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In common with other herpes viruses, bovine herpes virus 1 (BHV-1) induces strong virus-specific CD8 T-cell responses. However, there is a paucity of information on the antigenic specificity of the responding T-cells. The development of a system to generate virus-specific CD8 T-cell lines from BHV-1-immune cattle, employing Theileria-transformed cell lines for antigen presentation, has enabled us to address this issue. Use of this system allowed the study to screen for CD8 T-cell antigens that are efficiently presented on the surface of virus-infected cells. Screening of a panel of 16 candidate viral gene products with CD8 T-cell lines from 3 BHV-1-immune cattle of defined MHC genotypes identified 4 antigens, including 3 immediate early (IE) gene products (ICP4, ICP22 and Circ) and a tegument protein (UL49). Identification of the MHC restriction specificities revealed that the antigens were presented by two or three class I MHC alleles in each animal. Six CD8 T-cell epitopes were identified in the three IE proteins by screening of synthetic peptides. Use of an algorithm (NetMHCpan) that predicts the peptide-binding characteristics of restricting MHC alleles confirmed and, in some cases refined, the identity of the epitopes. Analyses of the epitope specificity of the CD8 T-cell lines showed that a large component of the response is directed against these IE epitopes. The results indicate that these IE gene products are dominant targets of the CD8 T-cell response in BHV-I-immune cattle and hence are prime-candidate antigens for the generation of a subunit vaccine.

As part of an international effort to develop vaccines for Theileria lestoquardi, we undertook a limited screen to test T. lestoquardi orthologues of antigens recognised by CD8+ T lymphocyte responses against T. annulata and T. parva in cattle. Five MHC defined sheep were immunized by live T. lestoquardi infection and their CD8+ T lymphocyte responses determined. Thirteen T. lestoquardi orthologues of T. parva and T. annulata genes, previously shown to be targets of CD8+ T lymphocyte responses of immune cattle, were expressed in autologous fibroblasts and screened for T cell recognition using an IFNγ assay. Genes encoding T. lestoquardi antigens Tl8 (putative cysteine proteinase, 349 aa) or Tl9 (hypothetical secreted protein, 293 aa) were recognise by T cells from one animal that displayed a unique MHC class I genotype. Antigenic 9-mer peptide epitopes of Tl8 and Tl9 were identified through peptide scans using CD8+ T cells from the responding animal. These experiments identify the first T. lestoquardi antigens recognised by CD8+ T cell responses linked to specific MHC class I alleles., Competing Interests: The authors have declared that no competing interests exist.

An infection and treatment protocol is used to vaccinate cattle against Theileria parva infection. Due to incomplete cross-protection between different parasite isolates, a mixture of three isolates, termed the Muguga cocktail, is used for vaccination. While vaccination of cattle in some regions provides high levels of protection, some animals are not protected against challenge with buffalo-derived T. parva. Knowledge of the genetic composition of the Muguga cocktail vaccine is required to understand how vaccination is able to protect against field challenge and to identify the potential limitations of the vaccine. The aim of the current study was to determine the extent of genetic and antigenic diversity within the parasite isolates that constitute the Muguga cocktail. High throughput multi-locus sequencing of antigen-encoding loci was performed in parallel with typing using a panel of micro- and mini-satellite loci. The former focused on genes encoding CD8(+) T cell antigens, believed to be relevant to protective immunity. The results demonstrate that each of the three component stocks of the cocktail contains limited parasite genotypic diversity, with single alleles detected at many gene/satellite loci and, moreover, that two of the components show a very high level of similarity. Thus, the vaccine incorporates very little of the genetic and antigenic diversity observed in field populations of T. parva. The presence of alleles at low frequency (<10%) within vaccine component populations also points to the possibility of variability in the content of vaccine doses and the potential for loss of allelic diversity during tick passage. The results demonstrate that there is scope to modify the content of the vaccine in order to enhance its diversity and thus its potential for providing broad protection. The ability to accurately quantify genetic diversity in vaccine component stocks will facilitate improved quality control procedures designed to ensure the long-term efficacy of the vaccine., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bovine Neonatal Pancytopenia (BNP) is a disease of calves characterised by haematopoietic depletion, mediated by ingestion of alloantibodies in colostrum. It has been linked epidemiologically to vaccination of the dams of affected calves with a particular vaccine (Pregsure) containing a novel adjuvant. Evidence suggests that BNP-alloantibodies are directed against MHC I molecules, induced by contaminant bovine cellular material from Madin-Darby Bovine Kidney (MDBK) cells used in the vaccine's production. We aimed to investigate the specificity of BNP-alloantibody for bovine MHC I alleles, particularly those expressed by MDBK cells, and whether depletion of particular cell types is due to differential MHC I expression levels. A complement-mediated cytotoxicity assay was used to assess functional serum alloantibody titres in BNP-dams, Pregsure-vaccinated dams with healthy calves, cows vaccinated with an alternative product and unvaccinated controls. Alloantibody specificity was investigated using transfected mouse lines expressing the individual MHC I alleles identified from MDBK cells and MHC I-defined bovine leukocyte lines. All BNP-dams and 50% of Pregsure-vaccinated cows were shown to have MDBK-MHC I specific alloantibodies, which cross-reacted to varying degrees with other MHC I genotypes. MHC I expression levels on different blood cell types, assessed by flow cytometry, were found to correlate with levels of alloantibody-mediated damage in vitro and in vivo. Alloantibody-killed bone marrow cells were shown to express higher levels of MHC I than undamaged cells. The results provide evidence that MHC I-specific alloantibodies play a dominant role in the pathogenesis of BNP. Haematopoietic depletion was shown to be dependent on the titre and specificity of alloantibody produced by individual cows and the density of surface MHC I expression by different cell types. Collectively, the results support the hypothesis that MHC I molecules originating from MDBK cells used in vaccine production, coupled with a powerful adjuvant, are responsible for the generation of pathogenic alloantibodies., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Infection with Theileria parva is asymptomatic in African buffalo but results in severe disease in cattle. Currently, vaccination relies on infection and treatment, using a mixture of three parasite isolates to overcome the strain specificity of immunity. Genotypic analyses of field populations of T. parva indicate a panmictic population structure, reflecting frequent sexual recombination. Profound immunodominance of protective CD8 T cell responses, together with polymorphism of the target antigens and frequent genetic recombination, contribute to the strain-restricted immunity. The dominant CD8 target antigens are highly polymorphic, but the live vaccine appears to contain limited diversity. A model to explain the ability of the vaccine to confer immunity against highly diverse field parasite challenge is discussed. Parasites in cattle exhibit much more limited antigenic diversity than parasites in buffalo, consistent with other evidence that the cattle-maintained population represents a subset of T. parva recently adapted to cattle.

Continuously growing cell lines infected with the protozoan parasite Theileria annulata can readily be established by in vitro infection of leukocytes with the sporozoite stage of the parasite. The aim of the current study was to determine whether such transformed cell lines could be used as antigen presenting cells to analyse the antigenic specificity of bovine CD8 T cell responses to viral infections. Bovine herpes virus 1 (BHV-1), which is known to induce CD8 T cell responses, was used as a model. T. annulata- transformed cells were shown to express high levels of CD40 and CD80 and were susceptible to infection with BHV-1, vaccinia and canarypox viruses. The capacity of the cells to generate antigen-specific CD8 T cell lines was initially validated using a recombinant canarypox virus expressing a defined immunodominant T. parva antigen (Tp1). Autologous T. annulata-transformed cells infected with BHV-1 were then used successfully to generate specific CD8 T cell lines and clones from memory T cell populations of BHV-1-immune animals. These lines were BHV-1-specific and class I MHC-restricted. In contrast to previous studies, which reported recognition of the glycoproteins gB and gD, the CD8 T cell lines generated in this study did not recognise these glycoproteins. Given the ease with which T. annulata-transformed cell lines can be established and maintained in vitro and their susceptibility to infection with poxvirus vectors, these cell lines offer a convenient and efficient in vitro system to analyse the fine specificity of virus-specific CD8 T cell responses in cattle.

Polymorphism of immunodominant CD8(+) T cell epitopes can facilitate escape from immune recognition of pathogens, leading to strain-specific immunity. In this study, we examined the TCR β-chain (TRB) diversity of the CD8(+) T cell responses of cattle against two immunodominant epitopes from Theileria parva (Tp1(214-224) and Tp2(49-59)) and investigated the role of TCR recognition and MHC binding in determining differential recognition of a series of natural variants of the highly polymorphic Tp2(49-59) epitope by CD8(+) T cell clones of defined TRB genotype. Our results show that both Tp1(214-224) and Tp2(49-59) elicited CD8(+) T cell responses using diverse TRB repertoires that showed a high level of stability following repeated pathogenic challenge over a 3-y period. Analysis of single-alanine substituted versions of the Tp2(49-59) peptide demonstrated that Tp2(49-59)-specific clonotypes had a broad range of fine specificities for the epitope. Despite this diversity, all natural variants exhibited partial or total escape from immune recognition, which was predominantly due to abrogation of TCR recognition, with mutation resulting in loss of the lysine residue at P8, playing a particularly dominant role in escape. The levels of heterozygosity in individual Tp2(49-59) residues correlated closely with loss of immune recognition, suggesting that immune selection has contributed to epitope polymorphism.

Although parasite strain-restricted CD8 T cell responses have been described for several protozoa, the precise role of antigenic variability in immunity is poorly understood. The tick-borne protozoan parasite Theileria annulata infects leukocytes and causes an acute, often fatal lymphoproliferative disease in cattle. Building on previous evidence of strain-restricted CD8 T cell responses to T. annulata, this study set out to identify and characterize the variability of the target antigens. Three antigens were identified by screening expressed parasite cDNAs with specific CD8 T cell lines. In cattle expressing the A10 class I major histocompatibility complex haplotype, A10-restricted CD8 T cell responses were shown to be focused entirely on a single dominant epitope in one of these antigens (Ta9). Sequencing of the Ta9 gene from field isolates of T. annulata demonstrated extensive sequence divergence, resulting in amino acid polymorphism within the A10-restricted epitope and a second A14-restricted epitope. Statistical analysis of the allelic sequences revealed evidence of positive selection for amino acid substitutions within the region encoding the CD8 T cell epitopes. Sequence differences in the A10-restricted epitope were shown to result in differential recognition by individual CD8 T cell clones, while clones also differed in their ability to recognize different alleles. Moreover, the representation of these clonal specificities within the responding CD8 T cell populations differed between animals. As well as providing an explanation for incomplete protection observed after heterologous parasite challenge of vaccinated cattle, these results have important implications for the choice of antigens for the development of novel subunit vaccines.

T cell receptor (TCR) recognition of peptide-MHC class I (pMHC) complexes is a crucial event in the adaptive immune response to pathogens. Peptide epitopes often display a strong dominance hierarchy, resulting in focusing of the response on a limited number of the most dominant epitopes. Such T cell responses may be additionally restricted by particular MHC alleles in preference to others. We have studied this poorly understood phenomenon using Theileria parva, a protozoan parasite that causes an often fatal lymphoproliferative disease in cattle. Despite its antigenic complexity, CD8+ T cell responses induced by infection with the parasite show profound immunodominance, as exemplified by the Tp1(214-224) epitope presented by the common and functionally important MHC class I allele N*01301. We present a high-resolution crystal structure of this pMHC complex, demonstrating that the peptide is presented in a distinctive raised conformation. Functional studies using CD8+ T cell clones show that this impacts significantly on TCR recognition. The unconventional structure is generated by a hydrophobic ridge within the MHC peptide binding groove, found in a set of cattle MHC alleles. Extremely rare in all other species, this feature is seen in a small group of mouse MHC class I molecules. The data generated in this analysis contribute to our understanding of the structural basis for T cell-dependent immune responses, providing insight into what determines a highly immunogenic p-MHC complex, and hence can be of value in prediction of antigenic epitopes and vaccine design.

Although immunodominance of CD8(+) T-cell responses is a well-recognised feature of viral infections, its role in responses to more antigenically complex pathogens is less clear. In previous studies we have observed that CD8(+) T-cell responses to Theileria parva exhibit different patterns of parasite strain specificity in cattle of different MHC genotypes. In the current study, we demonstrated that animals homozygous for the A10 and A18 MHC haplotypes have detectable responses to only one of 5 T. parva antigens. Over 60% of the responding T cells from the A18(+) and A10(+) animals recognised defined epitopes in the Tp1 and Tp2 antigens, respectively. Comparison of T-cell receptor beta chain expression profiles of CD8(+) T-cell lines and CD8(+) T cells harvested ex vivo confirmed that the composition of the T-cell lines was representative of the in vivo memory CD8(+) T-cell populations. Analysis of the Tp1 and Tp2 antigens revealed sequence polymorphism, which was reflected by differential recognition by T-cell lines. In conclusion, we have demonstrated a profound immunodominance in the CD8(+) T-cell response to T. parva, which we propose is a major determinant of the parasite strain specificity of the response and hence immune protection.

Theileria parva causes an acute lympho-proliferative disease in cattle, which can result in death of susceptible animals within 2-3 weeks of infection. Analyses of the cellular response in the lymph node draining the site of infection demonstrated an early T cell response, with the appearance of large numbers of uninfected lymphoblasts between 6 and 9 days p.i., coinciding with initial detection of parasitised cells. There was a marked increase in the representation of CD8(+) T cells and the emergence of a sizable sub-population of CD2(-) CD8(+) alpha/beta T cells during this period. Analysis of T cell receptor beta chain variable (TCR BV) gene expression did not reveal any evidence for the involvement of a superantigen in stimulating the response. Responding lymph node cells were found to produce increased quantities of IFNgamma and IL-10, and both the CD2(+) CD8(+) and CD2(-) CD8(+) populations expressed IFNgamma transcripts. Purified CD2(+) CD8(+) cells proliferated when stimulated in vitro with autologous parasitised cells or non-specific mitogens, whereas CD2(-) CD8(+) cells were refractory to these stimuli. In contrast to the parasite-specific cytotoxic activity associated with T cell responses in immune cattle, the responses to primary infection exhibited variable levels of non-specific cytotoxic activity. Stimulation of purified CD2(+) CD8(+) T cells in vitro with autologous parasitised cells also failed to reveal evidence of specific cytotoxic activity. These findings indicate that primary infection with T. parva induces an aberrant T cell response that lacks appropriate effector activity.

We report in this study the design and validation of a Pan-Vbeta primer that in combination with Cbeta-specific primers enables the amplification, in a single semi-nested PCR, of TCRbeta chains expressed by bovine T-cell clones irrespective of the expressed Vbeta sequence. Using the Pan-Vbeta primer we examined the TCRbeta chains expressed by 16 Theileria parva-specific clones that had not been previously analysed. TCRbeta chain sequence was obtained from 15 of the clones following direct sequencing of the PCR product, whilst the other clone appeared to express 2 different TCRbeta chains which were characterised following sub-cloning of the PCR product. We have also successfully used the Pan-Vbeta primer to amplify the TCRbeta chains expressed by 19 T-cell clones, on which previous analysis using Vbeta-subfamily-specific primers had failed to do. Sequencing of these TCRbeta chains has identified members of 2 novel bovine Vbeta subfamilies-Vbeta5 and VbetaX. This method offers a simple and rapid method of analyzing the TCRbeta chains of bovine T-cell clones that has many potential applications in the investigation of bovine T-cell responses.

The present study set out to examine the nature and specificity of the bovine CD8 T cell response at the clonal level in a group of eight animals immunized with a cloned population of Theileria annulata. The results demonstrated that immunized animals generated parasite-specific CD8 T cells that produced IFN in response to parasite stimulation but had highly variable levels of cytotoxicity for parasitized cells. The study also demonstrated that these parasite-specific CD8 T cells could be propagated and cloned in vitro from the memory T cell pool of cattle immunized with live T. annulata parasites. Within the small group of animals studied, there was evidence that responses were preferentially directed to antigens presented by an A10+ class I major histocompatibility complex (MHC) haplotype, suggesting that responses restricted by products of this haplotype may be dominant. The A10-restricted responses showed differential recognition of different parasite isolates and clones. By using a cloned population of parasites both for immunization of the animals and for in vitro analyses of the responses, we obtained unambiguous evidence that at least a proportion of CD8 T cells restricted by one MHC haplotype were parasite strain restricted.

Although techniques that permit analysis of the clonal composition of T cell populations have been used extensively to provide a better understanding of the mechanisms that influence efficacy of T cell responses in humans and mice, such methods are lacking for other animal species. In this paper we report the establishment and validation of a panel of Vbeta subfamily-specific semi-nested PCR assays, and a CDR3beta heteroduplex technique for analysing the clonal diversity of bovine alphabeta T cell responses. Development of these methods was based on available sequence data for 48 functional Vbeta genes classified within 17 subfamilies. These techniques were used to determine the clonal composition of parasite-reactive CD8(+) T cells obtained from two animals immunised with the protozoan parasite Theileria parva. Analyses of uncloned T cell lines as well as large panels of cloned T cells derived from each of these lines confirmed the specificity and sensitivity of the assays. Specific PCR products were obtained from 96% of the T cell clones examined, indicating that the currently identified Vbeta genes represent most of the functional Vbeta subfamilies in cattle. Heteroduplex analyses, coupled with sequencing of PCR products, identified over 20 clonal expansions within each of the T cell lines, distributed over a large number of Vbeta subfamilies, although a limited number of clonotypes numerically dominated the response in both animals. The development and validation of these methods provides for the first time a generic set of molecular tools that can be used to perform detailed analysis of the TCR diversity and clonal composition of bovine T cell responses.

Theileria parva is an intracellular protozoan parasite transmitted by ticks that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. Vaccination against the disease currently relies on inoculation of the infective sporozoite stage of the parasite and simultaneous treatment with long-acting formulations of oxytetracycline. Sporozoites are maintained as frozen stabilates of triturated infected ticks and the method requires accurate titration of stabilates to determine appropriate dose rates. Titration has traditionally been undertaken in cattle and requires large numbers of animals because of individual variation in susceptibility to infection. An alternative tissue culture-based method is laborious and time consuming. We have developed a flow cytometric method for quantifying the infectivity of sporozoite stabilates in vitro based on the detection of intracellular parasite antigen. The method allows clear identification of parasitized cells with a high degree of sensitivity and specificity. Analysis of infected cells between 48 and 72 h post-infection clearly defines the potential transforming capability of different stabilates.

Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes.

Major histocompatibility complex (MHC) class I genes play a crucial role in the immune defence against intracellular pathogens. An important evolutionary strategy is to generate and maintain a high level of diversity in these genes. Humans express three highly polymorphic classical MHC class I genes (HLA-A, HLA-B and HLA-C). In contrast, some species, for example rat and rhesus macaque, maintain diversity by generation of haplotypes that vary considerably with regard to the number and combination of transcribed genes. Cattle appear to use both strategies. We show that various combinations of six apparently classical genes, three of which are highly polymorphic, are transcribed on different haplotypes. Although additional sequences were identified in both cDNA and gDNA, it was not possible to assign them to any of these defined genes. Most were highly divergent or were non-classical class I genes. Thus, we found little evidence for frequent duplication and deletion of classical class I genes as reported in some other species. However, the maintenance of class I diversity in cattle may involve limited gene shuffling and deletion, possibly as a result of unequal crossing-over within the class I region.

Theileriaparva is an intracellular protozoan parasite that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. The parasite infects host lymphocytes causing their transformation and uncontrolled proliferation. Infiltration of major organs with parasitized lymphoblasts results in most cases in death within 3 weeks. Although both T and B lymphocytes are susceptible to infection, the majority of cell lines arising from infection of peripheral blood mononuclear cells in vitro are of T cell lineage. To explore the basis of this phenotypic bias we have followed the very early stages of parasite development in vitro at the single cell level. Peripheral blood mononuclear cells were infected and stained for both surface phenotype and intracellular parasite antigen and analysed by flow cytometry. Although the parasite antigen was detected intracellularly as early as 6h p.i., our data indicate that parasite infection does not lead to cell transformation in all instances. Rather, specific cell types appear to undergo selection very early after infection and expansion of particular cell subsets results in survival and growth of only a small proportion of the cells originally parasitized.

We investigated the repertoire of functional T-cell receptor beta-chain variable genes (TRBV genes) in cattle by analysing the nucleotide sequences and predicted amino acid sequences of a set of cDNA clones isolated from lymph node T cells. Thirty-nine distinct TRBV sequences were identified, bringing the total number of recognised bovine TRBV gene segments to more than 40. Sixteen TRBV subgroups were defined based on their sequence homology to each other and to human TRBV genes. All of the main phylogenetic lineages of BV gene subgroups described in humans and mice were represented. Eight of the subgroups were found to contain more than one member. The most striking feature of the results was the large number of sequences (more than half of the sequenced clones) in the BV9 and BV20 subgroups, which were found to contain 12 and 8 distinct sequences, respectively. In contrast, the corresponding human TRBV subfamilies contain a single member. The results indicate that, as in humans, there has been extensive gene duplication within the TRBV locus during evolution. However, duplication of different BV subgroups in cattle has resulted in a TRBV gene repertoire distinct from that found in other species.

Information on major histocompatibility complex (MHC) diversity in cattle is important to aid our understanding of immune responses and may contribute to maintenance of healthy cattle populations. Equally, understanding the mechanisms involved in generating this diversity may shed light on the complex nature of mammalian MHC evolution. The aim of this study was to assess molecular and serological variation within cattle MHC class I molecules and to study the mechanisms generating diversity. To address this aim, sequence variation was examined in 12 serologically assigned alleles from three putative loci and correlated with monoclonal antibody (mAb) binding data. The results demonstrate that both alloantisera and mAbs often fail to distinguish gene products that differ by a significant number of amino acids. Conversely, some mAbs could distinguish alleles differing by only one or two amino acids. Examination of the sequences demonstrates sharing of motifs between alleles, some encoded at distinct loci, supporting the occurrence of interlocus recombination within the cattle MHC class I region. The implications of this for MHC sequence diversity, and functional capability, are discussed.

The major histocompatibility complex of cattle (BoLA) contains the class II genes DYA and DIB which are transcribed with a dendritic cell restricted distribution. As part of the process to determine whether these genes have any functional significance, we demonstrate that they form a closely linked pair characteristic of other expressed class II MHC molecules. Accepted nomenclature convention suggests that BoLA-DIB should therefore be renamed BoLA-DYB. Analysis of the first full-length DYA and DYB transcripts revealed open reading frames with potential to translate 253 and 259 amino acid proteins, respectively. Comparative sequence analysis between the DY polypeptides and classical cattle, human and mouse class II MHC alpha and beta polypeptide chains revealed 16 unique amino acid residues at positions predicted to form and line the putative peptide-binding region. Expression of tagged constructs demonstrates for the first time that the DY genes of cattle are capable of translating distinctive class II MHC alpha and beta polypeptide chains.

African trypansosomes are tsetse-transmitted parasites of chief importance in causing disease in livestock in regions of sub-Saharan Africa. Previous studies have demonstrated that certain breeds of cattle are relatively resistant to infection with trypanosomes, and others are more susceptible. Because of its extracellular location, the humoral branch of the immune system dominates the response against Trypanosoma congolense. In the following study, we describe the humoral immune response generated against T. congolense in SCID mice reconstituted with a bovine immune system (SCID-bo). SCID-bo mice infected with T. congolense were treated with an agonistic anti-CD40 antibody and monitored for the development of parasitemia and survival. Anti-CD40 antibody administration resulted in enhanced survival compared with mice receiving the isotype control. In addition, we demonstrate that the majority of bovine IgM+ B cells in SCID-bo mice expresses CD5, consistent with a neonatal phenotype. It is interesting that the percentage of bovine CD5+ B cells in the peripheral blood of infected SCID-bo mice was increased following anti-CD40 treatment. Immunohistochemical staining also indicated increased numbers of Ig+ cells in the spleens of anti-CD40-treated mice. Consistent with previous studies demonstrating high IL-10 production during high parasitemia levels in mice and cattle, abundant IL-10 mRNA message was detected in the spleens and peripheral blood of T. congolense-infected SCID-bo mice during periods of high parasitemia. In addition, although detected in plasma when parasites were absent or low in number, bovine antibody was undetectable during high parasitemia. However, Berenil treatment allowed for the detection of VSG-specific IgG 14 days postinfection in T. congolense-infected SCID-bo mice. Overall, the data indicate that survival of trypanosome-infected SCID-bo mice is prolonged when an agonistic antibody against bovine CD40 (ILA156) is administered. Thus, stimulation of B cells and/or other cell types through CD40 afforded SCID-bo mice a slight degree of protection during T. congolense infection.

Cattle in Africa are a genetically diverse population that has resulted from successive introduction of Asian Bos indicus and European B. taurus cattle. However, analysis of mitochondrial genetic diversity in African cattle identified three lineages, one associated with Asian B. indicus, one with European B. taurus, and a third ascribed to an indigenous African sub-species of cattle. Due to their extended coevolution, indigenous African herbivores are generally tolerant to endemic African pathogens. We are interested in identifying alleles derived from the indigenous African cattle that may be associated with tolerance to African pathogens. An analysis of the locus which encodes the abundant plasma membrane-associated tyrosine phosphatase, CD45, identified three highly divergent allelic families in Kenya Boran cattle. Analysis of allelic distribution in a diverse range of cattle populations suggests a European B. taurus, an Asian B. indicus, and an African origin. This demonstrates not only significant allelic polymorphism at the CD45 locus in cattle but also convincing autosomal evidence for a distinct African sub-species of cattle. Furthermore, maximum-likelihood analysis of selection pressures revealed that the CD45 locus is subject to exceptionally strong natural selection which we suggest may be pathogen driven.

We describe a highly sensitive, non-radioactive assay for T cell activation, based on the rapid induction of class II MHC expression by constitutively negative bovine endothelial cells, when cultured in the presence of supernatants derived from activated bovine T cells. We demonstrate the effectiveness of this assay in detecting rBoIFNgamma and activation of immune CD4(+) and CD8(+) T cell lines and clones in response to specific antigen and transfected COS-7 cells, respectively. We also demonstrate its utility in identifying purified pathogen fractions that activate immune CD4(+) T cell clones.

Analysis of cattle major histocompatibility complex (MHC) (BoLA) class I gene expression using serological and biochemical methods has demonstrated a high level of polymorphism. However, analysis of class I cDNA sequences has failed to produce conclusive evidence concerning the number and nature of expressed genes. Such information is essential for detailed studies of cattle immune responses, and to increase our understanding of the mechanisms of MHC evolution. In this study a selective breeding programme has been used to generate a number of MHC homozygous cattle expressing common serologically defined class I specificities. Detailed analysis of five class I haplotypes was carried out, with transcribed class I genes identified and characterized by cDNA cloning, sequence analysis, and transfection/expression studies. Surface expression of the gene products (on lymphocytes) was confirmed using monoclonal antibodies of defined BoLA specificity. Phylogenetic analysis of available transcribed cattle MHC class I sequences revealed complex evolutionary relationships including possible evidence for recombination. The study of individual haplotypes suggests that certain groupings of related sequences may correlate with loci, but overall it was not possible to define the origin of individual alleles using this approach. The most striking finding of this study is that none of the cattle class I genes is consistently expressed, and that in contrast to human, haplotypes differ from one another in both the number and composition of expressed classical class I genes.

We describe the characterisation of a monoclonal antibody (mAb), designated MM1A, which reacts with an antigen molecule on the surface of bovine alphabeta and gammadeltaTcR+ T cells. The mAb immunoprecipitated a series of polypeptides of 21 kDa, 22 kDa, 32 kDa, 36 kDa and 44 kDa which is consistent with it recognising the TcR/CD3 complex. COS cells, transfected with a cDNA encoding the bovine CD3epsilon chain, reacted with mAb MM1A indicating that the epitope recognised is on the epsilon chain of the complex and confirming that the mAb recognised bovine CD3.

We describe the characterization of two subsets of bovine gamma delta T cells having distinct cell surface phenotype and tissue distribution. One population expresses the previously described 215,000 MW WC1 antigen and is negative for the cell-surface differentiation antigens CD2, CD4, and CD8. The second population expresses CD2 and CD8 but not WC1 and appears to have a T-cell receptor (TCR) rearrangement distinct from that of the WC1+ population. The WC1- population is found in large numbers in spleen and intestine. In addition, this subset is not recognized by a number of monoclonal antibodies (mAbs) specific for TCR families that are well represented in the WC1+ population. The results indicate that the gamma delta T-cell population in cattle is considerably larger than previously described and that this population can be subdivided into two distinct subsets based on cell-surface phenotype and tissue distribution.

Recombinant bovine IFN gamma is a potent inhibitor of Cowdria ruminantium growth in vitro irrespective of the rickettsial stock, or the origin of the endothelial cells. These results suggest an important role for IFN gamma in protective immune responses against C. ruminantium infections. Here we also show that IFN gamma can induce the expression of MHC class II molecules on the surface of endothelial cells. However, treatment of endothelial cells with IFN gamma following infection with Cowdria fails to induce MHC class II expression. The implications of this pathogen-specific effect on class II expression by endothelial cells with regard to its recognition by the host immune system are discussed.

Theileria parva has been shown to infect and transform B cells and T cells at similar frequencies in vitro. However, the majority of parasitized cells in the tissues of infected cattle are alpha/beta T cells. The aim of this study was to determine whether the cell type infected with T. parva influenced the pathogenicity of the parasite. The initial approach, which involved inoculation of cattle with autologous cloned cell lines of different phenotypes, failed to resolve the issue, because of prolonged period of culture required to clone and characterize the cell lines resulted in attenuation of the cells. As an alternative approach, cattle were inoculated with purified populations of autologous cells that had been incubated in vitro with T. parva sporozoites for 48 h. As few as 3 x 10(4) peripheral blood mononuclear cells (PBMC) treated in this way were found to produce severe clinical reactions with high levels of parasitosis. Infections of similar severity were produced with purified populations of CD2+, CD4+, and CD8+ T cells. By contrast, infected B cells gave rise to mild self-limiting infections even when administered at a 10-fold-higher dose. In animals that received infected CD4+ or CD8+ T cells, the parasitized cells in the lymph nodes on day 11 of infection were all within the CD4+ and CD8+ populations, respectively, indicating that there had been minimal transfer of the parasite between cell types. Phenotypic analyses of cultures of PBMC infected in vitro with saturating concentrations of sporozoites revealed that parasitized B cells were abundant in the cultures after 1 week but were subsequently overgrown by T cells. The results of these experiments indicate that the cell type infected by T. parva influences the pathogenicity of the parasite.

The 180,000 MW isoform of CD45 (CD45RO) has been identified in cattle with a novel monoclonal antibody (mAb) (IL-A116). This has allowed a more precise analysis of T-cell function in relation to CD45 isoform expression. Within the CD4+ and CD8+ T-cell populations, CD45RO+ and CD45RO- subsets were evident. Most CD4+ and CD8+ T cells that expressed the CD45RO isoform did not express the 220,000 and 205,000 MW isoforms recognized by mAb CC76. In contrast, the WC1+, CD2-, CD4-, CD8-, gamma delta T-cell receptor (TCR)+ T cells in bovine peripheral blood mononuclear cells (PBMC) were all CD45RO+. Monocytes and granulocytes were CD45RO+ but B cells were CD45RO-. Sorting experiments with CD4+ T cells from an immunized calf demonstrated that proliferative responses to ovalbumin (OVA) were entirely within the CD45RO+ subset. Following stimulation with concanavalin A (Con A) the CD45RO- subset of CD4+ T cells produced transcripts for interleukin-2 (IL-2) but not IL-4 or interferon-gamma (IFN-gamma), while the CD45RO+ subset produced mRNA for IL-2, IL-4 and IFN-gamma. Biologically active IL-2 was present in supernatants from both CD45RO+ and CD45RO-, CD4+ T cells, and IFN-gamma protein was identified by ELISA in supernatants from the CD45RO+ subset, confirming the production of cytokines implied by polymerase chain reaction (PCR). In contrast, sorting experiments with CD8+ T cells from animals immune to the protozoan parasite Theileria parva revealed substantial numbers of cytotoxic T-lymphocyte precursors in both the CD45RO+ and CD45RO- subsets. Thus it appears that although all antigenically primed CD4+ T cells remain CD45RO+, and expression of this molecule consequently identifies memory cells within PBMC, antigenically primed CD8+ T cells down-regulate CD45RO expression after activation.

The CD4-specific monoclonal antibody (mAb) CC26, when tested on a total of 143 cattle, failed to react with T cells from 16% of animals and gave reduced intensity staining in a further 35% of animals. The results of family studies with groups of half- and full-siblings indicated that CC26 recognizes an allele of CD4 which is co-dominantly expressed in heterozygous animals. This was confirmed by sequential immunoprecipitation and by selecting transfectants expressing the CC26+ and CC26- allelic forms of CD4 following transfection of genomic DNA from a heterozygous animal. Biochemical studies also revealed an allelic difference in the relative molecular weight (M(r)) of the CD4 molecule, one allele giving 49,000/52,000 MW bands and the other 52,000/57,000 MW bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of CD4+ cell lines with tunicamycin resulted in the appearance of a 47,000 MW band for both allelic forms indicating that the difference in M(r) is due to glycosylation. All of the CC26+ alleles examined were of the low molecular weight form (M(r)low) whereas both M(r)low and M(r)high alleles were represented in CC26- animals. Thus, on the basis of M(r) and reactivity with mAb CC26, three allelic forms of bovine CD4 can be identified, namely CC26+ M(r)low, CC26- M(r)low and CC26- M(r)high; it is proposed that these alleles are designated CD4.1, CD4.2 and CD4.3, respectively. The allelic difference detected by CC26 was present in both Bos taurus and B. indicus cattle indicating that it had arisen prior to divergence of these subspecies. The M(r)high allele (CD4.3) was detected only in B. indicus animals.

CD4-CD8- gamma delta T cells of ruminants uniquely express a 220-kDa surface Ag recognized by several mAbs clustered as WC1. We recently reported the isolation of a cDNA clone encoding a WC1 Ag. Southern blotting suggested that the bovine genome contains multiple sequences highly related to the isolated WC1 cDNA. Here, we demonstrate that some of the clustered WC1 mAbs stain predominantly nonoverlapping subsets of bovine CD4-CD8- gamma delta T cells. By the isolation of two additional cDNA clones encoding molecules highly related to the original WC1 Ag, we provide a molecular basis for this phenomenon. Cells transfected with cDNAs encoding individual WC1 Ags are differentially recognized by various WC1 mAbs. Thus, expression of members of the WC1 gene family divides bovine CD4-CD8- gamma delta T cells into phenotypical subsets. Field inversion gel electrophoresis revealed that all WC1 genes map to a single, large (> 1 Mbp) Notl fragment. Although the function of WC1 remains unknown, it likely involves interaction with ligands that originate from a similarly complex genetic system.

Evidence that class I major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) are involved in immunity to malaria has highlighted the potential importance of these cells in protection against intracellular parasites. Parasite-specific CTL are a prominent feature of the immune response of cattle to Theileria parva, a related apicomplexan parasite. The relationship between the appearance of these cells in the blood of immune cattle under challenge and the clearance of infection suggests that they are involved in the control of infection, but direct evidence is lacking that CTL can mediate protection. We have made a quantitative kinetic study of CTL responses in lymph originating from infected lymph nodes in a number of immune cattle under challenge with T. parva. Direct killing activity and the frequency of CTL precursors (CTLp) within responding cell populations were evaluated. A substantial increase in the proportion of CD8+ CTL was observed between days 8 and 11 after challenge. Frequencies of CTLp as high as 1:32 were observed and activity was essentially confined to the large blasting cell fraction. The analogous response in peripheral blood was of lower magnitude and delayed by 1-2 days. The high frequency of CTLp in efferent lymph permitted the adoptive transfer of this activity between immune and naive monozygotic twin calves. In separate experiments, naive calves lethally infected with T. parva were protected by inoculation of up to 10(10) responding CD8+ T cells derived from their immune twins. Elimination of CD8+ T cells within the inoculum abrogated this effect. These findings provide direct evidence that CD8+ T cells can control T. parva infections in immune cattle.

Mouse L cells and COS cells were transfected with either genomic DNA or cDNAs encoding leukocyte differentiation antigens. Positive transfectants were isolated by FACS and cloned by limiting dilution. Transfectants expressing CD4, CD5, CD8, CD25, CD44, two different WC1 gene products and a transfectant expressing an unknown gene product were isolated using these techniques. Antibodies from the various preliminary clusters were analysed for reactivity on these transfectants. The results confirm the fine specificity of mAbs for two alleles of CD4 and CD5; they also subdivide mAbs recognizing CD8 into two groups based on their specificity for the CD8 alpha chain or a combination of both alpha and beta chains. The data also provides support for the clustering of mAbs recognizing CD25 and CD44 based on transfection of cDNAs encoding these specificities. These results reflect the ability of transfection technology to elucidate the fine specificity of mAbs recognizing bovine CD antigens.

A monoclonal antibody (mAb), MM1A, that identifies a molecule expressed on a large percentage of bovine lymphocytes (60-80%) was examined to determine its specificity. Two and three colour immunofluorescence analysis using flow cytometry revealed the molecule is highly expressed on all CD4+ and CD8+ lymphocytes and lymphocytes that express WC1 and the gamma/delta TCR. In contrast to mAbs reactive with BoCD5, MM1A did not react with B lymphocytes. Biochemical analysis revealed that the mAb immunoprecipitates a molecular complex comprising a set of peptides with M(r) approximately 12, 16, 22, 32, 36, and 44 kDa under reducing conditions and an additional 96 kDa peptide complex under non-reducing conditions. The data indicate that MM1A recognizes the bovine orthologue of CD3 (BoCD3).

Ten monoclonal antibodies (mAbs), that comprised temporary cluster TC1, and included a previously published CD45R mAb, were compared together with mAb IL-A116 which did not cluster with any workshop mAb. The ten mAbs of TC1 immunoprecipitate molecules with an M(r) of 220 and 205 kDa; sequential precipitation with a CD45 mAb indicates that these mAbs recognize restricted isoforms of the leukocyte common antigen. Cellular and tissue distribution suggests that mAbs within TC1 recognize at least two different specificities of the CD45 family of molecules. mAb IL-A116 precipitated a molecule of 180 kDa suggesting that it may recognize the CD45RO isoform of the leukocyte common antigen.

Mouse L cell lines expressing two different bovine WC1 glycoproteins were produced by transfection of the cells with the corresponding cDNAs. The cell lines were used to analyze the reactivities of 67 monoclonal antibodies (mAbs) which recognize bovine gamma/delta T cells. The results indicated that preliminary clustering of mAbs can be achieved based on their recognition of epitopes expressed on all gene products, or of epitopes encoded by individual members of the gene family. The studies also showed that at least three members of the WC1 gene family are expressed, although it is not yet known how many can be expressed by individual bovine gamma/delta T cells. Final clustering of the WC1 mAbs will not be possible until the exact number of expressed gene products is known, and the reactivities of the mAbs with these products have been analyzed.

The expression of major histocompatibility complex class II (MHC II) non-polymorphic antigens detected by four monoclonal antibodies was investigated in Theileria parva-infected and non-infected cloned lymphoid cell lines, bulk cultures, and in peripheral blood mononuclear cells (PBMC) and lymph node cells (LNC) of experimentally infected calves. Compared with non-infected cell lines, both immunofluorescence microscopy and flow cytofluorometry analysis of infected lines of alpha beta T-cell, gamma delta T-cell and B-cell origin revealed high expression of MHC II MHC molecules. After T. parva infection in vitro, three alloreactive T cell clones, three interleukin-2 (IL-2)-dependent cell lines and a concanavalin A (Con A)-stimulated bulk culture all had an increase both in the proportion of MHC II+ cells and in their mean fluorescence intensity. Radioimmunoprecipitation of class II molecules biosynthesized in infected and non-infected cells revealed that they were constitutively produced in infected cells, and were a slightly larger relative mass than the MHC II molecules of uninfected cells. In a study of the serial expression of MHC II antigens in PBMC and LNC of six calves inoculated with a lethal dose of T.parva, MHC II expression by non-parasitized cells peaked at Days 7 (LNC) or 9 (PBMC) following inoculation and, subsequently, MHC II non-expressing parasitized lymphocytes progressively outnumbered MHC II-expressing parasitized cells. In two calves studied in detail, MHC II expression in PBMC and LNC generally, and in T cells particularly, increased during the course of the disease. Finally, among LNC sorted for MHC II expression at 11 and 17 days after parasite inoculation, the proportion of parasitized cells increased markedly in MHC II non-expressing populations and was reduced or increased only slightly in MHC II-expressing populations. These findings indicate that: (1) enhanced MHC II antigen expression by parasitized lymphocytes may be important in the pathogenesis of the lymphoproliferation that characterizes T. parva infection; (2) the in vivo preponderance of MHC II non-expressing over MHC II-expressing T. parva-infected cells may reflect host-mediated destruction or antigenic modulation of parasitized MHC II-expressing cells.

Although gamma delta T lymphocytes were identified several years ago, the functional importance of these cells remains to be established. gamma delta T cells of ruminants are unique in two respects. First, they are present at much higher levels compared to man and rodents. Second, ruminant CD4-CD8- gamma delta T cells uniquely express a 220 kD surface Ag recognized by a panel of mAb, recently clustered as WC1. WC1 has been most extensively studied in sheep with the use of the mAb T19. Here, we report on the isolation of a full length cDNA clone, encoding the WC1 Ag, from a COS cell cDNA expression library prepared from a bovine gamma delta T cell line. The protein encoded by the pWC1 cDNA clone was reactive with the bovine mAb CC15 and IL.A29, and with T19. The cDNA clone consisted of 4475 bp and contained a single long open reading frame of 1436 amino acids. The pWC1 cDNA clone encoded a type 1 integral membrane protein with an extracellular domain consisting of 11 scavenger receptor cysteine-rich-repeats with homology to CD5 and CD6. Southern blotting suggested that the bovine genome contained multiple sequences highly related to the isolated WC1 cDNA. Furthermore, WC1-like sequences were present in the genomes of all mammals tested including mouse and man. The molecular characterization of the WC1 Ag as reported here provides a starting point for the definition of its role in gamma delta T cell biology.

Veiled cells have been prepared from bovine afferent lymph and their surface phenotype has been compared with that of blood monocytes using a panel of mAb that define bovine leukocyte Ag. It has been observed that the expression of CD1c and a putative bovine CD45R Ag is restricted within these populations to afferent lymph veiled cells (ALVC), whereas CD11b is expressed only on monocytes. The CD11a and CD11c specificities and an additional Ag defined by mAb 1LA24 (p110/75) are expressed by both cell types, although CD11a and p110/75 are found only on subpopulations of ALVC. In a comparison of accessory function, we have found that ALVC are considerably superior to blood monocytes both as stimulators in mixed leukocyte cultures and as presenters of soluble Ag to primed T cells. This superiority appears to be associated with an increased capacity to form clusters with responding lymphocytes. In addition, we have established that the capacity of ALVC to present soluble Ag to Ag-specific T cell clones resides almost exclusively in a subpopulation defined by the phenotype p110/75+ CD11a-.

A murine IgG1 monoclonal antibody (mAb), CC76, has been produced that, based on findings of the relative molecular mass of polypeptides that it recognized, staining of leukocytes in blood and tissues, and the biological properties of the T lymphocyte subpopulations with which it reacts, is considered to identify an isoform of the leukocyte common antigen (LCA) family of molecules in cattle. The mAb is more similar to human CD45R which detect products requiring the presence of the B exon within the LCA gene and to the anti-rat mAb MRC-OX22, than to CD45RA or CD45R0. mAb CC76 reacts with an antigen expressed by subpopulations of cells in bovine blood that express BoCD2 and either the BoCD4 or BoCD8 antigens. T cells that express the gamma/delta T cell receptor identified with mAb to BoWC1 antigen did not react with CC76. The molecule detected is expressed on B cells but not on monocytes or granulocytes. Only 2% of cells in thymic suspensions stained with mAb CC76. Immature cortical thymocytes that were BoCD1+ did not react with CC76 and 90% of the cells in thymic suspensions that were CC76+ had the phenotype of mature thymocytes. These cells were primarily in the medulla. The LCA isoform detected thus appears to be acquired by mature cells shortly before emigration from the thymic medulla into the periphery. Expression of the molecule detected by mAb CC76 on cells from lymph nodes was similar to that in blood, but expression on cells from the gut mucosa was quite different. Almost all, 95% and 93% respectively, of the BoCD4+ cells in the gut mucosa or discrete Peyer's patches were CC76-. A greater proportion of BoCD8+ cells from these sites, 35% and 26%, expressed the antigen. Lymphocytes from animals that had been immunized with Trypanosoma brucei were sorted into BoCD4+, CC76+ and BoCD4+, CC76- populations and cultured in vitro with the variable surface glycoprotein antigen from the parasite. Lymphocyte transformation responses were entirely within the CC76- population indicating that the mAb distinguished naive from memory BoCD4+ T cells in cattle. Major histocompatibility complex (MHC) class I-restricted cytotoxic precursor cells that expressed the BoCD8 antigen sorted from cattle that were immune to Theileria parva were both CC76+ and CC76- indicating that different isoforms of the LCA may be expressed on MHC class I- and class II-restricted memory cells and that BoCD8 memory cells are heterogeneous with respect to the LCA isoform that they express.

The results support the view that these mAbs can be regarded as equivalent to CD1 mAbs in man, as has been concluded for certain of them previously. Although mAb 20-27 did not cluster with the other mAbs, the finding that it precipitated the same molecule from thymocytes indicates that it should be included in BoCD1. Studies in man indicate that more than one CD1 molecule exists (Boumsell and Knowles, 1988; Cattoretti et al., 1988) and a similar situation is apparent in cattle with 20-27 probably being equivalent to CD1c and the others to CD1b.

A cDNA library prepared from lymphocytes of a cow (E98), homozygous at major histocompatibility complex (MHC) loci (BoLA phenotype w10, KN104), was screened with a bovine MHC class I probe. Of the cDNA clones isolated, two, (2.1 and 5.1) were selected and showed divergence at both 5' and 3' termini. E98 DNA was digested with rare-cutter enzymes (Sfi I, Mlu I, Not I, and Cla I) and fragments were size-separated by field inversion gel electrophoresis (FIGE). Hybridization with an entire class I cDNA probe revealed multiple fragments generated by each enzyme. When the 3' untranslated regions (UT) of 2.1 and 5.1 were used as probes, only one fragment was revealed in each digested sample, showing locus specificity of these probes in cattle. Further, DNA of transfected mouse fibroblasts L4 (expressing KN104) and L10 (expressing w10) hybridized to the 3'UT regions of clones 2.1 and 5.1, respectively. Northern blot analysis of the mRNA of the L4 and L10 transfected cells provided further evidence that the cDNA clones 2.1 and 5.1 code for the BoLA-KN104 and BoLA-w10 class I molecules respectively, and thus these represent the products of two different genes. A long range physical mapping of the BoLA-w10 and KN104 genes was performed using FIGE analysis of DNA of an homozygous and an heterozygous animal. This analysis revealed that the BoLA-w10 and KN104 genes are separated by not more than 210 kilobases (kb) and that they are components of a multigene family spanning 1550 kb. As the w10 gene is at the BoLA-A locus we assign the KN104 gene to a B locus.

Mouse L-cells transfected with bovine CD8 and two Theileria parva-infected cloned T cell lines expressing bovine CD8 were used to screen the panel of ten monoclonal antibodies (mAbs) submitted to the workshop. Eight of the ten mAbs reacted with the transfectant and both the cloned T cell lines. However, two mAbs CC58 and BAT82A did not recognise the transfectant and only reacted with one of the T cell lines. Further biochemical studies indicated that the eight mAbs react with both homo- and heterodimeric forms of bovine CD8 whilst the two mAbs CC58 and BAT82A react with only heterodimeric forms. These data suggest that bovine DC8 is encoded by two genes as is the case in mouse and man.

Monoclonal antibodies specific for T cell differentiation antigens were tested on four cloned populations of lymphocytes derived from the peripheral blood mononuclear cells of an animal immunised with Theileria parva. The clones were defined functionally in terms of cytotoxic activity, MHC restriction and expression of messenger RNA for CD3 and T cell receptor (TCR). Two clones contained RNA transcripts for CD3, TCR-alpha and beta and were positive for CD2, CD5 and CD6; one of these was a typical CD4+ class II MHC-restricted non-cytotoxic clone while the other was a CD8+ class I MHC-restricted cytotoxic clone. By contrast, the remaining two clones had the characteristics of non-specific killer cells in that they exhibited moderate levels of non-MHC-restricted killing; they contained TCR-delta mRNA and a 1.2 kb truncated form of TCR-beta message, but they did not contain CD3 or TCR-alpha mRNA. One of these non-specific killer clones only expressed CD2 whereas the other clone only expressed CD8, but without the CD8 determinant recognised by monoclonal antibodies CC58 and BAT52. All four clones were negative for the WC1 antigen which is expressed on gamma/delta T lymphocytes.

w10 and KN104 are distinct class I major histocompatibility complex (MHC) serological specificities present in Boran (Bos indicus) cattle. Although these specificities are commonly expressed together, they may also be expressed independently. To establish whether w10 and KN104, when expressed together, are on the same or different molecules, and whether a second class I MHC locus exists in cattle, genomic DNA from an animal homozygous for a haplotype encoding the w10 and KN104 specificities was transfected into thymidine kinase-deficient mouse L cells (Ltk- cells), and the transfected cells were screened with monoclonal antibodies (mAb) specific for the w10 or KN104 allospecificities. Two different populations of transfectants were identified: the cells of one population reacted only with w10-specific mAb, whereas those of the other population were recognized only by the KN104-specific mAb. Alloreactive cytotoxic T lymphocytes (CTL) also distinguished between the two populations. Two CTL clones, shown to be restricted by the KN104 specificity, killed only those L cells expressing molecules recognized by the KN104-reactive mAb. Of eight CTL clones which recognized class I molecules associated with the w10 specificity, four killed the L cells expressing the w10 specificity. The remaining four clones did not kill either population of transfectants. Finally, immunoprecipitation studies revealed that both populations express full-length bovine class I MHC molecules. These results demonstrate that the w10 and KN104 specificities are on distinct class I molecules. As the genes encoding these molecules were derived from a MHC-homozygous animal, the findings also provide strong evidence that there are at least two classical class I loci in cattle.

In this study, two monoclonal antibodies, IL-A29 and CC15, are described that identify a novel bovine cell surface marker of 215/300 kDa. The antibodies reacted with a discrete population of resting lymphocytes in peripheral blood which, in young animals, constituted about 25% of the mononuclear cells. Thymus, lymph nodes and spleen contained less than 5% positive cells. These cells were negative for surface Ig, a monocyte/granulocyte marker, and the T lymphocyte antigens CD2, CD6, CD4 and CD8. Immunohistological analyses revealed the presence of IL-A29/CC15-positive lymphocytes in the thymic medulla, in the outer cortex of lymph nodes, in the marginal zones of the spleen, in the dermal and epidermal layers of the skin and in the lamina propria of the gut. The IL-A29/CC15+ cells in unfractionated blood mononuclear cells responded in autologous and allogeneic mixed lymphocyte cultures, and when purified they responded to concanavalin A in the presence of recombinant interleukin 2. These observations suggested this population of cells belonged to the T cell lineage. In order to unambiguously define their lineage, cDNA clones encoding bovine T cell receptor (TcR) and CD3 proteins were isolated. Northern blot analyses of IL-A29/CC15+ cell populations and of established cell lines of various lineages demonstrated that they expressed TcR delta and CD3 gamma, delta and epsilon mRNA: TcR alpha was not expressed, whereas only a truncated form of TcR beta mRNA was present. These results indicate that the IL-A29 and CC15 antibodies define a unique population of CD4-CD8-, gamma/delta T cells.