Cancer Immunology, Immunotherapy

, Volume 55, Issue 6, pp 672–683 | Cite as

Vaccination with mRNAs encoding tumor-associated antigens and granulocyte-macrophage colony-stimulating factor efficiently primes CTL responses, but is insufficient to overcome tolerance to a model tumor/self antigen

  • Paul R. HessEmail author
  • David Boczkowski
  • Smita K. Nair
  • David Snyder
  • Eli Gilboa
Original Article


Immunization of mice with dendritic cells transfected ex vivo with tumor-associated antigen (TAA)-encoding mRNA primes cytotoxic T lymphocytes (CTL) that mediate tumor rejection. Here we investigated whether direct injection of TAA mRNA, encapsulated in cationic liposomes, could function similarly as cancer immunotherapy. Intradermal and intravenous injection of ovalbumin (OVA) mRNA generated specific CTL activity and inhibited the growth of OVA-expressing tumors. Vaccination studies with DNA have demonstrated that co-administration of antigen (Ag)- and cytokine-encoding plasmids potentiate the T cell response; in analogous fashion, the inclusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA enhanced OVA-specific cytotoxicity. The ability of this GM-CSF-augmented mRNA vaccine to treat an established spontaneous tumor was evaluated in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mouse, using the SV40 large T Ag (TAg) as a model tumor/self Ag. Repeated vaccination elicited vigorous TAg-specific CTL activity in nontransgenic mice, but tumor-bearing TRAMP mice remained tolerant. Adoptive transfer of naïve splenocytes into TRAMP mice prior to the first vaccination restored TAg reactivity, and slowed tumor progression. The data from this study suggests that vaccination with TAA mRNA is a simple and effective means of priming antitumor CTL, and that immunogenicity of the vaccine can be augmented by co-delivery of GM-CSF mRNA. Nonetheless, limitations of such vaccines in overcoming tolerance to tumor/self Ag may mandate prior or simultaneous reconstitution of the autoreactive T cell repertoire for this form of immunization to be effective.


CTL mRNA vaccine GM-CSF SV40 large T antigen Tolerance 





Antigen-presenting cells


Adoptive transfer


Bone marrow-derived dendritic cells


Prostate carcinoma


Cytotoxic T lymphocyte


Cytotoxic T lymphocyte antigen-4


Effector: target


Green fluorescent protein


Granulocyte-macrophage colony-stimulating factor


Gene-modified tumor vaccine










In vitro transcribed




Plasmid DNA




Prostatic intraepithelial neoplasia


Prostate-specific antigen




Tumor-associated antigen


SV40 large T antigen


Transgenic adenocarcinoma of mouse prostate


Wild type



We are grateful to Duane Mitchell, Brenda Faiola, Carmen Wong, Charu Adlakha, Justin Hart, and Catherine McLaughlin. We also thank Erning Li for help with statistical analysis, and Xu Lin, Barbara Foster, and Norman Greenberg for their assistance with the TRAMP mouse model.


  1. 1.
    Iezzi G, Karjalainen K, Lanzavecchia A (1998) The duration of antigenic stimulation determines the fate of naive and effector T cells. Immunity 8:89–95PubMedCrossRefGoogle Scholar
  2. 2.
    van Stipdonk MJB, Hardenberg G, Bijker M, Lemmens EE, Droin NM, Green DR, Schoenberger SP (2003) Dynamic programming of CD8+ T lymphocyte responses. Nat Immunol 4:361–365PubMedCrossRefGoogle Scholar
  3. 3.
    Nair SK, Heiser A, Boczkowski D, Majumdar A, Naoe M, Lebkowski JS, Vieweg J, Gilboa E (2000) Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells. Nat Med 6:1011–1017PubMedCrossRefGoogle Scholar
  4. 4.
    Boczkowski D, Nair SK, Synder D, Gilboa E (1996) Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J Exp Med 184:465–472PubMedCrossRefGoogle Scholar
  5. 5.
    Martinon F, Krishnan S, Lenzen G, Magne R, Gomard E, Guillet J-G, Levy J-P, Meulien P (1993) Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA. Eur J Immunol 23:1719–1722PubMedCrossRefGoogle Scholar
  6. 6.
    Hoerr I, Obst R, Rammensee H-G, Jung G (2000) In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies. Eur J Immunol 30:1–7PubMedCrossRefGoogle Scholar
  7. 7.
    Zhou W-Z, Hoon DSB, Huang SKS, Fujii S, Hashimoto K, Morishita R, Kaneda Y (1999) RNA melanoma vaccine: induction of antitumor immunity by human glycoprotein 100 mRNA immunization. Hum Gene Ther 10:2719–2724PubMedCrossRefGoogle Scholar
  8. 8.
    Ying H, Zaks TZ, Wang R-F, Irvine KR, Kammula US, Marincola FM, Leitner WW, Restifo NP (1999) Cancer therapy using a self-replicating RNA vaccine. Nat Med 5:823–827PubMedCrossRefGoogle Scholar
  9. 9.
    Schirrmacher V, Forg P, Dalemans W, Chlichlia K, Zeng Y, Fournier P, von Hoegen P (2000) Intra-pinna anti-tumor vaccination with self-replicating infectious RNA or with DNA encoding a model tumor antigen and a cytokine. Gene Ther 7:1137–1147CrossRefGoogle Scholar
  10. 10.
    Colmenero P, Liljestrom P, Jondal M (1999) Induction of P815 tumor immunity by recombinant Semliki Forest virus expressing the P1A gene. Gene Ther 6:1728–1733PubMedCrossRefGoogle Scholar
  11. 11.
    Cheng WF, Hung CF, Chai CY, Hsu KF, He L, Ling M, Wu TC (2001) Enhancement of sindbis virus self-replicating RNA vaccine potency by linkage of herpes simplex virus type 1 VP22 protein to antigen. J Virol 75:2368–2376PubMedCrossRefGoogle Scholar
  12. 12.
    Ishii N, Fukushima J, Kaneko T, Okada E, Tani K, Tanaka S-I, Hamajima K, Xin K-Q, Kawamoto S, Koff WC (1997) Cationic liposomes are a strong adjuvant for a DNA vaccine of human immunodeficiency virus type 1. AIDS Res Hum Retrovir 13:1421–1427PubMedCrossRefGoogle Scholar
  13. 13.
    Li X-L, Boyanapalli M, Weihua X, Kalvakolanu DV, Hassel BA (1998) Induction of interferon synthesis and activation of interferon-stimulated genes by liposomal transfection reagents. J Interferon Cytokine Res 18:947–952PubMedGoogle Scholar
  14. 14.
    Xiang Z, Ertl HC (1995) Manipulation of the immune response to a plasmid-encoded viral antigen by coinoculation with plasmids expressing cytokines. Immunity 2:129–135PubMedCrossRefGoogle Scholar
  15. 15.
    Iwasaki A, Stiernholm BJ, Chan AK, Berinstein NL, Barber BH (1997) Enhanced CTL responses mediated by plasmid DNA immunogens encoding costimulatory molecules and cytokines. J Immunol 158:4591–4601PubMedGoogle Scholar
  16. 16.
    Geissler M, Gesien A, Tokushige K, Wands JR (1997) Enhancement of cellular and humoral immune responses to hepatitis C virus core protein using DNA-based vaccines augmented with cytokine-expressing plasmids. J Immunol 158:1231–1237PubMedGoogle Scholar
  17. 17.
    Charo J, Ciupitu AM, Le Chevalier De Preville A, Trivedi P, Klein G, Hinkula J, Kiessling R (1999) A long-term memory obtained by genetic immunization results in full protection from a mammary adenocarcinoma expressing an EBV gene. J Immunol 163:5913–5919PubMedGoogle Scholar
  18. 18.
    Bowne WB, Srinivasan R, Wolchok JD, Hawkins WG, Blachere NE, Dyall R, Lewis JJ, Houghton AN (1999) Coupling and uncoupling of tumor immunity and autoimmunity. J Exp Med 190:1711–1722CrossRefGoogle Scholar
  19. 19.
    Gabrilovich EI, Ciernik IF, Carbone DP (1996) Dendritic cells in antitumor immune responses. I. Defective antigen presentation in tumor-bearing hosts. Cell Immunol 170:101–110PubMedCrossRefGoogle Scholar
  20. 20.
    Bronte V, Chappelli DB, Appoloni E, Cabrelle A, Wang M, Hwu P, Restifo NP (1999) Unopposed production of granulocyte-macrophage colony stimulating factor by tumors inhibits CD8+ T cell response by dysregulating antigen-presenting cell maturation. J Immunol 162:5278–5283Google Scholar
  21. 21.
    Mizoguchi H, O’Shea JJ, Longo DL, Loefller CM, McVicar DW, Ochoa AC (1992) Alterations in signal transduction molecules in T lymphocytes from tumor-bearing mice. Science 258:1795–1798PubMedCrossRefGoogle Scholar
  22. 22.
    Kolenko V, Wang Q, Riedy MC, O’Shea J, Ritz J, Cathcart MK, Rayman P, Tubbs R, Edinger M, Novick A et al. (1997) Tumor-induced suppression of T lymphocyte proliferation coincides with inhibition of Jak3 expression and IL-2 receptor signaling: role of soluble products from human renal cell carcinomas. J Immunol 159:3057–3067PubMedGoogle Scholar
  23. 23.
    Correa MR, Ochoa AC, Ghosh P, Mizoguchi H, Harvey L, Longo DL (1997) Sequential development of structural and functional alterations in T cells from tumor-bearing mice. J Immunol 158:5292–5296PubMedGoogle Scholar
  24. 24.
    Dyall R, Bowne WB, Weber LW, LeMaoult J, Szabo P, Moroi Y, Piskun G, Lewis JJ, Houghton AN, Nikolic-Zugic J (1998) Heteroclitic immunization induces tumor immunity. J Exp Med 188:1553–1561PubMedCrossRefGoogle Scholar
  25. 25.
    Tsuboi A, Oka Y, Ogawa H, Elisseeva OA, Li H, Kawasaki K, Aozasa K, Kishimoto T, Udaka K, Sugiyama H (2000) Cytotoxic T-lymphocyte responses elicited to Wilms’ tumor gene WT1 product by DNA vaccination. J Clin Immunol 20:195–202PubMedCrossRefGoogle Scholar
  26. 26.
    Bronte V, Apolloni E, Ronca R, Zamboni P, Overwijk WW, Surman DR, Restifo NP, Zanovello P (2000) Genetic vaccination with “self” tyrosinase-related protein 2 causes melanoma eradication but not vitilligo. Canc Res 60:253–258Google Scholar
  27. 27.
    Tuting T, Bambotto A, De Leo A, Lotze MT, Robbins PD, Storkus WJ (1999) Induction of tumor antigen-specific immunity using plasmid DNA immunization in mice. Canc Gene Ther 6:73–80CrossRefGoogle Scholar
  28. 28.
    Greenberg NM, DeMayo F, Finegold MH, Medina D, Tilley WD, Aspinal JO, Cunha GR, Donjacour AA, Matusik RJ, Rosen JM (1995) Prostate cancer in a transgenic mouse. Proc Natl Acad Sci USA 92:3439–3443PubMedCrossRefGoogle Scholar
  29. 29.
    Gingrich JR, Barrios RJ, Morton RA, Boyce BF, DeMayo FJ, Finegold MH, Angelopoulou R, Rosen JM, Greenberg NM (1996) Metastatic prostate cancer in a transgenic mouse. Canc Res 56:4096–4102Google Scholar
  30. 30.
    Granziero L, Krajewski S, Farness P, Yuan L, Courtney MK, Jackson MR, Peterson PA, Vitiello A (1999) Adoptive immunotherapy prevents prostate cancer in a transgenic animal model. Eur J Immunol 29:1127–1138PubMedCrossRefGoogle Scholar
  31. 31.
    Hurwitz AA, Foster BA, Kwon ED, Truong T, Choi EM, Greenberg NM, Burg MB, Allison JP (2000) Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade. Canc Res 60:2444–2448Google Scholar
  32. 32.
    Mandleboim O, Berke G, Fridkin M, Feldman M, Eisenstein M, Eisenbach L (1994) CTL induction by a tumour-associated antigen octapeptide derived from a murine lung carcinoma. Nature 369:67–71CrossRefGoogle Scholar
  33. 33.
    Mylin LM, Deckhut AM, Bonneau RH, Kierstead TD, Tevethia MJ, Simmons DT, Tevethia SS (1995) Cytotoxic T lymphoctye escape variants, induced mutations, and synthetic peptides define a dominant H2-Kb-restricted determinant in simian virus 40 tumor antigen. Virology 208:159–17 2PubMedCrossRefGoogle Scholar
  34. 34.
    Rock KL, Rothstein L, Gamble S (1990) Generation of class I MHC-restricted T-T hybridomas. J Immunol 145:804–811PubMedGoogle Scholar
  35. 35.
    Gingrich JR, Barrios RJ, Kattan MW, Nahm HS, Finegold MJ, Greenberg NM (1997) Androgen-independent prostate cancer progression in the TRAMP model. Canc Res 57:4687–491Google Scholar
  36. 36.
    Lu D, Benjamin R, Kim M, Conry RM, Curiel DT (1994) Optimization of methods to achieve mRNA-mediated transfection of tumor cells in vitro and in vivo employing cationic liposome vectors. Canc Gene Ther 1:245–252Google Scholar
  37. 37.
    Speiser DE, Miranda R, Zakarian A, Bachman MF, McKall-Faienza K, Odermatt B, Hanahan D, Zinkernagel RM, Ohashi PS (1997) Self antigens expressed by solid tumors do not efficiently stimulate naive or activated T cells: implications for immunotherapy. J Exp Med 186:645–653PubMedCrossRefGoogle Scholar
  38. 38.
    Grossmann ME, Davila E, Celis E (2001) Avoiding tolerance against prostatic antigens with subdominant peptide epitopes. J Immunother 24:237–241CrossRefGoogle Scholar
  39. 39.
    Schell TD, Knowles BB, Tevethia SS (2000) Sequential loss of cytotoxic T lymphocyte responses to simian virus 40 large T antigen epitopes in T antigen transgenic mice developing osteosarcomas. Canc Res 60:3002–3012Google Scholar
  40. 40.
    Mounkes LC, Zhong W, Cipres-Palacin G, Heath TD, Debs RJ (1998) Proteoglycans mediate cationic liposome-DNA complex-based gene delivery in vitro and in vivo. J Biol Chem 273:26164–26170PubMedCrossRefGoogle Scholar
  41. 41.
    Farhood H, Serbina N, Huang L (1995) The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. Biochem Biophys Acta 1235:289–295PubMedCrossRefGoogle Scholar
  42. 42.
    Segal AW, Wills EJ, Richmond JE, Slavin G, Black CDV, Gregoriadis G (1974) Morphological observations on the cellular and subcellular destination of intravenously administered liposomes. Brit J Exp Path 55:320–326PubMedGoogle Scholar
  43. 43.
    Haddad D, Ramprakash J, Sedegah M, Charoenvit Y, Baumgartner T, Kumar S, Hoffman SL, Weiss WR (2000) Plasmid vaccine expressing granulocyte-macrophage colony-stimulating factor attracts infiltrates including immature dendritic cells into injected muscles. J Immunol 165:3772–3781PubMedGoogle Scholar
  44. 44.
    Bowne WB, Wolchok JD, Hawkins WG, Srinivasan R, Gregor P, Blachere NE, Moroi Y, Engelhorn ME, Houghton AN, Lewis JJ (1999) Injection of DNA encoding granulocyte-macrophage colony-stimulating factor recruits dendritic cells for immune adjuvant effects. Cytokines Cell Mol Ther 5:217–225PubMedGoogle Scholar
  45. 45.
    Kim JJ, Trivedi NN, Nottingham LK, Morrison L, Tsai A, Hu Y, Mahalingam S, Dang K, Ahn L, Doyle NK, et al. (1998) Modulation of amplitude and direction of in vivo immune responses by co-administration of cytokine gene expression cassettes with DNA immunogens. Eur J Immunol 28:1089–1103PubMedCrossRefGoogle Scholar
  46. 46.
    Kim JJ, Bagarazzi ML, Trivedi N, Hu Y, Kazahaya K, Wilson DM, Ciccarelli R, Chattergoon MA, Dang K, Mahalingam S et al. (1997) Engineering of in vivo immune responses to DNA immunization via codelivery of costimulatory molecule genes. Nat Biotech 15:641–646CrossRefGoogle Scholar
  47. 47.
    Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K, Jackson V, Hamada H, Pardoll D, Mulligan RC (1993) Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci USA 90:3539–3543PubMedCrossRefGoogle Scholar
  48. 48.
    Soo Hoo W, Lundeen KA, Kohrumel JR, Pham N-L, Brostoff SW, Bartholomew RM, Carlo DJ (1999) Tumor cell surface expression of granulocyte-macrophage colony-stimulating factor elicits antitumor immunity and protects from tumor challenge in the P815 mouse mastocytoma tumor model. J Immunol 162:7343–7349PubMedGoogle Scholar
  49. 49.
    Heiser A, Maurice MA, Yancey DR, Wu NZ, Dahm P, Pruitt SK, Boczkowski D, Nair SK, Ballo MS, Gilboa E, et al. (2001) Induction of polyclonal prostate cancer-specific CTL using dendritic cells transfected with amplified tumor RNA. J Immunol 166:2593–2960Google Scholar
  50. 50.
    Ludewig B, Ochsenbein AF, Odermatt F, Paulin D, Hengartner H, Zinkernagel RM (2000) Immunotherapy with dendritic cells directed against tumor antigen shared with normal host cells results in severe autoimmune disease. J Exp Med 191:795–803PubMedCrossRefGoogle Scholar
  51. 51.
    Kass E, Schlom J, Thompson J, Guadagni F, Graziano P, Greiner JW (1999) Induction of protective host immunity to carcinoembryonic antigen (CEA), a self-antigen in CEA transgenic mice, by immunizing with a recombinant vaccinia-CEA virus. Canc Res 59:676–683Google Scholar
  52. 52.
    Limmer A, Sacher T, Alferink J, Kretschmar M, Schonrich G, Nichterlein T, Arnold B, Hammerling GJ (1998) Failure to induce organ-specific autoimmunity by breaking of tolerance: importance of the microenvironment. Eur J Immunol 28:2395–2406PubMedCrossRefGoogle Scholar
  53. 53.
    Morgan DJ, Kreuwel HTC, Fleck S, Levitsky HI, Pardoll DM, Sherman LA (1998) Activation of low avidity CTL specific for a self epitope results in tumor rejection but not autoimmunity. J Immunol 160:643–651PubMedGoogle Scholar
  54. 54.
    Eck SC, Turka LA (2001) Adoptive transfer enables tumor rejection targeted against a self-antigen without the induction of autoimmunity. Canc Res 61:3077–3083Google Scholar
  55. 55.
    Wei C, Willis RA, Tilton BR, Looney RJ, Lord EM, Barth RK, Frelinger JG (1997) Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: implications for tolerance and immunotherapy. Proc Natl Acad Sci USA 94:6369–6374PubMedCrossRefGoogle Scholar
  56. 56.
    Heiser A, Dahm P, Yancey DR, Maurice MA, Boczkowski D, Nair SK, Gilboa E, Vieweg J (2000) Human dendritic cells transfected with RNA encoding prostate-specific antigen stimulate prostate-specific CTL responses in vitro. J Immunol 164:5508–5514PubMedGoogle Scholar
  57. 57.
    Zheng X, Gao J-X, Zhang H, Geiger TL, Liu Y, Zheng P (2002) Clonal deletion of simian virus 40 large T antigen-specific T cells in the transgenic adenocarcinoma of mouse prostate mice: an important role for clonal deletion in shaping the repertoire of T cells specific for antigens overexpressed in solid tumors. J Immunol 169:4761–4769PubMedGoogle Scholar
  58. 58.
    Morgan RA, Dudley ME, Yu YYL, Zheng Z, Robbins PF, Theoret MR, Wunderlich JR, Hughes MS, Restifo NP, Rosenberg SA (2003) High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens. J Immunol 171:3287–3295PubMedGoogle Scholar
  59. 59.
    Kessels HW, Wolkers MC, van den Boom MD, van der Valk MA, Schumacher TN (2001) Immunotherapy through TCR gene transfer. Nat Immunol 2:957–961PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Paul R. Hess
    • 1
    • 2
    • 3
    Email author
  • David Boczkowski
    • 2
  • Smita K. Nair
    • 2
  • David Snyder
    • 2
  • Eli Gilboa
    • 2
  1. 1.Department of Microbiology, Pathology, and Parasitology, College of Veterinary MedicineNorth Carolina State UniversityRaleighUSA
  2. 2.Department of Surgery, and Center for Genetic and Cellular TherapiesDuke University Medical CenterDurhamUSA
  3. 3.Department of Clinical Sciences, College of Veterinary MedicineNorth Carolina State UniversityRaleighUSA

Personalised recommendations