Porcine CD27: Identification, expression and functional aspects in lymphocyte subsets in swine

Developmental and Comparative Immunology 38 (2012) 321–331

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Porcine CD27: Identification, expression and functional aspects in lymphocyte subsets in swine Katharina Reutner a, Judith Leitner b, Sabine E. Essler a, Kirsti Witter c, Martina Patzl a, Peter Steinberger b, Armin Saalmüller a, Wilhelm Gerner a,⇑ a b c

Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Borschkegasse 8a, 1090 Vienna, Austria Institute of Anatomy, Histology and Embryology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria

a r t i c l e

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Article history: Received 8 May 2012 Revised 26 June 2012 Accepted 29 June 2012 Available online 31 July 2012 Keywords: Swine SWC2 CD27 T-cell subsets NK cells T-cell costimulation

a b s t r a c t 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 CD8a+ T helper and CD8a+ cd 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-c and TNF-a production. Hence, our data indicate an important role of porcine CD27 for T-cell differentiation and activation as described for humans and mice. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Monoclonal antibodies (mAbs) with defined specificity are an indispensable part of the immunological toolbox. For swine, numerous mAbs were generated against leukocyte surface molecules and characterized in three workshops (Haverson et al., 2001; Lunney et al., 1994; Saalmüller, 1996). This work allowed the identification of porcine Clusters of Differentiation (CD) in analogy to the nomenclature used in human and mouse immunology. However, not for all antigens recognized by clustered mAbs the orthologous human CD molecule could be identified. Such antigens were designated as Swine Workshop Cluster (SWC) and differentiated by consecutive numbering. SWC2 was defined during the ‘‘1st International Swine Cluster of Differentiation Workshop’’ in 1992 (Saalmüller et al., 1994). At that time the molecule was described as a cell surface antigen with a molecular mass of 49–51 kDa and was recognized by two mAbs,

Abbreviations: CD, Cluster of Differentiation; cDNA, complementary DNA; FACS, fluorescence activated cell sorting; FCM, flow cytometry; mAb, monoclonal antibody; PBMCs, peripheral blood mononuclear cells; PE, phycoerythrin; SWC, Swine Workshop Cluster; TNFR, tumor necrosis factor receptor; TRAF, TNF receptor associated factor. ⇑ Corresponding author. Tel.: +43 1 25077 2753; fax: +43 1 25077 2791. E-mail address: [email protected] (W. Gerner). 0145-305X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dci.2012.06.011

b30c7 (Saalmüller, Tübingen, Germany) and PG124A (Davis, Pullman, WA). Early work indicated that SWC2 was solely expressed on T cells and NK cells, whereas no expression was observed on B cells and monocytes (Saalmüller, unpublished results). Another study reported that SWC2 was expressed with lower density on activated T cells than on resting T cells (Sinkora et al., 2001). In this study it was also described that T cells proliferating in response to viral recall antigen appeared to lack SWC2. These findings pointed towards a functional relevance of this molecule but since the orthologous human CD molecule was still unknown, the interpretation of these early data was hampered. Recently, our laboratory established a retroviral complementary DNA (cDNA) expression library generated from in vitro activated porcine peripheral blood mononuclear cells (PBMCs). By the use of this library we could identify SWC1 as the orthologous molecule of human CD52 (Leitner et al., 2012). In the present study, this approach was also used and resulted in the identification of CD27 as orthologous molecule of SWC2. CD27 belongs to the tumor necrosis factor receptor (TNFR) super-family which, among others, comprises costimulatory molecules for T-cell activation. As is known from humans and mice, CD27 expression starts on early thymocytes and the molecule is expressed on naïve CD4+ and CD8+ T cells in the periphery. Costimulation of CD27 potentiates proliferation,

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survival as well as cytokine production of TcR-activated T cells. Nonetheless, following antigen-specific stimulation, CD27 expression undergoes down-regulation after clonal expansion and during differentiation towards effector cells (reviewed in Nolte et al., 2009). In swine, until now, discrimination of antigen-experienced T cells for the identification of functional properties is rather insufficient since only a restricted panel of specific antibodies for cell surface markers such as CD25, CD45RC and MHC class II is currently available. Nevertheless, the peculiar extrathymic up-regulation of CD8a expression on porcine T helper cells and cd T cells turned out to be in correlation with T-cell activation and maturation (Gerner et al., 2009; Saalmüller et al., 2002). Consequently, with a mAb at hand whose specificity for porcine CD27 could be demonstrated, we studied CD27 expression on cells from blood, primary and secondary lymphoid organs. In additional experiments the costimulatory capability of mAb b30c7 on CD3activated T cells was addressed. 2. Materials and methods 2.1. Isolation of lymphocytes Heparinized blood, thymus, spleen, and mediastinal lymph nodes of healthy six-month old pigs were obtained from an abattoir. Animals were subjected to electric high voltage anaesthesia followed by exsanguination. This procedure is in accordance to the Austrian Animal Welfare Slaughter Regulation. PBMCs were isolated by gradient centrifugation using lymphocyte separation medium (PAA Laboratories, Pasching, Austria) as described previously (Saalmüller et al., 1987). Cells from spleen, mediastinal lymph node and thymus were gained by sieving small pieces of the respective organs through steel meshes. Dead cells were separated from cell suspensions by cotton–wool filtration. Remaining erythrocytes from spleen were removed from cell suspensions by an additional gradient centrifugation step. For use in proliferation assays (see below), PBMCs were cryopreserved at 150 °C as described elsewhere (Leitner et al., 2012).

pH 7.4 [TBS]), the membrane was incubated with streptavidinhorseradish-peroxidase (Roche, Vienna, Austria; 1:2000 in TBS with Casein [1%, w/v] and Tween 20 [0.1%]). Detection was carried out with enhanced chemoluminescence Western Blotting Substrate chemoluminescence kit (Thermo Fisher Scientific) and visualised on Amersham Hyperfilm MP autoradiography film (GE Healthcare, Little Chalfont, UK). 2.3. Screening of a porcine cDNA expression library using anti-SWC2 mAb A retroviral porcine cDNA expression library from activated PBMCs was generated and the following screening and cloning steps were performed as described elsewhere in detail (Leitner et al., 2012). Briefly, 2  107 cells of the murine target cell line expressing the library (Bw5147; abbreviation within this work Bw) were incubated with anti-SWC2 mAb (clone b30c7) for 20 min at 4 °C. After a washing step, binding of primary antibody was detected with phycoerythrin (PE)-conjugated goat anti-mouse IgG-Fcc specific antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were subjected to two rounds of sorting by a FACSAria cell sorter (BD Biosciences) and enrichment of SWC2+ cells was assessed each time after expansion of sorted cells by flow cytometry (FCM). Thereafter, SWC2+ single cell clones were established by limiting dilution culturing. Genomic cDNA was isolated and retroviral inserts were retrieved by PCR as described (Leitner et al., 2011; Pfisterhammer et al., 2009). After gel-purification and PCR-reamplification the products were cloned into the vector pCJK2 and retrovirally expressed in Bw cells. Reactivity with anti-SWC2 mAb was confirmed by FCM using PE-labelled goat anti-mouse IgG1 secondary antibody (Southern Biotech, Birmingham, AL). Isotypematched control antibody (mouse IgG1; Dianova, Hamburg, Germany) served as negative control. Plasmid DNA that conferred reactivity with SWC2 mAb to Bw cells upon transduction was subjected to automatic sequencing (Eurofins MWG, Ebersberg, Germany). The nucleotide sequence encoding porcine CD27 has been submitted to Gene Bank (JQ993894). 2.4. FCM analyses and antibodies

2.2. Immunoprecipitation PBMCs were biotinylated with PierceÒ Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific Inc., Rockford, IL) according to manufacturer‘s instructions. Immunoprecipitation was performed using PierceÒ Direct IP Kit (Thermo Fisher Scientific). Briefly, cells were lysed for 5 min with periodic mixing using provided IP Lysis/Wash Buffer and subsequently precleared twice for 1 h using PierceÒ Protein A/G Plus Agarose (Thermo Fisher Scientific) and 4 lg unspecific isotype-matched antibody (mouse IgG1, clone MOPC-31C; BD Biosciences, San Jose, CA). For immunoprecipitation 8 lg of either anti-SWC2 mAb (mouse IgG1, clone b30c7; Saalmüller et al., 1994) or mouse IgG1 control mAb (BD Biosciences) were covalently bound to AminoLinkÒ Plus Coupling Resin following manufacturer‘s instructions (Thermo Fisher Scientific). Antigen immunoprecipitation was performed for 1 h at 4 °C and proteins were eluted (pH 2.8, Elution Buffer, Thermo Fisher Scientific) followed by a desalting and concentration step using Vivaspin columns 500 (3000 MWCO, Sartorius Stedim Austria GmbH, Vienna, Austria). Samples were boiled with sample buffer (Thermo Fisher Scientific) supplemented with 1,4-dithiothreitol (20 mmol final concentration; Carl Roth, Karlsruhe, Germany) for 5 min at 96 °C and finally separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis. After transfer onto a polyvinylidene fluoride membrane by semi-dry blotting and blocking with 5% (w/v) skim milk in tris-buffered saline (20 mM Tris, 0.5 M NaCl,

For phenotypical analyses of CD27 expression in blood, thymus, spleen and mediastinal lymph nodes, freshly isolated cells were resuspended in PBS (without Ca2+/Mg2+, PAA) containing 10% (v/ v) porcine plasma and adjusted to 1  106 cells per sample. All incubation steps for FCM staining were performed for 20 min on ice. Analyses of T cells and NK cells were performed by the use of primary antibodies directed against porcine CD3 (mouse IgG2b, clone BB23 8E6; Southern Biotech), CD4 (mouse IgG2b, clone 7412-4), CD8a (mouse IgG2a, clone 11/295/33), CD8b (mouse IgG2a, clone PG164A; VMRD, Pullman, WA), TcR-cd (mouse IgG2b, clone PPT16) and CD27 (mouse IgG1, clone b30c7). All non-commercial mAbs were produced in-house (Saalmüller, 1996). After a washing step, binding of primary antibodies was detected by isotype-specific goat anti-mouse fluorochrome-conjugated secondary antibodies: IgG1-PE (Southern Biotech), IgG2a-Alexa647 and IgG2bAlexa488 (Invitrogen, Carlsbad, CA). Unspecific binding was evaluated using isotype-matched control mAbs (mouse IgG1, mouse IgG2a and mouse IgG2b; Dianova). Myeloid cells were stained with anti-CD172a mAb (mouse IgG1, clone 74-22-15) and binding was detected by goat anti-mouse IgG1-Alexa488 secondary antibody (Invitrogen). After blocking free binding sites of secondary antibodies with mouse IgG molecules (2 lg per sample; Jackson ImmunoResearch) cells were incubated with directly labelled anti-CD27-Alexa647 mAb. Alexa fluorescent labelling of anti-CD27 mAb b30c7 was performed with

K. Reutner et al. / Developmental and Comparative Immunology 38 (2012) 321–331

a Fluor 647 Monoclonal Antibody Labelling Kit (Invitrogen), according to manufacturer’s instructions. For intracellular staining of CD79a, cells were incubated in a first round with anti-CD27 mAb followed by staining with isotype-specific goat anti-mouse IgG1-Alexa647 secondary antibody (Invitrogen). In a third step, free binding sites of the secondary antibody were blocked with mouse IgG molecules. Afterwards, cell were fixed, permeabilized and stained for CD79a expression using anti-CD79a-PE mAb (mouse IgG1, clone HM57; Dako, Glostrup, Denmark) as described elsewhere (Gerner et al., 2008). Isotypematched non-binding antibodies (mouse IgG1 and mouse IgG1PE, clone DAK-GO1; Dako) served as negative controls. FCM measurements (including chapters 2.3 and 2.6) were performed on FACSCalibur, FACSCanto II or FACSAria (all BD Biosciences) flow cytometers. Data were analysed using either FACSDiva (Version 6.1.3., BD Biosciences) or FlowJo software (Version 7.6.3., Tree Star, Ashland, OR). 2.5. Immunohistochemistry Tissue samples were taken in the slaughterhouse from two sows (age of 8 months). Jejunal lymph nodes and samples of thymus, spleen and tonsilla veli palatine were embedded in TissueTekÒ O.C.T.™ Compound (Sakura Finetek Europe B.V., Zoeterwoude, Netherlands) and snap frozen in liquid nitrogen. Samples were cut into 6 lm thick sections which were mounted on silanized SuperFrostÒ Plus slides (Menzel-Gläser, Braunschweig, Germany) and fixed for 10 min in cold (4 °C) buffered formalin according to Lillie (Romeis, 1989). The fixed cryosections were washed in distilled water and transferred to PBS. Endogenous peroxidase activity was blocked with 0.6% (v/v) H2O2 in distilled water and non-specific binding activity with normal goat serum (150 ll/10 ml PBS; DakoCytomation, Hamburg, Germany) at room temperature. All sections were incubated overnight with the anti-CD27 mAb (clone b30c7) at 4 °C. Immune reaction was detected using the BrightVision poly HRP-Anti-Mouse IgG kit (ImmunoLogic, Duiven, Netherlands) according to the manufacturer’s instructions. The reaction was visualised with diaminobenzidine (Sigma–Aldrich, Vienna, Austria) in 0.03% (v/v) H2O2 in TRIS-buffered saline (pH 7.4). Afterwards, the sections were dehydrated and mounted with a medium soluble in xylene. Samples were analysed with a Leica DMD108 system (Leica Microsystems GmbH, Vienna, Austria).

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For phenotypical analyses of proliferating T cells, defrosted PBMCs were labelled using CellTrace™ Violet Cell Proliferation Kit (Invitrogen). For this purpose, 2  107 cells were washed and resuspended in 1 ml PBS (without Ca2+/Mg2+, PAA) prior to addition of 1 ml of Violet-solution (5 lmol/l) in PBS (without Ca2+/ Mg2+). After vortexing and incubation for 10 min at 37 °C in a water-bath, excessive Violet dye was quenched by addition of 2 ml FCS for further 15 min at room temperature. Finally, cells were washed three times in pre-warmed culture medium. Subsequently, cells (2  105 per well) were cultivated either unstimulated in cell culture medium or in wells that had been coated either with anti-CD27 mAb (3 lg/ml) or anti-CD3 mAb (two concentrations: 3 and 0.75 lg/ml) or a combination of anti-CD3 mAb and anti-CD27 mAb (anti-CD3 mAb: 0.75 lg/ml, anti-CD27 mAb: 3 lg/ml). After four days of cultivation, T cells were analysed for proliferation (Cell Trace Violet dye), membrane integrity and cell surface marker expression in a five-colour staining by FCM. The following primary antibodies were used: anti-CD3 (clone BB23 8E6), anti-CD4 (clone 74-12-4), anti-CD8a (clone 11/295/33), anti-CD8b (clone PG164A), anti-TcR-cd (clone PPT16) and anti-CD27 (clone b30c7, Alexa647-conjugated; see above for sources of antibodies). Isotype-matched mouse IgG2a, mouse IgG2b (Dianova) and directly labelled mouse IgG1-Alexa-647 (Invitrogen) served as isotype control antibodies. After washing twice in PBS without proteins, cells were incubated with goat anti-mouse fluorochrome-conjugated secondary antibodies (IgG2a-PE, Southern Biotech and IgG2b-Alexa488, Invitrogen) and Live/Dead fixable NearIR Cell Stain Kit (Invitrogen). After two final washing steps cells were analysed by FCM. 2.7. Analyses of IFN-c and TNF-a production by ELISA In parallel to analysis of proliferation by Cell Trace Violet dye (see above), supernatants of CD3–, CD27– and CD3/CD27– stimulated PBMCs were collected after four days of cultivation. Supernatants were analysed for IFN-c and TNF-a levels with commercially available porcine IFN-c ELISA kit (Mabtech, Nacka Strand, Sweden) and porcine TNF-alpha DuoSet (R&D Systems, Minneapolis, MN), respectively, according to manufacturer’s protocols. Optical density was measured at 450/620 nm with a Sunrise ELISA reader (Tecan, Crailsheim, Germany). 3. Results

2.6. Proliferation assays 3.1. Identification of the SWC2 antigen as porcine CD27 Round-bottomed 96-well plates (Greiner Bio-One, Kremsmünster, Austria) were coated with anti-CD3 mAb at a concentration of 3, 1.5, 0.75 or 0.375 lg/ml (mouse IgG1, clone PPT7; Yang et al., 1996) in 30 ll PBS per well for 3 h at 37 °C. In addition, wells were coated with the anti-CD27 mAb (clone b30c7) at concentrations of 6, 3, 1.5 or 0.75 lg/ml. To test the combined effects of anti-CD3/CD27 mAb stimulation, wells were also coated with both antibodies at all aforementioned antibody concentrations (i.e. checkerboard titration). Wells coated only with PBS served as negative controls. Unbound mAbs were removed by washing plates three times with PBS prior to cultivation. Thereafter, defrosted 2  105 PBMCs per well were plated in triplicates and cultivated in RPMI 1640 with stable glutamine (PAA) supplemented with 10% (v/v) FCS (PAA), 100 IU/ml penicillin and 0.1 mg/ml streptomycin (PAA). Cells were incubated for either 1 or 2 days, subsequently pulsed with 1 lCi of 3H-thymidine (MP Biomedicals, Eschwege, Germany) per well and harvested 18 h later using a Filtermate Harvester (Perkin Elmer, Wellesley, MA). 3H-thymidine uptake was determined in counts per minute (cpm) using a Top Count 4.00 Scintillation Counter (Perkin Elmer).

Previous work with SWC2-specific antibodies revealed that the SWC2 antigen has a molecular mass of approximately 49–51 kDa (Saalmüller et al., 1994). By the use of biotinylated PBMCs we could confirm these data in immunoprecipitation experiments with the anti-SWC2 mAb b30c7 (Fig. 1A). To identify the orthologous CD molecule for SWC2 we used a recently established porcine eukaryotic retroviral cDNA expression library. This library is based on mRNA isolated from a mixture of porcine PBMCs that had been stimulated in vitro either with ConA or PMA/Ionomycin. Details on the establishment of the library can be found in Leitner et al. (2012). In a first set of experiments, mouse thymoma cells (Bw cells), expressing the library, were stained with anti-SWC2 mAb b30c7 and subjected to two rounds of sorting by fluorescence activated cell sorting (FACS) (Fig. 1B). Initially, the frequency of SWC2 expressing Bw cells was very low (0.026%; Fig. 1B, upper left dot plot). After the first round of sorting, Bw cells (5  103) were expanded and analysed for SWC2 expression. Of these cells 37.2% already expressed the SWC2 antigen and these cells were then used for a second round of sorting and cultivated for propagation.

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Fig. 1. Identification of porcine CD27 as the antigen recognized by mAb b30c7. (A) Immunoprecipitation of SWC2 from biotinylated PBMCs by mAb b30c7 (lane 2) and a corresponding IgG1 isotype control antibody (lane 1). Biotinylated mass standards are displayed on the right. (B) Bw cells expressing the porcine cDNA library were incubated with anti-SWC2 mAb b30c7 and SWC2+ cells were gated for sorting (upper left panel). Bw cells reacting with anti-SWC2 mAb were expanded, re-analysed for SWC2 expression (upper right panel) and subjected to a second round of sorting (lower left panel). After further expansion, SWC2 expression on Bw cells was analysed (lower right panel). (C) cDNA obtained from SWC2+ Bw single cell clones was cloned in a retroviral expression vector and re-expressed in Bw cells. These cells (right panel) as well as nontransfected Bw cells (left panel) were stained with mAb b30c7 (solid line) or an IgG1 isotype control antibody (dotted line). (D) Multiple protein sequence alignment of full length CD27 amino acid sequences of different mammalian species by using GeneDoc Version 2.7.000 (Nicholas et al., 1997). Pig⁄ and Pig⁄⁄ sequences are derived from Gene Bank. The sequence obtained from SWC2+ transfected Bw cells is designated as SWC2-antigen. Based on the human CD27 sequence (P26842 [CD27_HUMAN] reviewed, UniProtKB/Swiss-Prot), the signal peptide (grey) as well as the extracellular (yellow), transmembrane (green) and cytoplasmic (blue) domains are highlighted. The black box indicates the TRAF2 binding motif (PIQE). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

After the second expansion, 96.8% of sorted Bw cells stained positive for the SWC2 antigen which were then used for establishing single cell clones by limiting dilution culturing. These single cell clone cultures were tested for uniform, high SWC2 expression by FCM (data not shown). From 11 cultures fulfilling this criterion, genomic DNA was isolated and retroviral inserts were retrieved by PCR using vector-specific primers. A 1500 bp PCR product that was obtained from all SWC2-reactive clones (data not shown) was subsequently isolated, cloned and re-expressed in Bw cells.

Reactivity of these Bw cells with SWC2 mAb was tested again by FCM and compared to non-transfected Bw cells (Fig. 1C). Bw cells expressing the 1500 bp insert strongly reacted with the SWC2 mAb indicating that it encoded the SWC2 antigen (Fig. 1C, right panel). The insert was subjected to DNA sequence analysis and the deduced amino acid sequence was compared to known sequences by BLAST analysis (Fig. 1D). Sequences derived from SWC2 expressing Bw cells (SWC2-antigen) showed high homologies to CD27 sequences of other mammalian species including predicted

K. Reutner et al. / Developmental and Comparative Immunology 38 (2012) 321–331 Table 1 Amino acid homology of CD27 between swine and other mammalian species. Speciesa

Amino acid (%)b

Human Chimpanzee Rhesus monkey Dog Cattle Pigc Pigc Horse Rat Mouse

71.37 71.37 70.97 70.28 76.92 100.00 100.00 74.19 61.76 60.92

a For accession nos. of mammalian species examined in this study please refer to Fig. 1. b Obtained from pairwise p-distances computed with Mega version 5 (Tamura et al., 2011). c Sus scrofa Acc. Nos. XM_003126546.1 and AK238826.1.

sequences from the swine genome (Pig⁄- XM_003126546.1 and Pig⁄⁄- AK238826.1). Pairwise p-distances were calculated by using Mega version 5 (Tamura et al., 2011) and are displayed in Table 1. A homology of 100% was found for predicted sequences of porcine CD27. The next most closely related sequences were derived from cattle (76.92%) and horse (74.19%). Of note, within the cytoplasmic domain, the region spanning amino acid residues 261–270 was identical in all CD27 molecules analysed, including the porcine sequence identified in this study (Fig. 1D). Within this region, the amino acid sequence motif ‘‘PxQE’’ has been described as binding motif for TNF receptor associated factor (TRAF) 2 and TRAF5 (Aggarwal, 2003; Ye et al., 1999). 3.2. CD27 expression on porcine leukocytes In a next step, we investigated the expression of the SWC2 antigen – now identified as porcine CD27 – on porcine leukocytes derived from various lymphatic organs by two- and three-colour FCM. In the first experiments, we investigated CD27 expression in combination with CD4 and CD8a expression on thymocytes to gain insight in the regulation of CD27 during thymic T-cell development (Fig. 2A). Staining for CD4 and CD8a was used to distinguish four different subpopulations: CD4 CD8a , CD4+CD8a+, CD4 CD8a+ and CD4+CD8a thymocytes. These four populations were gated and analysed for CD27 expression. Within immature CD4 CD8a thymocytes (gate I) a substantial proportion was CD27 whereas CD27 expression was already increased after maturation to CD4+CD8a+ thymocytes (gate II). Single positive CD4+CD8a and CD4 CD8a+ cells (gates III and IV, respectively), which define the most mature thymocytes, were found to express high levels of CD27 on their cell surface. In blood the expression of CD27 was analysed on major leukocyte populations including CD172a+ myeloid cells, CD79a+ B cells and CD3+ T cells (Fig. 2B). CD27 could not be identified on CD172ahigh monocytes and CD172aintermediate dendritic cells. A few CD172adimCD27+ cells were observed, but CD172adim cells are currently not characterized in swine. CD79a+ B cells also lacked CD27 expression, whereas a major proportion of CD3+ T cells expressed high to intermediate levels of this molecule. A small but distinct population of CD3 CD27+ cells was also observed, most probably representing NK cells (see below). Consequently, CD27 expression was analysed within various T-cell subsets and NK cells in blood, spleen and mediastinal lymph nodes (Fig. 2C). T helper cells were divided into naïve CD4+CD8a (gate I) and activated/memory CD4+CD8a+ (gate II) cells as described previously (Saalmüller et al., 2002). In blood naïve T

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helper cells expressed the CD27 antigen at high levels, whereas CD4+CD8a+ T helper cells were further divided into CD27+ and CD27 cells (arrow heads 1). T helper cells from spleen showed similar CD27 expression patterns while only a small proportion of CD27 cells was found within CD4+CD8a+ T helper cells in mediastinal lymph nodes (arrow head 2). Next, CD4 CD8b+ CTLs (gate III) were analysed and overall a substantial population of these cells showed high levels of CD27 expression on the cell surface. Of note, within PBMCs and splenocytes a minor subset of CTLs lacked CD27 expression (arrow head 3) which was found to be associated with a moderate down-regulation of the CD8b molecule (arrow head 4). In contrast, this subset was absent in the lymph node (arrow head 5). TcR-cd T cells, which represent an additional major T-cell subpopulation in swine, were divided into CD8a /dim and CD8ahigh subsets and investigated for CD27 expression. TcRcd+CD8a /dim cells (gate IV) were all CD27+, regardless of the lymphatic organ analysed. In contrast, within TcR-cd+CD8ahigh cells (gate V) CD27high, CD27dim/intermediate and CD27 expression levels could be found (arrow heads 6) within PBMCs and splenocytes. This did not apply to the mediastinal lymph nodes, where TcRcd+CD8ahigh cells were found to homogeneously express high levels of CD27 (arrow head 7). NK cells were identified by a CD3 CD8a+ phenotype (gate VI) and analysed for CD27. The vast majority of NK cells derived from blood, spleen and lymph nodes expressed CD27, albeit at an overall lower antigen density compared to T cells. In general, CD27 was abundantly expressed on porcine T cells and NK cells, which might be indicative for an important functional role of this molecule on these cells. 3.3. In situ localization of CD27 antigen expressing cells To further extend our knowledge regarding CD27 expression, we examined tissue sections from thymus, spleen, lymph nodes and tonsil by immunohistochemistry (Fig. 3). In thymus scattered small CD27+ thymocytes were located in the medulla (Fig. 3A). In spleen an accumulation of CD27+ cells was visible in the periarterial lymphatic sheath (PALS), whereas few CD27 expressing cells were present in the centre of lymph follicles (Fig. 3B). In lymph nodes and tonsil CD27+ lymphocytes were predominantly found in the extrafollicular T-cell areas. Furthermore, similar to the spleen, few scattered positive cells were also detectable in the centre of follicles of both lymphoid organs (Fig. 3C and D). 3.4. Costimulatory capacity of anti-CD27 mAb on CD3-activated T cells Stimulation of the TcR combined with costimulation via CD27 has been shown to enable T-cell survival, proliferation, cytokine production and differentiation into effector cells (reviewed in Nolte et al., 2009). Therefore, we aimed to investigate whether the anti-CD27 mAb b30c7 has the capacity to costimulate CD3activated T cells in vitro by using plate-bound antibodies. PBMCs were stimulated with immobilized anti-CD3 mAb and anti-CD27 mAb as well as with combinations of these antibodies coated at different concentrations. Thereafter, proliferation was analysed by tritium incorporation on days 1–2 and days 2–3 (Fig. 4A). The optimal concentration of anti-CD3 mAb alone for stimulating T-cell proliferation was between 3 and 1.5 lg/ml whereas lower concentrations (0.75 and 0.375 lg/ml) induced little or no proliferation of T cells. Anti-CD27 mAb alone had no effect on T-cell proliferation, regardless of the concentration used for coating. However, addition of 3 or 1.5 lg/ml anti-CD27 mAb to cultures stimulated with suboptimal concentrations of anti-CD3 mAb (0.75 and 0.375 lg/ml) restored proliferation of T cells up to levels observed with optimal anti-CD3 mAb concentrations (black boxes, Fig. 4A). Addition of higher amounts of anti-CD27 mAb (6 lg/ml) showed an inhibitory effect on all CD3-stimulated fractions. Nevertheless, at optimal

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Fig. 2. CD27 expression in porcine blood and lymphoid tissues. (A) Using three-colour FCM, thymocytes were analysed for CD4 (x-axis) and CD8a (y-axis) expression (contour plot) and four different subpopulations were gated. Each subpopulation was further analysed for CD27 expression (histograms). (B) PBMCs were analysed by two-colour FCM for CD27 expression on myeloid cells (CD172a+, left panel), B cells (CD79a+, middle panel) and T cells (CD3+, right panel). (C) CD27 expression was examined within T-cell subsets and NK cells in blood (left panels), spleen (middle panels) and mediastinal lymph nodes (right panels) by three-colour FCM. Contour plots show the expression of CD4, TcR-cd and CD3 (x-axes) versus CD8a or CD8b (y-axes) for identification of T helper cells, CTLs, cd T cells and NK cells (from top to bottom). CD4+CD8a (gate I) and CD4+CD8a+ (gate II) T helper cells, CD4 CD8b+ (gate III) CTLs, TcR-cd+CD8a /dim (gate IV) and TcR-cd+CD8a+ (gate V) cd T cells as well as CD3 CD8a+ (gate VI) NK cells were sub-gated for CD27 expression (histograms). See main text for description of arrow heads and numbers. (A–C) Per sample at least 5  104 lymphocytes were acquired. Data of one representative animal (PBMCs: 13 animals tested, lymphatic organs: five animals tested) is shown.

concentrations anti-CD27 mAb clearly had the capacity to costimulate proliferation of CD3-activated T cells. To further examine which T cells started to proliferate and if there were any alterations of the CD27 expression pattern caused by anti-CD3/anti-CD27 mAb stimulation, PBMCs were labelled with a Violet cell proliferation dye and cultivated in the presence of plate-bound antibodies for 4 days. Thereafter, CD27 expression and proliferation were analysed within T helper cells (CD4+ cells gated), CTLs (CD3+CD8b+ cells gated) and cd T cells (TcR-cd+ cells gated) by FCM (Fig. 4B). For T helper cells and cd T cells also expression of CD8a was investigated. T helper cells showed minor proliferation rates after stimulation with a low dose (0.75 lg/ml) of anti-CD3 mAb alone compared to cells from PBS-coated microcultures (Fig. 4B, dot plot series ‘‘1’’, 4.2% vs. 1.4%, respectively). Also, 3 lg/ml anti-CD27 mAb alone did not induce T-helper cell proliferation above controls (1.3%). A strong proliferation was observed for

high dose (3 lg/ml) anti-CD3 mAb stimulated T helper cells and low dose anti-CD3/anti-CD27 mAb costimulated T helper cells (63.1% vs. 52.6%, respectively). Furthermore, both stimulation regimens led to a decrease of CD4+CD8a cells (Fig. 4B, arrow heads ‘‘1’’), a phenomenon described earlier for polyclonally stimulated porcine T helper cells (Saalmüller et al., 2002). Similar results were obtained for CTLs. Stimulated with the anti-CD3/anti-CD27 mAb combination, CTLs showed comparable proliferation rates to high dose anti-CD3 mAb stimulated cells (Fig. 4B, dot plot series ‘‘2’’, 79% vs. 67.6%, respectively). Moreover, CTLs stimulated with these two regimens showed partially a loss in CD3 expression levels compared to cultures in which no proliferation was induced (Fig. 4B, arrow heads ‘‘2’’). However, CD27 expression patterns in proliferating and resting T helper cells as well as CTLs was barely influenced by costimulation with anti-CD27 mAb. For cd T cells already a relatively high spontaneous proliferation was observed

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Fig. 3. In situ detection of CD27 expression in porcine tissues by immunohistochemistry. Cryo-conserved tissue sections were incubated with mAb b30c7 and binding was visualised by horseradish peroxidase/diaminobenzidine; no counterstaining was performed. Representative sections of thymus (A), spleen (B), jejunal lymph nodes (C) and tonsil (D) are shown. Cort, cortex; med, medulla; has, Hassall’s corpuscle; Fol, lymph follicle; PALS, periarterial lymphatic sheath; sh, sheathed capillary; trab, trabecula; ep, epithelium.

in samples derived from wells that were not coated with any antibody for stimulation (Fig. 4B, dot plot series ‘‘3’’, ‘‘PBS’’: 12.3%). This level of spontaneous proliferation was not exceeded in cultures stimulated with the high dose of anti-CD3 mAb only or the combination of anti-CD3 and anti-CD27 mAbs. Again, no obvious differences in CD27 expression of cd T cells between these two stimulation regimens were observed. Of note, in microcultures with T helper cells and CTLs that showed no or only minor proliferation rates, those cd T cells that had undergone most division cycles were CD27 /dim, whereas this phenotype was lacking in the CD3 high dose or CD3/CD27 costimulated cultures. In addition to proliferation and cell surface marker expression, supernatants of the respective microcultures were tested for differences in production of IFN-c and TNF-a by ELISA (Fig. 4C). High levels of these cytokines were measured after stimulation with high dose anti-CD3 mAb and the combination of anti-CD3/antiCD27 mAbs. In contrast, stimulation with low dose anti-CD3 mAb or anti-CD27 mAb alone caused only minor cytokine production, for IFN-c partially even below the detection limit. Overall, cytokine production levels showed the same pattern as proliferation rates described in Fig. 4B, demonstrating again a costimulation of porcine T cells via CD27.

4. Discussion 4.1. Identification of porcine CD27 Eukaryotic expression cloning has successfully been used in the past for the characterization of cell surface antigens, for example membrane-resident binding partners (Pfisterhammer et al., 2008; Yamanishi et al., 2010) or surface antigens detected by mAbs (Aruffo and Seed, 1987; Seed and Aruffo, 1987). By using a retrovi-

ral cDNA expression library derived from in vitro stimulated porcine PBMCs we could recently demonstrate that CD52 is the orthologous molecule of SWC1 (Leitner et al., 2012). In the work presented here, with the use of this library we could unequivocally identify SWC2 as the porcine CD27 orthologue. Therefore, although antibodies for SWC2 were available for nearly 20 years, with the identification of CD27 as the recognized antigen, new insights could be generated and old findings revisited. Immunoprecipitation analyses revealed porcine CD27 as a 49 to 51 kDa molecule (Saalmüller et al., 1994; Fig. 1A). In contrast, on human lymphocyte membranes CD27 is expressed as a disulphide-linked homodimer with a molecular weight of 55 kDa (Van Lier et al., 1987). These discrepancies in the molecular mass of CD27 antigens within different species might be due to different glycosylation patterns. Furthermore, a modulation of the mouse CD27 molecule has been described after activation by mitogens or alloantigens resulting in a 55–60 kDa variability of the homodimer chains (Bigler et al., 1988). In regard to the amino acid sequence, moderate homology between pig and examined mammalian species was observed for almost the entire CD27 sequence including signal peptide, extracellular, transmembrane and cytoplasmic domain. The porcine CD27 sequence showed similarities descending from cattle, horse, human, chimpanzee, rhesus monkey, dog, to rat and mouse. However, conserved loci were found within extracellular (amino acid residues 80–90) and cytoplasmic (amino acid residues 261–270) domain. As mentioned above, the latter contains the intracellular binding motif (PxQE; amino acid residues 261–264; Ye et al., 1999) of TRAF2 and TRAF5 (Aggarwal, 2003). TRAF2 and TRAF5 activate the canonical and the non-canonical nuclear factor-jB (NF-jB) pathways as well as the c-Jun-N-terminal kinase (JNK)-signalling cascade after interaction of CD27 with its ligand CD70 (Akiba et al., 1998; Gravenstein et al., 1998). These intracel-

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Fig. 4. In vitro costimulation of porcine PBMCs by plate-bound anti-CD27 mAb. (A) PBMCs were cultivated in 96-well plates which had been coated with four different concentrations of anti-CD3 mAb (0.375, 0.75, 1.5, and 3 lg/ml; graphs from top to bottom) or with four different concentrations of anti-CD27 mAb (0.75, 1.5, 3 and 6 lg/ml; xaxes of respective graphs) as well as combinations of these two mAbs. Wells treated without one or both of the respective antibodies (denoted ‘‘PBS’’) served as controls. Cultivated PBMCs were tested for tritium incorporation at day 1–2 (left column) and day 2–3 (right column). Results are displayed in counts per minute (cpm) on the y-axes, representing the mean + standard deviation of triplicate cultures. Overall, data are representative of experiments with PBMCs of four individuals. (B) PBMCs were labelled with a Violet proliferation dye and stimulated with plate-bound anti-CD3 mAb (3 lg/ml and 0.75 lg/ml) or anti-CD27 mAb alone (3 lg/ml) or with a combination of 0.75 lg/ ml anti-CD3 and 3 lg/ml anti-CD27 mAbs for four days. PBS coated wells without antibodies served as negative control. Five-colour FCM including a live/death discrimination dye was used to examine CD27 expression of proliferating T-cell subsets. T helper cells (CD4 versus CD8a expression, left), CTLs (CD3 versus CD8b, middle) and cd T cells (TcR-cd versus CD8a, right) were gated and further analysed for proliferation (violet dye dilution, x-axis) and CD27 expression (y-axis). Per sample at least 5  104 live cells were acquired. (C) PBMCs were cultivated for four days as described in (B). Cell culture supernatants were collected and IFN-c (upper diagram) and TNF-a levels (lower diagram) analysed by ELISA. Bars represent cytokine levels in ng/ml of supernatants of the respective micro-cultures (a0.75 lg/ml anti-CD3 and 3 lg/ml anti-CD27 mAb). Mean values + standard deviation of duplicate wells tested in ELISA are shown. (B + C) Data are representative of experiments with cells/supernatants of two individuals.

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lular signalling pathways induce gene expression of T cells supporting proinflammatory effector as well as anti-inflammatory regulatory functions. Therefore, ligation of porcine CD27 should activate the same signalling pathways und gene regulation as described for other mammalian species.

4.2. CD27 expression in the thymus, blood and secondary lymphatic organs In accordance with studies of the human and murine system, porcine CD27 expression on T cells appears to be acquired during CD4 CD8 to CD4+CD8+ thymocyte maturation which can be considered to accompany TcR development (Gravenstein et al., 1996; Hintzen et al., 1993). Furthermore, porcine CD27 was found on nearly all matured CD4+CD8 and CD4 CD8+ thymocytes. The tissue localization of the CD27 expressing thymocytes differs between human and mouse. Human CD27+ cells are mainly present in the thymic medulla whereas in mice these cells are found in cortex and medulla (Gravenstein et al., 1996; Van Lier et al., 1987). Similar to human, in swine CD27+ cells were only detectable in the medulla. Moreover, although the vast majority of thymocytes already showed CD27 expression, immature CD4 CD8 thymocytes contained a substantial proportion of CD27 cells (Fig. 2A). These data are in line with described thymocyte movement during different developmental stages, in which immature CD4 CD8 and CD4+CD8+ thymocytes from the cortex maturate to CD4+CD8 and CD4 CD8+ T cells in the thymic medulla (Van Ewijk, 1991). For blood and secondary lymphatic organs, the tissue localization of porcine CD27-antigen expressing cells was comparable to that of humans (Van Lier et al., 1987). FCM analyses showed that CD27 was abundantly expressed on T lymphocytes and NK cells while it was absent on myeloid cells and B cells. In human B cells CD27 expression is induced after activation and maintained on memory B cells (Klein et al., 1998; Maurer et al., 1990). On the contrary, murine B cells only express CD27 at the centroblast stage, followed by a rapid antigen loss (Xiao et al., 2004). In our study we could not identify CD27 expressing porcine B cells within PBMCs (Fig. 2B) and lymphoid organs tested (data not shown). Nevertheless, further experiments with B-cell receptor stimulated B cells should be carried out to examine whether CD27 can be transiently expressed on porcine B cells as described for murine B cells. In regard to porcine T helper cells, we could identify CD4+CD8a+CD27+ but also CD4+CD8a+CD27 cells in equal parts in PBMCs and spleen whereas lymph nodes predominantly harboured CD4+CD8a+CD27+ cells. Previous work on porcine T helper cells described an up-regulation of CD8a after polyclonal as well as super-antigen stimulation. Furthermore, CD4+CD8a+ T cells showed a proliferative response after pseudorabies virus, classical swine fever virus and foot-and-mouth disease virus in vitro restimulation when derived from virus-infected animals (Gerner et al., 2006; Saalmüller et al., 2002; Summerfield et al., 1996). Therefore, extrathymic CD4+CD8a+ porcine T helper cells are considered as a population of activated and/or memory cells (Saalmüller et al. 2002). Human memory T cells can be divided into central memory and effector memory T cells with the latter showing a CD62L CCR7 phenotype that is also accompanied by a loss of CD27 expression (reviewed in Appay et al., 2008). By considering this information, it is tempting to speculate that CD27 expression might also divide CD4+CD8a+ porcine T helper cells into two populations with similar properties to those described for central memory and effector memory T cells in the human immune system since (i) CD27 cells were only present within CD4+CD8a+ activated/memory T helper cells and (ii) CD27 cells where nearly completely absent in lymph nodes where CCR7 expression would

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be required for entry. Studies on these issues are currently performed in our group. For porcine CD8b+ CTLs we could also identify a CD27 subpopulation that was absent in lymph nodes (Fig. 2C). Of note, CD27 expression on CTLs was correlated with a reduced level of CD8b expression even though the functional relevance of this observation remains unclear so far. Nevertheless, from a comparison with the phenotype of human CTLs, where CD27 cells represent late differentiated cells (Appay et al., 2008), one might again speculate that porcine CD8bdimCD27 CTLs may represent specialised effector and/or memory cells. However, further experiments are needed addressing for example perforin expression, cytokine production as well as chemokine receptor expression to give more detailed information about functional properties of CD8bdimCD27 CTLs. We could also show that CD8a together with CD27 expression defines three subsets of cd T cells, namely CD8a /dimCD27+, CD8ahigh CD27+ and CD8ahighCD27 cells. In swine cd T cells represent a substantial T-cell subpopulation with diverse phenotypic subsets whose respective functions have not been clarified yet (Gerner et al., 2009). As reviewed by Ribot et al. (2011), human and murine IFN-c and IL-17 producing cd T cells are distinguished by their CD27+ and CD27 phenotype, respectively. In this context, CD27 might be of great value for a further characterization of porcine cd T-cell subsets in regard to their functional properties. Porcine NK cells have been previously described as perforin+CD2+CD3 CD4 CD5 CD6 CD8a+CD8b CD11b+CD16+ (Denyer et al., 2006). In this study we identified NK cells by a CD3 CD8a+ phenotype, and overall identified a CD27 expression even though at a lower level compared to T cells. For human NK cells in blood CD27 CD56dim and CD27+CD56bright. phenotypes have been described, with a higher cytotoxic potential of the former and more production of IFN-c and TNF-a of the latter (Vossen et al., 2008). In mice CD11bhigh NK cells have been described to differ in their CD27 expression levels with CD27high cells showing increased IFN-c production and higher cytotoxic activity (Hayakawa and Smyth, 2006). However, besides of few CD27high and CD27 cells the vast majority of porcine NK cells in blood and spleen in this study displayed a CD27dim phenotype. Hence, it has to be investigated whether porcine CD27 allows a characterization of functionally distinct NK cells. As recently reported (Mair et al., 2012), porcine NKp46 defines two NK-cell subsets, NKp46+ and NKp46 cells. Both subsets showed comparable killing activities but NKp46+ cells produced higher levels of IFN-c. Since porcine NKp46 has been discussed to play a crucial role in NK-cell activation and surveillance, analysing CD27 co-expression might provide additional information about the functional diversity of these cells. 4.3. CD27 costimulation of CD3-activated porcine T cells Costimulation of T cells via CD27 and other members of the TNFR family influenced responsiveness, differentiation and viability of T cells after activation (reviewed in Nolte et al., 2009). As demonstrated in this study, costimulation of porcine CD27 also supported proliferation and cytokine production such as IFN-c and TNF-a of CD3-activated T cells (Fig. 4A and C). However, higher amounts of anti-CD27 mAb resulted in an inhibition of proliferation which may have been caused by an overload of plate-coated antibodies causing steric hindrance. Intriguingly, we found no obvious differences in the proliferation of CD27+ and CD27 T helper cells regardless whether they were stimulated with high amounts of anti-CD3 or the anti-CD3/anti-CD27 mAb combination (Fig. 3B). One possible explanation for this could be a reduced activation threshold in CD27 T helper cells. On the contrary, CD27 CTLs displayed much lower proliferation rates compared to their CD27+ counterparts for both stimulation regimens. Likewise, a strong response of CD27+ naïve murine

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CTLs towards CD27 costimulation has been reported (Hendriks et al., 2000; Van Gisbergen et al., 2011). In contrast, CD27 CTLs might have differentiated towards a terminal stage at which a strong in vitro proliferation cannot be achieved (Appay et al., 2008). Overall, CD3 stimulation and CD3/CD27 costimulation in vitro did not cause an obvious loss of CD27+ cells in both CTLs and T helper cells. Therefore, it seems that both stimulation strategies did not induce a down-regulation of CD27 in these cells. However, as shown above ex vivo we readily observed CD27 CTLs and T helper cells in blood and secondary lymphatic organs. CD27 T helper cells were entirely CD8a+, i.e. displayed a phenotype of activated/memory cells (Fig. 2C, Saalmüller et al., 2002). Most probably, this reflects a difference between in vitro and in vivo stimulation. Indeed, we could corroborate our in vivo findings in a recent study where T helper cells from blood were analysed in a time course study in piglets from the day of birth until six months of age. CD8a+CD27 T-helper cells appeared in temporal coincidence and were absent at time of birth (Talker et al., in preparation). In regard to in vitro stimulation an early study on human T cells also could not identify a down-regulation of CD27 following CD3 or CD3/CD2 stimulation by plate bound mAbs (de Jong et al., 1991). Later it was shown for human T cells that LPS stimulated monocytes in combination with superantigen could induce a down-regulation of CD27 (Kai et al., 1999), therefore indicating that a more complex stimulation environment is required for the generation of CD27 T cells. On porcine cd T cells costimulation by anti-CD27 mAbs seemed to have no impact on proliferation (Fig. 4B). In contrast, CD27 costimulation has been shown to play a pivotal role in human and murine cd T-cell activation. However, administration of soluble recombinant CD70 enhanced expansion, whereas anti-CD27 mAb reduced proliferation of human cd T cells (Ribot et al., 2011). A previous study had shown that CD3-directed activation of porcine cd T cells by mAb PPT7 was insufficient in bulk and isolated cultures (Yang and Parkhouse, 1998). Therefore, signal 1 may have been lacked for the cd T cells in our study. Furthermore, it is also possible that a third signal generated by certain cytokines might be required for adequate stimulation of porcine cd T cells.

5. Conclusions The elucidation that SWC2 represents porcine CD27 opens up multiple possibilities for new functional investigations as well as comparative studies on porcine lymphocytes. In this study we provide insights into CD27 expression on major lymphocyte subsets and the potential costimulatory capacity of CD27 ligation on porcine T cells. These data provide first hints that, as described for humans and mice, CD27 expression might be used for a further discrimination of functional subsets of porcine T helper cells, CTLs, cd T cells and NK cells. Moreover, costimulation via porcine CD27 seems to activate central pathways for T-cell activation and differentiation.

Acknowledgments The authors thank Magdalena Helmreich for her excellent technical assistance, Maria Stadler, Claus Wenhardt and Irene Popow for general technical support, Dieter Printz for cell sorting, Prof. Garry P. Nolan and colleagues for providing the retroviral vector pBMNZ, Prof. Winfried F. Pickl for the pEAK12-MLV-gag-pol-env plasmid and Dr. Haru Takamatsu for the PPT7 hybridoma. Katharina Reutner was funded by the Comet programme ‘‘Preventive veterinary medicine: Improving pig health for safe pork production’’.

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