Cancer Immunology, Immunotherapy

, Volume 58, Issue 10, pp 1565–1576

Incubation of antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin in IL-7 + IL-15 increases yield of cells capable of inducing regression of melanoma metastases compared to culture in IL-2

  • Hanh K. Le
  • Laura Graham
  • Catriona H. T. Miller
  • Maciej Kmieciak
  • Masoud H. Manjili
  • Harry Douglas Bear
Original Article


Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). We hypothesized that B/I-activated T cells cultured in IL-7 + IL-15 might proliferate and survive in culture better than cells cultured in IL-2, and that these cells would have equal or greater anti-tumor activity in vivo. Tumor antigen-sensitized DLN lymphocytes from either wild-type or T cell receptor transgenic mice were harvested, activated with B/I, and expanded in culture with either IL-2, IL-7 + IL-15 or a regimen of alternating cytokines. Cell yields, proliferation, apoptosis, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for anti-tumor activity against melanoma lung metastases established by prior i.v. injection of B16 melanoma cells. IL-7 + IL-15 or alternating cytokines resulted in much faster and prolonged proliferation and much less apopotosis of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately tenfold greater yields of viable cells. Culture in IL-7 + IL-15 yielded higher proportions of CD8+ T cells and a higher proportion of cells with a central memory phenotype. Despite this, T cells grown in IL-7 + IL-15 had higher IFN-γ release responses to tumor antigen than cells grown in IL-2. Adoptive transfer of B/I-activated T cells grown in IL-7 + IL-15 or the alternating regimen had equal or greater efficacy on a “per-cell” basis against melanoma metastases. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in adoptive immunotherapy of cancer.


Adoptive immunotherapy Melanoma IL-7 IL-15 IL-2 T lymphocytes 


  1. 1.
    Alexander JP, Kudoh S, Melsop KA, Hamilton TA, Edinger MG, Tubbs RR, Sica D, Tuason L, Klein E, Bukowski RM, Finke JH (1993) T-cells infiltrating renal cell carcinoma display a poor proliferative response even though they can produce interleukin 2 and express interleukin 2 receptors. Cancer Res 53:1380–1387PubMedGoogle Scholar
  2. 2.
    Bathe OF, yot-Herman N, Malek TR (2001) IL-2 during in vitro priming promotes subsequent engraftment and successful adoptive tumor immunotherapy by persistent memory phenotypic CD8(+) T cells. J Immunol 167:4511–4517PubMedGoogle Scholar
  3. 3.
    Boyman O, Purton JF, Surh CD, Sprent J (2007) Cytokines and T-cell homeostasis. Curr Opin Immunol 19:320–326PubMedCrossRefGoogle Scholar
  4. 4.
    Cantrell D (1996) T cell antigen receptor signal transduction pathways. Annu Rev Immunol 14:259–274PubMedCrossRefGoogle Scholar
  5. 5.
    Carrio R, Bathe OF, Malek TR (2004) Initial antigen encounter programs CD8+ T cells competent to develop into memory cells that are activated in an antigen-free, IL-7- and IL-15-rich environment. J Immunol 172:7315–7323PubMedGoogle Scholar
  6. 6.
    Chang AE, Li Q, Jiang G, Sayre DM, Braun TM, Redman BG (2003) Phase II trial of autologous tumor vaccination, anti-CD3-activated vaccine-primed lymphocytes, and interleukin-2 in stage IV renal cell cancer. J Clin Oncol 21:884–890PubMedCrossRefGoogle Scholar
  7. 7.
    Chatila T, Silverman L, Miller R, Geha R (1989) Mechanisms of T cell activation by the calcium ionophore ionomycin. J Immunol 143:1283–1289PubMedGoogle Scholar
  8. 8.
    Chin CS, Miller CH, Graham L, Parviz M, Zacur S, Patel B, Duong A, Bear HD (2004) Bryostatin 1/ionomycin (B/I) ex vivo stimulation preferentially activates L-selectinlow tumor-sensitized lymphocytes. Int Immunol 16:1283–1294PubMedCrossRefGoogle Scholar
  9. 9.
    Cohen PA, Peng LM, Kjaergaard J, Plautz GE, Finke JH, Koski GK, Czerniecki BJ, Shu SY (2001) T-cell adoptive therapy of tumors: mechanisms of improved therapeutic performance. Crit Rev Immunol 21:215–248PubMedGoogle Scholar
  10. 10.
    Crossland KD, Lee VK, Chen W, Riddell SR, Greenberg PD, Cheever MA (1991) T cells from tumor-immune mice nonspecifically expanded in vitro with anti-CD3 plus IL-2 retain specific function in vitro and can eradicate disseminated leukemia in vivo. J Immunol 146:4414–4420PubMedGoogle Scholar
  11. 11.
    Dudley ME, Rosenberg SA (2003) Adoptive-cell-transfer therapy for the treatment of patients with cancer. Nat Rev Cancer 3:666–675PubMedCrossRefGoogle Scholar
  12. 12.
    Dudley ME, Wunderlich JR, Robbins PF, Yang JC, Hwu P, Schwartzentruber DJ, Topalian SL, Sherry R, Restifo NP, Hubicki AM, Robinson MR, Raffeld M, Duray P, Seipp CA, Rogers-Freezer L, Morton KE, Mavroukakis SA, White DE, Rosenberg SA (2002) Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Sci 298:850–854CrossRefGoogle Scholar
  13. 13.
    Fong TA, Mosman TR (1989) The role of IFN-gamma in delayed-type hypersensitivity mediated by Th1 clones. J Immunol 143:2887–2893PubMedGoogle Scholar
  14. 14.
    Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY (2005) A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol 6:1142–1151PubMedCrossRefGoogle Scholar
  15. 15.
    Fontenot JD, Rudensky AY (2005) A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nat Immunol 6:331–337PubMedCrossRefGoogle Scholar
  16. 16.
    Gattinoni L, Klebanoff CA, Palmer DC, Wrzesinski C, Kerstann K, Yu Z, Finkelstein SE, Theoret MR, Rosenberg SA, Restifo NP (2005) Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8(+) T cells. J Clin Invest 115:1616–1626PubMedCrossRefGoogle Scholar
  17. 17.
    Gattinoni L, Powell DJ Jr, Rosenberg SA, Restifo NP (2006) Adoptive immunotherapy for cancer: building on success. Nat Rev Immunol 6:383–393PubMedCrossRefGoogle Scholar
  18. 18.
    Gett AV, Sallusto F, Lanzavecchia A, Geginat J (2003) T cell fitness determined by signal strength. Nat Immunol 4:355–360PubMedCrossRefGoogle Scholar
  19. 19.
    Halaas O, Vik R, Espevik T (1998) Induction of Fas ligand in murine bone marrow NK cells by bacterial polysaccharides. J Immunol 160:4330–4336PubMedGoogle Scholar
  20. 20.
    Harada M, Okamoto T, Omoto K, Tamada K, Takenoyama M, Hirashima C, Ito O, Kimura G, Nomoto K (1996) Specific immunotherapy with tumour-draining lymph node cells cultured with both anti-CD3 and anti-CD28 monoclonal antibodies. Immunol 87:447–453CrossRefGoogle Scholar
  21. 21.
    Haux J, Johnsen AC, Steinkjer B, Egeberg K, Sundan A, Espevik T (1999) The role of interleukin-2 in regulating the sensitivity of natural killer cells for Fas-mediated apoptosis. Cancer Immunol Immunother 48:139–146PubMedCrossRefGoogle Scholar
  22. 22.
    Hunder NN, Wallen H, Cao J, Hendricks DW, Reilly JZ, Rodmyre R, Jungbluth A, Gnjatic S, Thompson JA, Yee C (2008) Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1. N Engl J Med 358:2698–2703PubMedCrossRefGoogle Scholar
  23. 23.
    Joshi NS, Kaech SM (2008) Effector CD8 T cell development: a balancing act between memory cell potential and terminal differentiation. J Immunol 180:1309–1315PubMedGoogle Scholar
  24. 24.
    Kazanietz MG, Lewin NE, Gao F, Pettit GR, Blumberg PM (1994) Binding of [26-3H] bryostatin 1 and analogs to calcium-dependent and calcium-independent protein kinase C isozymes. Mol Pharmacol 46:374–379PubMedGoogle Scholar
  25. 25.
    Keller AM, Borst J (2006) Control of peripheral T cell survival: a delicate division of labor between cytokines and costimulatory molecules. Hum Immunol 67:469–477PubMedCrossRefGoogle Scholar
  26. 26.
    Klebanoff CA, Finkelstein SE, Surman DR, Lichtman MK, Gattinoni L, Theoret MR, Grewal N, Spiess PJ, Antony PA, Palmer DC, Tagaya Y, Rosenberg SA, Waldmann TA, Restifo NP (2004) IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8+ T cells. Proc Natl Acad Sci U S A 101:1969–1974PubMedCrossRefGoogle Scholar
  27. 27.
    Klebanoff CA, Gattinoni L, Torabi-Parizi P, Kerstann K, Cardones AR, Finkelstein SE, Palmer DC, Antony PA, Hwang ST, Rosenberg SA, Waldmann TA, Restifo NP (2005) Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells. Proc Natl Acad Sci USA 102:9571–9576PubMedCrossRefGoogle Scholar
  28. 28.
    Lanzavecchia A, Sallusto F (2005) Understanding the generation and function of memory T cell subsets. Curr Opin Immunol 17:326–332PubMedCrossRefGoogle Scholar
  29. 29.
    Lian RH, Maeda M, Lohwasser S, Delcommenne M, Nakano T, Vance RE, Raulet DH, Takei F (2002) Orderly and nonstochastic acquisition of CD94/NKG2 receptors by developing NK cells derived from embryonic stem cells in vitro. J Immunol 168:4980–4987PubMedGoogle Scholar
  30. 30.
    Lipshy KA, Kostuchenko PJ, Hamad GG, Bland CE, Barrett SK, Bear HD (1997) Sensitizing T-lymphocytes for adoptive immunotherapy by vaccination with wild-type or cytokine gene-transduced melanoma. Ann Surg Oncol 4:334–341PubMedCrossRefGoogle Scholar
  31. 31.
    Liu S, Riley J, Rosenberg S, Parkhurst M (2006) Comparison of common gamma-chain cytokines, interleukin-2, interleukin-7, and interleukin-15 for the in vitro generation of human tumor-reactive T lymphocytes for adoptive cell transfer therapy. J Immunother 29:284–293PubMedCrossRefGoogle Scholar
  32. 32.
    Mackensen A, Meidenbauer N, Vogl S, Laumer M, Berger J, Andreesen R (2006) Phase I study of adoptive T-cell therapy using antigen-specific CD8+ T cells for the treatment of patients with metastatic melanoma. J Clin Oncol 24:5060–5069PubMedCrossRefGoogle Scholar
  33. 33.
    Melchionda F, Fry TJ, Milliron MJ, McKirdy MA, Tagaya Y, Mackall CL (2005) Adjuvant IL-7 or IL-15 overcomes immunodominance and improves survival of the CD8+ memory cell pool. J Clin Invest 115:1177–1187PubMedGoogle Scholar
  34. 34.
    Mitchell MS, Darrah D, Yeung D, Halpern S, Wallace A, Voland J, Jones V, Kan-Mitchell J (2002) Phase I trial of adoptive immunotherapy with cytolytic T lymphocytes immunized against a tyrosinase epitope. J Clin Oncol 20:1075–1086PubMedCrossRefGoogle Scholar
  35. 35.
    Morse MA, Clay TM, Lyerly HK (2002) Current status of adoptive immunotherapy of malignancies. Expert Opin Biol Ther 2:237–247PubMedCrossRefGoogle Scholar
  36. 36.
    Nijhuis EWP, Wiel-van Kemenade EVD, Figdor CG, Van Lier RAW (1990) Activation and expansion of tumour-infiltrating lymphocytes by anti-CD3 and anti-CD28 monoclonal antibodies. Cancer Immunol Immunother 32:245–250PubMedCrossRefGoogle Scholar
  37. 37.
    Oh S, Berzofsky JA, Burke DS, Waldmann TA, Perera LP (2003) Coadministration of HIV vaccine vectors with vaccinia viruses expressing IL-15 but not IL-2 induces long-lasting cellular immunity. Proc Natl Acad Sci USA 100:3392–3397PubMedCrossRefGoogle Scholar
  38. 38.
    Pettit GR, Herald SL, Doubek DL, Arnold E, Clardy J (1982) Isolation and structure of bryostatin 1. J Am Chem Soc 104:6846–6848CrossRefGoogle Scholar
  39. 39.
    Plautz GE, Cohen PA, Shu S (2003) Considerations on clinical use of T cell immunotherapy for cancer. Arch Immunol Ther Exp (Warsz) 51:245–257Google Scholar
  40. 40.
    Refaeli Y, Van Parijs L, London CA, Tschopp J, Abbas AK (1998) Biochemical mechanisms of IL-2-regulated Fas-mediated T cell apoptosis. Immunity 8:615–623PubMedCrossRefGoogle Scholar
  41. 41.
    Rolle CE, Carrio R, Malek TR (2008) Modeling the CD8+ T effector to memory transition in adoptive T-cell antitumor immunotherapy. Cancer Res 68:2984–2992PubMedCrossRefGoogle Scholar
  42. 42.
    Roychowdhury S, May KF Jr, Tzou KS, Lin T, Bhatt D, Freud AG, Guimond M, Ferketich AK, Liu Y, Caligiuri MA (2004) Failed adoptive immunotherapy with tumor-specific T cells: reversal with low-dose interleukin 15 but not low-dose interleukin 2. Cancer Res 64:8062–8067PubMedCrossRefGoogle Scholar
  43. 43.
    Sprent J, Cho JH, Boyman O, Surh CD (2008) T cell homeostasis. Immunol Cell Biol 86:312–319PubMedCrossRefGoogle Scholar
  44. 44.
    Tuttle TM, Bethke KP, Inge TH, McCrady CW, Pettit GR, Bear HD (1992) Bryostatin 1-activated T cells can traffic and mediate tumor regression. J Surg Res 52:543–548PubMedCrossRefGoogle Scholar
  45. 45.
    Tuttle TM, Fleming MF, Hogg PS, Inge TH, Bear HD (1994) Low-dose cyclophosphamide overcomes metastasis-induced immunosuppression. Ann Surg Oncol 1:53–58PubMedCrossRefGoogle Scholar
  46. 46.
    Tuttle TM, McCrady CW, Inge TH, Salour M, Bear HD (1993) γ-Interferon plays a key role in T-cell-induced tumor regression. Cancer Res 53:833–839PubMedGoogle Scholar
  47. 47.
    Van PL, Refaeli Y, Lord JD, Nelson BH, Abbas AK, Baltimore D (1999) Uncoupling IL-2 signals that regulate T cell proliferation, survival, and Fas-mediated activation-induced cell death. Immunity 11:281–288CrossRefGoogle Scholar
  48. 48.
    Waldmann TA (2006) The biology of interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design. Nat Rev Immunol 6:595–601PubMedCrossRefGoogle Scholar
  49. 49.
    Waldmann TA, Dubois S, Tagaya Y (2001) Contrasting roles of IL-2 and IL-15 in the life and death of lymphocytes: implications for immunotherapy. Immunity 14:105–110PubMedGoogle Scholar
  50. 50.
    Yee C (2003) Adoptive T cell therapy—immune monitoring and MHC multimers. Clin Immunol 106:5–9PubMedCrossRefGoogle Scholar
  51. 51.
    Yee C, Thompson JA, Byrd D, Riddell SR, Roche P, Celis E, Greenberg PD (2002) Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci USA 99:16168–16173PubMedCrossRefGoogle Scholar
  52. 52.
    Yoshizawa H, Chang AE, Shu S (1991) Specific adoptive immunotherapy mediated by tumor-draining lymph node cells sequentially activated with anti-CD3 and IL-2. J Immunol 147:729–737PubMedGoogle Scholar
  53. 53.
    Zheng SG, Wang J, Wang P, Gray JD, Horwitz DA (2007) IL-2 is essential for TGF-beta to convert naive CD4+CD25− cells to CD25 + Foxp3 + regulatory T cells and for expansion of these cells. J Immunol 178:2018–2027PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Hanh K. Le
    • 1
  • Laura Graham
    • 2
  • Catriona H. T. Miller
    • 3
  • Maciej Kmieciak
    • 3
  • Masoud H. Manjili
    • 3
  • Harry Douglas Bear
    • 2
  1. 1.Department of Physiology and BiophysicsVirginia Commonwealth University’s Medical College of VirginiaRichmondUSA
  2. 2.Division of Surgical Oncology, Department of Surgery and the Massey Cancer CenterVirginia Commonwealth University’s Medical College of VirginiaRichmondUSA
  3. 3.Department of Microbiology and ImmunologyVirginia Commonwealth University’s Medical College of Virginia and the Massey Cancer CenterRichmondUSA

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