Cancer Immunology, Immunotherapy

, Volume 59, Issue 4, pp 519–527 | Cite as

A B-cell lymphoma vaccine using a depot formulation of interleukin-2 induces potent antitumor immunity despite increased numbers of intratumoral regulatory T cells

  • Sofía Grille
  • Andreína Brugnini
  • Martha Nese
  • Esteban Corley
  • Frank W. Falkenberg
  • Daniela Lens
  • José A. Chabalgoity
Original Article


Therapeutic vaccination holds great potential as complementary treatment for non-Hodgkin’s lymphoma. Here, we report that a therapeutic whole cell vaccine formulated with IL-2 adsorbed onto aluminum hydroxide as cytokine-depot formulation elicits potent antitumor immunity and induces delayed tumor growth, control of tumor dissemination and longer survival in mice challenged with A20-lymphoma. Therapeutic vaccination induced higher numbers of tumor’s infiltrating lymphocytes (CD4+ and CD8+ T cells and NK cells), and the production of IFN-γ and IL-4 by intratumoral CD4+ T cells. Further, strong tumor antigen-specific cellular responses were detected at systemic level. Both the A20-derived antigenic material and the IL-2 depot formulation were required for induction of an effective immune response that impacted on cancer progression. All mice receiving any form of IL-2, either as part of the vaccine or alone as control, showed higher numbers of CD4+CD25+/highFoxp3+ regulatory T cells (Treg) in the tumor, which might have a role in tumor progression in these animals. Nevertheless, for those animals that received the cytokine as part of the vaccine formulation, the overall effect was improved immune response and less disseminated disease, suggesting that therapeutic vaccination overcomes the potential detrimental effect of intratumoral Treg cells. Overall, the results presented here show that a simple vaccine formulation, that can be easily prepared under GMP conditions, is a promising strategy to be used in B-cell lymphoma and may have enough merit to be tested in clinical trials.


Lymphoma Vaccine Interleukin-2 Aluminum hydroxide Regulatory T cells 



This work was partially supported by a grant from the Comisión Sectorial de Investigación Científica (CSIC). Universidad de la República. Uruguay. S. Grille was funded by a scholarship of ProInBio, Uruguay and CSIC. The authors thank Prof. Dr. Miguel Torres and Tech. Mr. Marcelo Curbelo of the Departament of Radiotherapy, Hospital de Clínicas for technical assistance. S.G. designed and performed the experiments, and analyzed data. D.L. and J.A.C. designed the experiments and analyzed data. A.B. performed the experiments, collected and analyzed data. E.C and F.W.F. contributed with vital reagents (IL-2-AL), and contributed to experimental design. M.N. analyzed the data and S.G., D.L., and J.A.C. wrote the manuscript.


  1. 1.
    Marcus R, Hagenbeek A (2007) The therapeutic use of rituximab in non-Hodgkin’s lymphoma. Eur J Haematol Suppl (67):5–14Google Scholar
  2. 2.
    Neelapu SS, Kwak LW (2007) Vaccine therapy for B-cell lymphomas: next-generation strategies. In: Gewirtz AM, Winter JN, Zuckerman K (eds) American Society of Hematology education program book. Atlanta, GA, pp 243–249Google Scholar
  3. 3.
    Neelapu SS, Lee ST, Qin H, Cha SC, Woo AF, Kwak LW (2006) Therapeutic lymphoma vaccines: importance of T-cell immunity. Expert Rev Vaccines 5(3):381–394CrossRefPubMedGoogle Scholar
  4. 4.
    Kwak LW, Campbell MJ, Czerwinski DK, Hart S, Miller RA, Levy R (1992) Induction of immune responses in patients with B-cell lymphoma against the surface-immunoglobulin idiotype expressed by their tumors. N Engl J Med 327(17):1209–1215PubMedGoogle Scholar
  5. 5.
    Hsu FJ, Caspar CB, Czerwinski D, Kwak LW, Liles TM, Syrengelas A, Taidi-Laskowski B, Levy R (1997) Tumor-specific idiotype vaccines in the treatment of patients with B-cell lymphoma—long-term results of a clinical trial. Blood 89(9):3129–3135PubMedGoogle Scholar
  6. 6.
    Timmerman JM, Singh G, Hermanson G, Hobart P, Czerwinski DK, Taidi B, Rajapaksa R, Caspar CB, Van BA, Levy R (2002) Immunogenicity of a plasmid DNA vaccine encoding chimeric idiotype in patients with B-cell lymphoma. Cancer Res 62(20):5845–5852PubMedGoogle Scholar
  7. 7.
    Timmerman JM, Czerwinski DK, Davis TA, Hsu FJ, Benike C, Hao ZM, Taidi B, Rajapaksa R, Caspar CB, Okada CY, Van BA, Liles TM, Engleman EG, Levy R (2002) Idiotype-pulsed dendritic cell vaccination for B-cell lymphoma: clinical and immune responses in 35 patients. Blood 99(5):1517–1526CrossRefPubMedGoogle Scholar
  8. 8.
    Chabalgoity JA, Baz A, Rial A, Grille S (2007) The relevance of cytokines for development of protective immunity and rational design of vaccines. Cytokine Growth Factor Rev 18(1–2):195–207CrossRefPubMedGoogle Scholar
  9. 9.
    Caporale A, Brescia A, Galati G, Castelli M, Saputo S, Terrenato I, Cucina A, Liverani A, Gasparrini M, Ciardi A, Scarpini M, Cosenza UM (2007) Locoregional IL-2 therapy in the treatment of colon cancer cell-induced lesions of a murine model. Anticancer Res 27(2):985–989PubMedGoogle Scholar
  10. 10.
    Liu K, Rosenberg SA (2003) Interleukin-2-independent proliferation of human melanoma-reactive T lymphocytes transduced with an exogenous IL-2 gene is stimulation dependent. J Immunother 26(3):190–201CrossRefPubMedGoogle Scholar
  11. 11.
    Toubaji A, Hill S, Terabe M, Qian J, Floyd T, Simpson RM, Berzofsky JA, Khleif SN (2007) The combination of GM-CSF and IL-2 as local adjuvant shows synergy in enhancing peptide vaccines and provides long term tumor protection. Vaccine 25(31):5882–5891CrossRefPubMedGoogle Scholar
  12. 12.
    Vaage J (1987) Local and systemic effects during interleukin-2 therapy of mouse mammary tumors. Cancer Res 47(16):4296–4298PubMedGoogle Scholar
  13. 13.
    Bubenik J, Indrova M, Perlmann P, Berzins K, Mach O, Kraml J, Toulcova A (1985) Tumour inhibitory effects of TCGF/IL-2/-containing preparations. Cancer Immunol Immunother 19(1):57–61CrossRefPubMedGoogle Scholar
  14. 14.
    Meziane K, Bhattacharyya T, Armstrong AC, Qian C, Hawkins RE, Stern PL, Dermime S (2004) Use of adenoviruses encoding CD40L or IL-2 against B cell lymphoma. Int J Cancer 111(6):910–920CrossRefGoogle Scholar
  15. 15.
    Atkins MB, Lotze MT, Dutcher JP, Fisher RI, Weiss G, Margolin K, Abrams J, Sznol M, Parkinson D, Hawkins M, Paradise C, Kunkel L, Rosenberg SA (1999) High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. J Clin Oncol 17(7):2105–2116PubMedGoogle Scholar
  16. 16.
    Fyfe G, Fisher RI, Rosenberg SA, Sznol M, Parkinson DR, Louie AC (1995) Results of treatment of 255 patients with metastatic renal cell carcinoma who received high-dose recombinant interleukin-2 therapy. J Clin Oncol 13(3):688–696PubMedGoogle Scholar
  17. 17.
    Fisher RI, Coltman CA Jr, Doroshow JH, Rayner AA, Hawkins MJ, Mier JW, Wiernik P, McMannis JD, Weiss GR, Margolin KA (1988) Metastatic renal cancer treated with interleukin-2 and lymphokine-activated killer cells. A phase II clinical trial. Ann Intern Med 108(4):518–523PubMedGoogle Scholar
  18. 18.
    Gluck WL, Hurst D, Yuen A, Levine AM, Dayton MA, Gockerman JP, Lucas J, Denis-Mize K, Tong B, Navis D, Difrancesco A, Milan S, Wilson SE, Wolin M (2004) Phase I studies of interleukin (IL)-2 and rituximab in B-cell non-Hodgkin’s lymphoma: IL-2 mediated natural killer cell expansion correlations with clinical response. Clin Cancer Res 10(7):2253–2264CrossRefPubMedGoogle Scholar
  19. 19.
    Eisenbeis CF, Grainger A, Fischer B, Baiocchi RA, Carrodeguas L, Roychowdhury S, Chen L, Banks AL, Davis T, Young D, Kelbick N, Stephens J, Byrd JC, Grever MR, Caligiuri MA, Porcu P (2004) Combination immunotherapy of B-cell non-Hodgkin’s lymphoma with rituximab and interleukin-2: a preclinical and phase I study. Clin Cancer Res 10(18 Pt 1):6101–6110CrossRefPubMedGoogle Scholar
  20. 20.
    Neelapu SS, Gause BL, Harvey L, Lee ST, Frye AR, Horton J, Robb RJ, Popescu MC, Kwak LW (2007) A novel proteoliposomal vaccine induces antitumor immunity against follicular lymphoma. Blood 109(12):5160–5163CrossRefPubMedGoogle Scholar
  21. 21.
    Neelapu SS, Baskar S, Gause BL, Kobrin CB, Watson TM, Frye AR, Pennington R, Harvey L, Jaffe ES, Robb RJ, Popescu MC, Kwak LW (2004) Human autologous tumor-specific T-cell responses induced by liposomal delivery of a lymphoma antigen. Clin Cancer Res 10(24):8309–8317CrossRefPubMedGoogle Scholar
  22. 22.
    Falkenberg Frank W, Krup Oliver C (1999) Compositions and methods for treatment of tumors and metastatic diseases. 261816[6406689]. 18-6-2002. 3-2-1999. Ref Type: PatentGoogle Scholar
  23. 23.
    Krup OC, Kroll I, Bose G, Falkenberg FW (1999) Cytokine depot formulations as adjuvants for tumor vaccines. I. Liposome-encapsulated IL-2 as a depot formulation. J Immunother 22(6):525–538CrossRefPubMedGoogle Scholar
  24. 24.
    Agorio C, Schreiber F, Sheppard M, Mastroeni P, Fernandez M, Martinez MA, Chabalgoity JA (2007) Live attenuated Salmonella as a vector for oral cytokine gene therapy in melanoma. J Gene Med 9(5):416–423CrossRefPubMedGoogle Scholar
  25. 25.
    Liu D, Yu J, Chen H, Reichman R, Wu H, Jin X (2006) Statistical determination of threshold for cellular division in the CFSE-labeling assay. J Immunol Methods 312(1–2):126–136CrossRefPubMedGoogle Scholar
  26. 26.
    Williams MA, Tyznik AJ, Bevan MJ (2006) Interleukin-2 signals during priming are required for secondary expansion of CD8+ memory T cells. Nature 441(7095):890–893CrossRefPubMedGoogle Scholar
  27. 27.
    Waldmann TA (2006) The biology of interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design. Nat Rev Immunol 6(8):595–601CrossRefPubMedGoogle Scholar
  28. 28.
    Caligiuri MA, Murray C, Robertson MJ, Wang E, Cochran K, Cameron C, Schow P, Ross ME, Klumpp TR, Soiffer RJ (1993) Selective modulation of human natural killer cells in vivo after prolonged infusion of low dose recombinant interleukin 2. J Clin Invest 91(1):123–132CrossRefPubMedGoogle Scholar
  29. 29.
    Kwak LW, Pennington R, Boni L, Ochoa AC, Robb RJ, Popescu MC (1998) Liposomal formulation of a self lymphoma antigen induces potent protective antitumor immunity. J Immunol 160(8):3637–3641PubMedGoogle Scholar
  30. 30.
    Popescu MC, Robb RJ, Batenjany MM, Boni LT, Neville ME, Pennington RW, Neelapu SS, Kwak LW (2007) A novel proteoliposomal vaccine elicits potent antitumor immunity in mice. Blood 109(12):5407–5410CrossRefPubMedGoogle Scholar
  31. 31.
    Curiel TJ (2008) Regulatory T cells and treatment of cancer. Curr Opin Immunol 20(2):241–246CrossRefPubMedGoogle Scholar
  32. 32.
    Beyer M, Schultze JL (2006) Regulatory T cells in cancer. Blood 108(3):804–811CrossRefPubMedGoogle Scholar
  33. 33.
    Heier I, Hofgaard PO, Brandtaeg P, Jahnsen FL, Karlsson M (2008) Depletion of CD4+ CD25+ regulatory T cells inhibits local tumour growth in a mouse model of B cell lymphoma. Clin Exp Immunol 152(2):381–387PubMedCrossRefGoogle Scholar
  34. 34.
    Tzankov A, Meier C, Hirschmann P, Went P, Pileri SA, Dirnhofer S (2008) Correlation of high numbers of intratumoral FOXP3+ regulatory T cells with improved survival in germinal center-like diffuse large B-cell lymphoma, follicular lymphoma and classical Hodgkin’s lymphoma. Haematologica 93(2):193–200CrossRefPubMedGoogle Scholar
  35. 35.
    Almeida AR, Legrand N, Papiernik M, Freitas AA (2002) Homeostasis of peripheral CD4+ T cells: IL-2R alpha and IL-2 shape a population of regulatory cells that controls CD4+ T cell numbers. J Immunol 169(9):4850–4860PubMedGoogle Scholar
  36. 36.
    Malek TR, Yu A, Vincek V, Scibelli P, Kong L (2002) CD4 regulatory T cells prevent lethal autoimmunity in IL-2Rbeta-deficient mice. Implications for the nonredundant function of IL-2. Immunity 17(2):167–178CrossRefPubMedGoogle Scholar
  37. 37.
    Malek TR, Bayer AL (2004) Tolerance, not immunity, crucially depends on IL-2. Nat Rev Immunol 4(9):665–674CrossRefPubMedGoogle Scholar
  38. 38.
    Nelson BH (2004) IL-2, regulatory T cells, and tolerance. J Immunol 172(7):3983–3988PubMedGoogle Scholar
  39. 39.
    Cesana GC, DeRaffele G, Cohen S, Moroziewicz D, Mitcham J, Stoutenburg J, Cheung K, Hesdorffer C, Kim-Schulze S, Kaufman HL (2006) Characterization of CD4+ CD25+ regulatory T cells in patients treated with high-dose interleukin-2 for metastatic melanoma or renal cell carcinoma. J Clin Oncol 24(7):1169–1177CrossRefPubMedGoogle Scholar
  40. 40.
    Kato Y, Yoshimura K, Shin T, Verheul H, Hammers H, Sanni TB, Salumbides BC, Van EK, Schulick R, Pili R (2007) Synergistic in vivo antitumor effect of the histone deacetylase inhibitor MS-275 in combination with interleukin 2 in a murine model of renal cell carcinoma. Clin Cancer Res 13(15 Pt 1):4538–4546CrossRefPubMedGoogle Scholar
  41. 41.
    Elpek KG, Lacelle C, Singh NP, Yolcu ES, Shirwan H (2007) CD4+ CD25+ T regulatory cells dominate multiple immune evasion mechanisms in early but not late phases of tumor development in a B cell lymphoma model. J Immunol 178(11):6840–6848PubMedGoogle Scholar
  42. 42.
    Yang ZZ, Novak AJ, Stenson MJ, Witzig TE, Ansell SM (2006) Intratumoral CD4+ CD25+ regulatory T-cell-mediated suppression of infiltrating CD4+ T cells in B-cell non-Hodgkin lymphoma. Blood 107(9):3639–3646CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Sofía Grille
    • 1
    • 2
  • Andreína Brugnini
    • 1
    • 2
  • Martha Nese
    • 3
  • Esteban Corley
    • 4
  • Frank W. Falkenberg
    • 5
  • Daniela Lens
    • 1
  • José A. Chabalgoity
    • 2
  1. 1.Departamento Básico de Medicina, Facultad de Medicina, Hospital de ClínicasUniversidad de la RepúblicaMontevideoUruguay
  2. 2.Laboratory for Vaccine Research, Department of Biotechnology, Facultad de Medicina, Instituto de HigieneUniversidad de la RepúblicaMontevideoUruguay
  3. 3.Cátedra de Hematología, Facultad de MedicinaHospital de ClínicasMontevideoUruguay
  4. 4.PCgenBuenos AiresArgentina
  5. 5.Cires Cell and Immune Research Services GmbHDortmundGermany

Personalised recommendations