Wilhelm Gerner

University of Veterinary Medicine in Vienna, Wien, Vienna, Austria

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Publications (44)102.73 Total impact

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    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.
    Developmental and comparative immunology. 07/2014;
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    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.
    Developmental and comparative immunology 04/2014; · 3.29 Impact Factor
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    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.
    Parasite Immunology 04/2014; · 2.21 Impact Factor
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    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.
    Developmental and comparative immunology 02/2014; · 3.29 Impact Factor
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    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 01/2014; 9(6):e100619. · 3.53 Impact Factor
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    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.
    Veterinary Immunology and Immunopathology 01/2014; · 1.88 Impact Factor
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    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.
    Clinical and vaccine Immunology: CVI 08/2013; · 2.60 Impact Factor
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    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.
    Journal of veterinary science (Suwŏn-si, Korea) 06/2013; 14(2):207-214. · 0.89 Impact Factor
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    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. · 3.43 Impact Factor
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    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. · 3.43 Impact Factor
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    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).
    Journal of comparative pathology 02/2013; · 1.73 Impact Factor
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    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.
    Developmental and comparative immunology 01/2013; · 3.29 Impact Factor
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    ABSTRACT: Up to now for Swine Workshop Cluster 2 (SWC2) the orthologous human CD molecule was unknown. By use of the SWC2-specific mAb b30c7 and a retroviral cDNA expression library derived from stimulated porcine peripheral blood mononuclear cells we could identify SWC2 as porcine CD27. Phenotypic analyses of lymphocytes isolated from blood and lymphatic organs revealed that mature T cells in thymus and T cells in the periphery with a naïve phenotype were CD27(+). However, within CD8α(+) T helper and CD8α(+) γδ T cells also CD27(-) cells were present, indicating a down-regulation after antigen contact in vivo. B cells lacked CD27 expression, whereas NK cells expressed intermediate levels. Furthermore, plate-bound mAb b30c7 showed a costimulatory capacity on CD3-activated T cells for proliferation, IFN-γ and TNF-α production. Hence, our data indicate an important role of porcine CD27 for T-cell differentiation and activation as described for humans and mice.
    Developmental and comparative immunology 07/2012; 38(2):321-31. · 3.29 Impact Factor
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    ABSTRACT: The porcine major histocompatibility complex (MHC) harbors the highly polymorphic swine leukocyte antigen (SLA) class I and II gene clusters encoding glycoproteins that present antigenic peptides to T cells in the adaptive immune response. In Austria, the majority of commercial pigs are F (2) descendants of F (1) Large White/Landrace hybrids paired with Pietrain boars. Therefore, the repertoire of SLA alleles and haplotypes present in Pietrain pigs has an important influence on that of their descendants. In this study, we characterized the SLA class I ( SLA-1 , SLA-2 , SLA-3 ) and class II ( SLA-DRB1 , SLA-DQB1 , SLA-DQA ) genes of 27 purebred Pietrain pigs using a combination of the high-resolution sequence-based typing (SBT) method and a low-resolution (Lr) PCR-based method using allele-group, sequence-specific primers (PCR-SSP). A total of 15 class I and 13 class II haplotypes were identified in the studied cohort. The most common SLA class I haplotype Lr-43.0 ( SLA-1 *11XX- SLA-3 *04XX- SLA-2 *04XX) was identified in 11 animals with a frequency of 20%. For SLA class II, the most prevalent haplotype, Lr-0.14 [ SLA-DRB1 *0901- SLA-DQB1 *0801- SLA-DQA *03XX], was found in 14 animals with a frequency of 26%. Two class II haplotypes, tentatively designated as Lr-Pie-0.1 [ SLA-DRB1 *01XX/be01/ha04- SLA-DQB1 *05XX- SLA - DQA*blank] and Lr-Pie-0.2 [ SLA-DRB1 *06XX- SLA-DQB1 *03XX- SLA-DQA *03XX], appeared to be novel and have never been reported so far in other pig populations. We showed that SLA genotyping using PCR-SSP-based assays represents a rapid and cost-effective way to study SLA diversity in outbred commercial pigs and may facilitate the development of more effective vaccines or identification of disease-resistant pigs in the context of SLA antigens to improve overall swine health.
    Animal Genetics 05/2012; · 2.58 Impact Factor
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    ABSTRACT: So far little is known about natural killer (NK) cells in the pig due to the lack of NK cell-specific markers. In this study, we identified the activating receptor NKp46 (CD335) in swine with newly developed monoclonal antibodies (mAbs) for more detailed studies on NK cells in this species. The NKp46 mAbs showed a specific reactivity with a distinct population of perforin(+) CD2(+) CD3(-) CD8α(+) CD16(+) lymphocytes. In spleen and liver, an additional subset of CD8α(dim/-) lymphocytes with increased NKp46 expression was observed. Surprisingly, we could identify NKp46(-) cells with an NK cell phenotype in all animals analyzed. These lymphocytes showed comparable cytolytic activity against xenogeneic and allogeneic target cells as NKp46(+) NK cells. In contrast, NKp46(+) NK cells produced several fold higher levels of interferon-γ (IFN-γ) than the NKp46(-) cells after cytokine stimulation. Furthermore, an activation-dependent induction of NKp46 expression in formerly NKp46(-) cells after stimulation with interleukin-2 (IL-2), IL-12, and IL-18 could be shown. In summary, our data indicate that NKp46 is not expressed by all porcine NK cells and that NKp46 discriminates porcine NK cells differing in regard to cytokine production, which challenges the paradigm of NKp46 as a comprehensive marker for NK cells across different mammalian species.
    European Journal of Immunology 05/2012; 42(5):1261-71. · 4.97 Impact Factor
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    ABSTRACT: During the last decades for several species--e.g. swine--many mAb to leukocyte-specific molecules have been developed and clusters of differentiation corresponding to human CD could be established. However, for a significant amount of the raised mAb the corresponding antigens were not characterized on the molecular level and therefore preliminary clusters--in swine so-called Swine workshop clusters (SWC)--were established. These clusters contain antigens with currently no obvious orthologs to human leukocyte differentiation antigens. In this study, we describe the generation of a eukaryotic cDNA expression library from in vitro activated porcine peripheral blood mononuclear cells. Screening of this library with an antibody recognizing SWC1 enabled isolation and sequencing of cDNAs coding for the porcine SWC1 molecule. A BLAST search of the obtained sequence revealed that SWC1 is the orthologous molecule of human CD52. Therefore, our study provides the basis for comparative studies on the role of CD52 in different mammalian species. In addition, the established cDNA library can be used for investigation of additional SWC-defined molecules.
    Veterinary Immunology and Immunopathology 03/2012; 146(1):27-34. · 1.88 Impact Factor
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    ABSTRACT: Fatty acids are essential for immune cell function. Maternal dietary fatty acid supply influences body fat composition of their offspring. As a first step to study immunonutritional interactions at an early age of pigs, four sows were fed a diet containing sunflower oil or oil from seal blubber during pregnancy and lactation. Corresponding piglets were sacrificed at three consecutive time points in the suckling period and their mesenteric lymph nodes and spleen were analysed by gas chromatography for levels of fatty acid. At the same time mononuclear cells of these organs and of the intestinal lymphoid tissue from the jejunum were isolated and subpopulations characterised by flow cytometry. Levels of fatty acids from the lymphatic organs of the piglets were significantly influenced by the maternal diet. The concentration of the fatty acids 20:5n-3, 22:5n-3 and 22:6n-3 were higher in the spleen and mesenteric lymph node of piglets suckled to sows of the test diet. Additionally, suckling time affected the levels of some long chain polyunsaturated fatty acids. Dietary effects were seen on some subpopulations including CD4-CD8alpha+ lymphocytes of the mesenteric lymph nodes and CD4+CD8alpha+ lymphocytes of the lamina propria, which were higher in the group fed seal blubber oil. The levels of CD21+ B-cells were higher in the group fed sunflower oil. The results indicate that the maternal diet and suckling time affect the fatty acid status of the investigated lymphatic tissues of piglets, but may have minor effects on the investigated lymphocyte subpopulations.
    Archives of animal nutrition 10/2011; 65(5):341-53. · 1.10 Impact Factor
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    ABSTRACT: Regulatory T cells (Tregs) are known in humans and mice from last 15 years, and several studies led to a detailed knowledge on their phenotype, functions, and role in various immune reactions. In swine, the existence of Tregs was first demonstrated in 2008 and research is still at the beginning. Nevertheless, basic information regarding phenotype, mechanisms and targets of suppression, as well as implications in transplantation and some diseases are available. Purpose of this review is to give a brief summary of the current knowledge about porcine Tregs.
    Veterinary Immunology and Immunopathology 06/2011; 148(1-2):136-8. · 1.88 Impact Factor
  • Tobias Käser, Wilhelm Gerner, Armin Saalmüller
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    ABSTRACT: Tregs are known for their suppressive capacity on various immune reactions. In swine, existence as well as suppressive activity of Foxp3(+) Tregs could be demonstrated but detailed functional investigations are lacking. Therefore, we analysed the functional properties of porcine Tregs. We observed that besides TCR stimulation Tregs require IL-2 for activation. Furthermore, we investigated the following mechanisms of suppression: (i) cell-cell contact dependency, (ii) production of soluble suppressive factors and (iii) competition for growth factors. Our experiments revealed that suppression by porcine Tregs is abrogated by blocking cell-cell contact or by supplementing excessive amounts of IL-2. Additionally it could be shown that porcine Tregs produce immunosuppressive IL-10. Thereby, we demonstrated that porcine Tregs can use all main mechanisms of suppression mentioned above. Further investigations on the suppressive activity of Tregs using CFSE proliferation assays demonstrated that suppression affects T-helper cells as well as cytotoxic T lymphocytes and TCR-γδ T cells.
    Developmental and comparative immunology 04/2011; 35(11):1166-72. · 3.29 Impact Factor
  • Cytokine 01/2011; 56(1):90-90. · 2.52 Impact Factor