[Show abstract][Hide abstract] ABSTRACT: In vitro generated monocyte-derived dendritic cells (moDCs) have frequently been used to study the influence of porcine reproductive and respiratory syndrome virus (PRRSV) infection on antigen presenting cells. However, obtained results have often been conflicting in regard to expression of co-stimulatory molecules and interaction with T cells. In this study we performed a detailed phenotypic characterisation of PRRSV-infected moDCs and non-infected moDCs. For CD163 and CD169, which are involved in PRRSV-entry into host cells, our results show that prior to infection porcine moDCs express high levels of CD163 but only very low levels for CD169. Following infection with either PRRSV-1 or PRRSV-2 strains after 24 h, PRRSV-nucleoprotein (N-protein)+ and N-protein− moDCs derived from the same microculture were analyzed for expression of swine leukocyte antigen-DR (SLA-DR) and CD80/86. N-protein+ moDCs consistently expressed higher levels of SLA-DR and CD80/86 compared to N-protein− moDCs. We also investigated the influence of PRRSV-infected moDCs on proliferation and frequency of Foxp3+ regulatory T cells present within CD4+ T cells in in vitro co-cultures. Neither CD3-stimulated nor unstimulated CD4+ T cells showed differences in regard to proliferation and frequency of Foxp3+ T cells following co-cultivation with either PRRSV-1 or PRRSV-2 infected moDCs. Our results suggest that a more detailed characterisation of PRRSV-infected moDCs will lead to more consistent results across different laboratories and PRRSV strains as indicated by the major differences in SLA-DR and CD80/86 expression between PRRSV-infected and non-infected moDCs present in the same microculture.
Veterinary Research 05/2015; 46(1). DOI:10.1186/s13567-015-0186-z · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although swine are natural hosts for influenza A viruses, the porcine T-cell response to swine influenza A virus (FLUAVsw) infection has been poorly characterized so far. We have studied Ki-67 expression and FLUAVsw-specific production of IFN-γ, TNF-α and IL-2 in CD4+ and CD8β+ T cells isolated from piglets that had been intratracheally infected with a H1N2 FLUAVsw isolate. IFN-γ+TNF-α+IL-2+ multifunctional CD4+ T cells were present in the blood of all infected animals at one or two weeks after primary infection and their frequency increased in four out of six animals after homologous secondary infection. These cells produced higher amounts of IFN-γ, TNF-α and IL-2 than did CD4+ T cells that only produced a single cytokine. The vast majority of cytokine-producing CD4+ T cells expressed CD8α, a marker associated with activation and memory formation in porcine CD4+ T cells. Analysis of CD27 expression suggested that FLUAVsw-specific CD4+ T cells included both central memory and effector memory populations. Three out of six animals showed a strong increase of Ki-67+perforin+ CD8β+ T cells in blood one week post infection. Blood-derived FLUAVsw-specific CD8β+ T cells could be identified after an in vitro expansion phase and were multifunctional in terms of CD107a expression and co-production of IFN-γ and TNF-α. These data show that multifunctional T cells are generated in response to FLUAVsw infection of pigs, supporting the idea that T cells contribute to the efficient control of infection.
Electronic supplementary material
The online version of this article (doi:10.1186/s13567-015-0182-3) contains supplementary material, which is available to authorized users.
Veterinary Research 05/2015; 46(1). DOI:10.1186/s13567-015-0182-3 · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Porcine circovirus type 2 (PCV2) is one of the economically most important pathogens for swine production worldwide. Vaccination is a powerful tool to control porcine circovirus diseases (PCVD). However, it is not fully understood how PCV2 vaccination interacts with the porcine immune system. Especially knowledge on the cellular immune response against PCV2 is sparse. In this study we analysed antigen-specific T cell responses against PCV2 in a controlled vaccination and infection experiment. We focused on the ability of CD4+ T cells to produce cytokines using multicolour flow cytometry (FCM). Vaccination with a PCV2 subunit vaccine (Ingelvac CircoFLEX®) induced PCV2-specific antibodies only in five out of 12 animals. Conversely, vaccine-antigen specific CD4+ T cells which simultaneously produced IFN-γ and TNF-α and had a phenotype of central and effector memory T cells were detected in all vaccinated piglets. After challenge, seroconversion occurred earlier in vaccinated and infected pigs compared to the non-vaccinated, infected group. Vaccinated pigs were fully protected against viremia after subsequent challenge. Therefore, our data suggests that the induction of IFN-γ/TNF-α co-producing T cells by PCV2 vaccination may serve as a potential correlate of protection for this type of vaccine.
Electronic supplementary material
The online version of this article (doi:10.1186/s13567-015-0157-4) contains supplementary material, which is available to authorized users.
Veterinary Research 03/2015; 46(1). DOI:10.1186/s13567-015-0157-4 · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The immune response of piglets to a vaccination against the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) was tested after feeding different feed additives. The trial included four groups of ten piglets and lasted 39 days. Feed additives were added to a control diet (C). Tested substances included an autolyzed yeast preparation from Saccharomyces cerevisiae (YA) at 0.2%, dried bovine colostrum (CM) at 0.9% and the probiotic B. cereus var. toyoi NCIMB 40112 (BC) at 109 colony forming units/kg diet. Three-week-old piglets were vaccinated against PRRS-Virus, blood samples were taken before vaccination and 7, 21, and 35 days after vaccination for complete blood counts, flow cytometry for phenotyping of different lymphocyte subsets in peripheral blood, measurement of specific IgG antibodies and ELISpot assay to detect specific interferon γ-producing lymphocytes.
[Show abstract][Hide abstract] ABSTRACT: In lagomorphs, lymphocyte subset distributions and the importance of CD4+ T cell levels has so far only been considered in the frame of rabbit disease models. In this study, the first assessment of CD4+ T lymphocytes in peripheral blood cells in brown hares (Lepus europaeus L., 1758), a further leporid species using a cross-reactive rabbit anti-CD4 antibody in flow cytometry, is presented. In addition, the entire coding region of the hare CD4 gene (1,380 bp) coding for a polypeptide of 459 amino acids has been sequenced. Using generalized least squares fitting by maximum likelihood (GLS) test, significantly (p = 0.0095) higher CD4+ T cell frequencies in males than in females and significantly (p = 0.0001) higher frequencies for leverets (younger than 2 months of age) than for subadult and adult (older than 7 months of age) individuals were detected. No significant age influence, however, was found for subadult and adult hares. The study is particularly meant to provide a first step in establishing a toolbox for the assessment of the immune response in this leporid species.
[Show abstract][Hide abstract] ABSTRACT: Background
Many dogs suffering from inflammatory bowel disease (IBD) are presented to veterinary clinics. These patients are diagnosed based on a history of chronic gastrointestinal signs and biopsy-confirmed histopathologic intestinal inflammation. Intestinal intraepithelial lymphocytes (IEL) are part of the first line of defense in the gastrointestinal immune system. Alterations in IEL subsets may play a role in the pathogenesis of IBD.HypothesisThe aim of this study was to characterize the phenotypes of IEL in dogs with IBD compared with healthy control dogs.AnimalsIntestinal intraepithelial lymphocytes subpopulations of control dogs (n = 5) obtained from endoscopic biopsies (EB) were compared to those obtained from full thickness biopsies (FTB) on the same day. In addition, the phenotypes of IEL from FTB of control dogs (n = 10) were compared with EB of IBD dogs (n = 10). Each participant was scored clinically using the canine inflammatory bowel disease activity index (CIBDAI), and all samples were graded histopathologically. Three-color flow cytometry of isolated IEL was performed using monoclonal antibodies against T- and B-lymphocyte subpopulations.ResultsNo significant differences in the composition of IEL subpopulations were found in control dogs based on method of biopsy. The IBD dogs had significantly higher CIBDAI and histopathologic scores compared with control dogs and their IEL contained a significantly higher frequency TCRγδ T-cells.Conclusions and Clinical ImportanceEndoscopic biopsies provide suitable samples for 3-color flow cytometry when studying canine intestinal IEL and IBD patients show significant changes of major T-cell subsets compared to healthy control dogs.
Journal of Veterinary Internal Medicine 09/2014; 28(6). DOI:10.1111/jvim.12456 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: γδ T cells are highly abundant in the blood and spleen of pigs but little is known about their functional differentiation. In this study the potential of the type-1 polarizing cytokines IL-12 and IL-18 in combination with IL-2 and Concanavalin A (ConA) to stimulate porcine γδ T cells was investigated. Stimulation of purified γδ T cells with ConA and IL-2 induced a strong proliferation of CD2(-) γδ T cells, whereas additional stimulation with IL-12 and IL-18 caused a stronger proliferation of CD2(+) γδ T cells. IFN-γ could only be detected in supernatants of γδ T-cell cultures supplemented with IL-12 and IL-18. Experiments with sorted CD2/SWC5-defined γδ T-cell subsets revealed that CD2(+)SWC5(-) γδ T cells are the main producers of IFN-γ following stimulation with IL-2/IL-12/IL-18. Additional stimulation with ConA led to an upregulation of CD2 within the CD2(-) γδ T cell subsets, indicating a previously unnoticed plasticity of CD2-defined γδ T cell subsets.
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells are important players in the innate immune response against influenza A virus and the activating receptor NKp46, which binds hemagglutinin on the surface of infected cells, has been assigned a role in this context. As pigs are natural hosts for influenza A viruses and pigs possess both NKp46- and NKp46+ NK cells, they represent a good animal model for studying the role of the NKp46 receptor during influenza. We explored the role of NK cells in piglets experimentally infected with 2009 pandemic H1N1 influenza virus by flow cytometric analyses of cells isolated from blood and lung tissue and by immunostaining of lung tissue sections. The number of NKp46+ NK cells was reduced while NKp46- NK cells remained unaltered in the blood 1-3 days after infection. In the lungs, the intensity of NKp46 expression on NK cells was increased during the first 3 days, and areas where influenza virus nucleoprotein was detected were associated with increased numbers of NKp46+ NK cells when compared to uninfected areas. NKp46+ NK cells in the lung were neither found to be infected with influenza virus nor to be undergoing apoptosis. The binding of porcine NKp46 to influenza virus infected cells was verified in an in vitro assay. These data support the involvement of porcine NKp46+ NK cells in the local immune response against influenza virus.
PLoS ONE 06/2014; 9(6):e100619. DOI:10.1371/journal.pone.0100619 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Over the last few years, we have seen an increasing interest and demand for pigs in biomedical research. Domestic pigs (Sus scrofa domesticus) are closely related to humans in terms of their anatomy, genetics, and physiology, and often are the model of choice for the assessment of novel vaccines and therapeutics in a preclinical stage. However, the pig as a model has much more to offer, and can serve as a model for many biomedical applications including aging research, medical imaging, and pharmaceutical studies to name a few. In this review, we will provide an overview of the innate immune system in pigs, describe its anatomical and physiological key features, and discuss the key players involved. In particular, we compare the porcine innate immune system to that of humans, and emphasize on the importance of the pig as model for human disease.
[Show abstract][Hide abstract] ABSTRACT: Infections of neonatal piglets with Cystoisospora suis are responsible for substantial economic losses in pig production. To investigate kinetics of T-cell populations which are possibly involved in this infection, lymphocytes from blood, spleen, mesenteric lymph nodes and the jejunal mucosa of infected and non-infected piglets were investigated by flow cytometry and immunohistochemistry at five time points during the acute phase of primary infection. Additionally, mRNA expression levels of pattern recognition receptors and immunomodulatory cytokines in the jejunum were investigated. T-cell receptor-γδ+ T cells were found to be increased in the gut mucosa four days after infection and were most likely involved in the primary local immune response. Five to eleven days later cytotoxic T-cells peaked in this location which was preceded by an expansion of this lymphocyte population in the mesenteric lymph nodes. In intestines of infected piglets mRNA expression of TLR-2, NOD2 and TNF-α were significantly up-regulated, suggesting an involvement in parasite recognition, immune response, and possibly also in immunopathology. Taken together, this study identifies cellular and molecular players involved in the early immune responses against Cystoisospora suis but their precise role in the pathogenesis and control of this neonatal disease requires further investigation.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: γδ T cells are a remarkably prominent T-cell subset in swine with a high prevalence in blood. Phenotypic analyses in this study showed that CD2(-) γδ T cells in their vast majority had a CD8α(-)SLA-DR(-)CD27(+) phenotype. CD2(+) γδ T cells dominated in spleen and lymph nodes and had a more heterogeneous phenotype. CD8α(+)SLA-DR(-)CD27(+) γδ T cells prevailed in blood, spleen and lymph nodes whereas in liver a CD8α(+)SLA-DR(+)CD27(-) phenotype dominated, indicating an enrichment of terminally differentiated γδ T cells. γδ T cells were also investigated for their potential to produce IFN-γ, TNF-α and IL-17A. Within CD2(+) γδ T cells, IFN-γ and TNF-α single-producers as well as IFN-γ/TNF-α double-producers dominated, which had a CD8α(+)CD27(+/-) phenotype. IL-17A-producing γδ T cells were only found within CD2(-) γδ T cells, mostly co-produced TNF-α and had a rare CD8α(+)CD27(-) phenotype. However, quantitatively TNF-α single-producers strongly dominated within CD2(-) γδ T cells. In summary, our data identifies CD2 and CD8α as important molecules correlating with functional differentiation.
[Show abstract][Hide abstract] ABSTRACT: Vaccination with live attenuated classical swine fever virus (CSFV) induces solid protection after only five days, which has been associated with virus-specific T cell IFN-γ responses. In this study, we employed flow cytometry to characterise T cell responses following vaccination and subsequent challenge infections with virulent CSFV. The CD3(+)CD4(-)CD8(hi) T cell population was the first and major source of CSFV-specific IFN-γ. A proportion of these cells showed evidence for cytotoxicity, as evidenced by CD107a mobilisation, and co-expressed TNF-α. To assess the durability and recall of these responses, a second experiment was conducted where vaccinated animals were challenged with virulent CSFV after five and again after a further 28 days. While virus-specific CD4 T cell (CD3(+)CD4(+)CD8α(+)) responses were detected, the dominant response was again from the CD8 T cell population, with the highest numbers of these cells being detected 14 and 7 days after the primary and secondary challenges, respectively. These CD8 T cells were further characterised as CD44(hi)CD62L(-) and expressed variable levels of CD25 and CD27, indicative of a mixed effector and effector memory phenotype. The majority of virus-specific IFN-γ(+) CD8 T cells isolated at the peaks of the response after each challenge displayed CD107a on their surface and subpopulations were identified that co-expressed TNF-α and IL-2. While it is hoped that this data will aid the rational design and/or evaluation of next-generation marker CSFV vaccines, the novel flow cytometric panels developed should also be of value in the study of porcine T cell responses to other pathogens/vaccines.
[Show abstract][Hide abstract] ABSTRACT: To evaluate radiosensitivity and the effects of radiation on the expression of vascular endothelial growth factor (VEGF) and VEGF receptors in the canine oral melanoma cell line, TLM 1, cells were irradiated with doses of 0, 2, 4, 6, 8 and 10 Gray (Gy). Survival rates were then determined by a MTT assay, while vascular endothelial growth factor receptor (VEGFR)-1 and -2 expression was measured by flow cytometry and apoptotic cell death rates were investigated using an Annexin assay. Additionally, a commercially available canine VEGF ELISA kit was used to measure VEGF. Radiosensitivity was detected in TLM 1 cells, and mitotic and apoptotic cell death was found to occur in a radiation dose dependent manner. VEGF was secreted constitutively and significant up-regulation was observed in the 8 and 10 Gy irradiated cells. In addition, a minor portion of TLM 1 cells expressed vascular endothelial growth factor receptor (VEGFR)-1 intracellularly. VEGFR-2 was detected in the cytoplasm and was down-regulated following radiation with increasing dosages. In TLM 1 cells, apoptosis plays an important role in radiation induced cell death. It has also been suggested that the significantly higher VEGF production in the 8 and 10 Gy group could lead to tumour resistance.
[Show abstract][Hide abstract] ABSTRACT: Differentiation of porcine T helper cells is still poorly investigated, partly due to a lack of monoclonal antibodies (mAbs) specific for molecules involved in this process. Recently, we identified a mAb specific for porcine CD27 and showed that CD27 is expressed by all naive CD8alpha- T helper cells but divides CD8alpha+ T helper cells into a CD27+ and a CD27- subset. In the present study, detailed phenotypical and functional analyses of these T-helper cell subpopulations were performed. Naive CD8alpha-CD27+ T helper cells predominantly resided in various lymph nodes, whereas higher proportions of CD8alpha+CD27+ and CD8alpha+CD27- T helper cells were found in blood, spleen and liver. Both CD8alpha+CD27+ and CD8alpha+CD27- T helper cells were capable of producing IFN-gamma upon in vitro polyclonal stimulation and antigen-specific restimulation. Experiments with sorted CD8alpha-CD27+, CD8alpha+CD27+ and CD8alpha+CD27- T-helper cell subsets following polyclonal stimulation revealed the lowest proliferative response but the highest ability for IFN-gamma and TNF-alpha production in the CD8alpha+CD27- subset. Therefore, these cells resembled terminally differentiated effector memory cells as described in human. This was supported by analyses of CCR7 and CD62L expression. CD8alpha+CD27- T helper cells were mostly CCR7- and had considerably reduced CD62L mRNA levels. In contrast, expression of both homing-receptors was increased on CD8alpha+CD27+ T helper cells, which also had a proliferation rate similar to naive CD8alpha-CD27+ T helper cells and showed intermediate levels of cytokine production. Therefore, similar to human, CD8alpha+CD27+ T helper cells displayed a phenotype and functional properties of central memory cells.
Veterinary Research 03/2013; 44(1):18. DOI:10.1186/1297-9716-44-18 · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Natural Killer (NK) cells play a crucial role in the early phase of immune responses against various pathogens. In swine so far only little information about this lymphocyte population exists. Phenotypical analyses with newly developed monoclonal antibodies (mAbs) against porcine NKp46 recently revealed that in blood NKp46- and NKp46+ cells with NK phenotype exist with comparable cytotoxic properties. In spleen a third NKp46-defined population with NK phenotype was observed that was characterised by a low to negative CD8alpha and increased NKp46 expression. In the current study it is shown that this NKp46high phenotype was correlated with an increased expression of CD16 and CD27 compared to the CD8alpha+NKp46- and NKp46+ NK-cell subsets in spleen and blood. Additionally NKp46high NK cells expressed elevated levels of the chemokine receptor CXCR3 on mRNA level. Functional analyses revealed that splenic NKp46high NK cells produced much higher levels of Interferon-gamma and Tumor Necrosis Factor-alpha upon stimulation with cytokines or phorbol-12-myristate-13-acetate/Ionomycin compared to the other two subsets. Furthermore, cross-linking of NKp46 by NKp46-specific mAbs led to a superior CD107a expression in the NKp46high NK cells, thus indicating a higher cytolytic capacity of this subset. Therefore porcine splenic NKp46high NK cells represent a highly activated subset of NK cells and may play a profound role in the immune surveillance of this organ.
Veterinary Research 03/2013; 44(1):13. DOI:10.1186/1297-9716-44-13 · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to characterize histologically and immunohistochemically the lung lesions developing in growing pigs, 10 and 21 days after experimental challenge with a field strain of porcine reproductive and respiratory syndrome virus (PRRSV). Lung lesions were scored for (1) pneumocyte hypertrophy and hyperplasia, (2) septal mononuclear infiltration, (3) intra-alveolar necrotic debris, (4) intra-alveolar inflammatory cell accumulation and (5) perivascular inflammatory cell accumulation. Immunohistochemistry was performed using antibodies specific for cytokeratin, Ki67, thyroid transcription factor (TTF)-1, the myelomonocytic marker MAC387 and PRRSV. Anti-TTF-1 identified type II pneumocytes and there was marked proliferation of these cells compared with control lung (P <0.05). Anti-cytokeratin labelled type I and II pneumocytes as well as bronchial epithelial cells; however, this labelling was not suitable for cell counting purposes. There was a correlation between lesion severity and the number of cells expressing Ki67 (P <0.05).
[Show abstract][Hide abstract] ABSTRACT: Detailed information concerning the development of the immune system in young pigs is still rudimental. In the present study, we analyzed changes in phenotype and absolute numbers of natural killer cells, γδ T cells, T helper cells, regulatory T cells and cytolytic T cells in the blood of pigs from birth to six months of age. For each lymphocyte subpopulation, a combination of lineage and differentiation markers was investigated by six-color flow cytometry. Major findings were: i) absolute numbers of γδ T cells strongly increased from birth until 19 to 25 weeks of age, indicating an important role for these cells during adolescence; ii) phenotype of T helper cells changed over time from CD8α(-)SLA-DR(-)CD27(+) towards CD8α(+)SLA-DR(+)CD27(-) but CD45RC(-) T helper cells were found immediately after birth, therefore questioning the role of this marker for the identification of T-helper memory cells; iii) for cytolytic T cells, putative phenotypes for early effector (CD3(+)CD8αβ(+)perforin(+)CD27(dim)) and late effector or memory cells (CD3(+)CD8αβ(+)perforin(+)CD27(-)) could be identified.