expressed on lymphocytes and platelets, but not on red blood cells (RBC). Therefore, these specificities do not appear to be products of any of the eight known blood group systems of the horse. The distribution of these specificities in 113 Thoroughbred horses and 57 Arabian horses is presented. Two specificities are subtypic to two other specificities reported here. Family studies indicated that all of these specificities are products of one genetic system. However, it is not clear whether the system consists of one or more loci.     

Evidence of a second polymorphic ELA class I (ELA-B) locus and gene order for three loci of the equine major histocompatibility complex

Two antisera, B-442 and R-2046, were produced by immunizing offspring with purified peripheral blood lymphocytes from a parent matched for the ELA-A specificity carried on the unshared haplotype. Absorption analysis demonstrated that these antisera contained at least two families of cytotoxic antibodies, one directed against antigens present on T and B cells, and a second directed preferentially against antigens present on surface Ig positive cells. Immunoprecipitation studies using these antisera demonstrated that both antisera contain antibodies specific for glycoproteins with molecular weights characteristic of class I and class II MHC antigens. In lymphocyte typing tests of unfractionated lymphocytes, only the class I activity was readily detectable since the class II activity killed less than 25% of the cells. Family studies demonstrated that these antisera recognize products of genes linked to the ELA system. Based on two recombinants in an extended family it became apparent that the specificities detected by B-442 and R-2046 are not products of the ELA-A locus, but rather they are products of at least one other locus, defined in this paper as ELA-B. In this family a third recombinant was found between the A blood group system and the ELA-A locus. Based on these three recombinants, the most probable linear relationship of the following genes is: A blood group system/ELA-A/ELA-B.     

Joint report1 of the First International Workshop on Lymphocyte Alloantigens of the Horse held 24–29 October 1981

Six equine lymphocyte alloantigen (ELA) specificities were defined by an international antiserum comparison test and workshop held in 1981. Twelve laboratories from four countries submitted 195 antisera for analysis. The antisera were exchanged among the 12 laboratories and tested in a standard lymphocyte microcytoxicity assay against the isolated lymphocytes at 1009 horses of several breeds. The data was pooled and analysed by a single computer analysis. The calculated χ² values of all cells with all antisera provided comparisons between antisera. Fifteen antisera clusters were formed by this analysis, but only six of these clusters met the criteria established by the workshop for the identification of ELA antigens. No horses of the cell panel positively reacted with more than two of these six specificities. The consensus of the participants, although not substantiated in this workshop, was that these six clusters of antisera define alleles of a single genetic region, the ELA region, and it is likely that this genetic region is the major histocompatibility complex of the horse.     

Joint report of the First International Workshop on Lymphocyte Alloantigens of the Horse held 24-29 October 1981

Six equine lymphocyte alloantigen (ELA) specificities were defined by an international antiserum comparison test and workshop held in 1981. Twelve laboratories from four countries submitted 195 antisera for analysis. The antisera were exchanged among the 12 laboratories and tested in a standard lymphocyte microcytoxicity assay against the isolated lymphocytes at 1009 horses of several breeds. The data was pooled and analysed by a single computer analysis. The calculated chi 2 values of all cells with all antisera provided comparisons between antisera. Fifteen antisera clusters were formed by this analysis, but only six of these clusters met the criteria established by the workshop for the identification of ELA antigens. No horses of the cell panel positively reacted with more than two of these six specificities. The consensus of the participants, although not substantiated in this workshop, was that these six clusters of antisera define alleles of a single genetic region, the ELA region, and it is likely that this genetic region is the major histocompatibility complex of the horse.     

Joint Report of the Fifth International Workshop on Lymphocyte Alloantigens of the Horse, Baton Rouge, Louisiana, 31 October-1 November 1987

Six laboratories participated in the Fifth International Workshop on Lymphocyte Alloantigens of the Horse, testing 132 alloantisera against lymphocytes of 880 horses chosen to represent different families and breeds. Most of the alloantisera were produced by lymphocyte immunization between horses matched at the ELA-A locus. All horses were also tested with antisera contributed to the workshop by participating laboratories which identified ELA specificities A1-A10 and W12-W21. Previously identified workshop specificities ELA-W14, W15 and W19 were accepted as products of the ELA-A locus based on family and population studies by the workshop. Their designations were changed to ELA-A14, ELA-A15 and ELA-A19, respectively. Two new specificities were identified, namely ELA-W22 (W22) and ELA-W23 (W23). Population and family studies indicated that W22 and W23 as well as W13 are products of an ELA locus other than ELA-A. The presence of these specificities was correlated with the presence of certain ELA-A locus specificities, e.g. W13 with A3, W22 with A2 and W23 with A5. However, the association was not complete and W13, W22 and W23 also segregated with other ELA-A specificities in some families. Evidence for recombination was found between the ELA-A locus and the locus or loci encoding these specificities resulting in seven recombinant haplotypes found among the data presented in this workshop. Further studies are required for definitive assignment of the specificities to a class I or class II locus.     

The human Ia system: Definition and characterization by xenogeneic antisera

The production of xenogeneic anti-Ia serum against Ia antigens in serum has been previously described in the mouse and we now describe the production of xenogeneic anti-human Ia antisera using similar methods. With an indirect resetting technique, Ia-like antibodies were shown to react with the majority of B cells (95%), a subpopulation of T cells, with carbonyl iron adherent cells, and with some E^–Ig^– null cells, but there was no reaction with red cells and platelets. These reactions were the same as those obtained with DRW antisera using cytotoxicity testing. In addition, antigens detected with xenogeneic antisera were also found in serum, where they were found to exist in a low molecular weight, dialyzable form. By the selective removal of different cell surface markers by cocapping, no association could be found with the specifities detected with the xenogeneic anti-Ia antisera and with surface Ig, ₂-microglobulin, or HLA-A and B specificities. Alloantibodies to DRW specificities (but not HLA-A, B specificities) were able to specifically block the binding of the rabbit anti-Ia antibodies to B cells, and reciprocal blocking of rabbit antisera by DRW antibodies was also observed. Several xenogenic antisera were produced by immunizing rabbits with the serum of different individuals. Each antiserum was shown to contain a number of different specificities, as they gave different reaction patterns with different individuals when testing was done both directly and by absorption. These xenogeneic anti-la sera also segregated in a family with HLA-A and B specificities. The detection of a polymorphic antigenic system segregating with the HLA complex, distinct from HLA-A and B specificities, and whose antigens occur predominantly on B cells is therefore described. Because of the similarity of the reactions of the xenogeneic antisera in man to those found in the mouse, and because of the close relationship to the DRW specificities, the system has been provisionally called the H.Ia system.Abbreviations used in this paper AET 2-aminoethyl isothiouronium bromide - ₂-M -2 microglobulin - BSA Bovine serum albumin - H.Ia Human Ia - HuRBC Human red blood cells - Ig Immunoglobulin - Ir Immune response - MHC Major histocompatibility complex - MLR Mixed lymphocyte reaction - NHS Normal human serum - NMS Normal mouse serum - PBL Peripheral blood lymphocytes - PBS Phosphate-buffered saline - RAHIg Rabbit anti-human immunoglobulin - RASIg Rabbit anti-sheep immunoglobulin - RFC Rosette-forming cells - SAHIg Sheep anti-human immunoglobulin - SARIy Sheep anti-rabbit immunoglobulin - SRBC Sheep red blood cells     

Joint Report of the Fourth International Workshop on Lymphocyte Alloantigens of the Horse, Lexington, Kentucky, 12–22 October 1985

Summary. The workshop consisted of 12 monthly cell exchanges of full-sibling families among the 10 participating laboratories. A total of 33 parents, 52 offspring and five unrelated horses were typed by each laboratory using local antisera. The raw data were submitted for central analysis before any identification of the animals was revealed.
Confidence derived from the consistent agreement between the laboratories on the assignment and segregation of the first 10 ELA-W specificities led to the removal of the W (workshop) notation and acceptance of full status as locus A antigens. The seemingly supertypic W11 specificity, however, remained unchanged.
Ten additional specificities were seen to segregate with the ELA system, suggesting either splits of previously described specificities or products of linked loci. The workshop (W) notation was given to the 10 specificities W12-W21, befitting their status as specificities under study.
The previously described ELY-1.1 specificity, characterized by segregation independent from the ELA system, was confirmed along with a new specificity, ELY-1.2, which behaves as an allele of ELY-1.1. For informative families, the two specificities showed codominant expression and appeared to constitute a closed, autosomal system.
The ELY-2.1 specificity was confirmed to segregate independently from the ELA-A and ELY-1 loci.     

HLA serum screening based on an heuristic solution of the set cover problem.

A computer program initially written by the Milwaukee Blood Bank has been modified to use a new algorithm for the assignment of HLA specificities to antisera. The assignment is based on the reactions of cells with known specificities. Specificities which are present only on cells which do not react are first ruled out. This step is followed by one or more steps in which the 'least reactive' specificities are ruled out. The rationale for the algorithm is discussed and an example is presented.     

Bovine lymphocyte antigens: serological relationships with erythrocyte and spermatozoan antigens.

Bovine lymphocytotoxicity tests with 20 unabsorbed bovine blood group sera revealed extensive reactivity which in the majority of cases had no indication of blood group relationship. Six of these sera were absorbed with selected lymphocytes to produce eleven antisera of reduced specificity. Again, most of the sera had reaction patterns which could not be related to the hemolytic patterns of any known blood group antibodies. However, five comparisons involving unabsorbed antisera and two comparisons involving absorbed antisera provided statistical evidence of similarities between their lymphocytotoxic reaction patterns and the hemolytic reaction patterns of certain blood group antibodies. Several of the sera appeared to contain related cytotoxic specificities, and three such absorbed sera may have contained an anti-J specificity. All six examined monospecific isoimmune blood group antisera contained lymphocytic reactivities not related to their hemolytic specificities. Two normal sera containing naturally occurring anti-J had no cytotoxic activity. Anti-semen sera likewise were devoid of lymphocytotoxic activity.     

A comparative study of major histocompatibility complex antigens in East African and European cattle breeds

An account is given of the serologically defined class I specificities encoded by the bovine MHC (expressed as the BoLA system) in two populations of African cattle and in European breeds. The BoLA typing was performed using alloantisera raised against tissue antigens of both European and African breeds of cattle. All of the specificities agreed in the first two international BoLA workshops were found in the African cattle, although there were significant differences in the frequency of some specificities between the African and European animals. Many of the European antisera, which are operationally monospecific in Bos taurus cattle, were multispecific in the African animals. Subgroups of two specificities (w8 and w10) were demonstrated. Five new BoLA-A locus alleles were detected by means of antisera raised against alloantigens of African cattle. Two of these occurred at an extremely high frequency in the African populations; one being unique to these cattle. Monoclonal antibodies proved to be useful typing reagents, particularly in the elucidation of subgroups.     

Identification of goat allotypic determinants and generation of goat-mouse hybridomas secreting goat IgG2

Five allotypic determinants controlled by independent genes have been identified in goat. Of these determinants, four have been detected with alloimmune antisera and one with monoclonal antibodies. The specificities A1, C1 and D1 are lipoproteins; B1 is possibly an alpha 2 macroglobulin and E1 and IgG2. The specificity B1 is not expressed until the age of 3-4 months. The gene controlling the specificity E1 is present at about the same frequency (0.38-0.41) in goat, sheep, cattle and water buffalo. Stable hybridomas secreting goat IgG2 have been obtained by the fusion of goat peripheral lymphocytes with mouse myeloma cells.     

Use of I region-restricted, antigen-specific T cell hybridomas to produce idiotypically specific anti-receptor antibodies

Murine T cell hybridomas bearing receptors for antigen plus I region gene products were used as immunogens in mice in an effort to raise anti-receptor antisera. The antisera were assayed for anti-receptor activity by the ability to inhibit interleukin 2 production by the T cell hybridomas stimulated by antigen and I region expressing antigen-presenting cells. The T cell hybridomas used in these experiments were made by fusing antigen-specific, I region-restricted BALB/c T cell blasts to the AKR thymoma, BW5147. Three groups of mice were immunized with the T cell hybridomas: (BALB/c X AKR)F1 animals, syngeneic to the hybridoma; (BALB.B X aKR)F1 animals, differing from the hybridomas at H2; and (C.B20 X AKR)F1 animals, differing from the hybridomas at Igh. Mice were immunized multiple times and sera from individual animals were assayed for anti-receptor antibodies. In all groups, some mice produced anti-receptor antibodies by the criterion that they were inhibitory in the assay mentioned above. The frequency of mice producing these inhibitory antibodies varied considerably between groups, with the (BALB.B X AKR)F1 animals producing these antibodies most frequently, and the (BALB/c X AKR)F1 animals producing them least often. All inhibitory antisera were idiotypically specific; they inhibited the response of the immunizing T cell hybridomas, but not the responses of closely related hybridomas with different specificities. Moreover, when they could be absorbed, the inhibitory antibodies could only be absorbed by the immunizing hybridoma. It is hoped that these antisera, and B cell hybridomas prepared from the immunized animals, will be useful in the elucidation of the structure of the receptors for antigen plus I region products on T cells.     

Production and characterization of alloantisera specific for bovine class II major histocompatibility complex antigens

Ten alloantisera defining five major histocompatibility complex (MHC) class II specificities of the bovine lymphocyte antigen (BoLA) complex were produced and characterized. Eight antisera defining four of the specificities were generated by immunizing cattle with class I compatible-class II incompatible lymphocytes. The alloantiserum defining the fifth class II specificity was produced by skin implant immunization. A pregnancy serum specific for one of the class II specificities was also identified. The class II antigens recognized by these antisera were designated 'Dx' antigens to indicate that they are BoLA-D region antigens encoded by one or more undetermined class II loci. The molecules identified by the alloantisera are heterodimers composed of a 34-kd alpha and a 26- to 28-kd beta chain, and are expressed on B-lymphocytes but not on resting T-lymphocytes. In family studies the BoLA-Dx antigens segregated in linkage with the BoLA-A locus alleles. Most of the BoLA-A alleles present in the Cornell Holstein herd at a high frequency were found to exist in gametic association with two or more serologically defined class II haplotypes. On the basis of a population study it was determined that three pairs of class I and class II alleles (w10-Dx4, w31-Dx5, and c3-Dx2) were present in the Cornell herd at significantly increased frequencies.     

Bovine lymphocyte antigens: serological relationships with erythrocyte and spermatozoan antigens

Bovine lymphocytotoxicity tests with 20 unabsorbed bovine blood group sera revealed extensive reactivity which in the majority of cases had no indication of blood group relationship. Six of these sera were absorbed with selected lymphocytes to produce eleven antisera of reduced specificity. Again, most of the sera had reaction patterns which could not be related to the hemolytic patterns of any known blood group antibodies. However, five comparisons involving unabsorbed antisera and two comparisons involving absorbed antisera provided statistical evidence of similarities between their lymphocytotoxic reaction patterns and the hemolytic reaction patterns of certain blood group antibodies. Several of the sera appeared to contain related cytotoxic specificities, and three such absorbed sera may have contained an anti-J specificity. All six examined monospecific isoimmune blood group antisera contained lymphocytic reactivities not related to their hemolytic specificites. Two normal sera containing naturally occurring anti-J had no cytotoxic activity. Anti-semen sera likewise were devoid of lymphocytotoxic activity.     

An immunocytotoxicity assay for neural cells in monolayer cultures

The immunological analysis of cell surface constituents which may characterize neuronal and glial populations, though still in its infancy, will greatly facilitate the investigation of several important problems in neurobiology. One critical component of such analyses is the way by which a given antiserum can be shown to be active on, and possibly selective for neurons and glial cells from normal neural tissues. This report describes the use of monolayer cultures of normal neural cells for recognition and quantitative titration of antisera directed against them. Sera were collected from rabbits immunized with chick embryo spinal cord cell susptnsions, and found to be reactive to the same cells in the initial cell dissociate as well as in subsequent monolayer cultures of different in vitro ages. A monolayer assay procedure was developed, which (i) uses small numbers of cells and small volumes of immune reagents, with the possibility of further scaling down; (ii) applies equally to cultures using different substrata; (iii) permits differential counts of morphologically different cultured cells; (iv) allows to recognize cytological damage imposed by the immune serum in the presence, though not in the absence, of complement; and (v) quantitatively titrates the immune activity with 10- to 20-fold higher sensitivity than other titration procedures. While the study was not intended to investigate the possible specificities of the new antisera, it provided the unexpected observation that non-neuronal cells in these spinal cell cultures were considerably less sensitive than neurons to the complement-dependent action of the antisera.     

Preferential expression of neo-CRP epitopes on the surface of human peripheral blood lymphocytes

Antibodies specific for C-reactive protein (CRP) have been reported to react with certain human peripheral blood lymphocytes (PBL); however, the nature of the antigen has not been clearly defined. In the present study we identified the CRP antigenicity on PBL as a CRP neoepitope not seen on the native-CRP molecule. Neo-CRP epitopes are expressed when the native pentameric form of CRP is dissociated into free subunits. Commercial anti-CRP antisera were found to possess a significant proportion of specificities (up to 16% of the total reactivity) directed against neo-CRP antigenicity. Since similar reagents had been used in previous studies on the reactivity of anti-CRP antisera with PBL, we set out to determine if either native- or neo-CRP epitopes were preferentially expressed on PBL. We prepared antisera monospecific for native-CRP and neo-CRP, respectively, and characterized these reactivities in both direct and indirect enzyme immunoassays. When analyzed by flow cytometry, anti-neo-CRP but not anti-native-CRP antiserum was found to react with normal PBL. F(ab')2 fragments of affinity-purified anti-neo-CRP had identical activity, and the reactivity against CRP was absorbed by reagents expressing neo-CRP but not native-CRP epitopes. Flow cytometric analyses of monocyte-depleted PBL from 25 normal donors detected a mean of 23.8 +/- 5.8% anti-neo-CRP-positive cells, a higher proportion of PBL expressing the CRP antigen than previously reported. Our findings indicate that a molecule identical to, or cross-reactive with, a neo-antigenic form of CRP is present on the surface of a significant proportion of normal human PBL.     

Blood groups of Old World monkeys,evolutionary and taxonomic implications

For practical purposes two classes of blood groups of Old World monkeys can be distinguished: human-type and simian-type, depending on the kind of reagents used for testing. Of the human-type blood groups, only the A-B-O groups, defined by saliva inhibition and serum tests, are polymorphic in some, but not all, monkey species. The distributions of those groups show wide differences not only among monkey species but also among troops of one and the same species. The tests for other human-type antigens give with the monkey red cells either uniformly positive or uniformly negative results. Thus, the human-type blood groups seem to be of limited use as taxonomic tools in the systematics of the Old World monkeys.On the other hand, the simian-type blood groups, defined by isoor crossimmune monkey sera, display highly polymorphic patterns in most species of Old World monkeys, and the capability of the antisera to react with combining groups on the red cells of monkeys of closely related species seems to reflect the taxonomic closeness of two or more species. The fact that some of the simian-type specificities, notably those belonging to the rhesus D^rh graded blood group system, are shared by many species of Old World monkeys, indicates that they were introduced into genotypes during early stages of evolution of the Cercopithecidae.     

Continuous cultivation of equine lymphocytes: evidence for occasional T cell-like maturation events in horses with hereditary severe combined immunodeficiency

Peripheral blood mononuclear cells (PBMC) from 14 foals with hereditary severe combined immunodeficiency (SCID) were studied to determine the extent of lymphocyte differentiation that occurs in this disorder. PBMC from all 14 horses had the morphologic characteristics of large granular lymphocytes (LGL). Cells from only one of 14 horses were responsive to phytolectin stimulation in a standard blastogenesis assay; however, PBMC from all 14 horses proliferated in continuous culture in the presence of partially purified interleukin 2. Furthermore, there were differences in the growth patterns of these cultured cells that correlated with their ability to respond to phytolectin stimulation. PBMC obtained from the 13 phytolectin-unresponsive foals survived in culture for only 4 to 5 wk, divided very slowly, developed large granules composed primarily of calcium phosphate, and accumulated high concentrations of histamine. In contrast, PBMC from the phytolectin-responsive SCID foal proliferated in continuous culture for over 100 days, divided as rapidly as normal equine PBMC under identical culture conditions, and did not accumulate granules or histamine. These observations indicate that lymphoid cell differentiation occurs in some horses with SCID even though the identity of the LGL is unresolved. Two possibilities are that LGL are products of a pathway separate from that of lymphocytes or that LGL are precursors of mature lymphocytes.     

Blood groups of anthropoid apes and their relationship to human blood groups

Affinity between blood groups of man and those of anthropoid apes is reflected not only in similarities or identities of reactions of the red cells with many specific typing reagents, but also in overall structures of some of the main blood group systems defined in man and in apes.Besides specificities of human-type, such as A-B-O, M-N, Rh-Hr, I-i, etc. known to be present on the red cells of various species of apes, specific reagents were produced by iso- or cross-immunization of chimpanzees that detect red cell specificities characteristic for apes only. Some of those specificities were found to be shared by several ape species and to fall into separate blood group systems that are counterparts of the human blood group systems. Recently obtained serological, as well as population data, indicate that the chimpanzee R-C-E-F blood group system is the counterpart of the human Rh-Hr system. Similarly to the Rh-Hr system, it is built around a main antigen, the R^c antigen, to which secondary specificities are attached by means of multiple allelic genes. The R^c is not only the principal factor of the chimpanzee R-C-E-F group system, but also constitutes a direct link with the human Rh-Hr blood group system, since anti-R^c reagents also detect Rh₀ specificity on the human red cells. Another chimpanzee blood group system, the V-A-B-D system, is counterpart of the M-N-S-s system, and is built around the central antigen V^c. the V^c is not only the principal specificity of the chimpanzee V-A-B-D system, but it also constitutes the direct link with the human M-N-S-s system since anti-V^c reagent gives with chimpanzee red cells reactions parralleling those obtained with anti-N lectin (N^v) while in tests with human red cells it detects specificity identical or closely related to the Mi^a specificity.