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Structure and characterization of hamster IL-12 p35 and p40
Molecular Immunology 40 (2003) 319–326
Structure and characterization of hamster IL-12 p35 and p40 Kouji Maruyama a , Yutaka Takigawa a , Yasuto Akiyama a,b,∗ , Takashi Hojo a , Noriko Nara-Ashizawa a , Jin-yan Cheng a , Morihiro Watanabe c , Ken Yamaguchi b a Growth Factor Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan Immunotherapy Division, Shizuoka Cancer Center, 1007 Shimonagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan Experimental Therapeutics Section, Laboratory of Experimental Immunology, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702-1201, USA
b c
Received 6 January 2003; received in revised form 16 June 2003; accepted 24 June 2003
Abstract Complementary DNAs coding for two subunits of hamster interleukin-12 (IL-12), p35 and p40, were cloned from a hamster dendritic cell (DC) cDNA library. The cloning demonstrated that hamster IL-12 consisted of a p35 subunit with 216 amino acid (aa) residues and a p40 subunit with 327 aa. Structural comparison of hamster p35 and p40 at the protein level showed the highest homologies with each counterpart of sigmodon (hispid cotton rat). The gene expressions of hamster IL-12 p35 and p40 in bone marrow (BM) cells cultured in the presence of mouse granulocyte macrophage-colony-stimulating factor (mGM-CSF) and IL-4 were up-regulated during culture. Immunoblot analysis of 293 cells transfected with hamster p35 and p40 expression vectors suggested the presence of a covalently linked p35/p40 heterodimer. Furthermore, supernatant from the 293 cells transfected with both expression vectors induced the up-regulation of interferon-gamma (IFN-␥) mRNA in hamster splenocytes, indicating that the p35/p40 heterodimer IL-12 protein present in the supernatant was functional. These results suggest that the vectors containing hamster IL-12 cDNA might be suitable tools for developing an immunotherapeutic approach against experimental cancer in a hamster model. © 2003 Elsevier Ltd. All rights reserved. Keywords: Hamster; Interleukin-12; IL-12 p35 subunit; IL-12 p40 subunit; Dendritic cell
1. Introduction Pancreatic cancer is one of the most intractable cancers with a dismal outcome. The mortality of pancreatic cancer is ranked as the fifth largest cause of cancer death. Even at the present time, the 5-year survival is less than a few percent despite intensive therapy. The development of a novel and effective approach for the treatment of the pancreatic cancer is strongly demanded. Syrian hamster is a unique model animal of pancreatic cancer. The hamster pancreatic cancer cell lines, such as HPDNR (Mori et al., 1994) and PGHAM-1 (Yanagi et al., 2000; Matsushita et al., 2001), have been established using chemical carcinogens. These cells have some benefit as experimental models in that they are very similar to their Abbreviations: IL-12, interleukin-12; DC, dendritic cell; BM, bone marrow; GM-CSF, granulocyte macrophage-colony-stimulating factor; IFN-␥, interferon-␥; G-CSF, granulocyte-colony-stimulating factor; RT-PCR, reverse transcription-polymerase chain reaction ∗ Corresponding author. Tel.: +81-55-989-5222x4355; fax: +81-55-989-5634. E-mail address: [email protected] (Y. Akiyama). 0161-5890/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0161-5890(03)00165-2
human counterpart; these cells are of duct cell origin, as is the case in most pancreatic cancer patients, and have a mutation of the k-ras gene. Furthermore, these cells can grow in immunocompetent animals, suggesting their availability in immunotherapeutic experiments against pancreatic cancers. However, a serious problem with the hamster system is its poorly understood immunological background. Thus, in the previous study, we cloned partial cDNAs for some dendritic cell (DC) marker molecules (Akiyama et al., 2002). To develop a novel immunotherapeutic modality against cancer, the use of genetically modified DCs might be a promising strategy. We have already reported on the remarkable anti-tumor effects induced by DCs transduced with the IL-12 gene in a mouse B16F10 melanoma model (Akiyama et al., 2000). Furthermore, we have also reported that DCs pulsed with tumor lysates exhibited remarkable anti-tumor effects against an established hamster subcutaneous pancreatic cancer (Akiyama et al., 2002). Therefore, we decided to clone hamster IL-12 cDNA for gene transduction into hamster DCs with the aim of potentiating the anti-tumor effects induced by DCs.
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IL-12 was initially identified and isolated as natural killer (NK) cell stimulatory factor (Kobayashi et al., 1989). Compared with other cytokines, it has a unique 70 kDa heterodimeric structure composed of two covalently linked p35 and p40 subunits, both of which are required for biological activities (Wolf et al., 1991; Lamont and Adorini, 1996). IL-12 is produced principally by antigen presenting cells (APC), such as monocytes, macrophages and DCs (Manetti et al., 1994; Trinchieri, 1997). The biological effects of IL-12 include the enhancement of lytic activity and IFN-␥ secretion by NK and T cells, the potent proliferation induction of both naive and memory T cells, and the induction of T-helper type 1 (Th1) differentiation (Trinchieri, 1994; Murphy et al., 994). The sequence similarity of the p35 subunit to IL-6 and granulocyte colony stimulating factor (G-CSF), and that of the p40 subunit to the IL-6 and G-CSF receptors is suggestive of evolution from a common primordial cytokine and a p70 structure akin to a soluble cytokine–cytokine receptor complex (Gately et al., 1998; Merberg et al., 1992; Gearing and Cosman, 1991). In the present study, we determined the cDNA sequence of two subunits of hamster IL-12, and characterized their biological activity in vitro. Hamster IL-12 p35 and p40 subunits have structures closely related to their counterparts in other species. The gene expression of these two subunits increased in the course of differentiation from bone marrow (BM) cells to DCs, in which IL-12 gene products were potently up-regulated by activating agents like TNF-␣ and lipopolysaccharide (LPS) (data not shown). Furthermore, the gene products of these two subunits seem to have specific biological activities similar to those reported for other species.
2. Materials and methods 2.1. Animals, cell line, preparation of RNA and Northern blotting Specific pathogen-free 4- to 6-week-old female Syrian hamsters were purchased from SLC (Shizuoka, Japan). The animals were sacrificed, and then the lower limbs were dissected to collect BM cells. A transformed primary human embryonal kidney cell line, 293, was purchased
from American Type Culture Collection (ATCC, Manassas, VA). Total RNAs from hamster tissues were isolated by the acid guanidinium–phenol–chloroform method, and poly(A)+ RNA was prepared using a poly(A) Quick mRNA Isolation Kit (Stratagene, La Jolla, CA). Northern blotting was performed as previously described (Maruyama et al., 2002). 2.2. Culture of hamster BM-derived DCs Adherent BM cells were resuspended in RPMI-1640 medium supplemented with antibiotics and 5% fetal bovine serum (FBS), and incubated with recombinant mouse (rm) GM-CSF (10 ng/ml) and rmIL-4 (50 ng/ml) at a concentration of 2 × 106 cells/ml in a 24-well culture plate. Cytokines used were purchased from Pepro Tech (Rocky Hill, NJ). DC-rich non-adherent cells were collected by gentle pipetting from cultured BM cells and used for RNA extraction. 2.3. Construction of a cDNA library from cultured BM cells and DNA sequencing Poly(A)+ RNA (5 g) extracted from cultured hamster DCs was used to construct a cDNA library using a Lamda ZAP-CMV XR Library Construction Kit (Stratagene). Screening of the library was performed using a 32 P-labeled partial hamster IL-12 p35 or p40 cDNA fragment as a probe. This fragment was generated as follows; hamster thymus total RNA (5 g) was reverse transcribed with oligo d(T) primer, and amplified by polymerase chain reaction (PCR) using the primers listed in Table 1, which were designed from regions conserved between human, mouse and rat. The fragment of the PCR product for p35 was 404 base pairs (bp) in length with 69% identity against mouse p35, and that for p40 was 495 bp in length and had 70% identity with mouse p40 (Table 2). The positive clones isolated were sequenced by the dye-terminator method using an ABI 310 automated sequencer (Perkin-Elmer, Foster City, CA). The longest clone for p35 was registered as AB085791, and that for p40 as AB085792. The identity and similarity of IL-12 proteins between hamster and other species were calculated using the BLAST program.
Table 1 Primers used for the present experiments related to hamster IL-12 p35 and p40 PCR primers for cloning of IL-12 partial cDNA p35F 5 -ACATCGATCATGAAGACATCAC-3 p40F 5 -GAGACTCTGAGCCACTCACATC-3
p35R 5 -GAAGGCGTGAAGCAGGATGCAG-3 p40R 5 -AGGGTACTCCCAGCTGACCTCC-3
PCR primers for detection of IL-12 mRNA p35F 5 -TGACCTTGTGCCTTAGTAGC-3 p40F 5 -GAAGTTCAACGTCAAGAGCAGC-3
p35R 5 -AGGTAGCTCATCACTCTGTGA-3 p40R 5 -CTAACTGCAGGGCACAGATACC-3
PCR primers for detection of IFN-␥ mRNA IFN-␥F 5 -ATGCACACCACACGTTGCATCTT-3
IFN-␥R 5 -ATTGCTGGCAAGAATATTCTTGT-3
K. Maruyama et al. / Molecular Immunology 40 (2003) 319–326 Table 2 Homologies for IL-12 p35 and p40 proteins between hamster and other species of rodentia Species
Accession No.
Identity (%)
Similarity (%)
IL-12 p35 Sigmodon Mouse Rat Gerbil Guinea pig
AF421396 NM 008351 NM 053390 AF288849 AB025723
81 69 67 67 60
87 82 80 80 70
IL-12 p40 Sigmodon Mouse Rat Gerbil Guinea pig
AF421395 S82426 AF133197 AF288612 AB025724
85 70 70 63 64
91 80 80 73 77
2.4. Hamster IL-12 p35 and p40 mRNA expressions on cultured BM-derived DCs and various tissues using RT-PCR Specific PCR primers for hamster IL-12 p35 and p40 were designed from partially cloned cDNA sequences (Table 1). Total RNAs from cultured DCs and various tissues were subjected to RT (reverse transcription)-PCR analysis to detect the gene expression of IL-12 mRNA according to the method previously described (Nara-Ashizawa et al., 2001). The PCR primer sequences of hamster -actin were as follows: antisense 5 -GCTGTCCCTGTATGCCTCTG-3 , sense 5 -CCATCTCTTGCTCGAAGTCC-3 .
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prepared using a similar procedure to that described before (Akiyama et al., 2000). The resultant constructs were confirmed by DNA sequencing. Transient transfection of 293 cells with these constructs was performed by the calcium phosphate method. 2.6. The expression of hamster IFN-γ mRNA in splenocytes stimulated by the supernatant of 293 cells transfected with IL-12 gene-containing expression vectors To evaluate the biological activity of hamster IL-12 p35 and p40 gene products, the spleen cells from normal hamster stimulated by the supernatant of 293 cells transfected with p35/SCRIPT and p40/SCRIPT were used for RT-PCR analysis of hamster IFN-␥ mRNA. Spleen cells resuspended with 5% FBS-containing RPMI-1640 medium were seeded into a 24-well culture plate at 1 × 106 cells/ml, and then the serially diluted supernatants of 293 cells transfected with p35/SCRIPT and/or p40/SCRIPT were added to the wells. After 24 h, the cells were collected, and their total RNAs were subjected to RT-PCR analysis to detect the gene expression of IFN-␥ mRNAs. Specific primers for hamster IFN-␥ are shown in Table 1. 2.7. Immunoblotting The procedure for immunoblotting was previously described (Maruyama et al., 2002). Cell lysate containing 10 g of protein was subjected to analysis. Monoclonal antibody for FLAG epitope was purchased from Sigma (St. Louis, MO).
2.5. Construction of hamster IL-12-expressing plasmid and gene transfection into 293 cells 3. Results The pCMV-Script EX vector containing hamster IL-12 p35 or p40 cDNA was obtained from lambda ZAP phage by in vivo excision, and used as an expression vector (referred to as p35/SCRIPT and p40/SCRIPT, respectively). The expression vector for p35 or p40 tagged with FLAG epitope at the C-terminus was prepared by PCR using KOD polymerase (Toyobo, Tokyo, Japan) as follows; the sense primers were designed to contain an artificial Kozak sequence (GCCACC) just before the start codon (ATG) and the subsequent sequence of the open reading frame (ORF) (nucleotide (nt) 110–132 of AB085791 for p35, and nt 39–61 of AB085792 for p40). The antisense primers contained a 22 bp sequence just before the stop codon (nt 736–757 of AB085791 for p35 and nt 998–1019 of AB085792 for p40), linker sequence, the sequences for FLAG epitope, and stop codon. The amplified products were subcloned once into ZeroBlunt vector (Invitrogen), digested by suitable restriction enzymes and cloned into pCMV-Script EX vector, and referred to as p35/FLAG and p40/FLAG. In the meantime, as a positive control for the detection of p70 protein, a heterodimer of p35 and p40, the expression vector for p70 single chain composed of linked p35 and p40 cDNA tagged with FLAG epitope was also
3.1. cDNA and amino acid sequences of hamster IL-12 p35 and p40 The longest cDNA clone corresponding to hamster IL-12 p35 (registered as AB085791) was 1239 bp in length. The deduced amino acid (aa) sequence consisted of 216 residues with an N-terminal 22 aa secretion signal sequence according to the human p35 protein. The mature p35 protein had a calculated molecular mass of 21.8 kDa. The alignment of the p35 aa sequence among the animal species of rodentia is depicted in Fig. 1A. Among six other species, the highest homology against the hamster molecule was observed in sigmodon (registered as AF421395), in which the identity and similarity with hamster were 81% and 87%, respectively (Table 2). The cDNA sequence corresponding to hamster IL-12 p40 (registered as AB085792) was 2276 bp in length. The alignment of the deduced aa sequence is shown in Fig. 1B. Hamster IL-12 p40 consisted of 327 aa with an N-terminal 22 aa secretion signal sequence according to the human protein. The mature hamster p40 protein had a calculated
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molecular mass of 34.8 kDa. As is the case with hamster p35 protein, hamster p40 showed the highest homology to sigmodon p40 (Table 2). The identity and similarity among these two species were 85 and 91%, respectively. 3.2. Expression of hamster IL-12 p35 and p40 mRNAs in cultured BM cells and various tissues The gene expressions of hamster IL-12 p35 and p40 in cultured BM cells and tissues were investigated by RT-PCR using the primer sets listed in Table 1. In the BM cells cultured in the presence of rmGM-CSF and rmIL-4, p35 and p40 mRNAs were detected from day 0 to day 2 of culture, and reached a plateau level on day 6 to day 10 (Fig. 2A). Using this analytical method, transcripts of hamster p35 were detected in the thymus, liver and spleen, and transcripts of p40 were detected in the thymus and spleen (Fig. 2B). 3.3. Expression of hamster IL-12 p35 and p40 proteins in 293 cells transfected with IL-12 p35 and/or p40 expression vectors The protein expressions of hamster IL-12 p35 and p40 were analyzed in 293 cells transfected with p35-FLAG
and/or p40-FLAG by immunoblotting using anti-FLAG antibody. In 293 cells transfected with p40-FLAG, a protein product with an apparent molecular weight of 40 kDa was detected under reducing conditions (Fig. 3). However, we could not detect a protein product corresponding to p35 under the same conditions. The protein products from these two expression vectors were supposed to have a FLAG epitope at their C-terminus. In the blotting analysis under non-reducing conditions, a protein product with an apparent molecular weight of 70 kDa in addition to a smaller 40 kDa product was detected in 293 cells transfected with both p35- and p40-FLAG or p70-FLAG (Fig. 3). 3.4. Induction of IFN−γ production by hamster IL-12 stimulation on the cultured splenocytes The supernatant of the 293 cells transfected with both p35/SCRIPT and p40/SCRIPT was added at serial dilutions to the cultures of hamster splenocytes, and its ability to induce IFN-␥ production from spleen cells was evaluated by RT-PCR using the specific primers listed in Table 1. In the hamster splenocytes treated with the supernatants, IFN-␥ production was detected in a dose-dependent manner
Fig. 1. Alignment of amino acid sequences of hamster, sigmodon, mouse, rat, gerbil and guinea pig IL-12 p35 (A) and p40 (B). Identity or similarity data of IL-12 proteins between hamster and other species are shown in Table 2. Accession numbers of hamster IL-12 p35 and p40 cDNAs are AB085791 and AB085792, respectively. Accession numbers of IL-12 DNAs from five other species (sigmodon, mouse, rat, gerbil and guinea pig) are also shown in Table 2.
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Fig. 1. (Continued ).
(Fig. 4) in the case of p35 alone or both p35 and p40 protein product-including supernatant. However, in p40 alone protein product-containing supernatant, no significant induction of IFN-␥ mRNA was seen. Meanwhile, in the splenocytes with no treatment or the mock treatment, IFN-␥ production was not detected.
4. Discussion First of all, in the present study, cDNAs coding for two subunits of hamster IL-12, p35 and p40, were cloned from a hamster DC cDNA library. The cloning demonstrated that the hamster IL-12 consisted of a p35 subunit with 216 aa and a p40 subunit with 327 aa residues. The structural com-
parison of hamster p35 and p40 at the protein level showed the highest homologies with each counterpart of sigmodon (hispid cotton rat). Specifically, in terms of structural observations, hamster IL-12 p35 cDNA includes a 5 untranslated region (UTR) of 109 bp, an ORF of 651 bp, and a 3 UTR of 462 bp and a poly(A) tail. In the 3 UTR, seven mRNA destabilization sequences (ATTTTA) and one poly-adenylation signal sequence (AATAAA) were contained. In the hamster molecule, seven out of eight cysteine residues are also conserved like those of other species. Hamster IL-12 p40 cDNA consisted of a 5 UTR of 38 bp, an ORF of 984 bp, a 3 UTR of 1235 bp and a poly(A) tail. The 3 UTR of hamster p40 contains 12 mRNA destabilization sequences and one poly-adenylation signal sequence.
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Fig. 2. IL-12 p35 and p40 mRNA expressions on cultured BM-derived DCs or various hamster tissues using RT-PCR. Specific PCR primers for hamster IL-12 p35 and p40 were designed from partially cloned cDNA sequences. Total RNAs from cultured DCs and various tissues were isolated by the acid guanidinium-phenol-chloroform method. The PCR primer sequences of hamster -actin were as follows: antisense 5 -GCTGTCCCTGTATGCCTCTG-3 , sense 5 -CCATCTCTTGCTCGAAGTCC-3 .
The 21 aa flanking sequence of the cleavage site is completely conserved in hamsters like in the other four species. The domain structure analyzed by the BLAST program showed that hamster IL-12 p40 protein also has a fibronectin type III domain (aa 236–319) and an immunoglobulin C-2 type domain (aa 43–90), as in the other
Fig. 3. Immunoblotting analysis of hamster IL-12 p35 and p40 proteins using anti-FLAG monoclonal antibody. Ten micrograms of 293 cell lysate transfected with IL-12 gene-containing expression vectors (p35/FLAG or p40/FLAG) was utilized for immunoblotting using anti-FLAG monoclonal antibody. p35/FLAG or p40/FLAG contained IL-12 p35 or p40 cDNA tagged with FLAG epitope at the C-terminus. IL-12 p70 single chain-containing expression vector (p70/FLAG) was also used for a positive control to detect a heterodimer protein of p35 and p40. Intact shows 293 cells with no transfection procedure.
species reported (Schoenhaut et al., 1992; Khalife et al., 1998). In fact, the partial cDNA sequence for hamster IL-12 p40 has already been reported by Melby et al. (1998), and their registered sequence (AF046211) is completely identical to our sequence, corresponding to nt 319–717 of ours. Secondly, RT-PCR using specific primers for hamster IL-12 p35 and p40 demonstrated that the IL-12 mRNA gene in BM cells cultured in the presence of mGM-CSF and IL-4 was up-regulated. We have also already reported that the gene expression of hamster DEC-205, one of the authentic
Fig. 4. Induction of IFN-␥ production by hamster IL-12 stimulation on cultured splenocytes. The supernatants of the 293 cells transfected with p35/SCRIPT and/or p40/SCRIPT were added at serial dilutions (1 to 1/625) to cultures of hamster splenocytes, and their abilities to induce IFN-␥ production from spleen cells were evaluated by RT-PCR using the specific primers for hamster IFN-␥ listed in Table 1. Intact shows supernatant from 293 cells with no transfection procedure.
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markers for DCs, increased during BM cell culture under the same conditions. It was obvious that cultured hamster BM cells contained a substantial amount of DCs. These findings suggest that hamster BM cells increase the expression of both IL-12 subunits in the course of differentiation to DCs. However, in Northern blot analysis using total RNA fractions, no positive signal was detected in the tissues, which suggested the low mRNA expression levels in these tissues (data not shown). Thirdly, the protein expressions of hamster IL-12 p35 and p40 were analyzed in 293 cells transfected with p35-FLAG and/or p40-FLAG by immunoblotting using anti-FLAG antibody. In 293 cells transfected with p40-FLAG, a protein product with an apparent molecular weight of 40 kDa was detected, but p35 product was not seen in reducing condition. In contrast, in the blotting analysis with non-reducing condition, the protein product with apparent molecular weight of 70 kDa in addition to smaller 40 kDa product was detected in 293 cells transfected with both p35- and p40-FLAG or p70-FLAG. The protein product of around 70 kDa might correspond to the homodimer of p40 and the heterodimer of p35/p40. The exact cause of the undetectability of p35 protein alone tagged with FLAG is not clear, however, findings from the transfection experiment using both expression vectors p35 and p40 suggest the expression of a p35 protein product, and the presence of a covalently linked p35/p40 heterodimer reported in other species. Additionally, with the aim of verifying the biological activities of hamster IL-12 gene products, the supernatant of 293 cells transfected with both IL-12 gene-containing vectors was added to the cultures of hamster splenocytes, and its ability to induce IFN-␥ production from spleen cells was investigated by RT-PCR using specific primers. IFN-␥ production was detected in a dose-dependent manner in the case of p35 alone or both p35 and p40 gene product-including supernatant. However, in the p40 alone gene product-containing supernatant, no significant induction of IFN-␥ mRNA was seen. These results were considered to be compatible with the findings reported by Kalinski et al. that the IL-12 p70 heterodimer, composed of p35 and p40, mediates IL-12 biological activity as a Th1-inducer, but the IL-12 p40 homodimer in the absence of p35 product acts as an IL-12 antagonist (Kalinski et al., 2001). Finally, taking these findings together, the two subunits of hamster IL-12 p35 and p40 have closely related structures to those of other animals (Schoenhaut et al., 1992; Khalife et al., 1998; Shiratori et al., 2001; Swinburne et al., 2000), and may have potent biological activity at least in terms of an induction of IFN-␥. At present, we have constructed an adenoviral vector which expresses a hamster IL-12 p35–p40 single chain product, and are trying to generate hamster IL-12-producing DCs using an adenoviral-mediated gene transduction procedure. Specific therapeutic experiments using IL-12-producing
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DCs (Gambotto et al., 1999; Furumoto et al., 2002) against hamster pancreatic cancer will be performed soon. Acknowledgements We would like to thank Ms. Hasegawa, Ms. Ohtsubo, Ms. Kajimura and Ms. Ebinuma for their excellent technical assistance. This work was supported by Grants in Aid from the Ministry of Health, Labour and Welfare for Cancer Research (9-32 and 10-28) and the Second-Term Comprehensive 10-year Strategy of Cancer Control. References Akiyama, Y., Watanabe, M., Maruyama, K., Ruscetti, F.W., Wiltrout, R.H., Yamaguchi, K., 2000. Enhancement of antitumor immunity against B16 melanoma tumor using genetically modified dendritic cells to produce cytokines. Gene Ther. 7, 2113–2121. Akiyama, Y., Maruyama, K., Nara, N., Hojo, T., Cheng, J.Y., Mori, T., Wiltrout, R.H., Yamaguchi, K., 2002. Antitumor effects induced by dendritic cell (DC)-based immunotherapy against established pancreatic cancer in hamsters. Cancer Lett. 184, 37–47. Furumoto, K., et al., 2002. Interleukin-12-gene transduction makes DCs from tumor-bearing mice an effective inducer of tumor-specific immunity in a peritoneal dissemination model. Immunol. Lett. 83, 13–20. Gambotto, A., et al., 1999. Induction of antitumor immunity by direct intratumoral injection of a recombinant adenovirus vector expressing interleukin-12. Cancer Gene Ther. 6, 45–53. Gately, M.K., Renzetti, L.M., Magram, J., Stem, A.S., Adorini, L., Gubler, U., Presky, D.H., 1998. The interleukin-12/interleukin-12 receptor system: role in normal and pathological immune responses. Annu. Rev. Immunol. 16, 495–521. Gearing, D.P., Cosman, D., 1991. Homology of the p40 subunit of natural killer cell stimulatory factor (NKSF) with the extracellular domain of the interleukin-6 receptor. Cell 66, 9–10. Kalinski, P., et al., 2001. Prostaglandin E(2) is a selective inducer of interleukin-12 p40 (IL-12 p40) production and an inhibitor of bioactive IL-12 p70 heterodimer. Blood 97, 3466–3469. Khalife, J., et al., 1998. Molecular cloning and sequencing of the rat interleukin-12 p40 gene. Eur. Cytokine Netw. 9, 69–73. Kobayashi, M., Fitz, L., Ryan, M., Hewick, R.M., Clark, S.C., Chan, S., Loudon, R., Sherman, F., Perussia, B., Trinchieri, G., 1989. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiplebiologic effects on human lymphocytes. J. Exp. Med. 170, 827–845. Lamont, A.G., Adorini, L., 1996. IL-12: a key cytokine in immune regulation. Immunol. Today 17, 214–217. Manetti, R., et al., 1994. Interleukin 12 induces stable priming for interferon gamma (IFN-gamma) production during differentiation of human T helper (Th) cells and transient IFN-gamma production in established Th2 cell clones. J. Exp. Med. 179, 1273–1283. Maruyama, K., et al., 2002. Hamster DEC205, its primary structure, tissue and cellular distribution. Cancer Lett. 181, 223–232. Matsushita, A., Onda, M., Uchida, E., Maekawa, R., Yoshida, T., 2001. Antitumor effect of a new selective matrix metalloproteinase inhibitor, MMI-166, on experimental pancreatic cancer. Int. J. Cancer 92, 434– 440. Melby, P.C., Tryon, V.V., Chandrasekar, B., Freeman, G.L., 1998. Cloning of Syrian hamster (Mesocricetus auratus) cytokine cDNAs and analysis of cytokine mRNA expression in experimental visceral leishmaniasis. Infect. Immun. 66, 2135–2142. Merberg, D.M., Wolf, S.F., Clark, S.C., 1992. Sequence similarity between NKSF and the IL-6/G-CSF family. Immunol. Today 13, 77–78.
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Structure and characterization of hamster IL-12 p35 and p40 Kouji Maruyama a , Yutaka Takigawa a , Yasuto Akiyama a,b,∗ , Takashi Hojo a , Noriko Nara-Ashizawa a , Jin-yan Cheng a , Morihiro Watanabe c , Ken Yamaguchi b a Growth Factor Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan Immunotherapy Division, Shizuoka Cancer Center, 1007 Shimonagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan Experimental Therapeutics Section, Laboratory of Experimental Immunology, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702-1201, USA
b c
Received 6 January 2003; received in revised form 16 June 2003; accepted 24 June 2003
Abstract Complementary DNAs coding for two subunits of hamster interleukin-12 (IL-12), p35 and p40, were cloned from a hamster dendritic cell (DC) cDNA library. The cloning demonstrated that hamster IL-12 consisted of a p35 subunit with 216 amino acid (aa) residues and a p40 subunit with 327 aa. Structural comparison of hamster p35 and p40 at the protein level showed the highest homologies with each counterpart of sigmodon (hispid cotton rat). The gene expressions of hamster IL-12 p35 and p40 in bone marrow (BM) cells cultured in the presence of mouse granulocyte macrophage-colony-stimulating factor (mGM-CSF) and IL-4 were up-regulated during culture. Immunoblot analysis of 293 cells transfected with hamster p35 and p40 expression vectors suggested the presence of a covalently linked p35/p40 heterodimer. Furthermore, supernatant from the 293 cells transfected with both expression vectors induced the up-regulation of interferon-gamma (IFN-␥) mRNA in hamster splenocytes, indicating that the p35/p40 heterodimer IL-12 protein present in the supernatant was functional. These results suggest that the vectors containing hamster IL-12 cDNA might be suitable tools for developing an immunotherapeutic approach against experimental cancer in a hamster model. © 2003 Elsevier Ltd. All rights reserved. Keywords: Hamster; Interleukin-12; IL-12 p35 subunit; IL-12 p40 subunit; Dendritic cell
1. Introduction Pancreatic cancer is one of the most intractable cancers with a dismal outcome. The mortality of pancreatic cancer is ranked as the fifth largest cause of cancer death. Even at the present time, the 5-year survival is less than a few percent despite intensive therapy. The development of a novel and effective approach for the treatment of the pancreatic cancer is strongly demanded. Syrian hamster is a unique model animal of pancreatic cancer. The hamster pancreatic cancer cell lines, such as HPDNR (Mori et al., 1994) and PGHAM-1 (Yanagi et al., 2000; Matsushita et al., 2001), have been established using chemical carcinogens. These cells have some benefit as experimental models in that they are very similar to their Abbreviations: IL-12, interleukin-12; DC, dendritic cell; BM, bone marrow; GM-CSF, granulocyte macrophage-colony-stimulating factor; IFN-␥, interferon-␥; G-CSF, granulocyte-colony-stimulating factor; RT-PCR, reverse transcription-polymerase chain reaction ∗ Corresponding author. Tel.: +81-55-989-5222x4355; fax: +81-55-989-5634. E-mail address: [email protected] (Y. Akiyama). 0161-5890/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0161-5890(03)00165-2
human counterpart; these cells are of duct cell origin, as is the case in most pancreatic cancer patients, and have a mutation of the k-ras gene. Furthermore, these cells can grow in immunocompetent animals, suggesting their availability in immunotherapeutic experiments against pancreatic cancers. However, a serious problem with the hamster system is its poorly understood immunological background. Thus, in the previous study, we cloned partial cDNAs for some dendritic cell (DC) marker molecules (Akiyama et al., 2002). To develop a novel immunotherapeutic modality against cancer, the use of genetically modified DCs might be a promising strategy. We have already reported on the remarkable anti-tumor effects induced by DCs transduced with the IL-12 gene in a mouse B16F10 melanoma model (Akiyama et al., 2000). Furthermore, we have also reported that DCs pulsed with tumor lysates exhibited remarkable anti-tumor effects against an established hamster subcutaneous pancreatic cancer (Akiyama et al., 2002). Therefore, we decided to clone hamster IL-12 cDNA for gene transduction into hamster DCs with the aim of potentiating the anti-tumor effects induced by DCs.
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IL-12 was initially identified and isolated as natural killer (NK) cell stimulatory factor (Kobayashi et al., 1989). Compared with other cytokines, it has a unique 70 kDa heterodimeric structure composed of two covalently linked p35 and p40 subunits, both of which are required for biological activities (Wolf et al., 1991; Lamont and Adorini, 1996). IL-12 is produced principally by antigen presenting cells (APC), such as monocytes, macrophages and DCs (Manetti et al., 1994; Trinchieri, 1997). The biological effects of IL-12 include the enhancement of lytic activity and IFN-␥ secretion by NK and T cells, the potent proliferation induction of both naive and memory T cells, and the induction of T-helper type 1 (Th1) differentiation (Trinchieri, 1994; Murphy et al., 994). The sequence similarity of the p35 subunit to IL-6 and granulocyte colony stimulating factor (G-CSF), and that of the p40 subunit to the IL-6 and G-CSF receptors is suggestive of evolution from a common primordial cytokine and a p70 structure akin to a soluble cytokine–cytokine receptor complex (Gately et al., 1998; Merberg et al., 1992; Gearing and Cosman, 1991). In the present study, we determined the cDNA sequence of two subunits of hamster IL-12, and characterized their biological activity in vitro. Hamster IL-12 p35 and p40 subunits have structures closely related to their counterparts in other species. The gene expression of these two subunits increased in the course of differentiation from bone marrow (BM) cells to DCs, in which IL-12 gene products were potently up-regulated by activating agents like TNF-␣ and lipopolysaccharide (LPS) (data not shown). Furthermore, the gene products of these two subunits seem to have specific biological activities similar to those reported for other species.
2. Materials and methods 2.1. Animals, cell line, preparation of RNA and Northern blotting Specific pathogen-free 4- to 6-week-old female Syrian hamsters were purchased from SLC (Shizuoka, Japan). The animals were sacrificed, and then the lower limbs were dissected to collect BM cells. A transformed primary human embryonal kidney cell line, 293, was purchased
from American Type Culture Collection (ATCC, Manassas, VA). Total RNAs from hamster tissues were isolated by the acid guanidinium–phenol–chloroform method, and poly(A)+ RNA was prepared using a poly(A) Quick mRNA Isolation Kit (Stratagene, La Jolla, CA). Northern blotting was performed as previously described (Maruyama et al., 2002). 2.2. Culture of hamster BM-derived DCs Adherent BM cells were resuspended in RPMI-1640 medium supplemented with antibiotics and 5% fetal bovine serum (FBS), and incubated with recombinant mouse (rm) GM-CSF (10 ng/ml) and rmIL-4 (50 ng/ml) at a concentration of 2 × 106 cells/ml in a 24-well culture plate. Cytokines used were purchased from Pepro Tech (Rocky Hill, NJ). DC-rich non-adherent cells were collected by gentle pipetting from cultured BM cells and used for RNA extraction. 2.3. Construction of a cDNA library from cultured BM cells and DNA sequencing Poly(A)+ RNA (5 g) extracted from cultured hamster DCs was used to construct a cDNA library using a Lamda ZAP-CMV XR Library Construction Kit (Stratagene). Screening of the library was performed using a 32 P-labeled partial hamster IL-12 p35 or p40 cDNA fragment as a probe. This fragment was generated as follows; hamster thymus total RNA (5 g) was reverse transcribed with oligo d(T) primer, and amplified by polymerase chain reaction (PCR) using the primers listed in Table 1, which were designed from regions conserved between human, mouse and rat. The fragment of the PCR product for p35 was 404 base pairs (bp) in length with 69% identity against mouse p35, and that for p40 was 495 bp in length and had 70% identity with mouse p40 (Table 2). The positive clones isolated were sequenced by the dye-terminator method using an ABI 310 automated sequencer (Perkin-Elmer, Foster City, CA). The longest clone for p35 was registered as AB085791, and that for p40 as AB085792. The identity and similarity of IL-12 proteins between hamster and other species were calculated using the BLAST program.
Table 1 Primers used for the present experiments related to hamster IL-12 p35 and p40 PCR primers for cloning of IL-12 partial cDNA p35F 5 -ACATCGATCATGAAGACATCAC-3 p40F 5 -GAGACTCTGAGCCACTCACATC-3
p35R 5 -GAAGGCGTGAAGCAGGATGCAG-3 p40R 5 -AGGGTACTCCCAGCTGACCTCC-3
PCR primers for detection of IL-12 mRNA p35F 5 -TGACCTTGTGCCTTAGTAGC-3 p40F 5 -GAAGTTCAACGTCAAGAGCAGC-3
p35R 5 -AGGTAGCTCATCACTCTGTGA-3 p40R 5 -CTAACTGCAGGGCACAGATACC-3
PCR primers for detection of IFN-␥ mRNA IFN-␥F 5 -ATGCACACCACACGTTGCATCTT-3
IFN-␥R 5 -ATTGCTGGCAAGAATATTCTTGT-3
K. Maruyama et al. / Molecular Immunology 40 (2003) 319–326 Table 2 Homologies for IL-12 p35 and p40 proteins between hamster and other species of rodentia Species
Accession No.
Identity (%)
Similarity (%)
IL-12 p35 Sigmodon Mouse Rat Gerbil Guinea pig
AF421396 NM 008351 NM 053390 AF288849 AB025723
81 69 67 67 60
87 82 80 80 70
IL-12 p40 Sigmodon Mouse Rat Gerbil Guinea pig
AF421395 S82426 AF133197 AF288612 AB025724
85 70 70 63 64
91 80 80 73 77
2.4. Hamster IL-12 p35 and p40 mRNA expressions on cultured BM-derived DCs and various tissues using RT-PCR Specific PCR primers for hamster IL-12 p35 and p40 were designed from partially cloned cDNA sequences (Table 1). Total RNAs from cultured DCs and various tissues were subjected to RT (reverse transcription)-PCR analysis to detect the gene expression of IL-12 mRNA according to the method previously described (Nara-Ashizawa et al., 2001). The PCR primer sequences of hamster -actin were as follows: antisense 5 -GCTGTCCCTGTATGCCTCTG-3 , sense 5 -CCATCTCTTGCTCGAAGTCC-3 .
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prepared using a similar procedure to that described before (Akiyama et al., 2000). The resultant constructs were confirmed by DNA sequencing. Transient transfection of 293 cells with these constructs was performed by the calcium phosphate method. 2.6. The expression of hamster IFN-γ mRNA in splenocytes stimulated by the supernatant of 293 cells transfected with IL-12 gene-containing expression vectors To evaluate the biological activity of hamster IL-12 p35 and p40 gene products, the spleen cells from normal hamster stimulated by the supernatant of 293 cells transfected with p35/SCRIPT and p40/SCRIPT were used for RT-PCR analysis of hamster IFN-␥ mRNA. Spleen cells resuspended with 5% FBS-containing RPMI-1640 medium were seeded into a 24-well culture plate at 1 × 106 cells/ml, and then the serially diluted supernatants of 293 cells transfected with p35/SCRIPT and/or p40/SCRIPT were added to the wells. After 24 h, the cells were collected, and their total RNAs were subjected to RT-PCR analysis to detect the gene expression of IFN-␥ mRNAs. Specific primers for hamster IFN-␥ are shown in Table 1. 2.7. Immunoblotting The procedure for immunoblotting was previously described (Maruyama et al., 2002). Cell lysate containing 10 g of protein was subjected to analysis. Monoclonal antibody for FLAG epitope was purchased from Sigma (St. Louis, MO).
2.5. Construction of hamster IL-12-expressing plasmid and gene transfection into 293 cells 3. Results The pCMV-Script EX vector containing hamster IL-12 p35 or p40 cDNA was obtained from lambda ZAP phage by in vivo excision, and used as an expression vector (referred to as p35/SCRIPT and p40/SCRIPT, respectively). The expression vector for p35 or p40 tagged with FLAG epitope at the C-terminus was prepared by PCR using KOD polymerase (Toyobo, Tokyo, Japan) as follows; the sense primers were designed to contain an artificial Kozak sequence (GCCACC) just before the start codon (ATG) and the subsequent sequence of the open reading frame (ORF) (nucleotide (nt) 110–132 of AB085791 for p35, and nt 39–61 of AB085792 for p40). The antisense primers contained a 22 bp sequence just before the stop codon (nt 736–757 of AB085791 for p35 and nt 998–1019 of AB085792 for p40), linker sequence, the sequences for FLAG epitope, and stop codon. The amplified products were subcloned once into ZeroBlunt vector (Invitrogen), digested by suitable restriction enzymes and cloned into pCMV-Script EX vector, and referred to as p35/FLAG and p40/FLAG. In the meantime, as a positive control for the detection of p70 protein, a heterodimer of p35 and p40, the expression vector for p70 single chain composed of linked p35 and p40 cDNA tagged with FLAG epitope was also
3.1. cDNA and amino acid sequences of hamster IL-12 p35 and p40 The longest cDNA clone corresponding to hamster IL-12 p35 (registered as AB085791) was 1239 bp in length. The deduced amino acid (aa) sequence consisted of 216 residues with an N-terminal 22 aa secretion signal sequence according to the human p35 protein. The mature p35 protein had a calculated molecular mass of 21.8 kDa. The alignment of the p35 aa sequence among the animal species of rodentia is depicted in Fig. 1A. Among six other species, the highest homology against the hamster molecule was observed in sigmodon (registered as AF421395), in which the identity and similarity with hamster were 81% and 87%, respectively (Table 2). The cDNA sequence corresponding to hamster IL-12 p40 (registered as AB085792) was 2276 bp in length. The alignment of the deduced aa sequence is shown in Fig. 1B. Hamster IL-12 p40 consisted of 327 aa with an N-terminal 22 aa secretion signal sequence according to the human protein. The mature hamster p40 protein had a calculated
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molecular mass of 34.8 kDa. As is the case with hamster p35 protein, hamster p40 showed the highest homology to sigmodon p40 (Table 2). The identity and similarity among these two species were 85 and 91%, respectively. 3.2. Expression of hamster IL-12 p35 and p40 mRNAs in cultured BM cells and various tissues The gene expressions of hamster IL-12 p35 and p40 in cultured BM cells and tissues were investigated by RT-PCR using the primer sets listed in Table 1. In the BM cells cultured in the presence of rmGM-CSF and rmIL-4, p35 and p40 mRNAs were detected from day 0 to day 2 of culture, and reached a plateau level on day 6 to day 10 (Fig. 2A). Using this analytical method, transcripts of hamster p35 were detected in the thymus, liver and spleen, and transcripts of p40 were detected in the thymus and spleen (Fig. 2B). 3.3. Expression of hamster IL-12 p35 and p40 proteins in 293 cells transfected with IL-12 p35 and/or p40 expression vectors The protein expressions of hamster IL-12 p35 and p40 were analyzed in 293 cells transfected with p35-FLAG
and/or p40-FLAG by immunoblotting using anti-FLAG antibody. In 293 cells transfected with p40-FLAG, a protein product with an apparent molecular weight of 40 kDa was detected under reducing conditions (Fig. 3). However, we could not detect a protein product corresponding to p35 under the same conditions. The protein products from these two expression vectors were supposed to have a FLAG epitope at their C-terminus. In the blotting analysis under non-reducing conditions, a protein product with an apparent molecular weight of 70 kDa in addition to a smaller 40 kDa product was detected in 293 cells transfected with both p35- and p40-FLAG or p70-FLAG (Fig. 3). 3.4. Induction of IFN−γ production by hamster IL-12 stimulation on the cultured splenocytes The supernatant of the 293 cells transfected with both p35/SCRIPT and p40/SCRIPT was added at serial dilutions to the cultures of hamster splenocytes, and its ability to induce IFN-␥ production from spleen cells was evaluated by RT-PCR using the specific primers listed in Table 1. In the hamster splenocytes treated with the supernatants, IFN-␥ production was detected in a dose-dependent manner
Fig. 1. Alignment of amino acid sequences of hamster, sigmodon, mouse, rat, gerbil and guinea pig IL-12 p35 (A) and p40 (B). Identity or similarity data of IL-12 proteins between hamster and other species are shown in Table 2. Accession numbers of hamster IL-12 p35 and p40 cDNAs are AB085791 and AB085792, respectively. Accession numbers of IL-12 DNAs from five other species (sigmodon, mouse, rat, gerbil and guinea pig) are also shown in Table 2.
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Fig. 1. (Continued ).
(Fig. 4) in the case of p35 alone or both p35 and p40 protein product-including supernatant. However, in p40 alone protein product-containing supernatant, no significant induction of IFN-␥ mRNA was seen. Meanwhile, in the splenocytes with no treatment or the mock treatment, IFN-␥ production was not detected.
4. Discussion First of all, in the present study, cDNAs coding for two subunits of hamster IL-12, p35 and p40, were cloned from a hamster DC cDNA library. The cloning demonstrated that the hamster IL-12 consisted of a p35 subunit with 216 aa and a p40 subunit with 327 aa residues. The structural com-
parison of hamster p35 and p40 at the protein level showed the highest homologies with each counterpart of sigmodon (hispid cotton rat). Specifically, in terms of structural observations, hamster IL-12 p35 cDNA includes a 5 untranslated region (UTR) of 109 bp, an ORF of 651 bp, and a 3 UTR of 462 bp and a poly(A) tail. In the 3 UTR, seven mRNA destabilization sequences (ATTTTA) and one poly-adenylation signal sequence (AATAAA) were contained. In the hamster molecule, seven out of eight cysteine residues are also conserved like those of other species. Hamster IL-12 p40 cDNA consisted of a 5 UTR of 38 bp, an ORF of 984 bp, a 3 UTR of 1235 bp and a poly(A) tail. The 3 UTR of hamster p40 contains 12 mRNA destabilization sequences and one poly-adenylation signal sequence.
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Fig. 2. IL-12 p35 and p40 mRNA expressions on cultured BM-derived DCs or various hamster tissues using RT-PCR. Specific PCR primers for hamster IL-12 p35 and p40 were designed from partially cloned cDNA sequences. Total RNAs from cultured DCs and various tissues were isolated by the acid guanidinium-phenol-chloroform method. The PCR primer sequences of hamster -actin were as follows: antisense 5 -GCTGTCCCTGTATGCCTCTG-3 , sense 5 -CCATCTCTTGCTCGAAGTCC-3 .
The 21 aa flanking sequence of the cleavage site is completely conserved in hamsters like in the other four species. The domain structure analyzed by the BLAST program showed that hamster IL-12 p40 protein also has a fibronectin type III domain (aa 236–319) and an immunoglobulin C-2 type domain (aa 43–90), as in the other
Fig. 3. Immunoblotting analysis of hamster IL-12 p35 and p40 proteins using anti-FLAG monoclonal antibody. Ten micrograms of 293 cell lysate transfected with IL-12 gene-containing expression vectors (p35/FLAG or p40/FLAG) was utilized for immunoblotting using anti-FLAG monoclonal antibody. p35/FLAG or p40/FLAG contained IL-12 p35 or p40 cDNA tagged with FLAG epitope at the C-terminus. IL-12 p70 single chain-containing expression vector (p70/FLAG) was also used for a positive control to detect a heterodimer protein of p35 and p40. Intact shows 293 cells with no transfection procedure.
species reported (Schoenhaut et al., 1992; Khalife et al., 1998). In fact, the partial cDNA sequence for hamster IL-12 p40 has already been reported by Melby et al. (1998), and their registered sequence (AF046211) is completely identical to our sequence, corresponding to nt 319–717 of ours. Secondly, RT-PCR using specific primers for hamster IL-12 p35 and p40 demonstrated that the IL-12 mRNA gene in BM cells cultured in the presence of mGM-CSF and IL-4 was up-regulated. We have also already reported that the gene expression of hamster DEC-205, one of the authentic
Fig. 4. Induction of IFN-␥ production by hamster IL-12 stimulation on cultured splenocytes. The supernatants of the 293 cells transfected with p35/SCRIPT and/or p40/SCRIPT were added at serial dilutions (1 to 1/625) to cultures of hamster splenocytes, and their abilities to induce IFN-␥ production from spleen cells were evaluated by RT-PCR using the specific primers for hamster IFN-␥ listed in Table 1. Intact shows supernatant from 293 cells with no transfection procedure.
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markers for DCs, increased during BM cell culture under the same conditions. It was obvious that cultured hamster BM cells contained a substantial amount of DCs. These findings suggest that hamster BM cells increase the expression of both IL-12 subunits in the course of differentiation to DCs. However, in Northern blot analysis using total RNA fractions, no positive signal was detected in the tissues, which suggested the low mRNA expression levels in these tissues (data not shown). Thirdly, the protein expressions of hamster IL-12 p35 and p40 were analyzed in 293 cells transfected with p35-FLAG and/or p40-FLAG by immunoblotting using anti-FLAG antibody. In 293 cells transfected with p40-FLAG, a protein product with an apparent molecular weight of 40 kDa was detected, but p35 product was not seen in reducing condition. In contrast, in the blotting analysis with non-reducing condition, the protein product with apparent molecular weight of 70 kDa in addition to smaller 40 kDa product was detected in 293 cells transfected with both p35- and p40-FLAG or p70-FLAG. The protein product of around 70 kDa might correspond to the homodimer of p40 and the heterodimer of p35/p40. The exact cause of the undetectability of p35 protein alone tagged with FLAG is not clear, however, findings from the transfection experiment using both expression vectors p35 and p40 suggest the expression of a p35 protein product, and the presence of a covalently linked p35/p40 heterodimer reported in other species. Additionally, with the aim of verifying the biological activities of hamster IL-12 gene products, the supernatant of 293 cells transfected with both IL-12 gene-containing vectors was added to the cultures of hamster splenocytes, and its ability to induce IFN-␥ production from spleen cells was investigated by RT-PCR using specific primers. IFN-␥ production was detected in a dose-dependent manner in the case of p35 alone or both p35 and p40 gene product-including supernatant. However, in the p40 alone gene product-containing supernatant, no significant induction of IFN-␥ mRNA was seen. These results were considered to be compatible with the findings reported by Kalinski et al. that the IL-12 p70 heterodimer, composed of p35 and p40, mediates IL-12 biological activity as a Th1-inducer, but the IL-12 p40 homodimer in the absence of p35 product acts as an IL-12 antagonist (Kalinski et al., 2001). Finally, taking these findings together, the two subunits of hamster IL-12 p35 and p40 have closely related structures to those of other animals (Schoenhaut et al., 1992; Khalife et al., 1998; Shiratori et al., 2001; Swinburne et al., 2000), and may have potent biological activity at least in terms of an induction of IFN-␥. At present, we have constructed an adenoviral vector which expresses a hamster IL-12 p35–p40 single chain product, and are trying to generate hamster IL-12-producing DCs using an adenoviral-mediated gene transduction procedure. Specific therapeutic experiments using IL-12-producing
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