Interleukin-12 (IL-12), consisting of p40 and p35 subunits, produces both p70 heterodimer and free p40. p70 is essential for the induction of T-helper 1 (Th1) and cytotoxic T-cell (CTL) immunity, whereas p40 inhibits p70-mediated function. Here, we found that mutations introduced into N-glycosylation sites (N220 of murine p40 and N222 of human p40) reduced secretion of p40 but not p70. Co-immunization of N220 mutant mIL-12 gene with hepatitis C virus (HCV) E2 DNA significantly enhanced long-term E2-specific CD8+ T-cell response and protection against tumor challenge compared with that of wild type. Our results indicate that the ratio of p70 to p40 is important for generating sustained long-term cell-mediated immunity. Thus, the mutant IL-12 could be utilized for the development of DNA vaccines as an adjuvant for the generation of long-term memory T-cell responses.
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Trinchieri, G. Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv. Immunol. 70, 83–243 (1998).
D'Andrea, A. et al. Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells. J. Exp. Med. 176, 1387–1398 (1992).
Ling, P. et al. Human IL-12 p40 homodimer binds to the IL-12 receptor but does not mediate biologic activity. J. Immunol. 154, 116–127 (1995).
Gately, M.K. et al. Interleukin-12 antagonist activity of mouse interleukin-12 p40 homodimer in vitro and in vivo. Ann. N.Y. Acad. Sci. 795, 1–12 (1996).
Lieschke, G.J., Rao, P.K., Gately, M.K. & Mulligan, R.C. Bioactive murine and human interleukin-12 fusion proteins which retain antitumor activity in vivo. Nat. Biotechnol. 15, 35–40 (1997).
Anderson, R. et al. Construction and biological characterization of an interleukin-12 fusion protein (Flexi-12): delivery to acute myeloid leukemic blasts using adeno-associated virus. Hum. Gene Ther. 8, 1125–1135 (1997).
Lee, Y.L., Tao, M.H., Chow, Y.H. & Chiang, B.L. Construction of vectors expressing bioactive heterodimeric and single-chain murine interleukin-12 for gene therapy. Hum. Gene Ther. 9, 457–465 (1998).
Lode, H.N. et al. Gene therapy with a single-chain interleukin 12 fusion protein induces T cell-dependent protective immunity in a syngeneic model of murine neuroblastoma. Proc. Natl. Acad. Sci. USA 95, 2475–2480 (1998).
Carra, G., Gerosa, F. & Trinchieri, G. Biosynthesis and posttranslational regulation of human IL-12. J. Immunol. 164, 4752–4761 (2000).
Podlaski, F.J. et al. Molecular characterization of interleukin 12. Arch. Biochem. Biophys. 294, 230–237 (1992).
Kuzushima, K. et al. Rapid determination of Epstein–Barr virus-specific CD8(+) T-cell frequencies by flow cytometry. Blood 94, 3094–3100 (1999).
Marth, T., Zeitz, M., Ludviksson, B.R., Strober, W. & Kelsall, B.L. Extinction of IL-12 signaling promotes Fas-mediated apoptosis of antigen-specific T cells. J. Immunol. 162, 7233–7240 (1999).
Kieper, W.C., Prlic, M., Schmidt, C.S., Mescher, M.F. & Jameson, S.C. IL-12 enhances CD8 T cell homeostatic expansion. J. Immunol. 166, 5515–5521 (2001).
Tough, D.F., Zhang, X. & Sprent, J. An IFN-γ-dependent controls stimulation of memory phenotype CD8+ T cell turnover in vivo by IL-12, IL-18, and IFN-γ. J. Immunol. 166, 6007–6011 (2001).
Zhang, X., Sun, S., Hwang, I., Tough, D.F. & Sprent, J. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity 8, 591–599 (1998).
Ku, C.C., Murakami, M., Sakamoto, A., Kappler, J. & Marrack, P. Control of homeostasis of CD8+ memory T cells by opposing cytokines. Science 288, 675–678 (2000).
Ha, S.J., Lee, S.B., Kim, C.M., Shin, H.S. & Sung, Y.C. Rapid recruitment of macrophages in interleukin-12-mediated tumour regression. Immunology 95, 156–163 (1998).
Lee, S.W., Cho, J.H. & Sung, Y.C. Optimal induction of hepatitis C virus envelope-specific immunity by bicistronic plasmid DNA inoculation with the granulocyte–macrophage colony-stimulating factor gene. J. Virol. 72, 8430–8436 (1998).
Haraguchi, M., Yamashiro, S., Furukawa, K., Takamiya, K. & Shiku, H. The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function. Biochem. J. 312, 273–280 (1995).
Stern, A.S. et al. Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells. Proc. Natl. Acad. Sci. USA 87, 6808–6812 (1990).
Wu, C., Warrier, R.R., Wang, X., Presky, D.H. & Gately, M.K. Regulation of interleukin-12 receptor β1-chain expression and interleukin-12 binding by human peripheral blood mononuclear cells. Eur. J. Immunol. 27, 147–154 (1997).
The authors acknowledge Sang-Chun Lee and Su-Il Park for devoted animal care. We are grateful to Hye-Ryun Kim and Se-Hwan Yang for faithful reading and comments. This work was supported by National Research Laboratory grant from Korea Institute of Science and Technology Evaluation and Planning (2000-N-NL-01-C-202) and grants from POSCO (2000Y013), Superior Research Center supported by Korea Science and Engineering Foundation, and ProGen Co. Ltd.
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Ha, S., Chang, J., Song, M. et al. Engineering N-glycosylation mutations in IL-12 enhances sustained cytotoxic T lymphocyte responses for DNA immunization. Nat Biotechnol 20, 381–386 (2002). https://doi.org/10.1038/nbt0402-381
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