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Promoting the accumulation of tumor-specific T cells in tumor tissues by dendritic cell vaccines and chemokine-modulating agents

Nature Protocols volume 13, pages 335–357 (2018)Cite this article

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Abstract

This protocol describes how to induce large numbers of tumor-specific cytotoxic T cells (CTLs) in the spleens and lymph nodes of mice receiving dendritic cell (DC) vaccines and how to modulate tumor microenvironments (TMEs) to ensure effective homing of the vaccination-induced CTLs to tumor tissues. We also describe how to evaluate the numbers of tumor-specific CTLs within tumors. The protocol contains detailed information describing how to generate a specialized DC vaccine with augmented ability to induce tumor-specific CTLs. We also describe methods to modulate the production of chemokines in the TME and show how to quantify tumor-specific CTLs in the lymphoid organs and tumor tissues of mice receiving different treatments. The combined experimental procedure, including tumor implantation, DC vaccine generation, chemokine-modulating (CKM) approaches, and the analyses of tumor-specific systemic and intratumoral immunity is performed over 30–40 d. The presented ELISpot-based ex vivo CTL assay takes 6 h to set up and 5 h to develop. In contrast to other methods of evaluating tumor-specific immunity in tumor tissues, our approach allows detection of intratumoral T-cell responses to nonmanipulated weakly immunogenic cancers. This detection method can be performed using basic laboratory skills, and facilitates the development and preclinical evaluation of new immunotherapies.

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Figure 1: Overview of the protocol, with associated timing.
Figure 2: Key features of type-1 polarized DCs and their functions.
Figure 3: Combinatorial strategy to increase the numbers of tumor-specific TILs.
Figure 4: Monitoring of the immunotherapy-associated changes in the numbers of tumor-specific T cells in TME versus the spleen.

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References

  1. Johnson, L.A. & June, C.H. Driving gene-engineered T cell immunotherapy of cancer. Cell Res. 27, 38–58 (2017).

    Article  CAS  PubMed  Google Scholar 

  2. Kalos, M. & June, C.H. Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Immunity 39, 49–60 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Maus, M.V. et al. Adoptive immunotherapy for cancer or viruses. Annu. Rev. Immunol. 32, 189–225 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Palucka, K. & Banchereau, J. Cancer immunotherapy via dendritic cells. Nat. Rev. Cancer 12, 265–277 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Topalian, S.L. et al. Immunotherapy: the path to win the war on cancer? Cell 161, 185–186 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Tumeh, P.C. et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 515, 568–571 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Herbst, R.S. et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 515, 563–567 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Brahmer, J.R. et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366, 2455–2465 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Taube, J.M. et al. Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy. Clin. Cancer Res. 20, 5064–5074 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Topalian, S.L. et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N. Engl. J. Med. 366, 2443–2454 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ji, R.R. et al. An immune-active tumor microenvironment favors clinical response to ipilimumab. Cancer Immunol. Immunother. 61, 1019–1031 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Gajewski, T.F., Schreiber, H. & Fu, Y.X. Innate and adaptive immune cells in the tumor microenvironment. Nat. Immunol. 14, 1014–1022 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Mailliard, R.B. et al. Alpha-type-1 polarized dendritic cells: a novel immunization tool with optimized CTL-inducing activity. Cancer Res. 64, 5934–5937 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Giermasz, A.S. et al. Type-1 polarized dendritic cells primed for high IL-12 production show enhanced activity as cancer vaccines. Cancer Immunol. Immunother. 58, 1329–1336 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lee, J.J., Foon, K.A., Mailliard, R.B., Muthuswamy, R. & Kalinski, P. Type 1-polarized dendritic cells loaded with autologous tumor are a potent immunogen against chronic lymphocytic leukemia. J. Leukoc. Biol. 84, 319–325 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Muthuswamy, R. et al. NF-kappaB hyperactivation in tumor tissues allows tumor-selective reprogramming of the chemokine microenvironment to enhance the recruitment of cytolytic T effector cells. Cancer Res. 72, 3735–3743 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Muthuswamy, R., Corman, J.M., Dahl, K., Chatta, G.S. & Kalinski, P. Functional reprogramming of human prostate cancer to promote local attraction of effector CD8(+) T cells. Prostate 76, 1095–1105 (2016).

    Article  CAS  PubMed  Google Scholar 

  18. Muthuswamy, R., Wang, L., Pitteroff, J., Gingrich, J.R. & Kalinski, P. Combination of IFNα and poly-I:C reprograms bladder cancer microenvironment for enhanced CTL attraction. J. Immunother. Cancer 3, 6 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Downs-Canner, S. et al. Complement inhibition: a novel form of immunotherapy for colon cancer. Ann. Surg. Oncol. 23, 655–662 (2016).

    Article  PubMed  Google Scholar 

  20. Bach, S. et al. A yeast-based assay to isolate drugs active against mammalian prions. Methods 39, 72–77 (2006).

    Article  CAS  PubMed  Google Scholar 

  21. Galon, J., Fridman, W.H. & Pages, F. The adaptive immunologic microenvironment in colorectal cancer: a novel perspective. Cancer Res. 67, 1883–1886 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Zhang, L. et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N. Engl. J. Med. 348, 203–213 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Sato, E. et al. Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. Proc. Natl. Acad. Sci. USA 102, 18538–18543 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Fridman, W.H. et al. Prognostic and predictive impact of intra- and peritumoral immune infiltrates. Cancer Res. 71, 5601–5605 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Fridman, W.H., Pages, F., Sautes-Fridman, C. & Galon, J. The immune contexture in human tumours: impact on clinical outcome. Nat. Rev. Cancer 12, 298–306 (2012).

    Article  CAS  PubMed  Google Scholar 

  26. Pages, F. et al. Effector memory T cells, early metastasis, and survival in colorectal cancer. N. Engl. J. Med. 353, 2654–2666 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Llosa, N.J. et al. The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints. Cancer Discov. 5, 43–51 (2015).

    Article  CAS  PubMed  Google Scholar 

  28. Le, D.T. et al. PD-1 blockade in tumors with mismatch-repair deficiency. N. Engl. J. Med. 372, 2509–2520 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Astsaturov, I. et al. Amplification of virus-induced antimelanoma T-cell reactivity by high-dose interferon-alpha2b: implications for cancer vaccines. Clin. Cancer Res. 9, 4347–4355 (2003).

    CAS  PubMed  Google Scholar 

  30. Rosenberg, S.A. et al. Tumor progression can occur despite the induction of very high levels of self/tumor antigen-specific CD8+ T cells in patients with melanoma. J. Immunol. 175, 6169–6176 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Fox, B.A. et al. Defining the critical hurdles in cancer immunotherapy. J. Transl. Med. 9, 214 (2012).

    Article  Google Scholar 

  32. Apetoh, L. et al. Consensus nomenclature for CD8+ T cell phenotypes in cancer. Oncoimmunology 4, e998538 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Kalinski, P., Muthuswamy, R. & Urban, J. Dendritic cells in cancer immunotherapy: vaccines and combination immunotherapies. Expert Rev. Vaccines 12, 285–295 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gajewski, T.F. The next hurdle in cancer immunotherapy: overcoming the non-T-cell-inflamed tumor microenvironment. Semin. Oncol. 42, 663–671 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Tang, H. et al. Facilitating T cell infiltration in tumor microenvironment overcomes resistance to PD-L1 blockade. Cancer Cell 29, 285–296 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Fuertes, M.B., Woo, S.R., Burnett, B., Fu, Y.X. & Gajewski, T.F. Type I interferon response and innate immune sensing of cancer. Trends Immunol. 34, 67–73 (2013).

    Article  CAS  PubMed  Google Scholar 

  37. Woo, S.R. et al. STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors. Immunity 41, 830–842 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ohkuri, T. et al. STING contributes to antiglioma immunity via triggering type I IFN signals in the tumor microenvironment. Cancer Immunol. Res. 2, 1199–1208 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kreutz, M., Tacken, P.J. & Figdor, C.G. Targeting dendritic cells—why bother? Blood 121, 2836–2844 (2013).

    Article  CAS  PubMed  Google Scholar 

  40. Yang, X. et al. Targeting the tumor microenvironment with interferon-β bridges innate and adaptive immune responses. Cancer Cell 25, 37–48 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Wong, J.L., Muthuswamy, R., Bartlett, D.L. & Kalinski, P. IL-18-based combinatorial adjuvants promote the intranodal production of CCL19 by NK cells and dendritic cells of cancer patients. Oncoimmunology 2, e26245 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Wong, J.L., Berk, E., Edwards, R.P. & Kalinski, P. IL-18-primed helper NK cells collaborate with dendritic cells to promote recruitment of effector CD8+ T cells to the tumor microenvironment. Cancer Res. 73, 4653–4662 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kalinski, P. & Gingrich, J.R. Toward improved effectiveness of bladder cancer immunotherapy. Immunotherapy 7, 1039–1042 (2015).

    Article  CAS  PubMed  Google Scholar 

  44. Lu, H. TLR agonists for cancer immunotherapy: tipping the balance between the immune stimulatory and inhibitory effects. Front. Immunol. 5, 83 (2014).

    PubMed  PubMed Central  Google Scholar 

  45. Liu, Z. et al. CXCL11-armed oncolytic poxvirus elicits potent antitumor immunity and shows enhanced therapeutic efficacy. Oncoimmunology 5, e1091554 (2016).

    Article  PubMed  CAS  Google Scholar 

  46. Francis, L. et al. Modulation of chemokines in the tumor microenvironment enhances oncolytic virotherapy for colorectal cancer. Oncotarget 7, 22174–22185 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Kantoff, P.W. et al. Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N. Engl. J. Med. 363, 411–422 (2010).

    Article  CAS  PubMed  Google Scholar 

  48. Fong, L. et al. Activated lymphocyte recruitment into the tumor microenvironment following preoperative sipuleucel-T for localized prostate cancer. J. Natl. Cancer Inst. 106 http://dx.doi.org/10.1093/jnci/dju268 (2014).

  49. Inaba, K., Young, J.W. & Steinman, R.M. Direct activation of CD8+ cytotoxic T lymphocytes by dendritic cells. J. Exp. Med. 166, 182–194 (1987).

    Article  CAS  PubMed  Google Scholar 

  50. Caux, C., Dezutter-Dambuyant, C., Schmitt, D. & Banchereau, J. GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells. Nature 360, 258–261 (1992).

    Article  CAS  PubMed  Google Scholar 

  51. Inaba, K. et al. Identification of proliferating dendritic cell precursors in mouse blood. J. Exp. Med. 175, 1157–1167 (1992).

    Article  CAS  PubMed  Google Scholar 

  52. Bender, A., Sapp, M., Schuler, G., Steinman, R.M. & Bhardwaj, N. Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood. J. Immunol. Methods 196, 121–135 (1996).

    Article  CAS  PubMed  Google Scholar 

  53. Schuler, G. & Steinman, R.M. Dendritic cells as adjuvants for immune-mediated resistance to tumors. J. Exp. Med. 186, 1183–1187 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Sallusto, F. & Lanzavecchia, A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179, 1109–1118 (1994).

    Article  CAS  PubMed  Google Scholar 

  55. Peters, J.H. et al. Signals required for differentiating dendritic cells from human monocytes in vitro. Adv. Exp. Med. Biol. 329, 275–280 (1993).

    Article  CAS  PubMed  Google Scholar 

  56. Brasel, K., De Smedt, T., Smith, J.L. & Maliszewski, C.R. Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures. Blood 96, 3029–3039 (2000).

    Article  CAS  PubMed  Google Scholar 

  57. Saunders, D. et al. Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 184, 2185–2196 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Maraskovsky, E. et al. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. J. Exp. Med. 184, 1953–1962 (1996).

    Article  CAS  PubMed  Google Scholar 

  59. Brasel, K. et al. Hematologic effects of flt3 ligand in vivo in mice. Blood 88, 2004–2012 (1996).

    Article  CAS  PubMed  Google Scholar 

  60. Albert, M.L., Jegathesan, M. & Darnell, R.B. Dendritic cell maturation is required for the cross-tolerization of CD8+ T cells. Nat. Immunol. 2, 1010–1017 (2001).

    Article  CAS  PubMed  Google Scholar 

  61. Sparwasser, T. et al. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur. J. Immunol. 28, 2045–2054 (1998).

    Article  CAS  PubMed  Google Scholar 

  62. Binder, R.J., Anderson, K.M., Basu, S. & Srivastava, P.K. Cutting edge: heat shock protein gp96 induces maturation and migration of CD11c+ cells in vivo. J. Immunol. 165, 6029–6035 (2000).

    Article  CAS  PubMed  Google Scholar 

  63. Kalinski, P. et al. Natural killer-dendritic cell cross-talk in cancer immunotherapy. Exp. Opin. Biol. Ther. 5, 1303–1315 (2005).

    Article  CAS  Google Scholar 

  64. Jonuleit, H. et al. Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions. Eur. J. Immunol. 27, 3135–3142 (1997).

    Article  CAS  PubMed  Google Scholar 

  65. Anguille, S., Smits, E.L., Lion, E., van Tendeloo, V.F. & Berneman, Z.N. Clinical use of dendritic cells for cancer therapy. Lancet Oncol. 15, e257–e267 (2014).

    Article  CAS  PubMed  Google Scholar 

  66. Vieira, P.L., de Jong, E.C., Wierenga, E.A., Kapsenberg, M.L. & Kalinski, P. Development of Th1-inducing capacity in myeloid dendritic cells requires environmental instruction. J. Immunol. 164, 4507–4512 (2000).

    Article  CAS  PubMed  Google Scholar 

  67. Kalinski, P., Schuitemaker, J.H., Hilkens, C.M. & Kapsenberg, M.L. Prostaglandin E2 induces the final maturation of IL-12-deficient CD1a+CD83+ dendritic cells: the levels of IL-12 are determined during the final dendritic cell maturation and are resistant to further modulation. J. Immunol. 161, 2804–2809 (1998).

    CAS  PubMed  Google Scholar 

  68. Kalinski, P., Vieira, P.L., Schuitemaker, J.H., de Jong, E.C. & Kapsenberg, M.L. Prostaglandin E(2) is a selective inducer of interleukin-12 p40 (IL-12p40) production and an inhibitor of bioactive IL-12p70 heterodimer. Blood 97, 3466–3469 (2001).

    Article  CAS  PubMed  Google Scholar 

  69. Whittaker, D.S., Bahjat, K.S., Moldawer, L.L. & Clare-Salzler, M.J. Autoregulation of human monocyte-derived dendritic cell maturation and IL-12 production by cyclooxygenase-2-mediated prostanoid production. J. Immunol. 165, 4298–4304 (2000).

    Article  CAS  PubMed  Google Scholar 

  70. Hilkens, C.M., Kalinski, P., de Boer, M. & Kapsenberg, M.L. Human dendritic cells require exogenous interleukin-12-inducing factors to direct the development of naive T-helper cells toward the Th1 phenotype. Blood 90, 1920–1926 (1997).

    Article  CAS  PubMed  Google Scholar 

  71. Kalinski, P., Hilkens, C.M., Snijders, A., Snijdewint, F.G. & Kapsenberg, M.L. IL-12-deficient dendritic cells, generated in the presence of prostaglandin E2, promote type 2 cytokine production in maturing human naive T helper cells. J. Immunol. 159, 28–35 (1997).

    CAS  PubMed  Google Scholar 

  72. Butterfield, L.H., Gooding, W. & Whiteside, T.L. Development of a potency assay for human dendritic cells: IL-12p70 production. J. Immunother. 31, 89–100 (2008).

    Article  CAS  PubMed  Google Scholar 

  73. Gustafsson, K. et al. Recruitment and activation of natural killer cells in vitro by a human dendritic cell vaccine. Cancer Res. 68, 5965–5971 (2008).

    Article  CAS  PubMed  Google Scholar 

  74. Wesa, A., Kalinski, P., Kirkwood, J.M., Tatsumi, T. & Storkus, W.J. Polarized type-1 dendritic cells (DC1) producing high levels of IL-12 family members rescue patient TH1-type antimelanoma CD4+ T cell responses in vitro. J. Immunother. 30, 75–82 (2007).

    Article  CAS  PubMed  Google Scholar 

  75. Carreno, B.M. et al. IL-12p70-producing patient DC vaccine elicits Tc1-polarized immunity. J. Clin. Invest. 123, 3383–3394 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Okada, H. et al. Induction of CD8+ T-cell responses against novel glioma-associated antigen peptides and clinical activity by vaccinations with α-type 1 polarized dendritic cells and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose in patients with recurrent malignant glioma. J. Clin. Oncol. 29, 330–336 (2011).

    Article  CAS  PubMed  Google Scholar 

  77. Zitvogel, L. et al. IL-12-engineered dendritic cells serve as effective tumor vaccine adjuvants in vivo. Ann. N. Y. Acad. Sci. 795, 284–293 (1996).

    Article  CAS  PubMed  Google Scholar 

  78. Xu, S. et al. Rapid high efficiency sensitization of CD8+ T cells to tumor antigens by dendritic cells leads to enhanced functional avidity and direct tumor recognition through an IL-12-dependent mechanism. J. Immunol. 171, 2251–2261 (2003).

    Article  CAS  PubMed  Google Scholar 

  79. Ten Brinke, A., Karsten, M.L., Dieker, M.C., Zwaginga, J.J. & van Ham, S.M. The clinical grade maturation cocktail monophosphoryl lipid A plus IFNgamma generates monocyte-derived dendritic cells with the capacity to migrate and induce Th1 polarization. Vaccine 25, 7145–7152 (2007).

    Article  CAS  PubMed  Google Scholar 

  80. Chiang, C.L. et al. Optimizing parameters for clinical-scale production of high IL-12 secreting dendritic cells pulsed with oxidized whole tumor cell lysate. J. Transl. Med. 9, 198 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Hokey, D.A., Larregina, A.T., Erdos, G., Watkins, S.C. & Falo, L.D. Jr. Tumor cell loaded type-1 polarized dendritic cells induce Th1-mediated tumor immunity. Cancer Res. 65, 10059–10067 (2005).

    Article  CAS  PubMed  Google Scholar 

  82. Budiu, R.A. et al. Immunobiology of human mucin 1 in a preclinical ovarian tumor model. Oncogene 32, 3664–3675 (2013).

    Article  CAS  PubMed  Google Scholar 

  83. Schreibelt, G. et al. Commonly used prophylactic vaccines as an alternative for synthetically produced TLR ligands to mature monocyte-derived dendritic cells. Blood 116, 564–574 (2010).

    Article  CAS  PubMed  Google Scholar 

  84. Zitvogel, L. et al. Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines. J. Exp. Med. 183, 87–97 (1996).

    Article  CAS  PubMed  Google Scholar 

  85. Mayordomo, J.I. et al. Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nat. Med. 1, 1297–1302 (1995).

    Article  CAS  PubMed  Google Scholar 

  86. Paglia, P., Chiodoni, C., Rodolfo, M. & Colombo, M.P. Murine dendritic cells loaded in vitro with soluble protein prime cytotoxic T lymphocytes against tumor antigen in vivo. J. Exp. Med. 183, 317–322 (1996).

    Article  CAS  PubMed  Google Scholar 

  87. Fields, R.C., Shimizu, K. & Mule, J.J. Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo. Proc. Natl. Acad. Sci. USA 95, 9482–9487 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Schnurr, M. et al. Apoptotic pancreatic tumor cells are superior to cell lysates in promoting cross-priming of cytotoxic T cells and activate NK and gammadelta T cells. Cancer Res. 62, 2347–2352 (2002).

    CAS  PubMed  Google Scholar 

  89. Strome, S.E. et al. Strategies for antigen loading of dendritic cells to enhance the antitumor immune response. Cancer Res. 62, 1884–1889 (2002).

    CAS  PubMed  Google Scholar 

  90. Chiang, C.L., Coukos, G. & Kandalaft, L.E. Whole tumor antigen vaccines: where are we? Vaccines 3, 344–372 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Wieckowski, E. et al. Type-1 polarized dendritic cells loaded with apoptotic prostate cancer cells are potent inducers of CD8(+) T cells against prostate cancer cells and defined prostate cancer-specific epitopes. Prostate 71, 125–133 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  92. Specht, J.M. et al. Dendritic cells retrovirally transduced with a model antigen gene are therapeutically effective against established pulmonary metastases. J. Exp. Med. 186, 1213–1221 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Mullins, D.W. et al. Route of immunization with peptide-pulsed dendritic cells controls the distribution of memory and effector T cells in lymphoid tissues and determines the pattern of regional tumor control. J. Exp. Med. 198, 1023–1034 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Okada, N. et al. Administration route-dependent vaccine efficiency of murine dendritic cells pulsed with antigens. Br. J. Cancer 84, 1564–1570 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Engleman, E.G. & Fong, L. Induction of immunity to tumor-associated antigens following dendritic cell vaccination of cancer patients. Clin. Immunol. 106, 10–15 (2003).

    Article  CAS  PubMed  Google Scholar 

  96. Fong, L., Brockstedt, D., Benike, C., Wu, L. & Engleman, E.G. Dendritic cells injected via different routes induce immunity in cancer patients. J. Immunol. 166, 4254–4259 (2001).

    Article  CAS  PubMed  Google Scholar 

  97. Bedrosian, I. et al. Intranodal administration of peptide-pulsed mature dendritic cell vaccines results in superior CD8+ T-cell function in melanoma patients. J. Clin. Oncol. 21, 3826–3835 (2003).

    Article  CAS  PubMed  Google Scholar 

  98. Grover, A. et al. Intralymphatic dendritic cell vaccination induces tumor antigen-specific, skin-homing T lymphocytes. Clin. Cancer Res. 12, 5801–5808 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Radomski, M. et al. Prolonged intralymphatic delivery of dendritic cells through implantable lymphatic ports in patients with advanced cancer. J. Immunother. Cancer 4, 24 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  100. Hu, J. et al. Induction of potent antitumor immunity by intratumoral injection of interleukin 23-transduced dendritic cells. Cancer Res. 66, 8887–8896 (2006).

    Article  CAS  PubMed  Google Scholar 

  101. Nishioka, Y., Hirao, M., Robbins, P.D., Lotze, M.T. & Tahara, H. Induction of systemic and therapeutic antitumor immunity using intratumoral injection of dendritic cells genetically modified to express interleukin 12. Cancer Res. 59, 4035–4041 (1999).

    CAS  PubMed  Google Scholar 

  102. Thompson, E.D., Enriquez, H.L., Fu, Y.X. & Engelhard, V.H. Tumor masses support naive T cell infiltration, activation, and differentiation into effectors. J. Exp. Med. 207, 1791–1804 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Melero, I. et al. Evolving synergistic combinations of targeted immunotherapies to combat cancer. Nat. Rev. Cancer 15, 457–472 (2015).

    Article  CAS  PubMed  Google Scholar 

  104. Vasaturo, A., Verdoes, M., de Vries, J., Torensma, R. & Figdor, C.G. Restoring immunosurveillance by dendritic cell vaccines and manipulation of the tumor microenvironment. Immunobiology 220, 243–248 (2015).

    Article  CAS  PubMed  Google Scholar 

  105. Pardoll, D.M. The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 12, 252–264 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Ribas, A. et al. Dendritic cell vaccination combined with CTLA4 blockade in patients with metastatic melanoma. Clin. Cancer Res. 15, 6267–6276 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Rosenblatt, J. et al. PD-1 blockade by CT-011, anti-PD-1 antibody, enhances ex vivo T-cell responses to autologous dendritic cell/myeloma fusion vaccine. J. Immunother. 34, 409–418 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Wilgenhof, S. et al. Phase II study of autologous monocyte-derived mRNA electroporated dendritic cells (TriMixDC-MEL) plus ipilimumab in patients with pretreated advanced melanoma. J. Clin. Oncol. 34, 1330–1338 (2016).

    Article  PubMed  Google Scholar 

  109. Melero, I., Hervas-Stubbs, S., Glennie, M., Pardoll, D.M. & Chen, L. Immunostimulatory monoclonal antibodies for cancer therapy. Nat. Rev. Cancer 7, 95–106 (2007).

    Article  CAS  PubMed  Google Scholar 

  110. Fujita, M. et al. COX-2 blockade suppresses gliomagenesis by inhibiting myeloid-derived suppressor cells. Cancer Res. 71, 2664–2674 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Wong, J.L., Obermajer, N., Odunsi, K., Edwards, R.P. & Kalinski, P. Synergistic COX2 induction by IFNγ and TNFα self-limits type-1 immunity in the human tumor microenvironment. Cancer Immunol. Res. 4, 303–311 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Obermajer, N., Muthuswamy, R., Lesnock, J., Edwards, R.P. & Kalinski, P. Positive feedback between PGE2 and COX2 redirects the differentiation of human dendritic cells toward stable myeloid-derived suppressor cells. Blood 118, 5498–5505 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Vacchelli, E. et al. Trial watch: IDO inhibitors in cancer therapy. Oncoimmunology 3, e957994 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  114. Vanneman, M. & Dranoff, G. Combining immunotherapy and targeted therapies in cancer treatment. Nat. Rev. Cancer 12, 237–251 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Li, J. et al. Chemokine expression from oncolytic Vaccinia virus enhances vaccine therapies of cancer. Mol. Ther. 19, 650–657 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  116. Jensen, S.M. et al. Signaling through OX40 enhances antitumor immunity. Semin. Oncol. 37, 524–532 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Dranoff, G. Experimental mouse tumour models: what can be learnt about human cancer immunology? Nat. Rev. Immunol. 12, 61–66 (2011).

    Article  PubMed  CAS  Google Scholar 

  118. Ngiow, S.F., Loi, S., Thomas, D. & Smyth, M.J. Mouse models of tumor immunotherapy. Adv. Immunol. 130, 1–24 (2016).

    Article  CAS  PubMed  Google Scholar 

  119. Betts, M.R. et al. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J. Immunol. Methods 281, 65–78 (2003).

    Article  CAS  PubMed  Google Scholar 

  120. Zaritskaya, L., Shurin, M.R., Sayers, T.J. & Malyguine, A.M. New flow cytometric assays for monitoring cell-mediated cytotoxicity. Expert Rev. Vaccines 9, 601–616 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Klenerman, P., Cerundolo, V. & Dunbar, P.R. Tracking T cells with tetramers: new tales from new tools. Nat. Rev. Immunol. 2, 263–272 (2002).

    Article  CAS  PubMed  Google Scholar 

  122. Karsunky, H., Merad, M., Cozzio, A., Weissman, I.L. & Manz, M.G. Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo. J. Exp. Med. 198, 305–313 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. Pulendran, B. et al. Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proc. Natl. Acad. Sci. USA 96, 1036–1041 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Kalinski, P., Schuitemaker, J.H., Hilkens, C.M., Wierenga, E.A. & Kapsenberg, M.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. Immunol. 162, 3231–3236 (1999).

    CAS  PubMed  Google Scholar 

  125. Watchmaker, P.B. et al. Independent regulation of chemokine responsiveness and cytolytic function versus CD8+ T cell expansion by dendritic cells. J. Immunol. 184, 591–597 (2010).

    Article  CAS  PubMed  Google Scholar 

  126. Hoffmann, T.K., Meidenbauer, N., Dworacki, G., Kanaya, H. & Whiteside, T.L. Generation of tumor-specific T-lymphocytes by cross-priming with human dendritic cells ingesting apoptotic tumor cells. Cancer Res. 60, 3542–3549 (2000).

    CAS  PubMed  Google Scholar 

  127. Albert, M.L. et al. Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188, 1359–1368 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. Snijders, A., Kalinski, P., Hilkens, C.M. & Kapsenberg, M.L. High-level IL-12 production by human dendritic cells requires two signals. Int. Immunol. 10, 1593–1598 (1998).

    Article  CAS  PubMed  Google Scholar 

  129. Wieckowski, E. et al. Type-1 polarized dendritic cells loaded with apoptotic prostate cancer cells are potent inducers of CD8(+) T cells against prostate cancer cells and defined prostate cancer-specific epitopes. Prostate 71, 125–133 (2011).

    Article  CAS  PubMed  Google Scholar 

  130. MartIn-Fontecha, A. et al. Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming. J. Exp. Med. 198, 615–621 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Wei, W.Z., Jones, R.F., Juhasz, C., Gibson, H. & Veenstra, J. Evolution of animal models in cancer vaccine development. Vaccine 33, 7401–7407 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Mac Keon, S., Ruiz, M.S., Gazzaniga, S. & Wainstok, R. Dendritic cell-based vaccination in cancer: therapeutic implications emerging from murine models. Front. Immunol. 6, 243 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  133. Westwood, J.A., Darcy, P.K. & Kershaw, M.H. The potential impact of mouse model selection in preclinical evaluation of cancer immunotherapy. Oncoimmunology 3, e946361 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  134. Devaud, C. et al. Tissues in different anatomical sites can sculpt and vary the tumor microenvironment to affect responses to therapy. Mol. Ther. 22, 18–27 (2014).

    Article  CAS  PubMed  Google Scholar 

  135. Devaud, C., John, L.B., Westwood, J.A., Darcy, P.K. & Kershaw, M.H. Immune modulation of the tumor microenvironment for enhancing cancer immunotherapy. Oncoimmunology 2, e25961 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  136. Rakoff-Nahoum, S. & Medzhitov, R. Toll-like receptors and cancer. Nat. Rev. Cancer 9, 57–63 (2009).

    Article  CAS  PubMed  Google Scholar 

  137. Lou, Y. et al. Antitumor activity mediated by CpG: the route of administration is critical. J. Immunother. 34, 279–288 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  138. Amos, S.M. et al. Adoptive immunotherapy combined with intratumoral TLR agonist delivery eradicates established melanoma in mice. Cancer Immunol. Immunother. 60, 671–683 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. John, L.B. et al. Oncolytic virus and anti-4-1BB combination therapy elicits strong antitumor immunity against established cancer. Cancer Res. 72, 1651–1660 (2012).

    Article  CAS  PubMed  Google Scholar 

  140. Bartlett, D.L. et al. Oncolytic viruses as therapeutic cancer vaccines. Mol. Cancer 12, 103 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  141. Kirn, D.H. & Thorne, S.H. Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer. Nat. Rev. Cancer 9, 64–71 (2009).

    Article  CAS  PubMed  Google Scholar 

  142. Kerkar, S.P. et al. IL-12 triggers a programmatic change in dysfunctional myeloid-derived cells within mouse tumors. J. Clin. Invest. 121, 4746–4757 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Zhang, L. et al. Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment. Mol. Ther. 19, 751–759 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  144. Lotze, M.T. et al. Cytokine gene therapy of cancer using interleukin-12: murine and clinical trials. Ann. N. Y. Acad. Sci. 795, 440–454 (1996).

    Article  CAS  PubMed  Google Scholar 

  145. Schlom, J. Therapeutic cancer vaccines: current status and moving forward. J. Natl. Cancer Inst. 104, 599–613 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  146. Datta, J. et al. Rationale for a multimodality strategy to enhance the efficacy of dendritic cell-based cancer immunotherapy. Front. Immunol. 6, 271 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  147. Mach, N. et al. Differences in dendritic cells stimulated in vivo by tumors engineered to secrete granulocyte-macrophage colony-stimulating factor or Flt3-ligand. Cancer Res. 60, 3239–3246 (2000).

    CAS  PubMed  Google Scholar 

  148. Lane, P., Burdet, C., McConnell, F., Lanzavecchia, A. & Padovan, E. CD40 ligand-independent B cell activation revealed by CD40 ligand-deficient T cell clones: evidence for distinct activation requirements for antibody formation and B cell proliferation. Eur. J. Immunol. 25, 1788–1793 (1995).

    Article  CAS  PubMed  Google Scholar 

  149. Thirunavukarasu, P. et al. A rationally designed A34R mutant oncolytic poxvirus: improved efficacy in peritoneal carcinomatosis. Mol. Ther. 21, 1024–1033 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  150. Wilhelm, K. et al. Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R. Nat. Med. 16, 1434–1438 (2010).

    Article  CAS  PubMed  Google Scholar 

  151. Kaka, A.S., Foster, A.E., Weiss, H.L., Rooney, C.M. & Leen, A.M. Using dendritic cell maturation and IL-12 producing capacity as markers of function: a cautionary tale. J. Immunother. 31, 359–369 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Nakamura, Y. et al. Helper function of memory CD8+ T cells: heterologous CD8+ T cells support the induction of therapeutic cancer immunity. Cancer Res. 67, 10012–10018 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  153. Musha, H. et al. Selective infiltration of CCR5(+)CXCR3(+) T lymphocytes in human colorectal carcinoma. Int. J. Cancer 116, 949–956 (2005).

    Article  CAS  PubMed  Google Scholar 

  154. Kunz, M. et al. Strong expression of the lymphoattractant C-X-C chemokine Mig is associated with heavy infiltration of T cells in human malignant melanoma. J. Pathol. 189, 552–558 (1999).

    Article  CAS  PubMed  Google Scholar 

  155. Ohtani, H., Jin, Z., Takegawa, S., Nakayama, T. & Yoshie, O. Abundant expression of CXCL9 (MIG) by stromal cells that include dendritic cells and accumulation of CXCR3+ T cells in lymphocyte-rich gastric carcinoma. J. Pathol. 217, 21–31 (2009).

    Article  CAS  PubMed  Google Scholar 

  156. Sampath, P. et al. Crosstalk between immune cell and oncolytic vaccinia therapy enhances tumor trafficking and antitumor effects. Mol. Ther. 21, 620–628 (2013).

    Article  CAS  PubMed  Google Scholar 

  157. Obermajer, N., Muthuswamy, R., Odunsi, K., Edwards, R.P. & Kalinski, P. PGE(2)-induced CXCL12 production and CXCR4 expression controls the accumulation of human MDSCs in ovarian cancer environment. Cancer Res. 71, 7463–7470 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We dedicate this article to Eva Wieckowski (who died unexpectedly on 14 December 2015). We thank T.J. Curiel for providing the ID8A ovarian cancer cell line, P. Lane for CD40L-expressing J558 cells, and M. Kronenberg for Flt3-L-expressing B16 cells. We thank K. Lemon for assistance with monitoring the mice and BLI, and P. Bailey for critical review and editorial comments. This work was supported by NIH grant CA132714 (to P.K.), by a 2015 CRI Clinical Strategy Team Grant (to P.K.), and by the David C. Koch Regional Therapy Cancer Center (D.L.B.).

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Author notes

  1. Eva Wieckowski: Deceased.

Authors and Affiliations

  1. Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Nataša Obermajer, Eva Wieckowski, Ravikumar Muthuswamy, Roshni Ravindranathan, David L Bartlett & Pawel Kalinski

  2. Immunotransplantation Center, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Julie Urban, Eva Wieckowski & Pawel Kalinski

  3. University of Pittsburgh Cancer Institute, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Pawel Kalinski

  4. Department of Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Pawel Kalinski

  5. Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Pawel Kalinski

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  1. Nataša Obermajer
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  3. Eva Wieckowski
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  4. Ravikumar Muthuswamy
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Contributions

N.O., J.U., and R.R. participated in data generation; N.O. and J.U. selected and evaluated the experimental data; N.O., J.U., R.M., R.R., E.W., P.K., and D.L.B. participated in the design of the protocol; N.O. and P.K. wrote the manuscript.

Corresponding author

Correspondence to Pawel Kalinski.

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Competing interests

Methods of production and clinical use of human variants of type-1 polarized DCs are a subject of two US patents (to P.K.). The remaining authors declare no competing financial interests.

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Obermajer, N., Urban, J., Wieckowski, E. et al. Promoting the accumulation of tumor-specific T cells in tumor tissues by dendritic cell vaccines and chemokine-modulating agents. Nat Protoc 13, 335–357 (2018). https://doi.org/10.1038/nprot.2017.130

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