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Blockade of CTLA-4 promotes the development of effector CD8+ T lymphocytes and the therapeutic effect of vaccination with an attenuated protozoan expressing NY-ESO-1

Abstract

The development of cancer immunotherapy has long been a challenge. Here, we report that prophylactic vaccination with a highly attenuated Trypanosoma cruzi strain expressing NY-ESO-1 (CL-14-NY-ESO-1) induces both effector memory and effector CD8+ T lymphocytes that efficiently prevent tumor development. However, the therapeutic effect of such a vaccine is limited. We also demonstrate that blockade of Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) during vaccination enhances the frequency of NY-ESO-1-specific effector CD8+ T cells producing IFN-γ and promotes lymphocyte migration to the tumor infiltrate. As a result, therapy with CL-14-NY-ESO-1 together with anti-CTLA-4 is highly effective in controlling the development of an established melanoma.

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Abbreviations

ACK:

Ammonium Chloride Potassium

CD8+TCM :

Central Memory CD8+ T cells

CD8+TE :

Effector CD8+ T cells

CD8+TEM :

Effector Memory CD8+ T cells

CTA:

Cancer Testis Antigens

CTLA-4:

Cytotoxic T Lymphocyte Antigen 4

FBS:

Fetal Bovine Serum

gzmB:

Granzyme B

HBSS:

Hank’s Balanced Salt Solution

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

i.p.:

Intraperitoneal injection

IFN:

Interferon

IL:

Interleukin

MHC:

Major Histocompatibility Complex

PBS:

Phosphate-buffered saline

TI:

Tumor Infiltrate

Tregs:

Regulatory T cells

References

  1. Siegel R, Ma J, Zou Z, Jemal A (2014) Cancer statistics, 2014. CA Cancer J Clin 64(1):9–29. doi:10.3322/caac.21208

    Article  PubMed  Google Scholar 

  2. Rosenberg SA, Yang JC, Restifo NP (2004) Cancer immunotherapy: moving beyond current vaccines. Nat Med 10(9):909–915. doi:10.1038/nm1100

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Chen J, Zhang L, Wen W, Hao J, Zeng P, Qian X, Zhang Y, Yin Y (2012) Induction of HCA587-specific antitumor immunity with HCA587 protein formulated with CpG and ISCOM in mice. PLoS One 7(10):e47219. doi:10.1371/journal.pone.0047219

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Bolhassani A, Safaiyan S, Rafati S (2011) Improvement of different vaccine delivery systems for cancer therapy. Mol Cancer 10:3. doi:10.1186/1476-4598-10-3

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Junqueira C, Guerrero AT, Galvao-Filho B, Andrade WA, Salgado AP, Cunha TM, Ropert C, Campos MA, Penido ML, Mendonca-Previato L, Previato JO, Ritter G, Cunha FQ, Gazzinelli RT (2012) Trypanosoma cruzi adjuvants potentiate T cell-mediated immunity induced by a NY-ESO-1 based antitumor vaccine. PLoS One 7(5):e36245. doi:10.1371/journal.pone.0036245

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Chen YT, Scanlan MJ, Sahin U, Tureci O, Gure AO, Tsang S, Williamson B, Stockert E, Pfreundschuh M, Old LJ (1997) A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening. Proc Natl Acad Sci USA 94(5):1914–1918

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Chen YT, Ross DS, Chiu R, Zhou XK, Chen YY, Lee P, Hoda SA, Simpson AJ, Old LJ, Caballero O, Neville AM (2011) Multiple cancer/testis antigens are preferentially expressed in hormone-receptor negative and high-grade breast cancers. PLoS One 6(3):e17876. doi:10.1371/journal.pone.0017876

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Jungbluth AA, Chen YT, Stockert E, Busam KJ, Kolb D, Iversen K, Coplan K, Williamson B, Altorki N, Old LJ (2001) Immunohistochemical analysis of NY-ESO-1 antigen expression in normal and malignant human tissues. Int J Cancer 92(6):856–860. doi:10.1002/ijc.1282

    Article  CAS  PubMed  Google Scholar 

  9. Nicholaou T, Chen W, Davis ID, Jackson HM, Dimopoulos N, Barrow C, Browning J, Macgregor D, Williams D, Hopkins W, Maraskovsky E, Venhaus R, Pan L, Hoffman EW, Old LJ, Cebon J (2011) Immunoediting and persistence of antigen-specific immunity in patients who have previously been vaccinated with NY-ESO-1 protein formulated in ISCOMATRIX. Cancer Immunol Immunother 60(11):1625–1637. doi:10.1007/s00262-011-1041-3

    Article  CAS  PubMed  Google Scholar 

  10. Odunsi K, Matsuzaki J, Karbach J, Neumann A, Mhawech-Fauceglia P, Miller A, Beck A, Morrison CD, Ritter G, Godoy H, Lele S, duPont N, Edwards R, Shrikant P, Old LJ, Gnjatic S, Jager E (2012) Efficacy of vaccination with recombinant vaccinia and fowlpox vectors expressing NY-ESO-1 antigen in ovarian cancer and melanoma patients. Proc Natl Acad Sci USA 109(15):5797–5802. doi:10.1073/pnas.1117208109

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Junqueira C, Santos LI, Galvao-Filho B, Teixeira SM, Rodrigues FG, DaRocha WD, Chiari E, Jungbluth AA, Ritter G, Gnjatic S, Old LJ, Gazzinelli RT (2011) Trypanosoma cruzi as an effective cancer antigen delivery vector. Proc Natl Acad Sci USA 108(49):19695–19700. doi:10.1073/pnas.1110030108

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Martin DL, Weatherly DB, Laucella SA, Cabinian MA, Crim MT, Sullivan S, Heiges M, Craven SH, Rosenberg CS, Collins MH, Sette A, Postan M, Tarleton RL (2006) CD8+ T-Cell responses to Trypanosoma cruzi are highly focused on strain-variant trans-sialidase epitopes. PLoS Pathog 2(8):e77. doi:10.1371/journal.ppat.0020077

    Article  PubMed Central  PubMed  Google Scholar 

  13. Padilla AM, Bustamante JM, Tarleton RL (2009) CD8+ T cells in Trypanosoma cruzi infection. Curr Opin Immunol 21(4):385–390. doi:10.1016/j.coi.2009.07.006

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Kwek SS, Dao V, Roy R, Hou Y, Alajajian D, Simko JP, Small EJ, Fong L (2012) Diversity of antigen-specific responses induced in vivo with CTLA-4 blockade in prostate cancer patients. J Immunol 189(7):3759–3766. doi:10.4049/jimmunol.1201529

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Maraskovsky E, Sjolander S, Drane DP, Schnurr M, Le TT, Mateo L, Luft T, Masterman KA, Tai TY, Chen Q, Green S, Sjolander A, Pearse MJ, Lemonnier FA, Chen W, Cebon J, Suhrbier A (2004) NY-ESO-1 protein formulated in ISCOMATRIX adjuvant is a potent anticancer vaccine inducing both humoral and CD8+ T-cell-mediated immunity and protection against NY-ESO-1 + tumors. Clin Cancer Res 10(8):2879–2890

    Article  CAS  PubMed  Google Scholar 

  16. DaRocha WD, Silva RA, Bartholomeu DC, Pires SF, Freitas JM, Macedo AM, Vazquez MP, Levin MJ, Teixeira SM (2004) Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols. Parasitol Res 92(2):113–120. doi:10.1007/s00436-003-1004-5

    Article  PubMed  Google Scholar 

  17. Streeck H, Frahm N, Walker BD (2009) The role of IFN-gamma Elispot assay in HIV vaccine research. Nat Protoc 4(4):461–469. doi:10.1038/nprot.2009.7

    Article  CAS  PubMed  Google Scholar 

  18. Mitsui J, Nishikawa H, Muraoka D, Wang L, Noguchi T, Sato E, Kondo S, Allison JP, Sakaguchi S, Old LJ, Kato T, Shiku H (2010) Two distinct mechanisms of augmented antitumor activity by modulation of immunostimulatory/inhibitory signals. Clin Cancer Res 16(10):2781–2791. doi:10.1158/1078-0432.CCR-09-3243

    Article  CAS  PubMed  Google Scholar 

  19. Atayde VD, Neira I, Cortez M, Ferreira D, Freymuller E, Yoshida N (2004) Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14. Int J Parasitol 34(7):851–860. doi:10.1016/j.ijpara.2004.03.003

    Article  CAS  PubMed  Google Scholar 

  20. Bixby LM, Tarleton RL (2008) Stable CD8+ T cell memory during persistent Trypanosoma cruzi infection. J Immunol 181(4):2644–2650. doi:10.4049/jimmunol.181.4.2644

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Huster KM, Busch V, Schiemann M, Linkemann K, Kerksiek KM, Wagner H, Busch DH (2004) Selective expression of IL-7 receptor on memory T cells identifies early CD40L-dependent generation of distinct CD8+ memory T cell subsets. Proc Natl Acad Sci USA 101(15):5610–5615. doi:10.1073/pnas.0308054101

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Barber DL, Wherry EJ, Ahmed R (2003) Cutting edge: rapid in vivo killing by memory CD8 T cells. J Immunol 171(1):27–31

    Article  CAS  PubMed  Google Scholar 

  23. Bannard O, Kraman M, Fearon DT (2009) Secondary replicative function of CD8+ T cells that had developed an effector phenotype. Science 323(5913):505–509. doi:10.1126/science.1166831

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Curran MA, Kim M, Montalvo W, Al-Shamkhani A, Allison JP (2011) Combination CTLA-4 blockade and 4-1BB activation enhances tumor rejection by increasing T-cell infiltration, proliferation, and cytokine production. PLoS One 6(4):e19499. doi:10.1371/journal.pone.0019499

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Curran MA, Montalvo W, Yagita H, Allison JP (2010) PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc Natl Acad Sci USA 107(9):4275–4280. doi:10.1073/pnas.0915174107

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F, Roddie C, Henry JY, Yagita H, Wolchok JD, Peggs KS, Ravetch JV, Allison JP, Quezada SA (2013) Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J Exp Med 210(9):1695–1710. doi:10.1084/jem.20130579

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Nasman A, Romanitan M, Nordfors C, Grun N, Johansson H, Hammarstedt L, Marklund L, Munck-Wikland E, Dalianis T, Ramqvist T (2012) Tumor infiltrating CD8+ and Foxp3+ lymphocytes correlate to clinical outcome and human papillomavirus (HPV) status in tonsillar cancer. PLoS One 7(6):e38711. doi:10.1371/journal.pone.0038711

    Article  PubMed Central  PubMed  Google Scholar 

  28. Mahmoud SM, Paish EC, Powe DG, Macmillan RD, Grainge MJ, Lee AH, Ellis IO, Green AR (2011) Tumor-infiltrating CD8+ lymphocytes predict clinical outcome in breast cancer. J Clin Oncol 29(15):1949–1955. doi:10.1200/JCO.2010.30.5037

    Article  PubMed  Google Scholar 

  29. Selby MJ, Engelhardt JJ, Quigley M, Henning KA, Chen T, Srinivasan M, Korman AJ (2013) Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells. Cancer Immunol Res 1(1):32–42. doi:10.1158/2326-6066.CIR-13-0013

    Article  CAS  PubMed  Google Scholar 

  30. Pan ZK, Ikonomidis G, Lazenby A, Pardoll D, Paterson Y (1995) A recombinant Listeria monocytogenes vaccine expressing a model tumour antigen protects mice against lethal tumour cell challenge and causes regression of established tumours. Nat Med 1(5):471–477

    Article  CAS  PubMed  Google Scholar 

  31. Moreno M, Kramer MG, Yim L, Chabalgoity JA (2010) Salmonella as live trojan horse for vaccine development and cancer gene therapy. Curr Gene Ther 10(1):56–76

    Article  CAS  PubMed  Google Scholar 

  32. Roskin G (1946) Toxin therapy of experimental cancer; the influence of protozoan infections upon transplanted cancer. Cancer Res 6:363–365

    CAS  PubMed  Google Scholar 

  33. Klyueva NG, Roskin G (1946) Cancerolytic substance of Schizotrypanum cruzi. Am Rev Sov Med 4(2):127–129

    CAS  PubMed  Google Scholar 

  34. Hauschka TS, Goodwin MB (1948) Trypanosoma cruzi endotoxin (KR) in the treatment of malignant mouse tumors. Science 107(2788):600–602. doi:10.1126/science.107.2788.600

    Article  CAS  PubMed  Google Scholar 

  35. Lima MT, Lenzi HL, Gattass CR (1995) Negative tissue parasitism in mice injected with a noninfective clone of Trypanosoma cruzi. Parasitol Res 81(1):6–12

    Article  CAS  PubMed  Google Scholar 

  36. Smyth MJ, Godfrey DI, Trapani JA (2001) A fresh look at tumor immunosurveillance and immunotherapy. Nat Immunol 2(4):293–299. doi:10.1038/86297

    Article  CAS  PubMed  Google Scholar 

  37. Schuler-Thurner B, Schultz ES, Berger TG, Weinlich G, Ebner S, Woerl P, Bender A, Feuerstein B, Fritsch PO, Romani N, Schuler G (2002) Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells. J Exp Med 195(10):1279–1288

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Bartholomeu DC, Ropert C, Melo MB, Parroche P, Junqueira CF, Teixeira SM, Sirois C, Kasperkovitz P, Knetter CF, Lien E, Latz E, Golenbock DT, Gazzinelli RT (2008) Recruitment and endo-lysosomal activation of TLR9 in dendritic cells infected with Trypanosoma cruzi. J Immunol 181(2):1333–1344

    Article  CAS  PubMed  Google Scholar 

  39. Caetano BC, Carmo BB, Melo MB, Cerny A, dos Santos SL, Bartholomeu DC, Golenbock DT, Gazzinelli RT (2011) Requirement of UNC93B1 reveals a critical role for TLR7 in host resistance to primary infection with Trypanosoma cruzi. J Immunol 187(4):1903–1911. doi:10.4049/jimmunol.1003911

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Kaech SM, Ahmed R (2001) Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nat Immunol 2(5):415–422. doi:10.1038/87720

    PubMed Central  CAS  PubMed  Google Scholar 

  41. Wherry EJ, Teichgraber V, Becker TC, Masopust D, Kaech SM, Antia R, von Andrian UH, Ahmed R (2003) Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat Immunol 4(3):225–234. doi:10.1038/ni889

    Article  CAS  PubMed  Google Scholar 

  42. Stemberger C, Huster KM, Koffler M, Anderl F, Schiemann M, Wagner H, Busch DH (2007) A single naive CD8+ T cell precursor can develop into diverse effector and memory subsets. Immunity 27(6):985–997. doi:10.1016/j.immuni.2007.10.012

    Article  CAS  PubMed  Google Scholar 

  43. Corse E, Allison JP (2012) Cutting edge: CTLA-4 on effector T cells inhibits in trans. J Immunol 189(3):1123–1127. doi:10.4049/jimmunol.1200695

    Article  CAS  PubMed  Google Scholar 

  44. Hodi FS, O’Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, Gonzalez R, Robert C, Schadendorf D, Hassel JC, Akerley W, van den Eertwegh AJ, Lutzky J, Lorigan P, Vaubel JM, Linette GP, Hogg D, Ottensmeier CH, Lebbe C, Peschel C, Quirt I, Clark JI, Wolchok JD, Weber JS, Tian J, Yellin MJ, Nichol GM, Hoos A, Urba WJ (2010) Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363(8):711–723. doi:10.1056/NEJMoa1003466

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Dillard T, Yedinak CG, Alumkal J, Fleseriu M (2010) Anti-CTLA-4 antibody therapy associated autoimmune hypophysitis: serious immune related adverse events across a spectrum of cancer subtypes. Pituitary 13(1):29–38. doi:10.1007/s11102-009-0193-z

    Article  CAS  PubMed  Google Scholar 

  46. Heger M (2012) Cancer immunotherapy shows promise in multiple tumor types. Nat Med 18(7):993. doi:10.1038/nm0712-993

    Article  CAS  PubMed  Google Scholar 

  47. Voskens CJ, Goldinger SM, Loquai C, Robert C, Kaehler KC, Berking C, Bergmann T, Bockmeyer CL, Eigentler T, Fluck M, Garbe C, Gutzmer R, Grabbe S, Hauschild A, Hein R, Hundorfean G, Justich A, Keller U, Klein C, Mateus C, Mohr P, Paetzold S, Satzger I, Schadendorf D, Schlaeppi M, Schuler G, Schuler-Thurner B, Trefzer U, Ulrich J, Vaubel J, von Moos R, Weder P, Wilhelm T, Goppner D, Dummer R, Heinzerling LM (2013) The price of tumor control: an analysis of rare side effects of anti-CTLA-4 therapy in metastatic melanoma from the ipilimumab network. PLoS One 8(1):e53745. doi:10.1371/journal.pone.0053745

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. Hurwitz AA, Yu TF, Leach DR, Allison JP (1998) CTLA-4 blockade synergizes with tumor-derived granulocyte-macrophage colony-stimulating factor for treatment of an experimental mammary carcinoma. Proc Natl Acad Sci USA 95(17):10067–10071

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  49. Quezada SA, Peggs KS, Curran MA, Allison JP (2006) CTLA4 blockade and GM-CSF combination immunotherapy alters the intratumor balance of effector and regulatory T cells. J Clin Invest 116(7):1935–1945. doi:10.1172/JCI27745

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  50. Martins GA, Tadokoro CE, Silva RB, Silva JS, Rizzo LV (2004) CTLA-4 blockage increases resistance to infection with the intracellular protozoan Trypanosoma cruzi. J Immunol 172(8):4893–4901

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank the Ludwig Institute of Cancer Research (LICR) Tetramer Facility for tetramer synthesis; the LICR–Cornell University for the recombinant NY-ESO-1 protein; Dr. James Allison from the Memorial Sloan-Kettering Cancer Center for providing the anti-CTLA-4 monoclonal antibody (9D9 hybridoma); Dr. Jonathan Cebon from LICRMelbourne for providing the B16-NY-ESO-1 cell lines; Dr. Luiz Travassos from São Paulo University and Dr. Kevin Maloy from University of Oxford for incentive, scientific discussions, and suggestions during the development of this work. Grant support: Atlantic Philanthropies/Program of Clinical Discoveries from the LICR, Fundação de Amparo a Pesquisa de Minas Gerais, Fundação Oswaldo Cruz, and the National Institute of Science and Technology for Vaccines/Conselho Nacional de Desenvolvimento Científico e Tecnológico.

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The authors declare no conflict of interest.

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Correspondence to Ricardo Tostes Gazzinelli.

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dos Santos, L.I., Galvão-Filho, B., de Faria, P.C. et al. Blockade of CTLA-4 promotes the development of effector CD8+ T lymphocytes and the therapeutic effect of vaccination with an attenuated protozoan expressing NY-ESO-1. Cancer Immunol Immunother 64, 311–323 (2015). https://doi.org/10.1007/s00262-014-1634-8

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Keywords

  • Cancer
  • Immunotherapy
  • NY-ESO-1
  • Anti-CTLA-4
  • Trypanosoma cruzi