Understanding how pathogens or vaccine antigens are targeted to dendritic cell (DC) subsets is important for disease pathogenesis studies and vaccine design. We characterised the sub-populations of migrating bovine DC with functional and phenotypic diversity present in pseudoafferent lymph draining the skin. These skin draining DC exist as a series of maturation dependent subsets with differential capacities for antigen uptake and cytokine expression, and include both Langerhans’ cells (LC) and dermal derived cells. Furthermore, Mycobacterium bovis Bacille Calmette Guerin, a vaccine which is administered by the intradermal route, was only taken up by a small number of the migrating DC, which were SIRPα+ and expressed the mannose receptor and CD1b. This was evident following in vitro infection and also in vivo following inoculation of green fluorescent BCG over the lymphatic cannulation site. Only the SIRPα+ DC were able to present antigen to T cells isolated from BCG vaccinated calves. Furthermore, presentation of BCG antigens by DC to T lymphocytes was ineffective compared to mycobacterial proteins. However, mycobacterial antigen 85 was delivered more effectively to DC via an adenoviral vector and the magnitude of the subsequent antigen-specific T cell response was significantly increased. This study further extends our understanding of the biology of migrating DC, identifies potential explanations for the modest success of BCG vaccination and demonstrates that targeted delivery of antigens via adenoviruses to DC can improve antigen presentation.

The portfolio of reagents for immunology research in veterinary species is limited compared to those available for humans and model biomedical species such as mice. For example, we still lack the reagents to fully ascertain if the immunological paradigms relating to the development, function and inter-relationships between the major T helper cell subsets (Th1, Th2, Th17) apply in ruminants, including goats. Such reagents are essential for rational and strategic approaches to vaccine design based on the identification of correlates of protection. Although a number of human immunological reagents such as monoclonal antibodies (mAb) and cytokines have been shown to cross-react with the farmed ruminant species (cattle, sheep and goats), a greater degree of cross-reactivity is found for reagents specifically developed against ruminant species. Thus, for goats, the most likely sources of cross-reactive reagents are those made against cattle and sheep. Nevertheless, although high inter-species homologies are evident at the gene level in ruminants, cross-reactivity of mAb, cytokines and molecular probes cannot be guaranteed and reagents may have to be developed specifically. Here we review the current availability of immunological reagents for caprine immunology and how those reagents can be used to understand T cell biology.

Tumour necrosis factor alpha (TNF-α) is an innate pro-inflammatory cytokine involved in protection against intracellular pathogens. Existing methods for measuring TNF-α production and function in ruminants are limited to ELISA and many rely on polyclonal antisera. With a view to developing improved detection methods for bovine (bov) TNF-α, monoclonal antibodies (mAb) were produced by immunising mice with a plasmid encoding bov TNF-α. Two of the resulting mAb, termed CC327 and CC328, were used to develop a sandwich ELISA capable of detecting both native and recombinant bov TNF-α. This ELISA did not detect recombinant ovine (ov) TNF-α. A luminometric method was applied to the ELISA to improve sensitivity for detection of native bov TNF-α in culture supernatants derived from bovine monocyte-derived dendritic cells (DC) infected with Mycobacterium bovis. Both CC327 and CC328 detected intracytoplasmic expression of TNF-α in mitogen-activated bovine T lymphocytes. However, only CC328 detected intracytoplasmic ovine TNF-α in transfected cells, explaining the failure of the sandwich ELISA to detect recombinant ov TNF-α. These mAbs have generated the capability to study the role of TNF-α in host immune protection and disease pathogenesis in ruminants.

Bovine tuberculosis (bTB) is increasing in incidence in the UK. Effective control strategies could involve vaccination; BCG, either alone or in prime-boost strategies, remains the most effective vaccine against bovine tuberculosis. However, BCG vaccination of cattle would require development of diagnostic tests able to accurately discriminate Mycobacterium bovis-infected from BCG-vaccinated animals. Herein, we demonstrate that the detection of secreted IFN-gamma following short term culture (4h) of whole blood with purified protein derived from M. bovis (PPD-B) allows such discrimination. This reflects, in part, the differential kinetics of IFN-gamma secretion in infected compared to vaccinated cattle. This is the first study to demonstrate that accurate, rapid distinction of BCG-vaccinated from M. bovis-infected cattle can be achieved in a short time period without the need for production of M. bovis-specific antigens, complex antigen mixtures or extensive laboratory procedures. We were also able to detect PPD-specific IFN-gamma release during short term culture of blood from a number of humans with active TB indicating that this test may have wider application and is potentially useful for the rapid diagnosis of disease in humans.

ABSTRACTMycobacterium bovisis the causative agent of bovine tuberculosis, a disease that is increasing in incidence in United Kingdom cattle herds. In addition to increasing economic losses, the rise in bovine tuberculosis poses a human health risk. There is an urgent requirement for effective strategies for disease eradication; this will likely involve vaccination in conjunction with current test and slaughter policies. A policy involving vaccination would require an accurate diagnosis ofM. bovis-infected animals and the potential to distinguish these animals from vaccinates. Currently used diagnostic tests, the skin test and gamma interferon (IFN-γ) blood test, have a sensitivity of up to 95%. A further complication is thatM. bovisBCG-vaccinated animals are also scored positive by these tests. We tested the hypothesis that the quantification of IFN-γ-producing lymphocytes by flow cytometric analysis of intracellular IFN-γ expression would provide a more accurate discrimination ofM. bovis-infected animals from BCG vaccinates. Significant numbers of IFN-γ-expressing CD4+T cells were detected following culture of heparinized blood fromM. bovis-infected animals, but not from BCG vaccinates, with purified protein derived fromM. bovis(PPD-B) or live mycobacteria. Only 1 of 17 BCG-vaccinated animals had a significant number of CD4+T lymphocytes expressing IFN-γ, compared with 21/22M. bovis-infected animals. This assay could allow an accurate diagnosis ofM. bovisand allow the discrimination of BCG-vaccinated cattle from infected cattle.

Recombinant bovine IL-4 (rbo IL-4) was transiently expressed in COS-7 cells. Mice were immunised with a plasmid encoding rbo IL-4 and boosted with rbo IL-4. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-4 in an ELISA and these cloned hybridomas were termed CC311, CC312, CC313 and CC314. A pair of mAb (CC313 and CC314) was identified that together could be used to detect both recombinant and native bovine IL-4 by ELISA and a luminometric detection method was applied to the ELISA. Using this method native bovine IL-4 was detected in supernatants of PBMC stimulated with mitogens. In addition, high level secretion of IL-4 by Fasciola hepatica specific Th2 clones, but not by a Babesia bovis specific Th1 clone, was confirmed. The ELISA was also able to detect recombinant ovine IL-4. The pair of mAb used for ELISA could also be used for the detection of IL-4 spot forming cells by ELISPOT. In addition intracytoplasmic expression of IL-4 could be detected. The ability to detect ruminant IL-4 by three methods: ELISA, ELISPOT and by flow cytometric analysis of intracytoplasmic expression will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

Dendritic cells are central to the initiation of primary immune responses. They are the only antigen-presenting cell capable of stimulating naive T cells, and hence they are pivotal in the generation of adaptive immunity. Dendritic cells also interact with and influence the response of cells of the innate immune system. The manner in which dendritic cells influence the responses in cells of both the innate and adaptive immune systems has consequences for the bias of the adaptive response that mediates immunity to infection after vaccination or infection. It also provides an opportunity to intervene and to influence the response, allowing ways of developing appropriate vaccination strategies. Mouse and human studies have identified myeloid, lymphoid and plasmacytoid dendritic cells. Studies in domesticated animals with agents of specific infectious diseases have confirmed the applicability of certain of the generic models developed from mice or from in vitro studies on human cells. In vivo and ex vivo studies in cattle have demonstrated the existence of a number of subpopulations of myeloid dendritic cells. These cells differ in their ability to stimulate T cells and in the cytokines that they produce, observations clearly having important implications for the bias of the T-cell response. Dendritic cells also interact with the innate immune system, inducing responses that potentially bias the subsequent adaptive response.

Theileria infection of bovine leucocytes induces uncontrolled proliferation and a transformed phenotype comparable to tumour cells. Infected cells have many characteristics of activated leucocytes and use autocrine loops to augment proliferation. We have shown previously that, in infected B cells, PI3-K controls a granulocyte-macrophage colony-stimulating factor (GM-CSF) autocrine loop to increase both proliferation and activation of the activator protein 1 (AP-1) transcription factor. We show here that the same infected B cells also use a tumour necrosis factor (TNF) alpha autocrine loop that again contributes to proliferation and augments nuclear factor (NF)-kappaB activation. Interestingly, both pharmacological inhibition of TNF synthesis and neutralizing anti-TNF antibodies lead to a reduction in proliferation and a 50% drop in NF-kappaB activation, without inducing apoptosis.

The objective of the study was to develop an assay for bovine IL-10 that could be applied to analyses of immune responses and advance understanding of a variety of diseases of cattle. Recombinant bovine IL-10 (rbo IL-10) was transiently expressed in Cos-7 cells and shown to inhibit the synthesis of IFNγ by bovine cells stimulated with antigen in vitro. Mice were immunised with a plasmid containing a cDNA insert encoding rbo IL-10 and inoculated with rbo IL-10. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-10 in an ELISA. Some of these mAb neutralised the ability of rbo IL-10 to inhibit IFNγ synthesis by antigen-stimulated bovine cells. A pair of mAb was identified that together could be used to detect both recombinant and natural bovine IL-10 present in supernatant of PBMC stimulated with ConA. A luminescent detection method was applied to the ELISA making it more sensitive. Using this method native IL-10 was detected in supernatants of PBMC, diluted blood and undiluted blood from cattle immunised with Mycobacterium bovis BCG or ovalbumin and incubated in vitro with antigen indicating the applicability of the assay to a number of in vitro culture systems.

Pre-exposure to environmental mycobacteria and induction of an inappropriately biased immune response may be major factors affecting the efficacy of BCG; vaccination of neonates that have not been exposed to environmental mycobacteria may induce more effective immunity. Responses of neonatal calves to mycobacterial antigens using dendritic cells (DC) as antigen-presenting cells were investigated. In nonvaccinated, immunologically naive calves as young as 1 day old, a population of CD8(+) cells proliferated and produced IFN-gamma in response to BCG-infected DC. CD3(-) CD8(+) NK-like and CD3(+) CD8(+) T cells were evident within the responding CD8(+) population. The response was not MHC-restricted. The NK-like CD3(-) cells were the major population producing IFN-gamma. The presence of mycobacteria-reactive, IFN-gamma-secreting CD8(+) NK cells in neonatal calves may have important consequences for the induction of a Th1-biased immune response.

The objective of the investigation was to provide data on how a prior exposure of cattle to Mycobacterium avium, used here as a model of exposure to an environmental mycobacterium, affected the cellular immune response that follows vaccination with Mycobacterium bovis BCG. The assessment of cellular immune responses included lymphocyte proliferation assays, the delayed hypersensitivity skin test and IFN-gamma synthesis in whole blood cultures. One group of calves was inoculated subcutaneously with M. avium followed 12 weeks later by M. bovis-BCG. The other group was vaccinated subcutaneously with BCG alone. Calves previously exposed to M. avium responded more rapidly, as assessed in the in vitro assays, to purified protein derivative (PPD) from M. avium (PPD-A) or M. bovis (PPD-B) than did calves inoculated with BCG only, indicating that the exposure to M. avium had primed the immune response in these calves. Following inoculation of BCG the intensity of the in vitro responses and the delayed hypersensitivity skin test to PPD-A was higher for the M. avium-primed animals while the responses to PPD-B were similar in the M. avium-primed and BCG-only groups. The results are consistent with a model in which prior exposure to environmental mycobacteria does not necessarily inhibit the immune response to the vaccine strain, BCG. They suggest that M. avium infection primes the immune system of calves and that the detection of an immune response specific for M. bovis BCG is masked by reactivity to antigens also present in M. avium.

Recombinant bovine IL-12 (rbo IL-12) was transiently expressed in COS-7 cells and shown to upregulate the synthesis of IFNgamma by bovine cells stimulated with a suboptimal concentration of mitogen in vitro. Mice were immunised with a plasmid encoding rbo IL-12 and boosted with rbo IL-12 and a number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-12 in an ELISA. Some of these mAb neutralised the ability of rbo IL-12 to induce IFNgamma synthesis by bovine cells. A pair of mAb was identified that together could be used to detect both recombinant and natural bovine IL-12 by ELISA and a luminometric detection method was applied to the ELISA making it more sensitive. Using this method native bovine IL-12 was detected in supernatants of dendritic cells (DC) cultured in vitro with a synthetic lipopeptide known to stimulate secretion of IL-12 by human DC. The ELISA was also able to detect recombinant ovine IL-12 and, less effectively, recombinant human IL-12. In contrast, bovine IL-12 was not detected by a commercial human IL-12 ELISA kit. Intracytoplasmic IL-12 was detected in bovine DC using the antibodies described herein. The ability to detect ruminant IL-12 by three methods: ELISA, bioassay with neutralising mAb and cytoplasmic staining, will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

The major subset of dendritic cells (DC) from bovine afferent lymph expresses the SIRP alpha MyD-1 antigen, but not CD11a or the antigen recognized by mAb CC81, and potently stimulates CD4+ and CD8+ T lymphocyte proliferation. The minor subpopulation, that is CD11a+ CC81+ MyD-1-, effectively stimulates CD4+ but not CD8+ T lymphocyte proliferation. CD11a+ CC81+ MyD-1- DC did not induce anergy or death or secrete an inhibitory factor. However, supernatant from cultures of CD8+ T cells with CD11a- CC81- MyD-1+ DC significantly enhanced proliferation of CD8+ T cells in response to CD11a+ CC81+ MyD-1- DC, an effect that was blocked by interleukin (IL)-1alpha, but not IL-1beta, specific mAb. The proliferation of CD8+ T cells with CD11a+ CC81+ MyD-1- DC was also enhanced by adding IL-1alpha. IL-1beta slightly enhanced proliferation, whereas IL-2, IL-6, IL-12, and IL-15 had no effect. We conclude that the failure to stimulate CD8+ T cell proliferation results from the lack of IL-1alpha synthesis by this population, which may have important consequences in vivo.

Twelve subpanels of monoclonal antibodies (MAb) included within the 6th International Workshop on Human Leukocyte Differentiation Antigens (6th HLDA) were assayed for reactivity with bovine peripheral blood leukocytes. Sixty-nine of the 807 MAb (8.6%) stained bovine cells. These MAb represented 30 different human CD groups. Nine of the MAb to different human CD antigens (CD19, CD23, CD39, CD47, CD86, CD117, CD120b, CDw149, CD165) potentially recognized antigens on cattle cells that had not previously been identified. These were investigated further by two-colour immunofluorescence to compare the cellular expression of the antigen on cattle cells with that reported for the different CD antigens in humans. Four of the MAb that belonged to CD23, CD39, CD47, and CDw149 stained bovine cells in a manner that indicated an almost identical cellular distribution of the antigen to that reported in humans. This implied that these MAb reacted with the homologous cattle molecules. Further work would be necessary to confirm specificity of CD19, CD86, CD117, CD120b and CD165 MAb. Other cross-reacting MAb either recognized antigens already defined in cattle or antigens not yet clustered in humans. The study has identified valuable new reagents for studies of cattle and confirmed that most common cross-reactive MAb are to epitopes on integrins.

The synthesis of IFNγ and IL-4 by CD4, CD8 and WC1 γδ TCR+ T cell sub-populations, and T cells stained with activation/memory-sub-set markers has been examined by flow cytometric analysis. Cells from blood, prescapular, bronchial and mesenteric lymph nodes and Peyer's patches were incubated with phorbol 12-myristate 13-acetate (PMA), ionomycin and brefeldin-A before staining. Lymphocytes that stained for cytoplasmic IFNγ were evident within the CD4 and CD8 populations from all tissues and also in the WC1 population from lymph nodes. IL-4 producing cells were primarily evident within the CD4 population. IFNγ synthesis was evident within both CD45RO+ and CD45RB+ populations, but IL-4 synthesis was predominantly by cells that were CD45RO+/CD45RB−. Expression of CD62L is not related to functional memory in CD4+ T cells from cattle and CD62L+ cells, particularly from the lymph nodes draining the skin and the lungs, stained with mAb to IFNγ and IL-4. The findings indicate that at least for CD4+ T cells, where CD45 isoform expression is related to functional memory, these two cytokines are produced predominantly by cells with a memory phenotype. The observation that some WC1+ cells produce IFNγ implies the presence of distinct sub-sets of this γδ TCR+ population cattle and suggests a functional role.

Few data are available regarding the induction of memory T‐lymphocyte responses in cattle following Bacille Calmette Guérin (BCG) vaccination. Studies of the immune response induced by BCG vaccination provide an insight into the basis of antimycobacterial immunity that could be exploited for the development of more effective vaccination strategies. We used autologous dendritic cells (DC) infected with Mycobacterium bovis Bacille Calmette Guérin (BCG) or pulsed with purified protein derivative from M. bovis (PPD‐B) or M. avium (PPD‐A) to assess responses of CD4+, CD8+ and WC1+γδ TCR+ lymphocytes from BCG vaccinated and nonvaccinated cattle. Mycobacteria‐specific CD4+ and CD8+, but not WC1+γδ TCR+, memory T lymphocytes were demonstrated in BCG‐vaccinated cattle. CD4+ and CD8+ lymphocytes proliferated and produced interferon (IFN)‐γ in response to BCG‐infected or PPD‐B‐pulsed DC. Proliferative responses were greater for CD4+ than CD8+ lymphocytes, although secretion of IFN‐γ was higher from the CD8+ T cells. Responses to PPD‐A‐pulsed DC were lower, with no CD8+ response. Lymphocytes from nonvaccinated calves were also stimulated to proliferate by BCG‐infected DC, although the magnitude of proliferation was lower. The findings suggest that immunity to M. bovis induced by BCG vaccination in cattle may involve CD8+ memory T cells which produce IFN‐γ, as well as CD4+ memory T cells.

This chapter discusses the T-cell responses and the influence of dendritic cells in cattle. The major T-cell populations in cattle have been identified with monoclonal antibodies (mAbs) to differentiation antigens expressed by the cells produced and characterized in the laboratories of origin or within a series of international workshops that have been held. The nomenclature used follows the human canine distemper (CD) nomenclature where there is sufficient evidence to conclude that the homologous molecule is being identified in cattle and humans. If human CD homologs are not evident WC (workshop cluster) numbers were assigned to the mAb and molecules recognized. Thus cattle leukocytes are defined in terms of the CD or WC antigens expressed. A question, asked in relation to specific infections—that is central to vaccine design—is whether a particular T-cell population is primarily responsible for recovery from infection or immunity to reinfection. Differences among different infections would influence the appropriate strategy selected for immunization.