CD2 and CD8α define porcine γδ T cells with distinct cytokine production profiles

Developmental and Comparative Immunology 45 (2014) 97–106

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CD2 and CD8a define porcine cd T cells with distinct cytokine production profiles Corinna Sedlak, Martina Patzl, Armin Saalmüller, Wilhelm Gerner ⇑ Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria

a r t i c l e

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Article history: Received 20 December 2013 Revised 12 February 2014 Accepted 13 February 2014 Available online 19 February 2014 Keywords: cd T cells Swine Phenotype IFN-c TNF-a IL-17A

a b s t r a c t cd 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 cd T cells in their vast majority had a CD8a SLA-DR CD27+ phenotype. CD2+ cd T cells dominated in spleen and lymph nodes and had a more heterogeneous phenotype. CD8a+SLA-DR CD27+ cd T cells prevailed in blood, spleen and lymph nodes whereas in liver a CD8a+SLADR+CD27 phenotype dominated, indicating an enrichment of terminally differentiated cd T cells. cd T cells were also investigated for their potential to produce IFN-c, TNF-a and IL-17A. Within CD2+ cd T cells, IFN-c and TNF-a single-producers as well as IFN-c/TNF-a double-producers dominated, which had a CD8a+CD27+/ phenotype. IL-17A-producing cd T cells were only found within CD2 cd T cells, mostly co-produced TNF-a and had a rare CD8a+CD27 phenotype. However, quantitatively TNF-a single-producers strongly dominated within CD2 cd T cells. In summary, our data identify CD2 and CD8a as important molecules correlating with functional differentiation. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction

cd T cells are an evolutionary highly conserved population of lymphocytes that can be found in all vertebrates (Hayday, 2000). They form a heterogeneous population of functionally specialized subsets that are involved in diverse immune responses. Depending on their expressed TCR, tissue location and activation status cd T cells (i) are capable to produce a wide range of immunomodulatory cytokines and chemokines, (ii) can directly kill infected or transformed target cells, (iii) promote recruitment and activation of innate cells, (iv) provide B cell help, (v) facilitate the activation of ab T cells by presenting antigens to CD4+ and CD8+ T cells and also (vi) show regulatory characteristics (Bonville et al., 2010; Vantourout and Hayday, 2013; Zheng et al., 2013). Also, for murine and bovine cd T cells the formation of memory cells in response to bacterial infection or vaccination, respectively, has been reported (Blumerman et al., 2007; Sheridan et al., 2013). However, the frequency of this T cell subpopulation varies greatly among different species. In humans and mice, cd T cells Abbreviations: BM, bone marrow; br. LN, bronchial lymph nodes; FCM, flow cytometry; med. LN, mediastinal lymph nodes; mes. LN, mesenteric lymph nodes. ⇑ Corresponding author. Address: Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria. Tel.: +43 1 25077 2753; fax: +43 1 25077 2791. E-mail address: [email protected] (W. Gerner). http://dx.doi.org/10.1016/j.dci.2014.02.008 0145-305X/Ó 2014 Elsevier Ltd. All rights reserved.

represent a minor fraction in the spleen, lymph nodes and peripheral blood (approximately 1–5% in blood) but show a tissue tropism for epithelial surfaces (Bonville et al., 2010). In contrast, cd T cells in swine and other ungulates are highly abundant in the blood and can constitute up to 85% of total lymphocytes in pigs (Takamatsu et al., 2006). Despite their high abundance, the knowledge about the biology of this prominent T cell subset in swine is still fragmentary. Phenotypically, total cd T cells in swine can be identified by a mAb (clone PGBL22A), recognizing an epitope on the porcine TCR-d-chain (Davis et al., 1998), or by a mAb (clone PPT16) binding to CD3 molecules which are only associated with the porcine TCR-cd (Yang et al., 2005). cd T cells in swine have been divided into three subsets on the basis of their CD2/CD8a expression, including CD2 CD8a , CD2+CD8a+ and CD2+CD8a cells. These three subsets are already established in the thymus and migrate into the periphery (Sinkora et al., 2005, 2007). Differences with regard to their homing characteristics have been observed, whereupon CD2+CD8a+ and CD2+CD8a cd T cells preferentially accumulate in spleen while CD2 CD8a cd T cells are enriched in blood (Saalmüller et al., 1990; Sinkora et al., 1998; Yang and Parkhouse, 1996). More recently, CD2+ and CD2 cd T cells have been suggested as independent lineages (Stepanova and Sinkora, 2013). As already outlined, cd T cells are potent producers of cytokines. For example, in healthy humans, 50–80% of blood-derived Vc9Vd2

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T cells are capable to produce IFN-c and TNF-a and less than 1% produce IL-17 (Caccamo et al., 2013). For murine cd T cells it was shown that these cells acquire the capacity to produce either IL-17 or IFN-c already in the thymus and that CD27 expression is a key determinant for this developmental differentiation (Ribot et al., 2009). In contrast, knowledge on cytokine production in porcine cd T cells is sparse and was mainly focused on IFN-c production in total cd T cells (Lee et al., 2004; Olin et al., 2005; Takamatsu et al., 2006; Wen et al., 2012). Only one recent report indicated that porcine cd T cells have the capacity to produce IL-17A (Stepanova et al., 2012). Therefore, in this study we aimed to correlate the production of the pro-inflammatory cytokines IFN-c, TNF-a and IL-17A with an enhanced phenotypic characterization of porcine cd T cells. This included the aforementioned markers CD2 and CD8a but also CD27. In addition we performed more detailed phenotypical studies of porcine cd T cells derived from different anatomical sites (blood, spleen, tonsils, bronchial, mediastinal and mesenteric lymph nodes, liver, bone marrow and thymus) addressing also the expression of SLA-DR and swine workshop cluster 5 (SWC5). SLA-DR expression is absent on blood-derived porcine T cells at birth but increases with age (Talker et al., 2013). SWC5 is exclusively expressed on porcine cd T cells and has a molecular weight of 180 kDa (Lunney et al., 1994). The orthologous human CD molecule is currently unknown, but SWC5 was previously shown to be differentially expressed on porcine CD2+ and CD2 cd T cells in blood (Talker et al., 2013). 2. Materials and methods 2.1. Animals and cell isolation All analyzed organs and blood were obtained from healthy six-month old pigs either from an abattoir or from animals kept in-house at the Clinic for Swine of the University of Veterinary Medicine, Vienna. Animals from slaughterhouse were subjected to electric high voltage anesthesia followed by exsanguination. This procedure is in accordance to the Austrian Animal Welfare Slaughter Regulation. In-house pigs where euthanized by a combination of Ketamine/Azaperone for anesthesia (NarketanÒ, Vètoquinol GmbH, Vienna, Austria + StresnilÒ, Janssen Pharmaceutica, Beerse, Belgium), followed by T61Ò application (Intervet GmbH, Vienna, Austria). This procedure was approved by the institutional ethics committee, the Advisory Committee for Animal Experiments (§12 of Law for Animal experiments, Tierversuchsgesetz – TVG) and the Federal Ministry for Science and Research (reference number BMWF-68.205/0021-II/3b/2011). PBMCs were isolated from heparinized blood using gradient centrifugation with lymphocyte separation medium (PAA Laboratories, Pasching, Austria) as described elsewhere (Saalmüller et al., 1987). Cells from various lymph nodes, spleen, tonsils and thymus were isolated as reported previously (Reutner et al., 2012). Intrahepatic lymphocyte isolation was performed according to a protocol by Crispe (2001). Cells from bone marrow were gained by flushing pieces of sternum with PBS (without Ca2+/Mg2+, PAA) followed by density gradient centrifugation to remove erythrocytes and further two washing steps in PBS. For the IL-17A blocking assay cells were cryopreserved at 150 °C as described elsewhere (Leitner et al., 2012). 2.2. Ex vivo FCM staining For phenotyping of cd T cells isolated from organs, freshly isolated cells were re-suspended in PBS (without Ca2+/Mg2+, PAA) with 10% (v/v) heat-inactivated porcine plasma (in-house preparation), adjusted to 1  106 cells per sample and plated in

round-bottom 96-well plates (Greiner Bio-one, Frickenhausen, Germany). During the entire staining process samples were kept at 4 °C and antibodies were incubated for 20 min. cd T cells were either identified by in-house biotinylated mAb PPT16 (mouse IgG2b), which was detected in a second incubation step by streptavidin-eFluor450 (eBioscience, San Diego, CA, USA), or by ZenonAlexa405-labeled PGBL22A (mouse IgG1, VMRD, Pullman, WA, USA; IgG1-Alexa405 Zenon labeling kit, Life Technologies, Carlsbad, CA, USA). In addition, the following antibodies were used: SWC5 (mouse IgG1, clone b37c10), which was detected in a second incubation step by rat anti-mouse IgG1-PerCP labeled antibody (BD Biosciences, San Jose, CA, USA), CD2-Alexa488 (mouse IgG2a, clone MSA4), CD27-Alexa647 (mouse IgG1, clone b30c7), SLA-DRQdot605 (mouse IgG2a, clone MSA3, custom conjugated to Qdot605 by Life Technologies), CD8a-PE (mouse IgG2a, clone 762-11; BD Biosciences). All non-commercial mAbs were produced in-house (Saalmüller, 1996). Directly labeled CD2 and CD27 antibodies had been purified as described elsewhere (Talker et al., 2013) and covalently conjugated to respective Alexa-fluorochromes by the use of commercially available Protein Labeling Kits (Life Technologies). Dead cells were excluded from analysis by the use of LIVE/DEADÒ Fixable Near-IR dead cell stain kit (Life Technologies). After incubation with primary antibodies samples were washed two times in 200 ll PBS (without Ca2+/Mg2+, PAA) per well and 0.05 ll of Near-IR dye was added to each sample in combination with fluorochrome-conjugated secondary antibodies. In a third step, free binding sites of secondary antibodies were blocked with whole mouse IgG molecules (2 lg per sample; Jackson ImmunoResearch, West Grove, PA, USA). Thereafter, incubation with directly conjugated antibodies (see above) was performed. Finally cells were fixed and permeablized to lyse remaining erythrocytes by a procedure with in-house prepared fixation and permeabilization buffers described previously (Gerner et al., 2008). Single-color stained samples were prepared for automatic compensation. 2.3. Intracellular cytokine staining of IFN-c, TNF-a and IL-17A Freshly isolated PBMCs and splenocytes were washed and resuspended in cell culture medium (RPMI 1640 with stable glutamine supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 100 IU/ml penicillin and 0.1 mg/ml streptomycin, all from PAA). For PMA/ionomycin stimulation 2  105 cells per well were cultivated in cell culture medium in the presence of 50 ng/ ml PMA, 500 ng/ml ionomycin (both Sigma–Aldrich, St.Louis, MO, USA) and 1 lg/ml Brefeldin A (BD GolgiPlug™, BD Biosciences) at 37°C. After four hours, stimulated cells were harvested, washed and re-suspended in PBS (without Ca2+/Mg2+) containing 3% (v/v) heat-inactivated FCS. In a first incubation step, primary cell surface antibodies directed against TCR-cd (mouse IgG1, clone PGBL22A, VMDR) and CD8a (mouse IgG2a, clone 11/295/33) were added to cell samples and incubated for 20 min at 4 °C. The isotype-specific fluorescent dye-conjugated secondary antibodies anti-IgG1-PerCP (BD Biosciences) and anti-IgG2a-PE-Cy7 (Southern Biotech, Birmingham, AL, USA) were used to detect primary antibodies. Cells were washed two times in PBS (without Ca2+/Mg2+), before blocking of free binding sites with whole mouse IgG molecules and staining of dead cells (details see above). In a fourth incubation step, directly labeled CD2-Alexa488 (mouse IgG2a, clone MSA4) and biotinylated CD27 (mouse IgG1, clone b30c7) mAbs were added. The latter was detected by a further incubation step with streptavidin-BrilliantViolet-421 conjugate (BioLegend, San Diego, CA, USA). Thereafter, cells were fixed and permeabilized by the use of BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (BD Biosciences) according to manufacturer’s instructions. Intracellular cytokine production was analyzed by the following mAbs: IFN-cPE (mouse IgG1, clone P2G10, BD Biosciences),

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TNF-a-BrilliantViolet605 (mouse IgG1, clone MAb11, BioLegend) and IL-17A-Alexa647 (mouse IgG1, clone SCPL1362, BD Biosciences). To distinguish negative from dully stained cells, the following three fluorescence minus one control samples (Roederer, 2001) were prepared: Staining panel as described above but minus PGBL22A (1), minus CD8a (2) and minus CD27 (3). Further, control samples without intracellular cytokine staining and unstained control samples only incubated with Near-IR LIVE/DEADÒ dye were prepared. In addition, again single-color stained samples were prepared for automatic compensation. 2.4. FCM analysis FCM analyses were performed on a FACSCanto II (BD Biosciences) flow cytometer equipped with a High Throughput Sampler and three lasers (405, 488 and 633 nm). For the measuring of samples containing either Qdot605 or BrilliantViolet605 fluorochromes, for the violet laser a 580 nm longpass filter and a 605/ 40 nm bandpass filter was used. Compensation was calculated after measurement of single-color stained samples by FACSDiva Software Version 6.1.3 (BD Biosciences). For samples that had been prepared for phenotyping at least 1  105 lymphocytes were recorded. This number was increased to at least 5  105 lymphocytes for intracellular cytokine staining samples. Data analysis, including boolean gating for CD2+SWC5 cd T cells and cytokine producing cd T cells, was also performed by FACSDiva Software. 3. Results 3.1. Porcine cd T cells are most abundant in blood, spleen and liver To provide more fundamental information about porcine cd T cells and their distribution throughout the body, we determined their frequency at different anatomical sites by flow cytometry (FCM) analysis. Initially, we investigated the frequency of TCR-cd expressing cells as percentage of lymphocytes within blood, spleen, tonsils, mediastinal, mesenteric and bronchial lymph nodes (med. LN, mes. LN and br. LN, respectively), liver, bone marrow (BM) and thymus of six-month old pigs (Fig. 1). High frequencies of cd T cells circulated in blood (average 31%) or resided in spleen (average 31%), liver (average 26%) and thymus (average 17%). Lower frequencies were found in tonsils (average 5%), lymph nodes (average 5%) and bone marrow (average 7%) (Fig. 1). Of note, the frequency of cd T cells in blood, spleen and thymus varied greatly between individual animals: 9–47% in blood, 18–57% in spleen and 5–31% in thymus.

Blood

31.3

med. LN counts

7.5

Liver

22.5

Spleen

40.2

mes. LN

3.3

BM

TCR-γδ

6.9

Tonsil

br. LN

In a previous study from our group SWC5 was found to be differentially expressed on circulating CD2+ and CD2 cd T cells in swine. Three distinct CD2/SWC5-defined subsets were identified in blood, comprising CD2+SWC5 , CD2 SWC5 and CD2 SWC5+ cd T cells (Talker et al., 2013). To reveal possible tissue tropisms of these subsets for different anatomical localizations, their distribution and frequency was analyzed. In blood, liver and br. LN (Fig. 2A) the three CD2/SWC5-defined subsets were detected. A fourth subset, expressing both molecules, was detectable in spleen and thymus (Fig. 2A). The distribution of the subsets appeared to be tissue-specific as their frequency varied within the analyzed organs (Fig. 2B). The CD2 subsets (CD2 SWC5 , CD2 SWC5+, gray and blue bars, respectively) clearly dominated in blood and liver. Of note, in blood CD2 SWC5 and CD2 SWC5+ subsets were present in similar proportions. In contrast, in liver CD2 SWC5+ cd T cells (blue) were nearly absent and ranged from 0.02% to 0.42% within total lymphocytes. The CD2+ subsets were preferentially found in spleen and thymus. Whereas the proportion of CD2+SWC5+ cd T cells (dark blue) was extremely low in all analyzed animals (around 0.5% in spleen and 0.22–2% in thymus) the CD2+SWC5 cd T cells (red) were highly abundant. In br. LN the ratio of the three CD2/SWC5-defined cd T-cell subsets varied greatly within the analyzed animals and ranged from an equal distribution of CD2 and CD2+ cd T cells (pig #8 and #9) to a dominance of CD2+ cd T cells (pig #6 and #7) whereas CD2 cd T cells dominated in pig #10. Further, in four out of five analyzed pigs the CD2 SWC5 subset (gray) was dominating among CD2 cd T cells, while in one pig (pig #8) same frequencies of the CD2 SWC5 (gray) and CD2 SWC5+ (blue) subsets were observed.

3.3. CD2+ cd T cells display a heterogeneous, tissue-specific expression pattern for CD8a, SLA-DR and CD27 To phenotypically characterize porcine cd T cells in more detail we analyzed the CD2/SWC5-defined subsets for their expression pattern of the activation and differentiation markers CD8a, SLADR and CD27 in blood, spleen, br. LN, liver and thymus (Fig. 3). Within the CD2 subsets the vast majority of cd T cells in all analyzed organs showed a CD8a SLA-DR CD27+ phenotype. Notably, compared to other organs, br. LN comprised a more prominent proportion of CD2 cd T cells which also expressed low levels of CD8a and CD27 but not SLA-DR (arrow #1).

B 3.4

5.3

Thymus

% TCR-γδ+ cells

A

3.2. CD2/SWC5-defined cd T-cell subsets show a tissue-specific distribution

60 50 40 30 20 10 0

14.3

pig #1

pig #2

pig #3

pig #4

pig #5

pig #7

pig #8

pig #9

pig #10

mean

pig #6

Fig. 1. Frequency of cd T cells in different organs. Ex vivo FCM analysis was performed for ten pigs and cd T cells were identified by mAbs against TCR-cd. (A) Histograms illustrate frequencies of cd T cells in different organs from pig #9. Indicated numbers show percentage of TCR-cd+ T cells within total lymphocytes. (B) The frequency of cd T cells within total lymphocytes derived from blood, spleen, tonsils, mediastinal lymph nodes (med. LN), mesenteric lymph nodes (mes. LN), bronchial lymph nodes (br. LN) liver, bone marrow (BM) and thymus is shown. From pigs # 1–5 all organs were analyzed, from pigs #6–10 only blood, spleen, med. LN and thymus. Black bars show mean frequency of either five or ten pigs.

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A

Spleen

br. LN

Liver

Thymus

SWC5

Blood

CD2

B % TCR-γδ+ cells

Spleen

Blood

60% 60 50% 50 40% 40 30% 30 20% 20 10% 10 0%

0 1 2 3 4 5 6 7 8 9 10 Pig#

br. LN

%

TCR-γδ+

cells

40% 40 30% 30 20% 20 10% 10 0%0

6

7

8

CD2+SWC5+

9 10

1

2

Liver

40% 30% 20% 10% 0%

3

4

5

6

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8

9 10

Thymus

40% 30% 20% 10% 0%

6

7

CD2+SWC5−

8 9 10 Pig#

6

CD2−SWC5+

7

8

9 10

CD2−SWC5−

Fig. 2. Distribution and frequency of CD2/SWC5-defined cd T cells in various lymphatic organs and liver. (A) Ex vivo TCR-cd+ T cells were gated (not shown) and further analyzed according to their CD2 (x-axis) and SWC5 (y-axis) expression in blood, spleen, br. LN, liver and thymus. Exemplary data of Pig# 8 are shown. (B) Stacked bar graphs show the frequency of CD2/SWC5-defined cd T cell subsets within total lymphocytes in indicated organs and individual animals.

The expression pattern for CD2+ cd T cells was more diverse and a tissue-dependent expression for the differentiation/activation markers was found. In general, within the CD2+SWC5 subset in blood, spleen, br. LN and liver the majority of cells co-expressed CD8a and CD27. However, CD27 cd T cells were also present in these organs and this population showed a high CD8a expression and was enriched in the liver (arrow #2). Worthy to note, in spleen a substantial population of cells with slightly reduced CD8a and CD27 expression levels was present (arrow #3). In thymus, the vast majority of CD2+ cd T cells were CD8a SLA-DR CD27+, but a smaller proportion of cd T cells with a CD8a+CD27+ phenotype (arrow #4) was also detectable. Similar to the CD2+SWC5 cd T cells, the rare CD2+SWC5+ cells present in spleen and thymus showed a CD8a SLA-DR CD27+ phenotype in thymus while in spleen the majority expressed CD8a and CD27 and to a lower extent SLA-DR. To determine the predominant phenotypes of CD2+ cd T cell subsets in more detail and to investigate their differentiation/activation status in the respective tissues, a combined expression analysis of CD8a, SLA-DR and CD27 was performed by boolean gating (Fig. 4A). The CD2+SWC5+ cd T cells from spleen and thymus were excluded from this analysis due to their low abundance and phenotypic similarities for marker expression. For boolean gating, living CD2+SWC5 cd T cells were gated according to their CD8a and SLA-DR expression and the absence of CD27. This phenotype (CD8a+SLA-DR+CD27 , red color) seems to represent highly differentiated cells, whereas the opposite phenotype CD8a SLA-DR CD27+ (light gray) indicates naïve cd T cells (Fig. 4B). In thymus, two thirds of the CD2+SWC5 cd T cells showed this putatively naïve CD8a SLA-DR CD27+ phenotype (light gray) and a smaller proportion of the CD8a+SLA-DR CD27+ (dark gray) phenotype was also present. Further, a small proportion which lost the CD27 molecule (CD8a SLA-DR CD27 , dark blue) was also ob-

served and was rather present in thymus than in the periphery. Minor frequencies of cells with a CD8a+SLA-DR+ phenotype (yellow) were also detected but these cells always expressed CD27. In blood and br. LN the vast majority of CD2+SWC5 cd T cells expressed CD8a but displayed a SLA-DR CD27+ phenotype (dark gray). In all analyzed organs a smaller proportion with a CD8a+SLA+ DR CD27+ (yellow) phenotype was observed, and cells with this phenotype were in general more abundant in br. LN than in other organs. The putatively highly differentiated phenotypes CD8a+SLADR+/ CD27 (orange and red) were preferentially found in liver, where almost two thirds of CD2+SWC5 cd T cells had lost the CD27 expression. These phenotypes were also present in blood and to a lower extent in br. LN and spleen. Moreover, in spleen rather naïve CD8a /+SLA-DR CD27+ phenotypes (light gray and dark gray) were dominating, whereas a small proportion with a unique CD8a SLA-DR+CD27+ (light blue) phenotype was almost exclusively found in spleen (Fig. 4B). 3.4. Porcine cd T cells produce IFN-c, TNF-a and IL-17A after polyclonal stimulation In further experiments we investigated porcine cd T cells for their capability to produce the pro-inflammatory cytokines IFN-c, TNF-a and IL-17A. Therefore, blood- and spleen-derived lymphocytes were stimulated with PMA/ionomycin in the presence of Brefeldin A and intracellular cytokine production was subsequently analyzed by seven-color flow cytometry. Initially, a blocking assay was performed to approve the cross-reactivity of anti-human IL17A mAb (clone SCPL1326, Supporting information Fig. 1) with porcine IL-17A as postulated by Stepanova et al. (2012). Pre-incubation of PMA/ionomycin stimulated PBMCs with increasing

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SLA-DR

CD27

CD2+SWC5− CD8α

SLA-DR

CD2−SWC5− CD8α

SLA-DR

CD2

CD8α

SWC5

CD2−SWC5+

Spleen Blood

2

2

br. LN

3

1

1

1

2

1

Thymus

Liver

2

4

Spleen

CD27

SLA-DR

CD2

CD8α

SWC5

Thymus

SLA-DR

CD2

CD8α

SWC5

CD2+SWC5+

CD27

Fig. 3. Phenotypic analyses of CD2/SWC5-defined cd T cell subsets. Ex vivo TCR-cd+ T cells were gated (not shown) and further sub-gated according to their CD2/SWC5 expression (left column). CD2 SWC5+ (2nd to 3rd column, light blue), CD2 SWC5 (4th to 5th column, gray), CD2+SWC5 (6th to 7th column, violet) and CD2+SWC5+ (lower panel, dark blue) cd T cells isolated from various organs (indicated on the left) were further analyzed for their CD8a, SLA-DR (y-axes) and CD27 (x-axis) expression. Data are representative for five individual animals analyzed in five independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

amounts of unlabeled polyclonal anti-swine IL-17A antibody blocked the binding of Alexa647-conjugated mAb clone SCPL1326 in a dose-dependent manner (Supporting information Fig. 1A), strongly indicating that this anti-human IL-17A antibody cross-reacts with porcine IL-17A. Moreover, the capability of porcine cd T cells and CD4+ T cells to produce IL-17A was compared. Both cell types produced IL-17A after PMA/ionomycin stimulation but frequencies of IL-17A-producing CD4+ T cells (0.4%) were higher than frequencies of IL-17A-producing TCR-cd+ cells (0.2%) (Supporting information Fig. 1B). In the following the potential to produce IFN-c, TNF-a and IL17A was investigated for total cd T cells and their CD2-defined subsets in blood and spleen from six individual animals. Overall, IFN-c, TNF-a and IL-17A was produced by cd T cells from blood and spleen as shown for one representative individual (pig #14) in Fig. 5A. Analysis of all six animals showed that the highest frequency of IFN-c-producing cd T cells was found in spleen (average 4.2%) while TNF-a and IL-17A-producing cd T cells dominated in blood (average 17.42% and 0.46%, respectively) (Fig. 5B). Additionally, CD2 expression of cytokine-producing cells among total cd T cells was analyzed, i.e. the frequency of CD2 or CD2+ cytokineproducing cells within total cd T cells was calculated (Fig. 5B). Similar frequencies of CD2 and CD2+ IFN-c-producing cd T cells were

found in blood. In contrast, in spleen more IFN-c-producing cd T cells were CD2+. Further, the vast majority of IL-17A-producing cd T cells was CD2 in both organs. Similarly, TNF-a producers were mainly CD2 in blood as well as in spleen. Taking into account that CD2 and CD2+ cd T cell are differentially distributed in the analyzed organs (Fig. 2), and in order to evaluate the potential of CD2 and CD2+ cd T cells to produce IFN-c, TNF-a or IL-17A, we calculated the frequency of cytokineproducing cells within their respective subset. Therefore, the percentage of CD2-defined cytokine-producing cd T cells was re-calculated and the respective CD2-defined subset was set to 100% (Fig. 5C, see also figure caption for calculation formula). Indeed, these analyses revealed that the higher frequency of CD2+ IFN-cproducing cells in spleen (Fig. 5B) was caused by a higher frequency of CD2+ cd T cells in this organ as similar fractions of CD2+ cd T cells in blood and spleen had the capacity to produce IFN-c (Fig. 5C, average 14.57% in blood and 10.48% in spleen). Within the CD2 cd T cell subset only few cells were able to produce IFN-c (average 0.71% in blood and 1.11% in spleen). Moreover, IL-17A-producing cells were enriched within CD2 cd T cells, (average 0.47% in blood and 0.32% in spleen) whereas only a very low proportion of CD2+ cd T cells was capable to produce this cytokine (average 0.13% in blood and 0.09% in spleen). The capability

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SSC

CD2

CD27

SLA-DR

SSC

CD8α

FSC

SWC5

SSC

near IR

TCR-γδ

A

CD2

B CD8α+ SLA-DR+ CD27−

Blood

x x x

Spleen

br. LN

x x

x x

x x

Liver

x x x

Thymus

#6

#7

#8

#9

#10 Fig. 4. Analysis of CD8a, SLA-DR and CD27 expression of CD2+SWC5 cd T cells by boolean gating. (A) Gating strategy for boolean gating of CD2+SWC5 cd T cells is shown. Lymphocytes (left panel, white ellipse) were gated according to light scatter properties and analyzed for the presence of dead cells (2nd panel, nearIR+ cells). Dead cells were excluded by further gating (nearIR cells) and cd T cells were identified by mAbs against TCR-cd (3rd panel). TCR-cd+ cells were gated and further analyzed for CD2 and SWC5 expression (right panel). CD2+SWC5 cd T cells were gated and analyzed for the expression of CD8a, SLA-DR and CD27 by boolean gating. (B) Boolean Gating for CD8a, SLADR and CD27 expression in CD2+SWC5 cd T cells results in eight possible subpopulations as illustrated in the table. Pie charts illustrate the frequency of these eight different subsets within CD2+SWC5 cd T cells in blood, spleen, bronchial lymph nodes, liver and thymus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

for TNF-a-production was present to a comparable extent in both subsets in blood, whereas in spleen more TNF-a producers were found within CD2 cd T cells (Fig. 5C). 3.5. CD2 and CD2+ cd T cells show distinct cytokine production profiles Next, we addressed the potential co-production of the three cytokines by CD2 and CD2+ cd T cells (Fig. 6). After PMA/ionomycin stimulation the vast majority of IL-17A-producing cells within the CD2 cd subset in blood and spleen co-produced TNF-a (Fig. 6A, arrow 1) but not IFN-c. However, boolean gating analysis revealed that the vast majority of cytokine-producing CD2 cd T cells are TNF-a single-producers (light blue), especially in blood (Fig. 6B) and that CD2 IL-17A/TNF-a co-producing cd T cells are a minor population (Fig. 6B, green). Due to the high frequency of CD2 cd T cells in blood (Fig. 2), TNF-a single-producing cells dominated also the pool of cytokine-producing cells within total cd T

cells (Fig. 6B). Furthermore, in the spleen of two pigs a notable proportion of CD2 IL-17A single-producers (dark blue) was also present (Fig. 6B, pig #15 and #16). Additionally, within this CD2 cd T cell subset in blood a low number of IFN-c singleproducers (dark gray) and IFN-c/TNF-a double-producers (Fig. 6A, arrow 2, Fig. 6B yellow) were detectable, but these subsets were more prominent in spleen. Within the CD2+ cd subset IFN-c-producing cd T cells dominated in blood and spleen. These cells owned the capability to produce IFN-c alone or in combination with TNF-a (Fig. 6A, arrow 3 and arrow 4; Fig. 6B, gray and yellow, respectively). In spleen, more IFN-c single-producers (dark gray) were present compared to blood. TNF-a single-producers (light blue) were also present within CD2+ cd T cells but in a much smaller proportion than in CD2 cd T cells and were preferentially found in blood. Interestingly, IFN-c/TNF-a double-producers (yellow) were equally present in both organs and accounted with two exceptions (spleen, animals #15 and #16) for over one third of all cytokine-producing CD2+ cd T cells (Fig. 6B). IFN-c/IL-17A

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Fig. 5. IFN-c, TNF-a and IL-17A production of CD2 and CD2+ cd T cells. PBMCs and splenocytes were stimulated ex vivo with PMA/ionomycin. Thereafter, gated cd T cells were analyzed for their intracellular IFN-c, TNF-a and IL-17A production. (A) Representative IFN-c, TNF-a and IL-17A (y-axes) versus CD2 (x-axes) expression of gated cd T cells in blood (left panel) and spleen (right panel) are shown (pig #14). (B) and (C) percentages of IFN-c, TNF-a and IL-17A producing cd T cells, and their CD2-defined subsets are shown for six pigs. Each symbol represents one pig. Black bars indicate mean values. (B). Percent values were calculated as follows: Number of cytokine-producing cells within the respective subset (total cd T cells, CD2+ or CD2 cd T cells) multiplied with 100 and divided by the total number of cd T cells. (C). Calculation of percent values: Number of CD2+ or CD2 cytokine producing cd T cells multiplied with 100 and divided by total number of CD2+ or CD2 cd T cells, respectively.

double-producers (orange) were nearly absent in both CD2-defined cd subsets of all analyzed individuals and organs. Also only very low numbers of triple-cytokine-producing cd T cells (red) were detected in both subsets in blood (0.21% CD2+ and 0.3% CD2 cytokine-producing cd T cells) and spleen (0.3% CD2+ and 0.61% CD2 cytokine-producing cd T cells). 3.6. CD8a is expressed in most cytokine-producing cd T cells In addition, multi-color flow cytometry analyses were performed to examine the CD8a/CD27 expression profile of CD2defined cytokine-producing cd T cells (Fig. 7). Almost all cytokine-producing cd T cells expressed CD8a (CD8adim/high), independent of their anatomical localization and CD2 expression. An exception were TNF-a single-producers within the CD2 subset, these displayed a very heterogeneous CD8a/CD27 expression pattern whereupon CD8a cells were also capable to produce TNF-a. Within the CD2 cd subset IL-17A/TNF-a double-producers as well as IL-17A single-producers showed a CD27 phenotype in blood and spleen (arrow 1). In contrast, the IFN-c single-producers as well as IFN-c/TNF-a double-producers within this subset were CD27 or CD27+ (arrow 2). Similar, among the CD2+ cd subset the IFN-c and TNF-a single-producers showed a heterogeneous CD27 /+ phenotype (arrow 3). In contrast, the vast majority of the IFN-c/TNF-a double-producers within this subset showed a CD27 phenotype (arrow 4). Further, the small proportion of IFN-c/TNF-a/IL-17A triple-producers displayed a CD27 /dim phenotype (arrow5). In phenotypic analyses of this study (Fig. 3) the CD8a+CD27 phenotype was rarely found among CD2 cd T cells but represented one major phenotype within the cytokine-producing cd T cell subsets (Fig. 7, arrows 1, 2 and 5). Hence, we additionally investigated whether this phenotype was induced by stimulation

with PMA/ionomycin or whether the underrepresentation was caused by a lower number of analyzed cells (Supporting information Fig. 2). For this purpose, defrosted PBMCs were either immediately stained or stimulated with PMA/ionomycin and 2  105 cd T cells were analyzed for their CD2, SWC5, CD8a and CD27 expression by flow cytometry. PMA/ionomycin stimulation had no influence on the phenotype, as no differences were detectable between stimulated and defrosted CD2 cd T cells or the CD2/ SWC5-defined cd subsets. In contrast, cd T cells with a CD8a+CD27 phenotype became more visible in samples where 2  105 cd T cells were analyzed for CD8a and CD27 expression than in samples where 4  104 cd T cells were taken into account for the analysis. These data indicate that CD8a+CD27 cd T cells represent a rare phenotype among CD2+ cd T cells and that this phenotype was not induced by PMA/ionomycin stimulation.

4. Discussion In the current study we could demonstrate that cd T cells in adolescent pigs are pronounced in blood, spleen, thymus and liver and are sparsely distributed in tonsils, lymph nodes and bone marrow. This observation is in consensus with previous studies made in swine (Takamatsu et al., 2006; Stepanova and Sinkora, 2012) and other cd-high-species like ruminants (Hein and Mackay, 1991). Notable is the high occurrence of porcine cd T cells in the liver, which also represents a site of accumulation for cd T cells in humans (Kenna et al., 2004). In most species, discrete subsets of cd T cells are differentially located in certain tissues and organs to exert various immunological functions (Holderness et al., 2013). Data reported here complement the existing knowledge of the tissue distribution of CD2-defined cd T cells in swine (Saalmüller et al., 1990). CD2 cd

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Fig. 6. Cytokine co-production and boolean gating of CD2 and CD2+ cytokine-producing cd T cells. Freshly isolated PBMCs (upper panel) and splenocytes (lower panel) were stimulated with PMA/ionomycin and IFN-c, TNF-a and IL-17A production was analyzed in CD2 and CD2+ cd T cells (gating not shown). (A) Dot plots show IL-17A (y-axis) vs. IFN-c (x-axis) (top), IL-17A (y-axis) vs. TNF-a (x-axis) (middle) and IFN-c (y-axis) vs. TNF-a (x-axis) (bottom) production in gated total cd T cells (left column, dark green), CD2 cd T cells (middle column, light green) and CD2+ cd T cells (right column, violet). Data from pig #14 are shown. (B) Boolean gating was performed for cytokineproducing cd T cells (left column), CD2 cd T cells (middle column) and CD2+ cd T cells (right column). Color codes of possible cytokine-(co-) producing subpopulations are depicted in the table below. Pie charts illustrate the frequency of the different types of cytokine-(co-) producing cells in blood (upper panel) and spleen (lower panel). Data of six individuals are shown, with one individual in one row. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

T cells are known to be enriched in blood but, as we demonstrated here, represent also the dominating subset in liver. CD2+ cd T cells are preferentially accumulated in lymphoid tissues like spleen, lymph nodes and thymus. This is similar to cattle, where CD2 CD8 WC1+ cd T cells are predominant in blood, while CD2+CD8 / + WC1 cd T cells are enriched in spleen and intestine (Machugh et al., 1997). In contrast, all human cd T cells in blood are CD2+ (Groh et al., 1989). Recently, a mAb specific for the porcine CD27 molecule was identified and initial phenotyping experiments revealed the existence of CD27+ and CD27 cd T cells (Reutner et al., 2012). For human Vc9Vd2 T cells it was shown that these cells can perform memory responses and that CD27 is not only present on naive cells but also expressed on central memory cd T cells, whereas effector

memory cd T cells lost this molecule (Dieli et al., 2003). Our studies allowed for the first time to investigate and compare the expression pattern of CD27 for porcine CD2-defined cd T-cell subsets. The analysis of CD2 cd T cells in distinct anatomical locations showed that most of these cells have a phenotype reminiscent of naïve porcine CD4+ ab T cells (Reutner et al., 2013; Saalmüller et al., 2002) as the vast majority expressed the CD27 molecule and lacked the differentiation markers CD8a and SLA-DR. CD2+ cd T cells showed a more heterogeneous expression pattern for CD27, hence boolean gating analysis was performed to examine their frequency and differentiation status in more detail. In thymus, the majority of CD2+ cd T cells expressed CD27 in the absence of CD8a and SLA-DR but a minor population of these cells also expressed CD8a. This is in line with studies from Sinkora et al. (2005)

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Fig. 7. Phenotype of cytokine-producing CD2 and CD2 cd T cells. Cytokine-producing cd T cells were analyzed for CD8a (y-axes) and CD27 (x-axes) expression. Blood- or spleen-derived total cd T cells (dark green, left column), CD2 (light green, middle column) or CD2+ (violet, right column) cd T cells are depicted. Rows show single (IFN-c, 1st row; TNF-a, 2nd row; IL-17A, 3rd row), double (TNF-a + IL-17A, 4th row; IFN-c + TNF-a, 5th row; IFN-c + IL-17A, 6th row) and triple (7th row) cytokine-producing cd T cells, highlighted by respective colors. Non-cytokine producing cd T cells are depicted in light gray. Data from pig #11 is shown and representative for six individual animals. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

where they hypothesized that CD8a+ cd T cells might either develop in the thymus or acquire CD8a upon activation in the periphery. Further, a small proportion of thymus-derived CD2+ cd T cells not only lacked CD8a and SLA-DR but also CD27. This is similar to studies in mice, where thymus-derived cd T cells have been shown to be either CD27+ or CD27 and that this differential expression initiates the development of distinct cd T cell lineages with the potential for IFN-c (CD27+) or IL-17A (CD27 ) cytokine production (Ribot et al., 2009). In the analyzed secondary lymphatic organs CD8a+ was expressed on the majority of CD2+ cd T cells. Moreover, in liver an enrichment of CD2+ cd T cells with a CD8a+SLA-DR+/ CD27 phenotype was observed. Assuming that phenotypical differentiation in porcine cd T cells due to antigen contact has similarities with porcine ab T cells and human Vc9Vd2 T cells, this indicates an accumulation of highly differentiated cd T cells in this organ. The notion that a CD2+CD8a+CD27 phenotype correlates with late differentiation in porcine cd T cells is also supported by findings from our group where an age-dependent increase of cells with this phenotype was observed in blood (Talker et al., 2013).

In the second part of this study we aimed to correlate the phenotype of porcine cd T cells with their capacity for the production of IFN-c, TNF-a and IL-17A. IL-17A-producing cd T cells, which mainly co-produced TNF-a, showed a CD8a+CD27 phenotype and were nearly entirely found among cytokine-producing CD2 cd T cells. This may indicate a special differentiation pathway of this cd T cell subset, similar to the development described in murine IL-17A-producing cd T cells (Ribot et al., 2009). Differently, TNF-a single-producing cd T cells showed a heterogeneous CD8a/CD27 phenotype and were quantitatively dominating within the CD2 subset, which may indicate that TNF-a production is less tightly regulated in regard to phenotypic differentiation. Within the CD2+ cd T cell subset IFN-c/TNF-a double-producers with a CD8a+CD27 phenotype and IFN-c and TNF-a single-producers, with a CD8a+CD27 /+ phenotype prevailed. This again may indicate a determined differentiation pathway where CD2+ cd T cells show a preferential co-production of IFN-c and TNF-a. In vivo studies in mice, primates and humans revealed the existence of cd T cells producing both IL-17A and IFN-c, which have also been designated as ‘‘multifunctional’’ (Fenoglio et al., 2009; Ryan-Payseur et al.,

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2012; Sheridan et al., 2013). The cd T cells isolated from blood and spleen of six-month old pigs of our study barely contained IL-17A/ IFN-c double-producing or IL-17A/IFN-c/TNF-a triple-producing cells. However, this might be different in cd T cells isolated from other organs or during acute infections and therefore requires further investigation. 5. Conclusions In summary, our study reports a comprehensive overview of the phenotype of porcine cd T cells in primary and secondary lymphatic tissues as well as the liver, but also their capacity for cytokine production. This revealed insights into homing preferences and potential differentiation pathways in regard to phenotype and cytokine production profiles. Thereby, a basis for the role of cd T cells in healthy adolescent pigs has been provided, that contributes to the overall paradigm of cd T cells in mammalian species. Acknowledgments The authors thank Maria Stadler and Hanna Koinig for general technical support. Corinna Sedlak was funded by the Comet programme ‘‘Preventive veterinary medicine: Improving pig health for safe pork production’’ sponsored by BMVIT, BMWFJ and the government of Lower Austria. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dci.2014.02.008. References Blumerman, S.L., Herzig, C.T.A., Baldwin, C.L., 2007. WC1+ cd T cell memory population is induced by killed bacterial vaccine. Eur. J. Immunol. 37, 1204– 1216. Bonville, M., O’Brien, R.L., Born, K.W., 2010. cd T cell effector functions: a blend of innate programming and acquired plasticity. Nat. Rev. Immunol. 10, 467–478. Caccamo, N., Todaro, M., Sireci, G., Meraviglia, S., Stassi, G., Dieli, F., 2013. Mechanisms underlying lineage commitment and plasticity of human cd T cells. Cell. Mol. Immunol. 10, 30–34. Crispe, I.N., 2001. Isolation of mouse intrahepatic lymphocytes. In: Coligan, J.E., Bierer, B.E., Margulies, D.H., Shevach, E.M., Stober, W. (Eds.), Current Protocols in Immunology. John Wiley & Sons, Inc., Somerset, New York (Unit 3.21.1). Dieli, F., Poccia, F., Lipp, M., Sireci, G., Caccamo, N., Di Sano, C., Salerno, A., 2003. Differentiation of effector/memory Vdelta2 T cells and migratory routes in lymph nodes or inflammatory sites. J. Exp. Med. 198, 391–397. Davis, W.C., Zuckermann, F.A., Hamilton, M.J., Barbosa, J.I., Saalmüller, A., Binns, R.M., Licence, S.T., 1998. Analysis of monoclonal antibodies that recognize cd T/ null cells. Vet. Immunol. Immunopathol. 60, 305–316. Fenoglio, D., Poggi, A., Catellani, S., Battaglia, F., Ferrera, A., Setti, M., Murdaca, G., Zocchi, M.R., 2009. Vd1 T lymphocytes producing IFN-c and IL-17 are expanded in HIV-1-infected patients and respond to Candida albicans. Blood 113, 6611– 6618. Groh, V., Porcelli, S., Fabbi, M., Lanier, L.L., Picker, L.J., Anderson, T., Warnke, R.A., Bhan, A.K., Strominger, J.L., Brenner, M.B., 1989. Human lymphocytes bearing T cell receptor c/d are phenotypically diverse and evenly distributed throughout the lymphoid system. J. Exp. Med. 169, 1277–1294. Gerner, W., Käser, T., Pintaric, M., Groiss, S., Saalmüller, A., 2008. Detection of intracellular antigens in porcine PBMC by flow cytometry: a comparison of fixation and permeabilisation reagents. Vet. Immunol. Immunopathol. 121, 251–259. Hayday, A.C., 2000. cd T cells: a right time and a right place for a conserved third way of protection. Annu. Rev. Immunol. 18, 975–1026. Hein, W.R., Mackay, R., 1991. Prominence of cd T cells in the ruminant immune system. Immunol. Today. 12, 30–34. Holderness, J., Hedges, J.F., Ramstead, A., Jutila, M.A., 2013. Comparative biology of cd T cell function in humans, mice and domestic animals. Annu. Rev. Anim. Biosci. 1, 99–124. Kenna, T., Golden-Mason, L., Norris, S., Heagarty, J.E., O’Farrelly, C., Doherty, D.G., 2004. Distinct subpopulations of cd T cells are present in normal and tumorbearing human liver. Clin. Immunol. 113, 56–63. Lee, J., Choi, K., Olin, M.R., Cho, S., Molitor, T.W., 2004. cd T cells in immunity induced by Mycobacterium bovis bacillus Calmette-Guérin vaccination. Infect. Immun. 72, 1504–1511.

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