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Dendritic cell antigen targeting primes robust immune responses in mouse models. Optimizing this immunization strategy in the actual hosts that require protection will advance development of efficacious contemporary vaccines. In a proof-of-concept study, we tested the immunogenicity of a single, low dose of a novel multi-component DNA construct expressing a CD205-targeted antigen fused to a CD40L minimal functional domain for linked DC activation. The DNA construct was formulated with DNA-encoded Flt3L and GM-CSF for DC recruitment and the formulation was evaluated in MHC class II-matched calves. Immunization of the calves with the CD205 antigen-targeting construct mixed with the cytokine constructs induced significant IFN-γ-secreting CD4(+) T-cells, CD4(+) T-cell proliferation, and antibody responses detectable within one week post-immunization. CD205 antigen-targeting significantly expanded IFN-γ-secreting CD4(+) T-cells, CD4(+) T-cell proliferation, and IgG antibody responses three weeks post-immunization. Nineteen weeks post-priming, the IFN-γ-secreting CD4(+) T-cells, CD4(+) T-cell proliferation, and the IgG titers were waning, but they remained significant. Following boosting at nineteen weeks post-immunization, the immune responses primed by the CD205-targeted antigen underwent rapid recall and the mean response tripled within one week post-boost. Comparative analysis of the immune responses observed one week post-priming versus the responses detected one week post-boost revealed that the average number of the IFN-γ-secreting CD4(+) T-cells observed in the calves immunized with the CD205 antigen targeting construct increased five-fold, the mean CD4(+) T-cell proliferation increased three-fold, whereas the mean IgG antibody titer increased two hundred-fold. These promising outcomes support testing the protective efficacy of CD205-targeted antigens in the calf model., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Dendritic cells (DCs) are central to the induction of immune responses and are a pivotal control point that determines the outcome of infectious challenge. Cannulation of afferent lymphatic vessels allows the isolation of large numbers of lymph DCs. First, lymph nodes that are draining the skin are surgically removed (takes approximately 1 h). Over a period of 6-8 weeks, afferent lymphatic vessels re-anastomose with the efferent duct, forming larger 'pseudoafferent' lymphatic vessels that can be surgically cannulated. Surgical cannulation takes 2 h to perform; daily maintenance of the catheter requires 30 min. Isolation of lymph cells requires 1 h and an additional 60-180 min to enrich or purify the DCs. The lymph can be harvested for up to 1 month, with relatively constant cell numbers and subset distribution throughout this period. This technique, although technically demanding, facilitates studies of DCs and other cells that traffic in the lymph in both the steady state and following antigenic exposure.

Dendritic cells (DC) have been reported to migrate in afferent lymph in the steady state. However, it is unknown whether DC traffic is modulated by the nature of the drained tissue. To analyze the influence of mucosal versus cutaneous microenvironments on the constitutive DC release, we exploited a novel technique of lymph cannulation in sheep, which allowed a comparison of afferent lymph DC migrating from the head mucosae [cervical DC (CerDC)] with DC migrating from skin [prescapular DC (PresDC)]. The migration rate was lower for CerDC than for PresDC. Compared with PresDC, CerDC contained a higher proportion of the CD26hi signal regulatory protein (SIRP)- DC subset. It is interesting that cytoplasmic apoptotic DNA as well as cytokeratin-positive inclusions were primarily detected among CD26hi SIRP- DC, an observation similar to that made in rats, which leads to the suggestion that this subset was involved in self-antigen presentation and tolerance induction. After the inoculation of cholera toxin (CT) onto the oro-nasal mucosae, migration of CD26hi SIRP- and CD26lo SIRP+ DC was accelerated in lymph, indicating that the effect of CT on DC mobilization is not subset-specific. Our results show that a mucosal environment influences DC output and the relative DC subset representation in lymph. This modulation of DC traffic to lymph nodes by mucosal surfaces is likely to affect the bias of the mucosal immune responses.

Toll-like receptors (TLR) have been described as partially sharing signalling pathways but showing unique ligand specificity and tissue distribution. Here, the response of bovine macrophages (Mphi) and dendritic cells (DC), both derived from monocytes, was compared by exposing them to the TLR-specific ligands lipopolysaccharide, poly(I:C)-double-stranded RNA, and CpG-DNA, as well as inactivated Gram-negative and Gram-positive bacteria, shown to bind to TLR. The production of NO, superoxide anion, interleukin-10 (IL-10), IL-12 and tumour necrosis factor (TNF) was determined. Compared to monocytes, Mphi expressed more TLR2 and similar levels of TLR4 mRNA transcripts, as analysed by quantitative polymerase chain reaction, whereas DC expressed reduced amounts. Although both DC and Mphi recognized the TLR ligands, dramatic differences were seen in their reaction pattern to them. Both cell types responded with the production of TNF, but DC produced more IL-12, whereas Mphi produced more IL-10, regardless of the TLR agonist used. Co-stimulation with interferon-gamma influenced the amount of cytokine production, but did not alter the cell type-specific response pattern. Compared to Mphi, DC produced > 10 times less NO upon triggering with TLR ligands. In addition, DC produced superoxide anion to opsonized and non-opsonized zymosan, but not to phorbol 12-myristate 13-acetate, a response pattern confirmed for human Mphi and DC, respectively. Different protein kinase C isoforms and extracellular signal-regulated kinase patterns were detected in cell lysates of resting and stimulated Mphi and DC. Collectively, our results point to profound differences in pathogen-derived signal-response coupling occurring commensurate with distinct functions carried out by Mphi or DC.

The response of DC, and the subsequent stimulation of T cells, is an essential part of the initiation of immune responses following microbial challenge. The response of human DC to bacterial lipopeptides is mediated by toll-like receptor 2, and is characterised by DC maturation and the enhanced capacity to stimulate of T cells. We report here that bovine DC are also induced to mature following lipopeptide stimulation. Exposure of DC to the model lipopeptide Pam3CSK4 was associated with increased expression of MHC, costimulatory molecules, and enhanced secretion of IL-12 and TNFalpha. Lipopeptide-matured DC were superior in their ability to induce T cell activation and IFNgamma secretion. In contrast, exposure of MPhi to lipopeptides induced down-regulation of MHC expression and much lower increases in IL-12 secretion. A lipopeptide derived from the sequence of a relevant mycobacterial lipoprotein, MPB83, also influenced bovine DC by stimulating increases in IL-12 and TNFalpha secretion. These different changes in bovine DC and MPhi may have important implications for immune responses induced following bacterial infection with uptake of microbes by DC resulting in potentiation of their immunostimulatory capacity and uptake by MPhi having a much less marked effect on immune responses.

Two phenotypically distinct subpopulations of dendritic cells (SIRPalpha+ CC81Ag- DC and SIRPalpha- CC81Ag+ DC) have previously been identified in bovine afferent lymph which show functional differences when assayed in vitro. The purpose of this study was to investigate whether differences in cytokine production between the two subpopulations might occur which could influence the bias of the immune response they stimulate. Qualitative and quantitative polymerase chain reactions were used to detect specific mRNA transcripts and flow cytometry and enzyme-linked immunosorbent assays were used to detect protein production. The SIRPalpha- CC81Ag+ DC produced considerably more interleukin-12 (IL-12) mRNA transcripts and protein than the SIRPalpha+ CC81Ag- DC. Conversely, SIRPalpha+ CC81Ag- DC contained more of both transcripts and protein for IL-1 and of transcripts for IL-6. A small percentage of both subpopulations produced interferon-gamma (IFN-gamma) as evidenced by cytoplasmic staining. Stimulation of DC by culture with CD40L+ cells increased the production of IL-1, IL-6 and IL-12 but quantitative differences between the subpopulations remained. Production of IL-10 was also evident following culture with CD40L+ cells or lipopolysaccharide primarily by the SIRPalpha+ CC81Ag- DC. No evidence was found for type 1 IFN production, and hence plasmacytoid DC, by DC in afferent lymph; both subpopulations appear to be myeloid in origin. These different cytokine repertoires of the two subpopulations of ex vivo DC isolated from afferent lymph imply functional differences that could influence the presentation of antigen to T cells and bias of the immune response following vaccination or infection.

Regulation of humoral responses involves multiple cell types including the requirements for cognate interactions between T and B cells to drive CD40-dependent responses to T-dependent antigens. A third cell type has also been shown to play an essential role, the dendritic cell (DC). We demonstrate that bovine peripheral blood-derived (PB)-DC are similar in function to features described for human interstitial DC including the production of signature type 2 cytokines [interleukin (IL)-13, IL-10]. PB-DC express moderate-to-high costimulatory molecule expression, and major histocompatibility complex class II is negative for CD14 expression and has low or no expression of CD11c. Consistent with the interstitial phenotype is the ability of PB-DC to influence B cell activation and differentiation via direct expression of CD40L and type 2 cytokines. Collectively, these results suggest that direct B cell-DC interactions may promote an immunoglobulin-isotype expression pattern consistent with type 2 responses, independent of direct T cell involvement.

DNA-based immunization is a contemporary strategy for developing vaccines to prevent infectious diseases in animals and humans. Translating the efficacy of DNA immunization demonstrated in murine models to the animal species that represent the actual populations to be protected remains a significant challenge. We tested two hypotheses directed at enhancing DNA vaccine efficacy in outbred animals. The first hypothesis, that DNA-encoding fetal liver tyrosine kinase 3 ligand (Flt3L) and GM-CSF increases dendritic cell (DC) recruitment to the immunization site, was tested by intradermal inoculation of calves with plasmid DNA encoding Flt3L and GM-CSF followed by quantitation of CD1(+) DC. Peak DC recruitment was detected at 10-15 days postinoculation and was significantly greater (p < 0.05) in calves in the treatment group as compared with control calves inoculated identically, but without Flt3L and GM-CSF. The second hypothesis, that DNA encoding Flt3L and GM-CSF enhances immunity to a DNA vector-expressed Ag, was tested by analyzing the CD4(+) T lymphocyte response to Anaplasma marginale major surface protein 1a (MSP1a). Calves immunized with DNA-expressing MSP1a developed strong CD4(+) T cell responses against A. marginale, MSP1a, and specific MHC class II DR-restricted MSP1a epitopes. Administration of DNA-encoding Flt3L and GM-CSF before MSP1a DNA vaccination significantly increased the population of Ag-specific effector/memory cells in PBMC and significantly enhanced MSP1a-specific CD4(+) T cell proliferation and IFN-gamma secretion as compared with MHC class II DR-matched calves vaccinated identically but without Flt3L and GM-CSF. These results support use of these growth factors in DNA vaccination and specifically indicate their applicability for vaccine testing in outbred animals.

We compared the effects of live or inactivated bovine respiratory syncytial virus (BRSV) on cytokine production by bovine monocyte-derived dendritic cells (MoDC). We also investigated the response of resting memory CD4(+) T cells to MoDC exposed to both viral preparations. Although BRSV did not appear to replicate in MoDC or to affect expression of major histocompatibility complex (MHC) class I, MHC class II, or CD80/86, a higher percentage of cells exposed to live virus appeared to undergo apoptosis/necrosis. To investigate how the interaction of BRSV with MoDC affects the immune response, a multiplex, real-time, polymerase chain reaction was established to analyze transcription of bovine cytokines. Exposure of MoDC to live BRSV induced more interleukin (IL)-10 mRNA and markedly less IL-12p40 and IL-15 mRNA than did heat-inactivated virus. To determine whether these differences might influence the T cell response, CD4(+) memory T cells primed in vivo were restimulated in vitro by MoDC pulsed with heat-inactivated or live BRSV. Stimulation of CD4(+) T cells induced similar levels of IL-2-and IL-4-like activity and interferon-gamma. These observations suggest that while IL-10, produced by MoDC as a result of exposure to live BRSV, may affect IL-12 and IL-15 synthesis by MoDC, it does not appear to affect the cytokine response of BRSV-specific memory CD4(+) T cells. It is possible, however, that differences in the pattern of cytokines produced by MoDC exposed to live or inactivated virus may influence the development of the primary CD4(+) T cell response in vivo.

Two major sub-populations of dendritic cells (DC) are present in afferent lymph draining the skin of cattle distinguished by expression of signal regulator protein alpha (SIRPalpha). The SIRPalpha(-) population expresses the uncharacterized bovine WC10 antigen (Ag). Initial N-terminal sequencing of the WC10 protein purified by affinity chromatography showed significant homology with human CD26. A cDNA encoding bovine CD26 was cloned and the recombinant molecule expressed in COS-7 cells. Transfectants abrogated the ability of macrophage-derived chemokine (MDC) to cause a calcium flux in bovine PBMC indicating enzymatic activity characteristic of CD26. They also stained with WC10 monoclonal antibody confirming that the Ag is CD26. This is the first description of CD26 expression by DC in vivo or in vitro. It is expressed on a sub-population of ex vivo DC in afferent lymph draining the skin and on sub-populations of DC isolated from prescapular and mesenteric lymph nodes draining the skin or intestine, respectively. CD26 is an exopeptidase with specificity for motifs within the receptor-binding domain of several chemokines including MDC. CD26 mediated truncation of MDC affects the Th cell response effected by the chemokine and may produce a Th1 bias. Transcripts for MDC were present in both CD26(+) and CD26(-) DC, thus CD26 mediated modification of MDC may bias the immune response induced in naive T cells by DC.

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.

Abstract: Dendritic cell antigen targeting primes robust immune responses in mouse models. Optimizing this immunization strategy in the actual hosts that require protection will advance development of efficacious contemporary vaccines. In a proof-of-concept study, we tested the immunogenicity of a single, low dose of a novel multi-component DNA construct expressing a CD205-targeted antigen fused to a CD40L minimal functional domain for linked DC activation. The DNA construct was formulated with DNA-encoded Flt3L and GM-CSF for DC recruitment and the formulation was evaluated in MHC class II-matched calves. Immunization of the calves with the CD205 antigen-targeting construct mixed with the cytokine constructs induced significant IFN-γ-secreting CD4+ T-cells, CD4+ T-cell proliferation, and antibody responses detectable within one week post-immunization. CD205 antigen-targeting significantly expanded IFN-γ-secreting CD4+ T-cells, CD4+ T-cell proliferation, and IgG antibody responses three weeks post-immunization. Nineteen weeks post-priming, the IFN-γ-secreting CD4+ T-cells, CD4+ T-cell proliferation, and the IgG titers were waning, but they remained significant. Following boosting at nineteen weeks post-immunization, the immune responses primed by the CD205-targeted antigen underwent rapid recall and the mean response tripled within one week post-boost. Comparative analysis of the immune responses observed one week post-priming versus the responses detected one week post-boost revealed that the average number of the IFN-γ-secreting CD4+ T-cells observed in the calves immunized with the CD205 antigen targeting construct increased five-fold, the mean CD4+ T-cell proliferation increased three-fold, whereas the mean IgG antibody titer increased two hundred-fold. These promising outcomes support testing the protective efficacy of CD205-targeted antigens in the calf model. [Copyright &y& Elsevier]