Porcine interleukin-12 fusion protein and interleukin-18 in combination induce interferon-γ production in porcine natural killer and T cells

Veterinary Immunology and Immunopathology 86 (2002) 11–21

Porcine interleukin-12 fusion protein and interleukin-18 in combination induce interferon-g production in porcine natural killer and T cells K. Domeika*, M. Berg, M.-L. Eloranta, G.V. Alm Department of Veterinary Microbiology, Section of Immunology, Swedish University of Agricultural Sciences, Biomedical Centre, P.O. Box 588, SE-75123 Uppsala, Sweden Received 14 December 2000; received in revised form 15 November 2001; accepted 29 November 2001

Abstract A pcDNA3 vector containing a gene encoding a porcine interleukin-12 (poIL-12) fusion protein was constructed, with the p40 chain and its signal peptide positioned first, followed by a linker and the p35 domain. When expressed in COS cells, secreted poIL-12 fusion protein showed high activity in terms of ability to induce interferon-g (IFN-g) production in porcine peripheral blood mononuclear cells (PBMCs) in vitro. The IFN-g production induced by poIL-12 fusion protein, as well as heterodimeric poIL-12 and human IL-12, was markedly dependent on the presence of human IL-18 (huIL-18). Furthermore, huIL-18 showed a dose-dependent induction of IFN-g production in PBMC in the presence of a constant concentration of huIL-12. A marked synergism between poIL-12 and IL-18 was consequently observed in poPBMC. The actual IFN-g producing cells were identified as probable NK cells (about 30%) and T lymphocytes (about 70%), using flow cytometry. Furthermore, a histidinetagged poIL-12 fusion protein was expressed in Drosophila melanogaster Schneider 2 cells, using a modified pMT/V5-His vector lacking the V5 epitope. Such poIL-12 fusion protein was easily purified using Ni–NTA agarose and retained high biological activity. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Pig; Cytokines; Interleukin-12; Interleukin-18; Interferon-g

1. Introduction Interleukin-12 (IL-12) is secreted by especially monocytes, macrophages and dendritic cells (D’Andrea et al., 1992; Macatonia et al., 1995; Kato et al., 1996; Sousa et al., 1997) in response to different microbial factors. IL-12 plays an important role in the induction Abbreviations: DES, Drosophila expression system; MLC, mixed leucocyte culture * Corresponding author. Tel.: þ46-18-471-4533; fax: þ46-18-471-4382. E-mail address: [email protected] (K. Domeika).

of a cell-mediated immune response. It enhances the activity of cytolytic T lymphocytes (CTLs) and NK cells (Trinchieri and Scott, 1994; Cho et al., 1996) and is involved in the differentiation of naive T cells to the Th1 subset (Hsieh et al., 1993; Schopf et al., 1999). The IL-12 also induces production of interferon-g (IFN-g) in T lymphocytes and NK cells (Kobayashi et al., 1989). Another IFN-g inducing cytokine is interleukin-18 (IL-18), which is mainly produced by activated macrophages (Okamura et al., 1995). IL-18 acts synergistically with IL-12 in the induction of IFN-g production by antigen stimulated T cells in mice and humans (Micallef et al., 1996; Kohno et al., 1997; Tominaga

0165-2427/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 1 ) 0 0 4 3 1 - 7

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et al., 2000). Both IL-12 and IL-18 are released at interaction between Th1 cells and splenic antigen presenting cells (APCs) in presence of antigen (Kohno et al., 1997). The bioactive heterodimer form of IL-12 consists of 35 and 40 kDa proteins (p35 and p40, respectively), linked by disulphide bonds (Podlaski et al., 1992). The p35 subunit is transcribed constitutively (D’Andrea et al., 1992; Bost and Clements, 1995). Production of p40 in excess of p35 can result in formation of p40 homodimers that antagonise the activity of IL-12 (Mattner et al., 1993), demonstrating the need for the two subunits of IL-12 in equimolar amounts to produce biologically active protein. Porcine IL-12 (poIL-12) has been cloned (Foss and Murtaugh, 1997) and expressed as a fusion protein in a baculoviral system (Kokuho et al., 1999). It has also been expressed in Pichia pastoris using a plasmid as vector (Foss et al., 1999). In both studies the activity of IL-12 was determined by its ability to stimulate proliferation of lymphoblasts. The ability of IL-12 to stimulate antigen-specific Th1 cells and CTL indicates that IL-12 can be an important adjuvant in vaccines. In the pig, an increased number of IFN-g producing cells was found when human recombinant IL-12 was used as adjuvant in a pseudorabies vaccine (Zuckermann et al., 1998, 1999). Plasmids expressing IL-12 can also have adjuvant effects on DNA vaccine antigen-expressing plasmids in murine systems (Kim et al., 1997; Chow et al., 1998). The aim of the present study was to construct plasmids expressing biologically active poIL-12 and to study its ability to induce IFN-g production. We prepared several pcDNA3 plasmids encoding the poIL-12 p40 and p35 chains separately or as a fusion protein, with the two chains joined by a flexible peptide linker. Such poIL-12 was transfected in COS cells and culture supernatants were examined for biologic activity. Furthermore, we expressed a histidine-tagged porcine IL-12 fusion protein in a Drosophila expression system (DES) in order to facilitate its purification and characterisation. The biological activity of poIL-12 was measured as the ability of IL-12 to induce IFN-g production in porcine peripheral blood mononuclear cell (PBMC), alone or in combination with rhuIL-18. A synergistic effect between poIL-12 and rhuIL-18 was shown, with

higher IFN-g levels when the two cytokines were used in combination. Finally, the identity of the IFN-g producing cells was determined by means of flow cytometry, using combined staining for intracellular IFN-g and cell surface antigens.

2. Materials and methods 2.1. Preparation of mRNA and synthesis of cDNA Porcine PBMC were purified from heparinized blood using Ficoll Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. The cells were stimulated for 24 h at 37 8C with 1 mg/ml of LPS (Sigma, St Louis, MO) and 5 mg/ml PHA (Purex Diagnostics, Dartford, UK) in HEPES-buffered (20 mM) RPMI 1640 medium with 5% FCS (MyocloneTM; Gibco, Paisley, UK), 2-mercaptoethanol (5105 M), L-glutamine (2 mM), penicillin (200 U/ml) and streptomycin (100 mg/ml). The mRNA from such stimulated porcine PBMC was purified using the Dynabeads1 mRNA direct micro kit (Dynal, Oslo, Norway). In brief, 3  105 stimulated cells in 100 ml lysis buffer were mixed with 20 ml of Dynabeads Oligo(dT)25 and incubated for 5 min at room temperature (RT). The mRNA-Dynabeads were resuspended in reverse transcriptase buffer (Qiagen, Hilden, Germany), 1 mM dNTP mix, 10U RNase inhibitor, 4U Omniscript reverse transcriptase (Qiagen), and incubated for 1 h at 37 8C. 2.2. Construction of plasmids 2.2.1. The pcDNA3/p40 Amplification of the gene for the p40 subunit of porcine IL-12 including its native signal peptide was performed using Taq-polymerase (Amersham Pharmacia Biotech) and primers with a Xho I and Xba I restriction sites included in each 50 end for subsequent cloning. The primer sequences were: 50 -CCC CTC GAG ATG GCC CTT CAG CAG CTG GTTand 50 -ACATTC TAG AAT TGC AGG ACA CAG ATG C. For each PCR reaction, 10 ml Dynabeads-cDNA-mixture was used as template. The vector and the PCR product were digested with Xho I and Xba I, ligated using T4 DNA ligase (New England Biolabs) and then used to transform competent E. coli using standard methods.

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2.2.2. The pcDNA3/p35 To construct a plasmid expressing the p35 domain of IL-12, the open reading frame including signal peptide was amplified using 50 -CCG CTC GAG ATG GGC CCG CTG CGC AAC CTC as the forward primer and 50 -CCC GAG CTC TAG ATT AGG AAG AAT TCA GAT AGC T as the reverse primer, introducing 30 Xba I and Sac I restriction sites. As template in the PCR we used an IL-12 containing plasmid (pGEM35/40), kindly donated by Dr. Kokuho (National Institute of Animal Health, Ibaraki, Japan). The PCR product and pcDNA3 were cut with Xho I and Xba I, ligated and introduced into E. coli as described earlier. 2.2.3. The pcDNA3/p40-L-p35 The open reading frames for p40 and p35 were amplified using the previously described plasmids as templates. The same pairs of primers were used, except for the p35 for which a new forward primer omitting the signal peptide was designed (50 -CCC GAG CTC AGG AGC CTC CCT GCA ACC), introducing a 50 Sac I restriction site. First, the p40 part was ligated into a Xho I/Xba I cut pGEM plasmid, giving a pGEM p40 plasmid. Then, the double stranded sequence encoding the (Gly4Ser)3 linker (L) (Huston et al., 1988), containing a 50 Xba I overhang and a Sac I restriction site in the 30 end was ligated into the Xba I/Sac I digested pGEMp40 plasmid. This ligation destroyed the Xba I site and the p40 stop codon. The p35 domain was then digested with Sac I and ligated into the pGEMp40L construct. The orientation of the p35 fragment was determined by restriction analysis. Finally, the whole cassette was moved into pcDNA3 using Xho I and Xba I. 2.2.4. The pMT/His-p40-L-p35 The whole open reading frame from the pcDNA3/ p40-L-p35 was PCR amplified using the original primer of p40 and a new p35 reverse primer with an Age I restriction site. The PCR product was purified, digested with Xho I and Age I, and ligated into a similarly digested pMT/V5-HisA plasmid. This procedure resulted in a plasmid encoding a porcine IL-12 fusion protein (designated pMT/IL-12 fusion protein) with a polyhistidine (his) tag at the C-terminus but without the V5 epitope.

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2.3. Expression of plasmids in COS and Drosophila melanogaster Schneider 2 cells COS cells, grown in Dulbecco’s modified Eagles medium, 200 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 5% FCS, were transfected with plasmids using the FuGENETM 6 transfection reagent according to the manufacturer’s instructions (Boehringer Mannheim, Mannheim, Germany). Culture supernatants were collected after 24 and 48 h. D. melanogaster Schneider 2 (S2) cells were grown at RT in DES expression medium supplemented with 100 U/ml penicillin, 50 mg/ml streptomycin, 2 mM L-glutamine and 10% FCS (Invitrogen). Transfection of S2 cells was performed using the Ca-phosphate method as described in the DES manual (Invitrogen, Carlsbad, CA). Expression was induced by the addition of 0.5 mM CuSO4 and supernatants were collected 24 h later. Stable selection of S2 cells required cotransfection of a plasmid expressing the hygromycin resistance gene (pCoHYGRO) in combination with the plasmid expressing recombinant protein. After 5 days, 300 mg/ml of hygromycin-B was added to the medium and the selection continued for about 4 weeks. Cells were then expanded and protein was induced as described above. 2.4. Purification of poIL-12 fusion protein Purification of the histidine-tagged pMT/IL-12 fusion protein on Ni–NTA agarose (Qiagen, Hilden, Germany) was performed as recommended by the manufacturer. In brief, the supernatant from S2 cells was dialysed against lysis buffer (50 mM Na2PO4, 300 mM NaCl, 10 mM imidazole). Ni–NTA agarose was added to the supernatant and incubated rotating for 2 h. The agarose was then transferred into a small column (Econo-column; BioRad, Hercules, CA), washed and the fusion protein was eluted using a buffer containing 50 mM Na2PO4, 300 mM NaCl and 250 mM imidazole. The purity and concentration was estimated from a SDS-PAGE of the eluate. The gel was stained with Coomassie Brilliant Blue. 2.5. Western blots Recombinant human IL-12 (0.1 ng; R&D Systems, Minneapolis, MN) and 5 ml of the Ni–NTA purified

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pMT/IL-12 fusion protein were run on a 10% SDS polyacrylamide gel and the proteins were transferred to Hybond N nitrocellulose membrane (Amersham Pharmacia Biotech). Transfer of proteins to the membrane was verified using prestained molecular weight standard (New England Biolabs, Beverly, MA). The membrane was blocked with 5% blocking reagent (Amersham Pharmacia Biotech) in PBS. The IL-12 was detected by incubation at RT overnight with a 1:1000 dilution of a monoclonal anti-human IL-12 antibody, known to crossreact with porcine IL-12 (C8.6; Pharmingen, San Diego, CA). A horseradish peroxidase labelled goat anti-mouse IgG antibody (Dako, Glostrup, Denmark) diluted 1:2000 was used for detection, followed by staining with the ECL chemiluminescent detection system (Amersham Pharmacia Biotech). As a negative control, Ni–NTA agarose purified material from S2 cells transfected with pMT plasmid without the IL-12 gene was used. 2.6. Bioassay for IL-12 and IL-18 For the bioassay of IL-12 and IL-18, porcine PBMC were grown in RPMI 1640 medium (see above) at 5  106 cells/ml in 0.1 ml volumes in 96-well flatbottomed microtiter plates (Nunc, Roskilde, Denmark) at 37 8C and in 7% CO2. Unless otherwise indicated, PBMC from two pigs were mixed (1:1) to achieve a mixed leucocyte culture (MLC). The cultures contained different concentrations of recombinant human IL-12 (rhuIL-12; R&D Systems), rpoIL12 (Endogen, Woburn, MA), dilutions of supernatants from transfected COS cells, S2 cells or purified poIL12 from such supernatants, in combination or not with 100 ng/ml of rhuIL-18 (PeproTech EC, London, UK). In addition, the concentration of huIL-18 was varied in the presence of a constant level of huIL-12 (5 ng/ml). After 4 days, the concentration of poIFN-g in the medium was determined by a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) as previously described (Wattrang et al., 1997). The results were expressed as ng/ml using as standard a rpoIFN-g expressed in E. coli (kindly provided by Dr. Claude La Bonnadiere, INRA, France). 2.7. Phenotyping of IFN-g producing cells The cells that produce IFN-g after stimulation with poIL-12 and huIL-18 were stained for surface antigens

and intracellular IFN-g and characterised using flow cytometry. Porcine PBMC were cultured with or without rpoIL-12 (25 ng/ml) and rhuIL-18 (100 ng/ml) for 22 h in 24-well plates (Nunc), using 5  106 cells and 1.3 ml volumes per well. Brefeldin A (10 mg/ml; Sigma), was present for the last 4 h. The cells were harvested using PBS supplemented with 5 mM EDTA and washed in ice cold PBS. For staining of cell surface antigens, the following mAbs were used; a biotin-conjugated anti-poCD2 (2.5 mg/ml; clone MSA4; VMRD, Pullman, WA, USA), an FITC-conjugated anti-poCD3 (2.5 mg/ml; clone BB23-8E6-8C8; Pharmingen) or an antipoCD14 (dilution 1/20; clone MIL-2; Serotec, Oxford, UK). For detection of NK-cells, a combination of mAbs recognising CD2 and CD3 was used. Each mAb was compared to an isotype control mAb. The staining was performed for 30 min on ice in PBS containing 0.01 M Hepes, 0.1% human serum albumin (HSA) and 5% normal mouse serum (NMS). The number of cells stained in each reaction was 3  106 in 0.1 ml. The cells were washed and then incubated for 30 min on ice with the secondary reagents streptavidinRPE-Cy5 (dilution 1/250; Dako) for anti-poCD2 or an FITC-conjugated goat-anti-mouse Ig (5 mg/ml; Dako) for anti-poCD14. In the latter case, blocking NMS was omitted. Staining for intracellular IFN-g was carried out after staining for cell surface antigens. The cells were fixed using 2% paraformaldehyde for 15 min at RT, washed in PBS and then permeabilised using PBS supplemented with 0.1% saponin, 0.01 M Hepes, 0.1% HSA and 5% NMS. Intracellular staining was performed with a phycoerythrin (PE)-conjugated antipoIFN-g mAb (2.5 mg/ml; clone P2G10; Pharmingen) for 30 min at RT. The cells were washed, suspended in PBS and kept on ice until analysed using a FACStar plus and CellQuest software (Becton Dickinson, San Jose, CA, USA).

3. Results 3.1. Evaluation of the IL-12/IL-18 bioassay We found that huIL-12 induced poIFN-g production in PBMC in a dose-dependent fashion. This response was weak and varied between individuals (Fig. 1A),

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Fig. 1. Dose-dependent induction of IFN-g production in porcine PBMC by huIL-12 (A), and huIL-18 (B). In (A), the IL-12 concentration was varied in the presence (filled symbols) or not (open symbols) of 100 ng/ml IL-18. In (B), the IL-18 concentration was varied in the presence (filled symbols) or not (open symbols) of 5 ng/ml IL-12. The PBMC from two pigs were cultured separately (pig number 1 (circles); pig number 2 (triangles)) or mixed so as to create an MLC (squares). The IFN-g levels (ng/ml) were determined by immunoassay. Results are shown as mean of triplicate cultures.

but was increased in MLC (Fig. 1A and B). Presence of a constant concentration of huIL-18 markedly increased the IFN-g levels induced by IL-12 (Fig. 1A). The huIL-18 alone induced little or no IFN-g, but caused a dose-dependent increase of the IFN-g production by PBMC in the presence of a constant concentration (5 ng/ml) of either huIL-12 (Fig. 1B) or rpoIL-12 (results not shown). Consequently, both IL-12 and IL-18 can be measured in the present bioassay. 3.2. Construction and evaluation of porcine IL-12 vectors We constructed plasmids expressing the porcine IL-12 p40 and p35, transfected COS cells, collected supernatants 48 h post transfection and tested them in the IL-12/IL-18 bioassay for ability to induce IFN-g in poPBMC. As shown in Fig. 2, a significant biological activity was detected when COS cells were co-transfected with plasmids expressing p40 and p35, but not when they were transfected with these plasmids separately. This indicates that a functional heterodimer of p40 and p35 was necessary for biological activity.

Furthermore, we constructed a fusion protein of porcine IL-12 with the p40 domain positioned first, followed by a linker and the p35 domain. This plasmid construct (pcDNA3/p40-L-p35) was transfected in COS cells and supernatants were tested in the IL-12/IL-18 bioassay. One plasmid with a known deletion disrupting the open reading frame of the poIL-12 fusion protein (clone 1) was inactive, while one plasmid with the correct fusion protein coding sequence (clone 2) resulted in high levels of IFN-g inducing activity in the supernatants (Fig. 3). To obtain larger quantities of poIL-12 fusion protein, we expressed the pMT/His-p40-L-p35 plasmid in S2 cells in the DES. In this plasmid construct, the V5 epitope is deleted but the His tag remains and can be used as a purification handle. As shown in Fig. 4A, S2 cells showed a transient expression of poIL-12 fusion protein that induced IFN-g production in the IL-12/IL-18 bioassay. This indicates that poIL-12 with the His tag at the C-terminus is biologically active. We then selected for stable S2 cell transformants and scaled up the cell cultures. Expression of protein was induced by Cu2SO4, resulting in supernatants that contained approximately 30 mg/ml His-tagged poIL-12 fusion protein as measured by bioassay.

Fig. 2. Measurement of poIL-12 in culture supernatant of COS cells transfected with pcDNA3/p40, pcDNA3/p35 or the combination of these plasmids. The supernatants were designated IL-12p40, IL-12p35 and IL-12p35 þ p40, respectively. The activity of the IL-12 was measured as ability to induce IFN-g production in poPBMC cultures (MLC) in the presence of huIL-18 (100 ng/ml), using final supernatant concentrations of 1.7, 5 and 15%. The IFN-g levels (ng/ml) were determined by immunoassay. Results are shown as mean of triplicate cultures.

Fig. 3. Ability of the poIL-12 fusion protein (pcDNA3/p40-L-p35) to induce IFN-g production by poPBMC in the IL-12/IL-18 bioassay (MLC). Two plasmid clones encoding a potentially biologically inactive (clone 1) and active (clone 2) poIL-12-fusion protein were used to transfect COS cells for transient expression. Dilutions of COS cell culture supernatant (%) and for comparison rhuIL-12 (ng/ml) were analysed in the bioassay in the presence of huIL-18 (100 ng/ml). Levels of induced IFN-g (ng/ml) were determined by immunoassay. Results are shown as mean of triplicate cultures.

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Fig. 4. Determination of the bioactivity of His-tagged poIL-12 fusion protein produced by Drosophila Schneider (S2) cells transfected with the pMT/His-p40-L-p35 vector. (A) Appearance of poIL-12 bioactivity in culture supernatants after transient expression of pMT/His-p40-Lp35 (pMT/IL-12), using pMT/V5-His vector without insert as control (pMT). (B) Stable expression of the pMT/His-p40-L-p35 vector in S2 cells, with poIL-12 bioactivity in culture supernatants (S2/IL-12) and after purification on Ni–NTA agarose (IL-12pur). In both (A) and (B), the ability of dilutions of supernatants or purified poIL-12 (%) to induce IFN-g production in poPBMC cultures (MLC) was determined in the absence (first six bars in each graph) and presence of 100 ng/ml huIL-18 (next six bars in each graph). IFN-g levels (ng/ml) were measured by immunoassay. Results are shown as mean of triplicate cultures.

This poIL-12 was then purified using Ni–NTA agarose. The eluted poIL-12 was analysed by SDS-PAGE, as well as by Western blotting using an anti-huIL-12 mAb. One band with the expected molecular weight of the IL-12 fusion protein was detected (Fig. 5). The purified poIL-12 fusion protein was bioactive (Fig. 4B). While this procedure resulted in a pure poIL-12, the yield was low (5–10%). 3.3. Identification of IFN-g producing cells The cells producing IFN-g after stimulation of porcine PBMC in MLC by poIL-12 and huIL-18 for 22 h were identified by flow cytometry. When such cells were stained for intracellular IFN-g, 3:0  1:1% (n ¼ 5) were positive. In control MLC cultures not stimulated by these cytokines, the frequency of positive cells was 0:34  0:19. A similarly small proportion of cells was also defined as positive using an isotype control for the anti-IFN-g antibody, both in cultures with and without added poIL-12 and huIL-18 (0:32  0:28 and 0:23  0:17%, respectively).

The phenotype of the IFN-g producing cells was determined by the use of a triple-staining procedure for the surface antigens CD2 and CD3 and intracellular IFN-g. The cells gated as positive for IFN-g (gate R2 in Fig. 6A) were analysed for expression of CD2 and CD3. For comparison, the results of staining with PE-conjugated isotype control (Fig. 6B) and the distribution of all CD2 and CD3 cells (Fig. 6C) are shown. As shown in Fig. 6D, the IFN-g positive cells among PBMC stimulated by the combination of poIL-12 and huIL-18 are located in gates containing the CD2þCD3 cells (gate R3; 0.47% of all analysed cells) and the CD3þCD2þ cells (gate R4; 1.03%). While no IFN-g producers were detected in the CD3þCD2 T-cells (gate R6), 0.30% positive cells were present in gate R5 that contains the CD2CD3 cells. However, a similar proportion IFN-g positive cells predominantly located in gate R5 (0.22%; Fig. 6E) was also seen with PBMC not stimulated by poIL-12 and huIL-18. Furthermore, approximately the same proportion of PE-isotype staining cells in both unstimulated and poIL-12/IL-18 stimulated

Fig. 5. Analysis by SDS-PAGE (A) and Western blot (B) of His-tagged poIL-12 fusion protein purified from S2 cell culture supernatants using Ni–NTA agarose. (A) Coomassie-stained SDS-PAGE, lane1 showing the molecular weight standard and lane 2 results with control Ni–NTApurified supernatant from S2 cells transfected with pMT/V5-His without insert. Lanes 3 and 4 show two separate purified poIL-12 fusion protein preparations, with arrows indicating the poIL-12. (B) The purified poIL-12 fusion protein (lane 1, arrow) detected by the C8.6 antihuIL-12 mAb in Western blot. The same mAb produced a weak band with approximately 0.1 ng of rhuIL-12 (lane 2, arrow).

Fig. 6. Three colour flow cytometry of IFN-g producing poPBMC in MLC and their expression of CD2 and CD3. The cells were stained after 22 h in culture, using anti-CD2 (RPE-Cy5) and anti-CD3 (FITC), followed by anti-IFN-g (PE) to detect intracellular IFN-g. (A) The proportion IFN-g positive cells (1.92%, in gate R2) among poPBMC stimulated in MLC by the combination of poIL-12 (25 ng/ml) and huIL18 (100 ng/ml). (B) The same type of cells as in (A) stained with control isotype-matched mAbs (0.25% positive cells in gate R2). (C) Distribution according to their expression of CD2 and CD3 for all poPBMC stimulated in MLC by poIL-12 and huIL-18. (D) Distribution of the IFN-g positive cells (gate R2 in (A)) according to their expression of CD2 and CD3. (E) Distribution of IFN-g positive poPBMC in cultures not receiving poIL-12 and huIL-18, most positive cells located in gate R5. The proportions of cells in the gate R2 and the four gates R3–R6 are expressed as percent of all analysed cells and shown in the upper right part of each diagram. Data from one of the two experiments with similar results are shown.

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PBMC were concentrated to CD2 and CD3 cells (results not shown). Therefore, the low proportion IFN-g positive cells in the CD2/CD3 population is most likely due to a background staining. Consequently, the IFN-g producing cells were only seen in CD2þCD3 cells (31%) and in the CD2þCD3þ cells (69%). As expected from these data, significant numbers of IFN-g positive cells were not seen among CD14 positive PBMC (results not shown).

4. Discussion A vector expressing a porcine IL-12 fusion protein was constructed by joining the cDNA for the poIL-12 p40 and p35 subunits with DNA encoding a flexible peptide linker. Similar plasmid constructs have been used for expression of biologically active murine and human IL-12 fusion proteins (Lieschke et al., 1997). In order to evaluate the expression of the bioactive poIL-12 fusion protein in the present study, we developed a bioassay based on the ability of IL-12 to induce IFN-g production in porcine PBMC. Initial results showed that huIL-12 could induce poIFN-g production in this bioassay. The IFN-g levels were however low and varied between PBMC donors, but the induction of IFN-g by IL-12 was increased by mixing PBMC from two or more pigs. This was probably due to the enhancing effect of antigen stimulation in the resulting MLR, and such MLR was demonstrated as increased uptake of 3 H-thymidine (data not shown). In contrast, addition of huIL-18 strongly potentiated a dose-dependent induction of IFN-g production by huIL-12, allowing measurements of even low levels of bioactive IL-12. In fact, when IL-18 was added to the MLC, as little as 0.02 ng/ml of huIL-12 could be reproducibly detected, which makes the sensitivity of the assay comparable to a previously described proliferation bioassay (Kokuho et al., 1999). While IL-18 alone had little or no IFN-g inducing effect, the potentiating effect of huIL-18 in the presence of a constant IL-12 concentration was dose-dependent, indicating the possibility to also measure IL-18 bioactivity using this assay. Such synergistic effects of IL-12 and IL-18 have previously been described with human and murine cells (Ahn et al., 1997; Chang et al., 2000; Tominaga et al., 2000), and are probably related to the ability of IL-18 to up-regulate the IL-12Rb2

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(Chang et al., 2000) and of IL-12 to increase the IL-18Rb expression (Yoshimoto et al., 1998). We used intracellular staining for IFN-g and flow cytometry to determine the frequency of IFN-g producing cells in cultures where PBMC from two unrelated pigs had been mixed. We found that approximately 3% of the PBMC were IFN-g producers in cultures stimulated by poIL-12 and huIL-18, but no significant number of IFN-g positive cells in the absence of these cytokines. These results show that the MLR failed to induce detectable IFN-g production, despite a proliferative response detectable by 3H-thymidine uptake (results not shown). A characterisation of the phenotype of the IFN-g producing cells revealed that 31% were CD2þCD3, suggesting they were NK cells, or less likely B cells that to some extent also can express CD2 (Yang and Parkhouse, 1996). The remaining 69% of the IFN-g producers were CD2þCD3þ T cells. There was no evidence for an IFN-g production by cells positive for CD14, known to be expressed on porcine monocytes and myeloid dendritic cells (Carrasco et al., 2001). Furthermore, the CD2CD3þ T cells that constituted about 50% of the CD3þ cells and to a large extent should be CD4CD8TCRgdþ cells (Yang and Parkhouse, 1996) completely failed to produce IFN-g. Our results suggesting that porcine NK and T cells are the predominant IFN-g producers are in accord with findings in other species (Okamura et al., 1998; Tominaga et al., 2000; Nakanishi et al., 2001). Further studies are however required to determine the precise identity of the IFN-g producers in the pig. We found that poIL-12 from COS cells cotransfected with pcDNA3 expressing IL-12 p40 and p35 induced IFN-g production in poPBMC. Because supernatants from cells separately transfected with p40 and p35 encoding plasmids were inactive, poIL-12 was only active as a heterodimer, in agreement with earlier studies of poIL-12 (Foss et al., 1999). Furthermore, transfection of COS cells with the pcDNA3/p40-L-p35 plasmid construct resulted in supernatants that induced IFN-g production in poPBMC. This indicates that the linker (L) sequence does not interfere with the folding of the poIL-12 subunits, required for biologic activity. A relatively good expression of the poIL-12 fusion protein with a His-tag was obtained in Drosophila S2 cells, about 30 mg/ml medium. This His-tagged poIL-12 protein could be purified on Ni–NTA agarose,

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had high biologic activity and should be valuable as research reagent and as adjuvant. The yield at purification was low, probably because of presence of copper ions bound to the protein. This problem can probably be eliminated by the use of an alternative procedure recently reported, employing chelating sepharose (Lehr et al., 2000). The in vivo bioactivity of a similar poIL-12 p40-L-p35 fusion protein has recently been demonstrated in a cholera toxin vaccination model in swine (Foss et al., 1999). The pcDNA3/p40-L-p35 expression vector is now being evaluated as adjuvant to enhance the immune response to DNA vaccines in pigs, an approach previously proven successful in mice (Rao et al., 1996; Kim et al., 1997; Okada et al., 1997; Chow et al., 1998; Song et al., 2000). The marked synergistic effects of IL-12 and IL-18 seen on porcine cells in the present study and synergistic adjuvant effects observed in vivo in mice (Eberl et al., 2000) suggest that combinations of plasmids expressing IL-12 and IL-18 must be evaluated. Acknowledgements We thank Anne Riesenfeld for skilful technical assistance, Elin Johansson for help with collection of blood samples, Dr. Anders Johannisson for assistance with the flow cytometry, and Dr. Caroline Fossum for critical reading of the manuscript. The work was supported by grants from the Swedish Council for Forestry and Agricultural Research and the European Commission (FAIR3 PL96 1317). References Ahn, H.J., Maruo, S., Tomura, M., Mu, J., Hamaoka, T., Nakanishi, K., Clark, S., Kurimoto, M., Okamura, H., Fujiwara, H., 1997. A mechanism underlying synergy between IL-12 and IFNgamma-inducing factor in enhanced production of IFN-gamma. J. Immunol. 159, 2125–2131. Bost, K.L., Clements, J.D., 1995. In vivo induction of interleukin12 mRNA expression after oral immunization with Salmonella dublin or the B subunit of Escherichia coli heat-labile enterotoxin. Infect. Immun. 63, 1076–1083. Carrasco, C.P., Rigden, R.C., Schaffner, R., Gerber, H., Neuhaus, V., Inumaru, S., Takamatsu, H., Bertoni, G., McCullough, K.C., Summerfield, A., 2001. Porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties. Immunology 104, 175–184.

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