Infection of cattle with Theileria parva induces an early CD8 T cell response lacking appropriate effector function.

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.

Demonstration of strain-specific CD8 T cell responses to Theileria annulata.

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.

A rapid and sensitive intracellular flow cytometric assay to identify Theileria parva infection within target cells.

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.

Analysis of T-cell receptor BV gene sequences in cattle reveals extensive duplication within the BV9 and BV20 subgroups.

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.

Enhancing effects of anti-CD40 treatment on the immune response of SCID-bovine mice to Trypanosoma congolense infection.

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.

The CD45 locus in cattle: allelic polymorphism and evidence for exceptional positive natural selection.

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.

Transcription of the unique ruminant class II major histocompatibility complex-DYA and DIB genes in dendritic cells.

Dendritic cells (DC) constitute the most effective immune cell population for priming and recalling T cell responses to foreign antigens. DC patrol the peripheral tissues collecting foreign antigen for subsequent presentation by classical class II MHC molecules to T cells in the draining lymph nodes. Since the description of the DYA and DIB class II MHC genes, which are unique to ruminants, no transcript or protein have been reported. Here we provide evidence that these genes are transcribed in cattle and that paired transcription is restricted in afferent lymph to a functionally distinct population of DC. Analysis of lymph node, lung and thymus suggests that tissue DC also transcribe both genes. Cytokine-induced differentiation of cultured monocytes to a DC phenotype is linked with induction of both DYA and DIB transcription. This is consistent with an association of their products with the potent antigen presenting capacity of these cells in cattle.

A highly sensitive, non-radioactive assay for T cell activation in cattle: applications in screening for antigens recognised by CD4(+) and CD8(+) T cells.

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.

Variation in the number of expressed MHC genes in different cattle class I haplotypes.

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.

Characterisation of a monoclonal antibody recognising the CD3epsilon chain of the bovine T cell receptor complex.

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.

Identification of two distinct subsets of bovine gamma delta T cells with unique cell surface phenotype and tissue distribution.

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.

Expression of beta 2 integrins on blood leukocytes of cows with or without bovine leukocyte adhesion deficiency.

Peripheral blood leukocytes of 11 normal cows, 7 cows heterozygous and 2 heifers homozygous for bovine leukocyte adhesion deficiency (BLAD) were analysed by flow cytometry for the intensity of their beta 2 integrin expression (LFA-1(CD11a/CD18), CR3 (CD11b/CD18) and CR4 (CD11c/CD18)). BLAD-homozygotes revealed no or a very weak expression of the beta 2 integrins and had a 10-fold and 4- to 5-fold increase in absolute number of neutrophils and monocytes, respectively, whereas the absolute number of lymphocytes remained normal. The mean fluorescence intensity (MFI) of the beta 2 integrins (CD18) in heterozygous animals was 56 to 90% of this in the normal cows (MFI between 14 and 512). The difference in the expression level was most pronounced for LFA-1 on the small cluster of lymphocytes with the highest MFI for LFA-1. Repeated analysis and phorbol myristate acetate stimulation revealed that the LFA-1 expression on this high-expressing cell population of the peripheral blood allowed a ready identification of BLAD-heterozygotes by flow cytometry.

Recombinant bovine interferon gamma inhibits the growth of Cowdria ruminantium but fails to induce major histocompatibility complex class II following infection of endothelial cells.

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.

Pathogenicity of Theileria parva is influenced by the host cell type infected by the parasite.

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.

CD45RO expression on bovine T cells: relation to biological function.

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.

An assay for the identification of antigens recognized by cytotoxic T cells, based on transient transfection of COS cells.

The studies reported here describe methodology permitting the direct identification of antigens recognized by cytotoxic T lymphocytes. We demonstrated that bovine alloreactive CTL can detect a bovine MHC molecule transiently expressed in a COS cell population in a standard microcytotoxicity assay. We then showed that alloreactive CTL can detect cells expressing the bovine class I MHC molecule in a population of cells transfected with the plasmid containing the corresponding gene plus 100-fold as many plasmids containing an irrelevant gene. In addition, the transiently transfected COS cells can specifically restimulate CTL as detected by a standard microcytotoxicity assay using the target cell line. Overall, the results suggest that COS cells could be employed for the direct screening of an antigen or antigen gene library by immune CTL.

Identification of three distinct allelic forms of bovine CD4.

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.

Members of the novel WC1 gene family are differentially expressed on subsets of bovine CD4-CD8- gamma delta T lymphocytes.

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.

Adoptive transfer of immunity to Theileria parva in the CD8+ fraction of responding efferent lymph.

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.

Comparison of monoclonal antibodies with potential specificity for restricted isoforms of the leukocyte common antigen (CD45R).

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.