Characterization of efferent lymph cells and their function following immunization of cattle with an allogenic Theileria annulata infected cell line.

Immunization of cattle with in vitro propagated bovine mononuclear cells infected with Theileria annulata induces a protective immune response. Activation and effector function of T cells exiting the lymph node draining the site of cell line immunization were investigated to understand the mechanisms involved in the generation of immunity. Immunized animals exhibited a biphasic immune response in efferent lymph as well as peripheral blood. The first phase corresponded to allogenic responses against MHC antigens of the immunizing cell line and the second was associated with parasite specific responses. An increase in the output of CD2(+) cells and MHC class II(+) cells in efferent lymph was observed after cell line immunization with a corresponding decrease in WC1(+) cells. Although the percentage of CD4(+) T cells did not change significantly over the course of the experiment, they became activated. Both CD25 and MHC class II expressing CD4(+) T cells were detected from day 7 onwards, peaking around day 13. Efferent lymph leukocytes (ELL) exhibited sustained responses to IL-2 in vitro following cell line immunization. Antigen specific proliferation was also detected first to the immunizing cell line and then to parasite antigens. The two peaks of CD2(+) cells were observed, which corresponded to similar peaks of CD8(+) cells. The increase in CD8(+) cells was more pronounced during the second parasite specific phase than the first allogenic phase. Activated CD8(+) T cells mainly expressed MHC class II and some expressed CD25. Significantly the peak of activated CD4(+) T cells preceded the peak of activated CD8(+) T cells, highlighting the role of T. annulata specific CD4(+) T cells in inducing parasite specific CD8(+) cytotoxic responses. A biphasic cytotoxic response also appeared in efferent lymph and peripheral blood, the first directed against MHC antigens of the immunizing cell line followed by MHC class I restricted parasite specific cytotoxicity. The cytotoxic responses in efferent lymph appeared earlier than peripheral blood, suggesting that activated CD8(+) cells exiting the draining lymph node following immunization with T. annulata infected schizonts play an important role in the development of protective immune responses.

Proinflammatory cytokine expression by Theileria annulata infected cell lines correlates with the pathology they cause in vivo.

Control of Theileria annulata is currently best achieved by the use of live attenuated cell line vaccines. However, the mechanisms underlying attenuation are unclear and there is a need to rapidly produce new cell line vaccines, which could safely and effectively vaccinate cattle against tropical theileriosis. There is increasing evidence to suggest that proinflammatory cytokines produced by T. annulata infected cells play a central role in both pathology and immune evasion. This study aimed to test this hypothesis and to evaluate cytokine expression as a marker of virulence. The pathogenicity and protective efficacy of cloned T. annulata cell lines that expressed different levels of proinflammatory cytokines were compared. In two independent trials using different stocks of T. annulata, cell lines that expressed higher levels of proinflammatory cytokines induced severe reactions, and in some cases death, when used to vaccinate groups of cattle. In contrast, low cytokine expressing lines induced low post-vaccinal reactions. The results clearly demonstrated that cytokine expression by T. annulata infected cells could be used as a marker of virulence and provided strong evidence to support a role for cytokines in the induction of pathology. Both high and low cytokine expressing cell lines protected cattle against heterologous challenge infection, offering the possibility of using cytokine expression to rapidly select new safe, potent vaccines against tropical theileriosis without the need for culture attenuation.

Innate and adaptive immune responses co-operate to protect cattle against Theileria annulata.

For many years it was assumed that Theileria annulata resembled T. parva, parasitizing lymphocytes and causing lymphoproliferative disease, with the two species being controlled by similar protective immune responses. Patricia Preston et al. here review the evidence that has led to a different view of T. annulata. It is now thought that the schizonts of T. annulata inhabit macrophages and B cells, and that tropical theileriosis is not a lymphoproliferative disease. Both innate and adaptive responses contribute to recovery from infection and resistance to challenge and cytokines produced by infected and uninfected cells influence the outcome of infection. Partial protection has been stimulated recently by defined recombinant antigens; efficacy depended upon the delivery system.

Macrophages behaving badly: infected cells and subversion of immune responses to Theileria annulata.

The protozoan parasite Theileria annulata is the causative agent of the tick-borne disease tropical theileriosis, responsible for morbidity and mortality of cattle in many developing countries. Here, John Campbell and Roger Spooner discuss how the parasite might evade immune destruction during an acute primary infection. Theileria annulata macroschizont-infected macrophages act as over-efficient antigen-presenting cells within the infected draining lymph node. Infected cells activate CD4+ and CD8+ T cells abnormally, giving rise to a cascade of cytokine production. This altered immune response does not reject the parasitized cells, and might actively participate in the growth of the developing parasite.

In vivo development of Theileria annulata: major changes in efferent lymph following infection with sporozoites or allogeneic schizont-infected mononuclear cells.

The object of these experiments was to study the pathogenesis and kinetics of Theileria annulata infection in the efferent lymph of the draining lymph nodes of calves. Efferent lymphatics of calves were cannulated prior to infection with T. annulata sporozoite or an allogeneic schizont cell line. Potentially lethal sporozoite challenge induced cell shut-down from days 4-6 and then a massive increase in output of blasting cells (both infected and non-infected) in the efferent lymph. The rate of lymph flow and total cell output increased to 5 to 10-fold from day 6 onwards. Sporozoites were never isolated from the efferent lymph. However, large numbers of parasite-infected cells were seen in efferent lymph from the sixth day of infection. The animals inoculated with an allogeneic T. annulata-infected cell line exhibited only a small increase in flow rate and cell output. Parasite-infected cells of recipient origin were seen in efferent lymph from day 11 onwards. However, cells of donor origin were never isolated either from efferent lymph or peripheral blood. Thus the parasite transferred from the inoculated donor cell line to the cells of the recipient before schizonts appeared in efferent lymph.

Diminished IL-2 responses and alteration of CD2 expression on CD8+ T cells are associated with a lack of cytotoxic T cell responses during Theileria annulata infection.

Theileria annulata is a tick-borne protozoan parasite which causes the disease bovine tropical theileriosis. In immunized or drug-treated animals, the pathogenic macroschizont stage of the parasite is destroyed by MHC class I-restricted cytotoxic T lymphocytes (CTL). Here we show that although CD8+ T cells increase greatly in number and display activation markers during an acute infection, they exhibit no killing of infected cells. During the ineffectual response, efferent lymph cells' ability to proliferate to IL-2 drops, coinciding with loss of MoAb binding to CD2 by CD8+ cells. When animals were treated with the anti-parasite drug 'Butalex', IL-2 responses, anti-CD2 antibody binding by CD8+ cells and strong CTL activity were restored within 24 h. The initial activation of CD4+ T cells by parasite-infected cells altering the IL-2 production in the draining lymph node is the likely cause of the failure of CTL responses.

Evidence for strain specificity in cytotoxic T-lymphocyte-mediated, major histocompatibility complex class I-dependent killing of Theileria annulata-infected cells.

Cattle immunised against Theileria annulata with one parasite strain have been found to be immune to re-challenge with different strains of the parasite. However, recent evidence of apparent strain specificity has been documented in cattle immunised with attenuated parasite-infected cells. In this study the strain specificity of major histocompatibility complex class I-restricted cytotoxic T-lymphocytes (CTL), a major anti-parasite effector mechanism, was examined. CTL generated following challenge with the Hissar (Indian) strain effectively lysed autologous cells infected with this strain of the parasite. However, CTL were less effective against cells infected with the Gharb (Moroccan) strain and showed virtually no reactivity against the Ankara (Turkish) strain, providing the first direct evidence for strain specificity in immune responses against T. annulata.

A stage-specific, parasite-induced, "window" of in vivo interferon-gamma production is associated with pathogenesis in Theileria annulata infection.

The tick-borne protozoan parasite Theileria annulata causes tropical theileriosis, a severe leukoproliferative disease of cattle, which naive susceptible animals fail to control. The parasite infects and transforms macrophages, developing in the local draining lymph node. IFN gamma has been shown to block parasite development in newly infected cells, and inhibits the growth of fully differentiated macroschizont stage-infected cells in vitro. However, the parasite has been found to specifically induce IFN gamma production by T cells and appears to flourish in the face of this T cell-derived response in vivo. Here we show that the production of IFN gamma in vivo is tightly controlled by the parasite. Induction of cytokine production by T cells is not initiated until the parasite has developed beyond the IFN gamma sensitive trophozoite stage. Cytokine production is kept high as infected macrophages develop, and IFN gamma appears to play an active role in maintaining the growth of these cells. Once the infection is fully established, IFN gamma is down regulated, avoiding potential inhibitory effects. Thus by controlling T cell IFN gamma production, the parasite induces a "window" of cytokine expression which promotes its own growth, but avoids potential inhibitory effects of the cytokine.

A novel cell surface proliferation-associated marker expressed on T cells and up-regulated on germinal center B cells.

In this study we present data on a novel cell surface antigen recognized by monoclonal antibody (mAb) VPM30, originally thought to recognize only bovine and ovine sIg+ B cells from peripheral blood. Here we show that the antigen, molecular mass 28 kDa, is not only found in B cell follicles in frozen sections, but when used on paraffin sections VPM30 specifically stains B cells in the light zone of germinal centers but not in the mantle or dark zones. In addition we show that the antigen is also expressed by 90% of T cells after activation, with kinetics of antigen expression mirroring those of proliferation. By both size and distribution, the antigen appears to be novel, corresponding to no known cluster of differentiation, and will be of great use in the study of ruminant cellular immune responses.

A non-protective T helper 1 response against the intra-macrophage protozoan Theileria annulata.

Theileria annulata is a protozoan parasite which infects and transforms bovine macrophages. Infected macrophages possess augmented antigen presentation capabilities, as they are able to activate the majority of T cells from unexposed animals. In vivo, T cells in the draining lymph node (principal site of parasite development) are activated 'non-specifically' by the parasite. This event is followed by failure of the immune response to control the infection. Protective immune responses against intra-macrophage protozoa are usually mediated by T helper 1 (Th1) T cell responses. Here we examine the cytokine responses made by T. annulata-activated T cells. We show that the outcome of in vitro activation of T cells by parasitized macrophages is a skewing of their cytokine responses towards preferential expression of interferon-gamma (IFN-gamma) mRNA. The in vitro response is mirrored during in vivo infection, as greatly elevated amounts of IFN-gamma protein are found in lymph efferent from infected lymph nodes, while expression of IL-4 mRNA within the node stops. IFN-gamma production does not correlate with protection against the parasite, as infected cells flourish during peak IFN-gamma production, and only very small amounts of IFN-gamma are produced during the effective immune response of an immunized animal. Overproduction of IFN-gamma and loss of IL-4 expression are also likely to account for the failure of B cells to reach the light zone of germinal centres, a developmental step which is tightly regulated by cytokines.

Allograft responses can interfere with the development of immunity against Theileria annulata following vaccination with parasite infected cell lines.

Theileria annulata macroschizont-infected cell lines are successfully used as vaccines in several countries. The inoculated animals produce a strong allogeneic response against the MHC antigens of the immunizing cell line followed by an anti-parasite response. Immunity against the parasite wanes in the absence of challenge and re-immunization is sometimes recommended. However, it is not known if allogeneic responses generated by the first immunization with a T. annulata infected cell line will interfere with the boosting of immunity against the parasite at the time of re-immunization with the same cell line. Animals were primed against MHC antigens by skin grafting, followed by immunization with a T. annulata infected cell line prepared from the skin donor. A strong anti-MHC response was produced. This interfered with parasite transfer and the development of an anti-parasite immune response; the effect was more marked when a low vaccine cell dose was used. There was a negative correlation between the ease of isolating infected cells from the animals after cell line immunization, and the subsequent response to challenge. Where no cell lines could be isolated, the animals were fully susceptible to sporozoite challenge. These observations are of immediate importance in endemic areas where cell lines of T. annulata schizonts are being used as vaccines to control the disease.

Revaccination with the same Theileria annulata infected cell line may not be feasible for boosting immunity against tropical theileriosis.

A model for studying re-immunisation using skin grafting was developed as the allogeneic responses produced by T. annulata cell lines were similar to those produced by skin grafting. Appearance of schizonts and piroplasms post-immunisation was either delayed or prevented by already existing allogeneic responses. Isolation of parasite infected cell lines from lymph node biopsies and peripheral blood after cell line immunisation was also delayed or prevented by already existing allogeneic responses. Ability to isolate cell lines after immunisation correlated with protection i.e. if no parasite infected cell line of donor origin was isolated after immunisation, there was no protection. Allogeneic responses delayed or prevented the appearance of MHC I restricted parasite specific cytotoxic T lymphocytes post-immunisation. If the parasite transfer was prevented after immunisation; animals were fully susceptible to challenge. These experiments showed that allogeneic responses, generated in animals after immunisation with T. annulata schizont cell culture vaccine, can block parasite transfer and further development or enhancement of immunity against the parasite at the time of second immunisation with the same cell line. The observations are of immediate importance in endemic areas where T. annulata infected cell culture vaccines are being used. They are even more relevant in countries where animals are regularly moved between theileriosis free and endemic areas. It may not be advisable to re-immunise animals with the same cell line as that used for first vaccination.

Parasite-mediated steps in immune response failure during primary Theileria annulata infection.

"Exotic" European cattle are highly susceptible to T. annulata infection. In immunised animals, several effective anti-parasite responses can be demonstrated, such as anti-macroschizont cytotoxic T cells (CTL), and nitric oxide killing of parasites. The failure of infected animals to mount an effective primary immune response suggests that the presence of the parasite directly interferes with the development of immunity. When the activation pathways of CD4+ T cells in draining lymph nodes were examined during the course of a primary infection it was found that the development of this essential arm of the immune response was altered. Instead of interacting with antigen presenting cells in the paracortex, the majority of CD4+ T cells were rapidly activated by developing infected cells in the medulla of the node. Activation of T cells by infected cells also drastically alters the cytokines produced by the T cells. During effective immune responses, the principal cytokine involved appears to be IL-2, with only small, controlled "bursts" of IFN gamma production. However, IL-2 responsiveness is only transient in animals undergoing primary infection, while IFNg production is greatly elevated. IFN gamma does not appear to control parasitised cells, and may even aid the growth of infected macrophages--large numbers of macrophages enter the cell cycle during the peak period of IFN gamma production. Uncontrolled parasite-induced IFN gamma production is also likely to account for the local failure of antibody responses. Germinal centres in infected lymph nodes lose normal morphology, with IFN gamma sensitive zones failing to develop. A third strategy which the parasite uses to evade immune response destruction is through affecting CTL activity. CTL in infected draining lymph nodes lose expression of the adhesion molecule CD2--a molecule is essential in adherence to target cells for lysis. CD2- CTL are unable to lyse macroschizont infected cells.

Protective immune responses to Theileria annulata of relevance to vaccine development.

A series of projects on Theileria annulata funded by the European Union (STD1/STD2/STD3) have provided convincing evidence that macrophage and natural killer (NK) cell-dependent immune mechanisms may directly control the proliferation of different stages of T. annulata in cattle. The evidence for this conclusion and the implications for vaccine development are discussed in the following paper.

A novel expression vector for transfection of bovine MHC class I genes.

A vector is described for the expression of genomic or cDNA copies of bovine major histocompatibility complex (MHC) class I genes in transfected mouse Ltk- cells. Class I gene fragments are amplified by the polymerase chain reaction, using primers in conserved parts of exon 2 and the 3'-untranslated region of the gene. Amplified class I gene fragments can then be subcloned into the expression vector, pBoLA-21, which contains the necessary 5'- and 3'-sequences for correct expression. The vector was tested by subcloning and expressing genomic and cDNA clones.

Functional expression of a cattle MHC class II DR-like antigen on mouse L cells.

Cattle DRA and DRB genes, cloned by reverse-transcription polymerase chain reaction, were transfected into mouse L cells. The cattle DR-expressing L-cell transfectant generated was analyzed serologically, biochemically, and functionally. Sequence analysis of the transfected DRB gene clearly showed showed that it was DRB3 allele DRB3(*)0101 , which corresponds to the 1D-IEF-determined allele DRBF3. 1D-IEF analysis of the transfectant confirmed that the expressed DR product was DRBF3. Functional integrity of the transfected gene products was demonstrated by the ability of the transfectant cell line to present two antigens (the foot-and-mouth disease virus-derived peptide FMDV15, and ovalbumin) to antigen-specific CD4(+) T cells from both the original animal used to obtain the genes, and also from an unrelated DRBF3(+) heterozygous animal. Such transfectants will be invaluable tools, allowing us to dissect the precise contributions each locus product makes to the overall immune response in heterozygous animals, information essential for rational vaccine design.

Factors influencing the transmission and incidence of tropical theileriosis (Theileria annulata infection of cattle) in Morocco.

A longitudinal epidemiological investigation of tropical theileriosis was carried out in an endemic region of Morocco during the 1991 disease season. This involved approximately 220 cross-bred cattle on 15 farms. Data were collected on the frequency of existing infections, the size of the vector tick population and the incidences of new infections and clinical disease, and these were then analysed using statistical models. The prevalence of subclinically infected carriers of Theileria annulata, the number of adult Hyalomma detritum, the vector, and the probability of becoming newly infected with T. annulata increased significantly with the age of cattle, although the age effect on new infections may be a result of increased tick numbers on older animals. The probability of clinical disease in newly infected cattle was not significantly influenced by age or by the number of adult ticks, but was significantly positively associated with the cattle population on the farm. The number of H. detritum nymphs counted on cattle in the autumn was related significantly to the previous adult tick count on the same animal.

Expression of multiple DQB genes in Bos indicus cattle.

Polymorphism of expressed bovine major histocompatibility complex (MHC) class II DQB genes was investigated in a group of nine MHC-homozygous Kenya Boran cattle (Bos indicus). DQB second exon fragments were amplified by reverse transcription-polymerase chain reaction of total mononuclear cell RNA, cloned and sequenced. While a single DQB sequence was obtained from some animals, two DQB exon 2 sequences were found in others, implying expression of duplicated DQB genes. Two pairs of duplicated DQB genes were found in this group of homozygotes, and sequence analysis showed that both pairs contained distinct DQB alleles. One DQB duplication was observed in two related animals with the same MHC haplotype (F100 and F188), while the second was detected in three animals (F187, G166 and G277) that expressed the same class II DRB3 and DQB alleles but had different class I (BoLA-A) types, suggesting that this DR/DQ haplotype may be widespread in Boran cattle.