Dendritic cells (DCs) mediate the generation of strong cytotoxic T-lymphocyte (CTL) responses by functioning in antigen presentation and exerting adjuvant properties. We compared several activation markers and parameters of biological activity of DNA- and mRNA-transfected DCs in vitro and in vivo.
Methods
CpG-matured, bone marrow derived C57BL/6 mouse DCs were electroporated either with enhanced green fluorescence protein (EGFP) or human papillomavirus type 16 (HPV16) E7 expression plasmids or in vitro transcribed mRNAs encoding for the codon-optimized E7 or a shuffled version thereof. Activation marker expression and antigen presentation was analysed by fluorescence-activated cell sorting. The migratory behaviour of transfected DCs were investigated by in vitro chemotaxis experiments and cytokine expression by ELISA. CTL-priming capacity of transfected DCs were determined by vaccination of mice.
Results
mRNA transfection produced a two- to fourfold increase of the activation markers CD40, CD80, CD86 and MHC I and MHC II molecules. Predominately antigen-expressing DCs migrated after mRNA transfection. Furthermore, mRNA-transfected DCs were capable of inducing a chemokine gradient. After maturation, electroporation and activation with soluble CD40 ligand and interferon-γ, DCs displayed a T-helper cell type 2 cytokine expression pattern. Nevertheless, E7-transfected DCs were able to prime E7-specific CTL responses in vivo. The highest E7-specific CTL frequencies were found in mice immunized with mRNA-transfected DCs. The in vitro expanded CTLs exerted functional E7-specific cytotoxic activity.
Conclusions
Genetically modified DCs are suitable vehicles for the induction of E7-specific CTL responses in mice and hence could help to eradicate HPV-associated lesions in humans.
References
1.
zur HausenH.Papillomaviruses and cancer: from basic studies to clinical application.Nat Rev Cancer2002; 2: 342–350.
2.
FigdorC.G., de VriesI.J., LesterhuisW.J., MeliefC.J.Dendritic cell immunotherapy: mapping the way.Nat Med2004; 10: 475–480.
3.
WangT.L., LingM., ShihI.M.Intramuscular administration of E7- transfected dendritic cells generates the most potent E7- specific anti-tumor immunity.Gene Ther2000; 7: 726–733.
4.
TutingT., DeLeoA.B., LotzeM.T., StorkusW.J.Genetically modified bone marrow-derived dendritic cells expressing tumor-associated viral or ‘self’ antigens induce antitumor immunity in vivo.Eur J Immunol1997; 27: 2702–2707.
5.
SchaftN., DorrieJ., ThumannP.Generation of an optimized polyvalent monocyte-derived dendritic cell vaccine by transfecting defined RNAs after rather than before maturation.J Immunol2005; 174: 3087–3097.
6.
SchulerG., Schuler-ThurnerB., SteinmanR.M.The use of dendritic cells in cancer immunotherapy.Curr Opin Immunol2003; 15: 138–147.
7.
SteinbergT., OhlschlagerP., SehrP., OsenW., GissmannL.Modification of HPV 16 E7 genes: correlation between the level of protein expression and CTL response after immunization of C57BL/6 mice.Vaccine2005; 23: 1149–1157.
8.
OhlschlagerP., PesM., OsenW.An improved rearranged human papillomavirus type 16 E7 DNA vaccine candidate (HPV-16 E7SH) induces an E7 wildtype-specific T cell response.Vaccine2006; 24: 2880–2893.
9.
StepinskiJ., WaddellC., StolarskiR., DarzynkiewiczE., RhoadsR.E.Synthesis and properties of mRNAs containing the novel ‘anti-reverse’ cap analogs 7-methyl(3’-O-methyl) GpppG and 7-methyl (3’-deoxy) GpppG.RNA2001; 7: 1486–1495.
ClarkeS.R.The critical role of CD40/CD40L in the CD4-dependent generation of CD8+ T cell immunity.J Leukocyte Biol2000; 67: 607–614.
12.
Van MeirvenneS., StraetmanL., HeirmanC.Efficient genetic modification of murine dendritic cells by electroporation with mRNA.Cancer Gene Ther2002; 9: 787–797.
13.
PulendranB.Modulating vaccine responses with dendritic cells and Toll-like receptors.Immunol Rev2004; 199: 227–250.
14.
Reis e SousaC.Toll-like receptors and dendritic cells: for whom the bug tolls.Semin Immunol2004; 16: 27–34.
15.
Martin-FontechaA., SebastianiS., HopkenU.E.Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming.J Exp Med2003; 198: 615–621.
16.
RandolphG.J., Sanchez-SchmitzG., AngeliV.Factors and signals that govern the migration of dendritic cells via lymphatics: recent advances.Springer Semin Immunopathol2005; 26: 273–287.
17.
MoserM., MurphyK.M.Dendritic cell regulation of TH1-TH2 development.Nat Immunol2000; 1: 199–205.
18.
TrinchieriG.Interleukin-12 and the regulation of innate resistance and adaptive immunity.Nat Rev Immunol2003; 3: 133–146.
19.
DillonS., AgrawalA., Van DykeT.A Toll-like receptor 2 ligand stimulates Th2 responses in vivo, via induction of extracellular signal-regulated kinase mitogen-activated protein kinase and c- Fos in dendritic cells.J Immunol2004; 172: 4733–4743.
20.
EdwardsA.D., ManickasinghamS.P., SporriR.Microbial recognition via Toll-like receptor-dependent and -independent pathways determines the cytokine response of murine dendritic cell subsets to CD40 triggering.J Immunol2002; 169: 3652–3660.
21.
Perona-WrightG., JenkinsS.J., CrawfordA., GrayD., PearceE.J., MacDonaldA.S.Distinct sources and targets of IL-10 during dendritic cell-driven Th1 and Th2 responses in vivo.Eur J Immunol2006; 36: 2367–2375.
22.
LavelleE.C., McNeelaE., ArmstrongM.E., LeavyO., HigginsS.C., MillsK.H.Cholera toxin promotes the induction of regulatory T cells specific for bystander antigens by modulating dendritic cell activation.J Immunol2003; 171: 2384–2392.
23.
McGuirkP., McCannC., MillsK.H.Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis.J Exp Med2002; 195: 221–231.
24.
McGuirkP., MillsK.H.Pathogen-specific regulatory T cells provoke a shift in the Th1/Th2 paradigm in immunity to infectious diseases.Trends Immunol2002; 23: 450–455.
25.
GrommeM., NeefjesJ.Antigen degradation or presentation by MHC class I molecules via classical and non-classical pathways.Mol Immunol2002; 39: 181–202.
26.
BullockT.N., ColellaT.A., EngelhardV.H.The density of peptides displayed by dendritic cells affects immune responses to human tyrosinase and gp100 in HLA-A2 transgenic mice.J Immunol2000; 164: 2354–2361.
27.
WherryE.J., McElhaughM.J., EisenlohrL.C.Generation of CD8(+) T cell memory in response to low, high, and excessive levels of epitope.J Immunol2002; 168: 4455–4461.
28.
WherryE.J., PuorroK.A., PorgadorA., EisenlohrL.C.The induction of virus-specific CTL as a function of increasing epitope expression: responses rise steadily until excessively high levels of epitope are attained.J Immunol1999; 163: 3735–3745.
29.
SpeidelK., OsenW., FaathS.Priming of cytotoxic T lymphocytes by five heat-aggregated antigens in vivo: conditions, efficiency, and relation to antibody responses.Eur J Immunol1997; 27: 2391–2399.
30.
LjunggrenH.G., StamN.J., OhlenC.Empty MHC class I molecules come out in the cold.Nature1990; 346: 476–480.
31.
DellK., KoestersR., GissmannL.Transcutaneous immunization in mice: induction of T- helper and cytotoxic T lymphocyte responses and protection against human papillomavirus-induced tumors.Int J Cancer2006; 118: 364–372.
32.
LutzM.B., KukutschN., OgilvieA.L.An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow.J Immunol Methods1999; 223: 77–92.
33.
KarikoK., NiH., CapodiciJ., LamphierM., WeissmanD.mRNA is an endogenous ligand for Toll-like receptor 3.J Biol Chem2004; 279: 12542–12550.
34.
HondaK., SakaguchiS., NakajimaC., WatanabeSelective contribution of IFN-alpha/beta signaling to the maturation of dendritic cells induced by double-stranded RNA or viral infection.Proc Natl Acad Sci U S A2003; 100: 10872–10877.
35.
WeissmanD., NiH., ScalesD.HIV gag mRNA transfection of dendritic cells (DC) delivers encoded antigen to MHC class I and II molecules, causes DC maturation, and induces a potent human in vitro primary immune response.J Immunol2000; 165: 4710–4717.
36.
WarrenT.L., BhatiaS.K., AcostaA.M.APC stimulated by CpG oligodeoxynucleotide enhance activation of MHC class I- restricted T cells.J Immunol2000; 165: 6244–6251.
37.
Van MeirvenneS., DullaersM., HeirmanC., StraetmanL., MichielsA., ThielemansK.In vivo depletion of CD4+CD25+ regulatory T cells enhances the antigen-specific primary and memory CTL response elicited by mature mRNA-electroporated dendritic cells.Mol Ther2005; 12: 922–932.
38.
Van TendelooV.F., SnoeckH.W., LardonF.Nonviral transfection of distinct types of human dendritic cells: high-efficiency gene transfer by electroporation into hematopoietic progenitor- but not monocyte-derived dendritic cells.Gene Ther1998; 5: 700–707.
39.
CeppiM., RuggliN., TacheV., GerberH., McCulloughK.C., SummerfieldA.Double-stranded secondary structures on mRNA induce type I interferon (IFN alpha/beta) production and maturation of mRNA-transfected monocyte-derived dendritic cells.J Gene Med2005; 7: 452–465.
40.
RothoeftT., BalkowS., KrummenM.Structure and duration of contact between dendritic cells and T cells are controlled by T cell activation state.Eur J Immunol2006; 36: 3105–3117.
41.
MatsumotoK., WassarmanK.M., WolffeA.P.Nuclear history of a pre-mRNA determines the translational activity of cytoplasmic mRNA.EMBO J1998; 17: 2107–2121.
42.
TsengW.C., HaseltonF.R., GiorgioT.D.Transfection by cationic liposomes using simultaneous single cell measurements of plasmid delivery and transgenic expression.J Biol Chem1997; 272: 25641–25647.
43.
MorelS., LevyF., Burlet-SchiltzO., BrasseurProcessing of some antigens by the standard proteasome but not by the immunoproteasome results in poor presentation by dendritic cells.Immunity2000; 12: 107–117.
44.
KalinskiP., SchuitemakerJ.H., HilkensC.M., WierengaE.A., KapsenbergM.L.Final maturation of dendritic cells is associated with impaired responsiveness to IFN-gamma and to bacterial IL- 12 inducers: decreased ability of mature dendritic cells to produce IL-12 during the interaction with Th cells.J Immunol1999; 162: 3231–3236.
45.
LangenkampA., MessiM., LanzavecchiaA., SallustoF.Kinetics of dendritic cell activation: impact on priming of TH1, TH2 and nonpolarized T cells.Nat Immunol2000; 1: 311–316.
46.
JanssenE.M., LemmensE.E., WolfeT., ChristenU., von HerrathM.G., SchoenbergerS.P.CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes.Nature2003; 421: 852–856.
