Restricted accessResearch articleFirst published online 2010-08
Fusion of the Human Cytomegalovirus pp65 Antigen with Both Ubiquitin and Ornithine Decarboxylase Additively Enhances Antigen Presentation to CD8 + T Cells in Human Dendritic Cells
Antigenic molecules are modified for targeting to the proteasome by ubiquitin (Ub) or by a Ub-independent system such as ornithine decarboxylase (ODC) to be presented by MHC class I molecules. In this study, we compared the immunogenicity of human cytomegalovirus pp65 antigen fused with Ub and/or ODC, using RNA electroporation of human dendritic cells. Among the C-terminal mutants of Ub (G76, A76, and V76), Ub(G) showed the best ability to enhance the degradation of a target protein and stimulate T cells. The pp65 antigens fused with either Ub(G) or ODC enhanced the stimulation to CD8+ T cells, and the effects of Ub(G) and ODC were similar. Furthermore, the fusion of both Ub and ODC additively increased immunogenicity compared with the single-fusion proteins. The fusion of Ub(G) and ODC enhanced primarily the stimulation of CD8+ rather than CD4+ T cells and more efficiently induced pp65-specific T cells in vitro. These additive effects of Ub and ODC in antigen processing may provide improved strategies to stimulate CD8+ T cells for the development of immunotherapies against the variety of viral diseases and cancers.
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References
1.
AnderssonH.A., BarryM.A.2004. Maximizing antigen targeting to the proteasome for gene-based vaccines. Mol. Ther., 10:432–446.
2.
BaumeisterW., WalzJ., ZühlF., SeemüllerE.1998. The proteasome: Paradigm of a self-compartmentalizing protease. Cell, 92:367–380.
3.
BenaroudjN., TarcsaE., CascioP., GoldbergA.L.2001. The unfolding of substrates and ubiquitin-independent protein degradation by proteasomes. Biochimie, 83:311–318.
4.
BercovichZ., Rosenberg-HassonY., CiechanoverA., KahanaC.1989. Degradation of ornithine decarboxylase in reticulocyte lysate is ATP-dependent but ubiquitin-independent. J. Biol. Chem., 264:15949–15852.
5.
ButtT.R., KhanM.I., MarshJ., EckerD.J., CrookeS.T.1988. Ubiquitin–metallothionein fusion protein expression in yeast: A genetic approach for analysis of ubiquitin functions. J. Biol. Chem., 263:16364–16371.
6.
ColemanC.S., StanleyB.A., ViswanathR., PeggA.E.1994. Rapid exchange of subunits of mammalian ornithine decarboxylase. J. Biol. Chem., 269:3155–3158.
7.
DavisR.H., MorrisD.R., CoffinoP.1992. Sequestered end products and enzyme regulation: The case of ornithine decarboxylase. Microbiol. Rev., 56:280–290.
8.
HershkoA., CiechanoverA.1998. The ubiquitin system. Annu. Rev. Biochem., 67:425–479.
9.
HoytM.A., ZhangM., CoffinoP.2003. Ubiquitin-independent mechanisms of mouse ornithine decarboxylase degradation are conserved between mammalian and fungal cells. J. Biol. Chem., 278:12135–12143.
10.
ImaiT., DuanX., HisaedaH., HimenoK.2008. Antigen-specific CD8+ T cells induced by the ubiquitin fusion degradation pathway. Biochem. Biophys. Res. Commun., 365:758–763.
11.
JohnsonE.S., BartelB., SeufertW., VarshavskyA.1992. Ubiquitin as a degradation signal. EMBO J., 11:497–505.
12.
KrappmannD., WulczynF.G., ScheidereitC.1996. Different mechanisms control signal-induced degradation and basal turnover of the NF-κB inhibitor IκB α in vivo. EMBO J., 15:6716–6726.
13.
LevitskayaJ., CoramM., LevitskyV., ImrehS., Steigerwald-MullenP.M., KleinG., KurillaM.G., MasucciM.G.1995. Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1. Nature, 375:685–688.
14.
LiX., CoffinoP.1993. Degradation of ornithine decarboxylase: Exposure of the C-terminal target by a polyamine-inducible inhibitory protein. Mol. Cell. Biol., 13:2377–2383.
15.
MatsuzawaS., CuddyM., FukushimaT., ReedJ.C.2005. Method for targeting protein destruction by using a ubiquitin-independent, proteasome-mediated degradation pathway. Proc. Natl. Acad. Sci. U.S.A., 102:14982–14987.
16.
MichalekM.T., GrantE.P., GrammC., GoldbergA.L., RockK.L.1993. A role for the ubiquitin-dependent proteolytic pathway in MHC class I-restricted antigen presentation. Nature, 363:552–554.
17.
MichielsA., TuyaertsS., BonehillA., CorthalsJ., BreckpotK., HeirmanC., van MeirvenneS., DullaersM., AllardS., BrasseurF., van der BruggenP., ThielemansK.2005. Electroporation of immature and mature dendritic cells: Implications for dendritic cell-based vaccines. Gene Ther., 12:772–782.
18.
MicklethwaiteK., HansenA., FosterA., SnapeE., AntonenasV., SartorM., ShawP., BradstockK., GottliebD.2007. Ex vivo expansion and prophylactic infusion of CMV-pp65 peptide-specific cytotoxic T-lymphocytes following allogeneic hematopoietic stem cell transplantation. Biol. Blood Marrow Transplant., 13:707–714.
19.
MoscaF., PugniL.2007. Cytomegalovirus infection: The state of the art. J. Chemother., 2:46–48.
20.
MossP., KhanN.2004. CD8+ T-cell immunity to cytomegalovirus. Hum. Immunol., 65:456–464.
21.
MurakamiY., MatsufujiS., KamejiT., HayashiS., IgarashiK., TamuraT., TanakaK., IchiharaA.1992. Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination. Nature, 360:597–599.
22.
MurakamiY., MatsufujiS., HayashiS., TanahashiN., TanakaK.2000. Degradation of ornithine decarboxylase by the 26S proteasome. Biochem. Biophys. Res. Commun., 267:1–6.
23.
PerretR., RoncheseF.2008. Memory T cells in cancer immunotherapy: Which CD8 T-cell population provides the best protection against tumours?Tissue Antigens, 72:187–194.
24.
ReusserP., RiddellS.R., MeyersJ.D., GreenbergP.D.1991. Cytotoxic T lymphocyte response to cytomegalovirus after human allogeneic bone marrow transplantation: Pattern of recovery and correlation with cytomegalovirus infection and disease. Blood, 78:1373–1380.
25.
RockK.L., GrammC., RothsteinL., ClarkK., SteinR., DickL., HwangD., GoldbergA.L.1994. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell, 78:761–771.
26.
RodriguezF., ZhangJ., WhittonJ.L.1997. DNA immunization: Ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction. J. Virol., 71:8497–8503.
27.
Rosenberg-HassonY., BercovichZ., CiechanoverA., KahanaC.1989. Degradation of ornithine decarboxylase in mammalian cells is ATP dependent but ubiquitin independent. Eur. J. Biochem., 185:469–474.
28.
RouardH., KlonjkowskiB., MarquetJ., LahetC., MercierS., AndrieuM., MaisonP., Molinier-FrenkelV., EloitM., FarcetJ.P., Langlade-DemoyenP., Delfau-LarueM.H.2003. Adenoviral transgene ubiquitination enhances mouse immunization and class I presentation by human dendritic cells. Hum. Gene Ther., 14:1319–1332.
29.
SasawatariS., TadakiT., IsogaiM., TakaharaM., NiedaM., KakimiK.2006. Efficient priming and expansion of antigen-specific CD8+ T cells by a novel cell-based artificial APC. Immunol. Cell Biol., 84:512–521.
30.
SeligerB., HammersS., HöhneA., ZeidlerR., KnuthA., GerharzC.D., HuberC.1997. IFN-γ-mediated coordinated transcriptional regulation of the human TAP-1 and LMP-2 genes in human renal cell carcinoma. Clin. Cancer Res., 3:573–578.
31.
SheaffR.J., SingerJ.D., SwangerJ., SmithermanM., RobertsJ.M., ClurmanB.E.2000. Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination. Mol. Cell, 5:403–410.
StrehlB., SeifertU., KrügerE., HeinkS., KuckelkornU., KloetzelP.M.2005. Interferon-γ, the functional plasticity of the ubiquitin-proteasome system, and MHC class I antigen processing. Immunol. Rev., 207:19–30.
34.
SunQ., BurtonR.L., DaiL.J., BrittW.J., LucasK.G.2000. B lymphoblastoid cell lines as efficient APC to elicit CD8+ T cell responses against a cytomegalovirus antigen. J. Immunol., 165:4105–4111.
35.
VarshavskyA.2000. Recent studies of the ubiquitin system and the N-end rule pathway. Harvey Lect., 96:93–116.
36.
WalterE.A., GreenbergP.D., GilbertM.J., FinchR.J., WatanabeK.S., ThomasE.D., RiddellS.R.1995. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N. Engl. J. Med., 333:1038–1044.
37.
WhitesideT.L., StansonJ., ShurinM.R., FerroneS.2004. Antigen-processing machinery in human dendritic cells: Up-regulation by maturation and down-regulation by tumor cells. J. Immunol., 173:1526–1534.
