Tobias Käser

Porcine T lymphocytes and NK cells - an update.

Natural killer (NK) cells represent an important cell population of the innate immune system with the ability to attack spontaneously pathogen-infected and malignant body cells as well as to produce immune-regulatory cytokines. T lymphocytes belong to the adaptive immune system and perform a wide array of functions in immune regulation, inflammation and protective immune responses. In this review we summarize the current knowledge about the phenotype and functional characteristics of these two cell populations in swine. Porcine NK cells can be distinguished from T cells by the complex phenotype perforin+ CD3(-)CD4(-)CD5(-)CD6(-)CD8alpha+CD8beta(-)CD11b+CD16+. Investigations so far show that these cells have the capacity to lyse virus-infected target cells and respond to various regulatory cytokines. Such cytokines can induce interferon-gamma (IFN-gamma) production in porcine NK cells, as well as the up-regulation of effector/activation molecules like perforin and CD25. Porcine T cells can be divided into a number of subpopulations, including a prominent fraction of T cells expressing T-cell receptors (TCR) with gammadelta-chains. Like TCR-alphabeta T cells, these TCR-gammadelta T cells can express CD8alpha and MHC class II, two molecules which in swine seem to be correlated with an activation status of T cells. Functional properties of these cells seem to include cytolytic activity as well as antigen presentation; however, both aspects require further investigation. Like in other species, TCR-alphabeta T cells in swine comprise MHC class-I restricted cytolytic T cells, T-helper cells and recently identified regulatory T cells. We summarize data on the phenotype and function of these cells including memory cell formation. Current knowledge suggests that MHC class-I restricted cytolytic T cells can be identified by the expression of CD8alphabeta heterodimers. T-helper cells express CD4 as well as other activation-related markers, including CD8alpha, MHC class II and CD45RC. Porcine regulatory T cells have a phenotype similar to that of mouse and humans: CD4+CD25+Foxp3+. First results indicate that these cells can suppress proliferation of other T cells and produce IL-10. Finally, the abundant expression of swine-specific activation markers CD8alpha and MHC class II on T cells and NK cells is discussed in more detail.

The porcine innate immune system: an update.

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.

Detection of Foxp3 protein expression in porcine T lymphocytes.

Regulatory T cells (Tregs) are potent regulators of various immune reactions. Due to the lack of Treg-specific markers their analysis had often been challenging until the discovery of the transcription factor Forkhead-box p3 (Foxp3) which serves as this highly demanded marker. So far, antibodies staining human and murine Foxp3 have been developed. This study describes the analysis of four commercially available anti-Foxp3 antibodies for reactivity with their specific antigen in cells derived from porcine lymphoid tissues. One out of the four antibodies showed selective reactivity with porcine CD25(+) T lymphocytes. The intracellular antigen was expressed on a small subset of CD25(dim) cells and the majority of the CD25(high) positive T-cell subpopulation. Intracellular antigen positive cells showed a heterogeneous expression of other leukocyte differentiation antigens. The majority belonged to the CD4(+)CD8(+) T-lymphocyte subpopulation, but were also found in the CD4(+)CD8(-) subpopulation. Another small minority was included in the CD4(-)CD8(+) T-lymphocyte subpopulation. Additionally, a small fraction of the putative Foxp3(+) cells showed an expression of MHC-II molecules. These staining patterns in three and four colour flow cytometry analyses indicated that the cells detected by a rat anti-mouse/rat-Foxp3 antibody expressed the porcine Foxp3. The expression of the putative Foxp3 protein in distinct leukocyte subsets was confirmed by molecular analysis of Foxp3 mRNA expression. Using Western blot analysis specific protein bands could only be detected in fractions that also exhibited the corresponding Foxp3 mRNA expression. These experiments also revealed that the antibody recognized a single chain protein with a molecular mass of about 45kDA similar to Foxp3 described for other species. In summary, these data strongly indicate the reactivity of this antibody with porcine Foxp3. Thereby, this rat anti-mouse/rat Foxp3 antibody presents a powerful tool for the identification of porcine regulatory T cells.

Phenotypic maturation of porcine NK- and T-cell subsets

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 19-25 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.

Detection of intracellular antigens in porcine PBMC by flow cytometry: A comparison of fixation and permeabilisation reagents.

The staining of intracellular antigens is a standard method in flow cytometry but still only occasionally used for immunologic studies in veterinary species. In order to improve information about suitable fixation/permeabilisation protocols in porcine lymphocytes we tested six fixation/permeabilisation variants for the staining of different intracellular antigens: CD79alpha, perforin, interferon-gamma and the cell cycle associated protein Ki-67. For the fixation/permeabilisation procedure, four commercial kits from BD Biosciences, ADG Bio Research, Dako and AbD Serotec were tested in comparison to non-commercial fixation solutions of formaldehyde together with either saponin or Tween-20 for permeabilisation. Perforin and Ki-67 expressing cells were optimally stained only with permeabilisation reagents containing saponin, which includes the BD kit. In contrast, labelling of CD79alpha and IFN-gamma was possible with all investigated fixation/permeabilisation variants; however, here saponin and Tween protocols induced a higher degree of unspecific binding. Also, scatter properties of cells treated with saponin were worse compared to samples prepared with the kits from ADG, Dako and Serotec. Simultaneous staining of cell surface antigens was not negatively affected by any of the fixation/permeabilisation variants. A universal recommendation for a single fixation/permeabilisation strategy could not be deduced from our data but our work provides a useful guideline for optimal staining of common intracellular antigens analyzed in swine lymphocytes.

Porcine regulatory T cells: mechanisms and T-cell targets of suppression.

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.

Molecular characterisation of porcine Forkhead-box p3 (Foxp3).

In swine the phenotypical identification of regulatory T cells (Tregs) was limited so far to the surface expression of CD4 and CD25. However, with the discovery of the Treg-specific transcription factor forkhead-box p3 (Foxp3) in mice and humans a powerful marker for the identification of Tregs is available. Recently, we published data on a murine anti-mouse/rat Foxp3 antibody (FJK-16s) showing cross-reactivity with the putative porcine Foxp3 protein in lymphoid cells but the final proof for the specific cross-reactivity of this antibody was missing. By performing RACE-experiments, we have sequenced the entire porcine Foxp3 cDNA which is 1296 nucleotides in length and codes for a polypeptide of 432 amino acids. The porcine Foxp3 nucleotide and amino acid sequences show high homology to all known orthologues from other mammals, with the greatest homology with the bovine sequence. To demonstrate the specificity of the FJK-16s antibody for the porcine Foxp3 protein, HEK293T cells were transfected with porcine Foxp3 containing the FJK-16s-specific binding region and the expression of the epitope was identified by immuno-staining. In conclusion, this study represents the final proof for the specificity of the murine FJK-16s antibody for the porcine Foxp3 homologue and therefore strengthens future work on porcine Tregs.

Porcine T-helper and regulatory T cells exhibit versatile mRNA expression capabilities for cytokines and co-stimulatory molecules

T-helper (TH) and regulatory T cells (Tregs) are important modulators of immune responses. Aim of this study was to analyse their expression potential for cytokines and other immune-relevant molecules. Therefore, porcine PBMC, CD4(-) cells, CD4(+)CD25(-) resting, CD4(+)CD25(dim) activated TH cells, and CD4(+)CD25(high) Tregs were analysed on their mRNA expression potential ex vivo or after in vitro stimulation with CD3 and IL-2 by RT-qPCR. In vitro stimulation led to an increased production of pro-inflammatory (IL-6, TNFα) and TH (IL-2, IL-4, IL-17, IFN-γ) cytokines and a diverse production of immunosuppressive cytokines (IL-10 and TGF-β) in PBMC, CD4(-), and CD4(+) cells. Resting and activated TH cells showed an increased expression of various immune-modulatory molecules indicating that porcine TH cells possess distinct immunological skills in order to react on the actual immune situation. In contrast, Tregs appear to fulfil mainly immunosuppressive functions characterized by increased production of IL-10, IL-35, CD40L, and CD25.

Current knowledge on porcine regulatory T cells.

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.

Human Placental Alkaline Phosphatase as a Tracking Marker for Bone Marrow Mesenchymal Stem Cells

Abstract Currently, adult mesenchymal stem cells (MSCs) are being evaluated for a wide variety of therapeutic approaches. It has been suggested that MSCs possess regenerative properties when implanted or injected into damaged tissue. However, the efficacy of MSCs in several of the proposed treatments is still controversial. To further explore the therapeutic potential of these cells, it is necessary to trace the fate of individual donor or manipulated cells in the host organism. Recent studies from our lab showed that human placental alkaline phosphatase (hPLAP) is a marker with great potential for cell tracking. However, a potential concern related to this marker is its enzymatic activity, which might alter cell behavior and differentiation by hydrolyzing substrates in the extracellular space and thereby changing the cellular microenvironment. Therefore, the aim of this study was to characterize bone marrow MSCs (BMSCs) derived from hPLAP-transgenic inbred F344 rats (hPLAP-tg) in comparison to wild type (wt) BMSCs. Here, we show that BMSCs from wt and hPLAP-tg donors are indistinguishable in terms of cell morphology, viability, adhesion, immune phenotype, and proliferation as well as in their differentiation capacity over six passages. The expression of the hPLAP marker enzyme was not impaired by extensive in vitro cultivation, osteogenic, adipogenic, or chondrogenic differentiation, or seeding onto two- or three-dimensional biomaterials. Thus, our study underscores the utility of genetically labeled BMSCs isolated from hPLAP-tg donors for long-term tracking of the fate of transplanted MSCs in regenerative therapies.