Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Engineering N-glycosylation mutations in IL-12 enhances sustained cytotoxic T lymphocyte responses for DNA immunization

Abstract

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.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Western blot analysis of human p40 and its derivatives with mutations at putative N-glycosylation sites.
Figure 2: Comparison of receptor binding and IFN-γ induction between wild-type and N222L mutant p70 on Con A blast cells.
Figure 3: Kinetics of E2-specific antibody responses in DNA immunization.
Figure 4: Effect of mIL-12N220L on E2-specific CTL responses and protection against challenge with modified tumors.

References

  1. Trinchieri, G. Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv. Immunol. 70, 83–243 (1998).

    CAS  Article  Google Scholar 

  2. 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).

    CAS  Article  Google Scholar 

  3. 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).

    CAS  PubMed  Google Scholar 

  4. 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).

    CAS  Article  Google Scholar 

  5. 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).

    CAS  Article  Google Scholar 

  6. 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).

    CAS  Article  Google Scholar 

  7. 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).

    CAS  Article  Google Scholar 

  8. 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).

    CAS  Article  Google Scholar 

  9. Carra, G., Gerosa, F. & Trinchieri, G. Biosynthesis and posttranslational regulation of human IL-12. J. Immunol. 164, 4752–4761 (2000).

    CAS  Article  Google Scholar 

  10. Podlaski, F.J. et al. Molecular characterization of interleukin 12. Arch. Biochem. Biophys. 294, 230–237 (1992).

    CAS  Article  Google Scholar 

  11. Kuzushima, K. et al. Rapid determination of Epstein–Barr virus-specific CD8(+) T-cell frequencies by flow cytometry. Blood 94, 3094–3100 (1999).

    CAS  PubMed  Google Scholar 

  12. 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).

    CAS  PubMed  Google Scholar 

  13. 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).

    CAS  Article  Google Scholar 

  14. 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).

    CAS  Article  Google Scholar 

  15. 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).

    CAS  Article  Google Scholar 

  16. 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).

    CAS  Article  Google Scholar 

  17. 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).

    CAS  Article  Google Scholar 

  18. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 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).

    CAS  Article  Google Scholar 

  20. 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).

    CAS  Article  Google Scholar 

  21. 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).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Young C. Sung.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0402-381

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing