Flow cytometric detection of IFN-γ production and Caspase-3 activation in CD4+ T lymphocytes to discriminate between healthy and Mycobacterium bovis naturally infected water buffaloes.

Tuberculosis has a negative economic impact on buffalo farming, and it poses a potential threat to human health. Interferon-gamma (IFN-γ) plays a central role in protection against mycobacterial diseases, illustrating the importance of T-cell mediated immune responses in tuberculosis infection. Recently, the expression of Caspase-3, a critical executor of apoptosis, in M. tuberculosis-specific IFN-γ+CD4+ T cells was used as a new marker to distinguish active from latent tuberculosis infection in humans. The aims of this work were to develop a whole blood flow cytometric assay to detect the production of IFN-γ and the activation of Caspase-3 by CD4+ T lymphocytes from water buffalo and to evaluate whether these parameters can discriminate between healthy and M. bovis naturally infected buffaloes. A total of 35 Italian Mediterranean buffaloes were grouped in two groups: uninfected and M. bovis infected (based on the results of antemortem diagnostic tests: single intradermal tuberculin (SIT) and ELISA IFN-γ tests). Whole blood was incubated for 6 h with tubercular antigens: PPD-B, PPD-A, ESAT-6/CFP-10 and a new mix of precocious secreted antigens (PA). Our results showed a significant increase in the percentage of IFN-γ+CD4+ T cells in infected compared to the uninfected animals after each stimulus. Improved sensitivity of the assay was obtained by including the stimulation with the new mix of PA. Interestingly, we observed a concomitant decrease in percentage of Caspase-3+CD4+ T cells in M. bovis infected animals compared to the control healthy ones, regardless of the stimulus used. Overall, these results showed that M. bovis infection activates CD4+ T lymphocytes to produce IFN-γ and at the same time causes a concomitant decrease of Caspase-3 activation in CD4+ T cells. This study for the first time in water buffalo describes the development of a whole blood flow cytometric assay for the detection of IFN-γ producing CD4+ T cells and proposes the expression of active Caspase-3 as an additional bovine TB biomarker. Although further studies are needed to better understand the mechanisms of Caspase-3-mediated cell death during tuberculosis, our data can help to better understand the cellular immune response to M. bovis infection in buffalo species.

Comparative analysis of the specificity of monoclonal antibodies developed against the bottlenose dolphin, Tursiops truncatus, TNF-α, IL1-ß, IL-6, IL-8, IL-10 with monoclonal antibodies made against ovine IFN-γ bovine IL-17A and IL-1ß revealed they recognize epitopes conserved on dolphin and bovine orthologues.

Opportunities to include Cetancodontamorpha in the study of the evolution of the immune system in the clades of Artiodactylamorpha, Ruminantiamorpha, Suinamorpha, and Camelidamorpha have increased with the use of the bottlenose dolphin, Tursiops truncatus, as a sentinel species to study the effects of environmental pollutants on the health of marine mammals. Efforts are currently underway to increase the number reagents needed for detailed studies. Thus far, screening of monoclonal antibodies (mAbs) made to leukocyte differentiation molecules (LDM) and the major histocompatibility (MHC) class I and class II molecules in Ruminantiamorpha have yielded some mAbs that recognize conserved epitopes expressed on orthologues in the bottlenose dolphin. More direct approaches are in progress to identify additional mAbs to bottlenose LDM and cytokines. As reported here, both direct and indirect approaches were used to identify mAbs specific for cytokines useful in monitoring the effects of environmental pollutants on the immune system. Immunization of mice with expressed bottlenose dolphin cytokines yielded mAbs specific for IFN-γ, TNF-α, IL-6, IL-8, IL-10, and IL-17A. Screening of previously developed mAbs used in livestock immunology research revealed mAbs developed against ovine IFN-γ and bovine IL-17 and IL-1ß recognize conserved epitopes in bottlenose dolphin orthologues. The mAbs identified in the present study expand the reagents available to study the function of the immune system in bottlenose dolphins and cattle.

Ex vivo Platforms to Study the Primary and Recall Immune Responses to Intracellular Mycobacterial Pathogens and Peptide-Based Vaccines.

Progress in the study of the immune response to pathogens and candidate vaccines has been impeded by limitations in the methods to study the functional activity of T-cell subsets proliferating in response to antigens processed and presented by antigen presenting cells (APC). As described in this review, during our studies of the bovine immune response to a candidate peptide-based vaccine and candidate rel deletion mutants in Mycobacterium avium paratuberculosis (Map) and Mycbacterium bovis (BCG), we developed methods to study the primary and recall CD4 and CD8 T-cell responses using an ex vivo platform. An assay was developed to study intracellular killing of bacteria mediated by CD8 T cells using quantitative PCR to distinguish live bacteria from dead bacteria in a mixed population of live and dead bacteria. Through use of these assays, we were able to demonstrate vaccination with live rel Map and BCG deletion mutants and a Map peptide-based vaccine elicit development of CD8 cytotoxic T cells with the ability to kill intracellular bacteria using the perforin-granzyme B pathway. We also demonstrated tri-directional signaling between CD4 and CD8 T cells and antigen-primed APC is essential for eliciting CD8 cytotoxic T cells. Herein, we describe development of the assays and review progress made through their use in the study of the immune response to mycobacterial pathogens and candidate vaccines. The methods obviate some of the major difficulties encountered in characterizing the cell-mediated immune response to pathogens and development of attenuated and peptide-based vaccines.

Advances in Understanding of the Immune Response to Mycobacterial Pathogens and Vaccines through Use of Cattle and Mycobacterium avium subsp. paratuberculosis as a Prototypic Mycobacterial Pathogen.

Lack of understanding of the immune response to mycobacterial pathogens has impeded progress in development of vaccines. Infection leads to development of an immune response that controls infection but is unable to eliminate the pathogen, resulting in a persistent infection. Although this puzzle remains to be solved, progress has been made using cattle as a model species to study the immune response to a prototypic mycobacterium, Mycobacterium a. paratuberculosis (Map). As chronicled in the review, incremental advances in characterizing the immune response to mycobacteria during the last 30 years with increases in information on the evolution of mycobacteria and relA, a gene regulating the stringent response, have brought us closer to an answer. We provide a brief overview of how mycobacterial pathogens were introduced into cattle during the transition of humankind to nomadic pastoralists who domesticated animals for food and farming. We summarize what is known about speciation of mycobacteria since the discovery of Mybacterium tuberculsis Mtb, M. bovis Mbv, and Map as zoonotic pathogens and discuss the challenges inherent in the development of vaccines to mycobacteria. We then describe how cattle were used to characterize the immune response to a prototypic mycobacterial pathogen and development of novel candidate vaccines.

Flow Cytometric Analysis of the Cytotoxic T-Cell Recall Response to Theileria parva in Cattle Following Vaccination by the Infection and Treatment Method.

The apicomplexan hemoparasite, Theileria parva, causes East Coast fever (ECF), a frequently fatal disease of African cattle. Vaccine development has been impeded by incomplete understanding of protective immunity following natural exposure or the infection and treatment method (ITM) of immunization. This is attributable to a paucity of methods to characterize the memory T-cell repertoire following infection. To overcome this impediment, assays developed to study the immune response to other intracellular pathogens were adapted for use in studies with T. parva to enable definition of the phenotype and function of effector T cells in T. parva-immune cattle, facilitating vaccine development. As reported herein, stimulation of peripheral blood mononuclear cells (PBMC) from ITM-immunized steers with irradiated, autologous, T. parva-infected cell lines elicited a proliferative recall response comprised of CD45R0+/CCR7- CD4+ and CD8+ T cells. Subsequent co-incubation of stimulated cultures with infected cells demonstrated the presence of cytotoxic T cells (CTLs) with the ability to kill infected cells. Comparison of CTL activity in cultures depleted of CD4+ or CD8+ T cells demonstrated CTL activity was primarily attributed to CD8+ T cells. Importantly, stimulation of PBMC from vaccinated steers always elicited proliferation of CD4+ and CD8+ T cells. This was the first important observation obtained from the use of the assay described herein.

relA is Achilles' heel for mycobacterial pathogens as demonstrated with deletion mutants in Mycobacterium avium subsp. paratuberculosis and mycobacterium bovis bacillus Calmette-Guérin (BCG).

Studies with Mycobacterium avium subsp. paratuberculosis (Map) in cattle revealed deletion of relA, a global regulator gene, abrogated ability of the mutant to establish a persistent infection, attributed to development of an immune response that cleared infection. Analysis of the recall response demonstrated presence of CD8 cytotoxic T cells that kill intracellular bacteria. Replication of the primary response demonstrated the CTL response could be elicited with the ΔMap/relA mutant or the target of the immune response, a 35 kD membrane protein. Follow up comparative studies with Mycobacterium bovis bacillus Calmette-Guérin (BCG) and a BCG relA (ΔBCG/relA) deletion mutant revealed deletion of relA enhanced the CTL response compared to BCG. Analysis of the cytokine profile of cells proliferating in response to stimulation with BCG or BCG/relA showed increased expression of IFN-γ, TNF-α, and IL-17 by cells stimulated with ΔBCG/relA in comparison with BCG. The proliferative and CTL responses were markedly reduced in response to stimulation with heat killed BCG or ΔBCG/relA. Intracellular bacterial killing was mediated through the perforin, granzyme B (GnzB), and the granulysin pathway. The data indicate relA is the Achilles' heel for pathogenic mycobacteria and deletion may be key to improving efficacy of attenuated vaccines for mycobacterial pathogens.

Simultaneous cognate epitope recognition by bovine CD4 and CD8 T cells is essential for primary expansion of antigen-specific cytotoxic T-cells following ex vivo stimulation with a candidate Mycobacterium avium subsp. paratuberculosis peptide vaccine.

Studies in cattle show CD8 cytotoxic T cells (CTL), with the ability to kill intracellular bacteria, develop following stimulation of monocyte-depleted peripheral blood mononuclear cells (mdPBMC) with antigen presenting cells (APC, i.e. conventional dendritic cells [cDC] and monocyte-derived DC [MoDC]) pulsed with MMP, a membrane protein from Mycobacterium avium subsp. paratuberculosis (Map) encoded by MAP2121c. CTL activity was diminished if CD4 T cells were depleted from mdPBMC before antigen (Ag) presentation by APC, suggesting simultaneous cognate recognition of MMP epitopes presented by MHC I and MHC II molecules to CD4 and CD8 T cells is essential for development of CTL activity. To explore this possibility, studies were conducted with mdPBMC cultures in the presence of monoclonal antibodies (mAbs) specific for MHC class I and MHC class II molecules. The CTL response of mdPBMC to MMP-pulsed APC was completely blocked in the presence of mAbs to both MHC I and II molecules and also blocked in the presence of mAbs to either MHC I or MHC II alone. The results demonstrate simultaneous cognate recognition of Ag by CD4 and CD8 T cells is essential for delivery of CD4 T cell help to CD8 T cells to elicit development of CTL.

Isolation of canine adipose-derived mesenchymal stem cells from falciform tissue obtained via laparoscopic morcellation: A pilot study.

OBJECTIVE: To evaluate the feasibility of stem cell isolation from falciform fat harvested via laparoscopic morcellation. STUDY DESIGN: Pilot study. ANIMALS: Eleven client-owned dogs. METHODS: Falciform was harvested traditionally via laparotomy and laparoscopically via tissue morcellation. Harvested tissue was processed with a commercially available adipose tissue dissociation kit to obtain a stromal vascular fraction (SVF). Cells were subsequently labeled for CD90, CD45, and CD44 cell surface antigens by using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting flow cytometry. CD90+ cells were quantitated, and their viability was assessed with a hemocytometer and a trypan blue exclusion test of cell viability. RESULTS: No perioperative complications occurred in dogs undergoing laparoscopic morcellation. Laparoscopically and traditionally harvested samples yielded an average of 0.39 (±0.1) × 106 and 0.33 (±0.1) × 106 CD90+ cells, respectively, per 10 million SVF cells. CD90+ cell viability after MACS was 89% (±11%) for morcellated and 86% (±7%) for traditionally harvested samples. Neither CD90+ cell quantity nor viability was different between samples obtained via traditional laparotomy vs laparoscopic morcellation (P = .38 and P = .63, respectively). Populations of CD90+ cells isolated with each harvest technique had similar CD44 and CD45 expression profiles. CONCLUSION: Viable populations of CD90+ cells with similar CD44/CD45 expression profiles were isolated from laparoscopically morcellated and traditionally harvested falciform tissue. No appreciable morbidity was associated with laparoscopic falciform morcellation. CLINICAL SIGNIFICANCE: Laparoscopic morcellation is a safe and effective minimally invasive approach to falciform tissue harvest for adipose-derived mesenchymal stem cell isolation.

Pattern of CD14, CD16, CD163 and CD172a expression on water buffalo (Bubalus bubalis) leukocytes.

Previous studies on the immune system of water buffalo (Bubalus bubalis) using cross-reactive monoclonal antibodies (mAbs) revealed significant similarities and differences to the bovine immune system. Herein, we extend these studies and document the pattern of expression of CD14, CD16, CD163 and CD172a on buffalo leukocytes using a set of cross-reactive mAbs that are known to recognize conserved epitopes within orthologous molecules in cattle, sheep and goats. Buffalo leukocytes were isolated and subjected to mAb labelling for flow cytometry. Single color flow cytometry confirmed mAbs recognition of buffalo orthologues of CD14, CD16, CD163 and CD172a, and revealed consistent patterns of expression similar to that reported in other ruminants. Multicolor flow cytometry revealed that buffalo CD14+ monocytes uniquely co-express CD16, CD163 and CD172a, whereas buffalo granulocytes co-express CD16 and CD172a. This study expands mAbs available to define and study the buffalo monocytes, and also extends information available on the unique features of the buffalo immune system.

A peptide-based vaccine for Mycobacterium avium subspecies paratuberculosis.

Recent efforts to develop a live attenuated vaccine against Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of Johne's disease (JD), revealed relA is important in Map virulence. Deletion of the relA gene impairs the ability of Map to establish a persistent infection. Analysis of the basis for this observation revealed infection with a relA deletion mutant (ΔrelA) elicits development of cytotoxic CD8 T cells (CTL) with the ability to kill intracellular bacteria. Further analysis of the recall response elicited by ΔrelA vaccination showed a 35 kDa membrane peptide (MMP) is one of the targets of the immune response, suggesting it might be possible to develop a peptide-based vaccine based on MMP. To explore this possibility, ex vivo vaccination studies were conducted with MMP alone and incorporated into a nanoparticle (NP) vector comprised of poly (D, L-lactide-co-glycolide) and monophosphoryl lipid A (PLGA/MPLA). As reported, ex vivo vaccination studies showed CD8 CTL were elicited with classic and monocyte derived dendritic cells (cDC and MoDC) pulsed with MMP alone and incorporated into a PGLA/MPLA vector. Incorporation of MMP into a NP vector enhanced the ability of CD8 CTL to kill intracellular bacteria. The findings indicate incorporation of MMP into a PGLA/MPLA nanoparticle vector is one of the possible ways to develop a MMP based vaccine for Johne's disease.

Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells.

Studies focused on development of an attenuated vaccine against Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of paratuberculosis (Ptb) in cattle and other species, revealed that deletion of relA, a global gene regulator, abrogates the ability of Map to establish a persistent infection. In the absence of relA, cattle develop CD8 cytotoxic T cells (CTL) with the ability to kill intracellular bacteria. Analysis of the recall response to a relA mutant, Map/ΔrelA, with cells from a vaccinated steer demonstrated that a 35-kDa membrane peptide (MMP) is one of the targets of the response. This observation suggested that it might be possible to develop a peptide-based vaccine. As reported here, the gene encoding the hypothetical MMP ORF, MAP2121c, was modified for expression in mammalian cells as a first step in developing an expression cassette for incorporation into a mammalian expression vector. The modified sequence of MMP, tPA-MMP, was mutated to generate two additional sequences for the study, one with substitutions to replace five potential residues that could be glycosylated, tPA-MMP-5mut, and one with substitutions to replace the first two potential residues that could be glycosylated, tPA-MMP-2mut. The sequences were placed in an expression cassette to produce peptides for analysis. An ex vivo platform was used with flow cytometry and a bacterium viability assay to determine if modifications in the gene encoding MMP for expression in mammalian cells altered its capacity to elicit development of CD8 CTL, essential for its use in a peptide-based vaccine. Monocyte-depleted PBMC (mdPBMC) were stimulated with antigen-presenting cells (APC) pulsed with different MMP constructs. CD4 and CD8 T cells proliferated in response to stimulation with MMP (control) expressed in Escherichia coli (eMMP), tPA-MMP, and tPA-MMP-2mut. CD8 T cells retained the capacity to kill intracellular bacteria. The tPA-MMP-5mut failed to elicit a proliferative response and was not included in further studies. The data show that the expression cassettes containing MMP and MMP-2mut can be used to screen and select a mammalian expression vector for the development of an efficacious peptide-based vaccine against Ptb.

A Mycobacterium avium subsp. paratuberculosis relA deletion mutant and a 35 kDa major membrane protein elicit development of cytotoxic T lymphocytes with ability to kill intracellular bacteria.

Efforts to develop live attenuated vaccines against Mycobacterium avium subspecies paratuberculosis (Map), using indirect methods to screen Map deletion mutants for potential efficacy, have not been successful. A reduction in the capacity to survive in macrophages has not predicted the ability of mutants to survive in vivo. Previous studies for screening of three deletion mutants in cattle and goats revealed one mutant, with a deletion in relA (ΔMap/relA), could not establish a persistent infection. Further studies, using antigen presenting cells (APC), blood dendritic cells and monocyte derived DC, pulsed with ΔMap/relA or a 35 kDa Map membrane protein (MMP) revealed a component of the response to ΔMap/relA was directed towards MMP. As reported herein, we developed a bacterium viability assay and cell culture assays for analysis and evaluation of cytotoxic T cells generated against ΔMap/relA or MMP. Analysis of the effector activity of responding cells revealed the reason ΔMap/relA could not establish a persistent infection was that vaccination elicited development of cytotoxic CD8 T cells (CTL) with the capacity to kill intracellular bacteria. We demonstrated the same CTL response could be elicited with two rounds of antigenic stimulation of APC pulsed with ΔMap/relA or MMP ex vivo. Cytotoxicity was mediated through the perforin granzyme B pathway. Finally, cognate recognition of peptides presented in context of MHC I and II molecules to CD4 and CD8 T cells is required for development of CTL.

Characterization of αß and γδ T cell subsets expressing IL-17A in ruminants and swine.

As part of our ongoing program to expand immunological reagents available for research in cattle, we developed a monoclonal antibody (mAb) to bovine interleukin-17A (IL-17A), a multifunctional cytokine centrally involved in regulating innate and adaptive immune responses. Initial comparative studies demonstrated the mAb recognizes a conserved epitope expressed on orthologues of IL-17A in sheep, goats and pigs. Comparative flow cytometric analyses of lymphocyte subsets stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin revealed differences in expression of IL-17A by CD4, CD8, and γδ T cells across ruminants and swine species. Results in cattle showed the largest proportion of IL-17A+ cells were CD4+ followed by γδ and CD8+ T cells. Further analysis revealed the IL-17A+ γδ T cell subset was comprised of WC1.1+, WC1.2+, and WC1- subsets. Analysis of the IL-17A+ CD8+ T cell subset revealed it was comprised of αß and γδ T cell subsets. Results in sheep and goats revealed IL-17A is expressed mainly by CD4+ and CD8+ T cells, with little expression by γδ T cells. Analysis of IL-17A+ CD8+ T cells showed the majority were CD8+ αß in sheep, whereas they were CD8+ γδ in goats. The majority of the sheep and goat IL-17A+ γδ T cells were WC1+. Results obtained in swine showed expression of IL-17A by CD4, CD8, and γδ T cell subsets were similar to results reported in other studies. Comparison of expression of IL-17A with IFN-γ revealed subsets co-expressed IL-17A and IFN-γ in cattle, sheep, and goats. The new mAb expands opportunities for immunology research in ruminants and swine.

Identification of monoclonal antibodies cross-reactive with bottlenose dolphin orthologues of the major histocompatibility complex and leukocyte differentiation molecules.

The slow progress in understanding immunotoxic effects of environmental contaminants and their influence on disease susceptibility in whales is largely due to the limited information available on the immune systems and immune function of species included in the Cetancodontamorpha clade. Studies in species in the other major clades included in the Artiodactylamorpha, Ruminantiamorpha, Suinamorpha, and Camelidamorpha have revealed the immune systems are similar, but not identical. The present study was undertaken to expand the available monoclonal antibody reagents needed to gain insight into the composition, function, and evolution of the immune system in Cetancodontamorpha, using the dolphin (Tursiops truncatus) as a model cetacean species. Screening of a set of mAbs that recognize highly conserved epitopes expressed on the major histocompatibility complex (MHC) and leukocyte differentiation molecules (LDMs) in cattle by flow cytometry revealed some of the mAbs recognize epitopes conserved on dolphin orthologues of MHC class I, MHC class II, CD11a, CD14, CD16, CD18, CD163 and CD172a. Comparison of the amino acid sequences of dolphin and bovine orthologues revealed limited changes in sequence have occurred during speciation, suggesting an approach for developing cross-reactive mAbs for use in cetacean research.

Evaluation of antigen specific interleukin-1ß as a biomarker to detect cattle infected with Mycobacterium bovis.

Bovine tuberculosis (bTB) is a major world-wide health problem that has been difficult to control, due to the lack of an effective vaccine and limited ability of the tuberculin skin test (TST) and the ancillary whole blood interferon-gamma (IFN-γ) release assay (IGRA) to detect all infected animals. A 6 h cytokine flow cytometric IFN-γ (CFC) assay was developed in effort to overcome these limitations and expand methods for studying the mechanisms of bTB immunopathogenesis. The present study was conducted to evaluate IL-1ß as a biomarker to use in conjunction with the IFN-γ CFC assay to improve the diagnostic accuracy for bTB. Three animal groups with predefined Mbv infection status were used for analysis of IL-1ß in plasma from whole blood cultures stimulated with ESAT-6/CFP-10 for 20-24 h. Parallel stimulations were performed for enumeration of IFN-γ producing T cells. Data analysis showed that Mbv infected animals have a higher frequency of IFN-γ producing CD4+ T cells and plasma IL-1ß than animals exposed to non-tuberculous mycobacteria (NTM) or uninfected control animals, with a significant correlation between the two readouts, thus allowing differentiation between the three animal groups. IL-1ß has the potential to serve as an additional biomarker for detecting cattle infected with Mbv.

Characterization of leukocyte subsets in buffalo (Bubalus bubalis) with cross-reactive monoclonal antibodies specific for bovine MHC class I and class II molecules and leukocyte differentiation molecules.

Although buffaloes (Bubalus bubalis) are a major component of the livestock industry worldwide, limited progress has been made in the study of the mechanisms regulating the immune response to pathogens and parasites affecting their health and productivity. This has been, in part, attributable to the limited availability of reagents to study immune responses in buffalo. As reported here, a set of cross-reactive monoclonal antibodies (mAbs), developed against bovine, ovine and caprine leukocyte differentiation molecules (LDM) and major histocompatibility complex (MHC) molecules, were identified and used to compare expression of LDM in Italian and Egyptian buffalo. The results show most of the epitopes identified with the mAbs are conserved on LDM and MHC I and II molecules in both lineages of buffalo. Comparison of the composition of lymphocyte subsets between buffalo and cattle revealed they are similar except for expression of CD2 and CD8 on workshop cluster one (WC1) positive γδ T cells. In cattle, CD8 is expressed on a subset of CD2+/WC1- γδ T cells that are present in low frequency in blood of young and old animals, whereas, CD8-/CD2-/WC1+ γδ T cells are present in high frequency in young animals, decreasing with age. In the buffalo, CD2 is expressed on a subset of WC1+ γδ T cells and CD8 is expressed on all WC1+ γδ T cells. The availability of this extensive set of mAbs provides opportunities to study the immunopathogenesis of pathogens and parasites affecting the health of buffalo.

Correction: Phenotype and Function of CD209+ Bovine Blood Dendritic Cells, Monocyte-Derived-Dendritic Cells and Monocyte-Derived Macrophages.

[This corrects the article DOI: 10.1371/journal.pone.0165247.].

Phenotype and Function of CD209+ Bovine Blood Dendritic Cells, Monocyte-Derived-Dendritic Cells and Monocyte-Derived Macrophages.

Phylogenic comparisons of the mononuclear phagocyte system (MPS) of humans and mice demonstrate phenotypic divergence of dendritic cell (DC) subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny and function: conventional DC (cDC1 and cDC2), plasmacytoid DC (pDC), and monocyte derived DC (MoDC). DC of Artiodactyla (pigs and ruminants) can also be sub-classified using this system, allowing direct functional and phenotypic comparison of MoDC and other DC subsets trafficking in blood (bDC). Because of the high volume of blood collections required to study DC, cattle offer the best opportunity to further our understanding of bDC and MoDC function in an outbred large animal species. As reported here, phenotyping DC using a monoclonal antibody (mAb) to CD209 revealed CD209 is expressed on the major myeloid population of DC present in blood and MoDC, providing a phenotypic link between these two subsets. Additionally, the present study demonstrates that CD209 is also expressed on monocyte derived macrophages (MoΦ). Functional analysis revealed each of these populations can take up and process antigens (Ags), present them to CD4 and CD8 T cells, and elicit a T-cell recall response. Thus, bDC, MoDC, and MoΦ pulsed with pathogens or candidate vaccine antigens can be used to study factors that modulate DC-driven T-cell priming and differentiation ex vivo.

Characterization and use of new monoclonal antibodies to CD11c, CD14, and CD163 to analyze the phenotypic complexity of ruminant monocyte subsets.

The sequencing of the bovine genome and development of mass spectrometry, in conjunction with flow cytometry (FC), have afforded an opportunity to complete the characterization of the specificity of monoclonal antibodies (mAbs), only partially characterized during previous international workshops focused on antibody development for livestock (1991, Leukocyte Antigens in Cattle, Sheep, and Goats; 1993, Leukocyte Antigens of Cattle and Sheep; 1996, Third Workshop on Ruminant Leukocyte Antigens). The objective of this study was to complete the characterization of twelve mAbs incompletely characterized during the workshops that reacted with molecules predominantly expressed on bovine monocytes and use them to provide further information on the phenotypic complexity of monocyte subsets in ruminants. Analysis revealed that the mAbs could be grouped into three clusters that recognize three different molecules: CD11c, CD14, and CD163. Following characterization, comparison of the patterns of expression of CD14 and CD163 with expression of CD16, CD172a, and CD209 revealed the mononuclear cell population is comprised of multiple subsets with differential expression of these molecules. Further analysis revealed the epitopes recognized by mAbs to CD14 and CD163 are conserved on orthologues in sheep and goats. In contrast to CD14 that is also expressed on sheep and goat granulocytes, CD163 is a definitive marker for their monocytes.