Immunologic Monitoring of Cancer Vaccine Trials Using the ELISPOT Assay

  • Lisa H. Butterfield
  • Mary Jo Buffo
Part of the Methods in Molecular Biology book series (MIMB, volume 1102)


Cancer vaccines are designed to activate an immune response to tumor-specific or tumor-associated antigens expressed by the tumor. Cancer vaccines take many forms, including synthetic peptides, tumor cells and lysates, cell lines, and autologous antigen presenting cells like dendritic cells. The target antigens may be known, or “defined” in the vaccine, or unknown. In melanoma, more so than in other cancers, a large number of immunogenic “shared” antigens (tumor-specific or tumor-associated) have been identified. This allows for vaccination of groups of patients with the same vaccine, and also allows for testing for melanoma tumor immunity even when the vaccine does not include defined antigens. For the cancer vaccine field, the goal of a prognostic or predictive biomarker has yet to be achieved. However, the primary immunologic goal of any cancer vaccine is the induction (or amplification) of an immune response against the tumor, therefore the primary goal of immunologic monitoring in this setting, is testing for that response. In this chapter, we present standardized methodology from a central immunologic monitoring laboratory for melanoma cancer vaccine immune response assessment by the Enzyme-Linked Immunosorbant Spot (ELISPOT) assay. This assay allows for enumeration of antigen-specific cells in a plate format. We present the Interferon (IFN)-γ-producing lymphocyte assay, but the platform is easily adjusted to several cell types and several secreted molecules.

Key words

Cancer vaccine Immunologic monitoring Dendritic cells ELISPOT T cells 


  1. 1.
    Butterfield LH, Disis ML, Khleif SN et al (2010) Immuno-oncology biomarkers 2010 and beyond: perspectives from the iSBTc/SITC biomarker task force. J Transl Med 8:130CrossRefPubMedGoogle Scholar
  2. 2.
    Fox BA, Schendel DJ, Butterfield LH et al (2011) Defining the critical hurdles in cancer immunotherapy. J Transl Med 9:214CrossRefPubMedGoogle Scholar
  3. 3.
    Butterfield LH, Palucka AK, Britten CM et al (2011) Recommendations from the iSBTc-SITC/FDA/NCI workshop on immunotherapy biomarkers. Clin Cancer Res 17:3064–3076CrossRefPubMedGoogle Scholar
  4. 4.
    Czerkinsky CC, Nilsson LA, Nygren H et al (1983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 65:109–121CrossRefPubMedGoogle Scholar
  5. 5.
    Asai T, Storkus WJ, Whiteside TL (2000) Evaluation of the modified ELISPOT assay for gamma interferon production in cancer patients receiving antitumor vaccines. Clin Vaccine Immunol 7:145–154CrossRefGoogle Scholar
  6. 6.
    Cox JH, Ferrari G, Janetzki S (2006) Measurement of cytokine release at the single cell level using the ELISPOT assay. Methods 38:274–282CrossRefPubMedGoogle Scholar
  7. 7.
    Snyder JE, Bowers WJ, Livingstone AM et al (2003) Measuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing. Nat Med 9:231–235CrossRefPubMedGoogle Scholar
  8. 8.
    Cui Y, Chang L-J (1997) Computer-assisted, quantitative cytokine enzyme-linked immunospot analysis of human immune effector cell function. Biotechniques 22:1146–1149PubMedGoogle Scholar
  9. 9.
    Herr W, Linn B, Leister N et al (1997) The use of computer-assisted video image analysis for the quantification of CD8+ T lymphocytes producing tumor necrosis factor α spots in response to peptide antigens. J Immunol Methods 203:141–152CrossRefPubMedGoogle Scholar
  10. 10.
    Moodie Z, Price L, Gouttefangeas C et al (2010) Response definition criteria for ELISPOT assays revisited. Cancer Immunol Immunother 59:1489–1501CrossRefPubMedGoogle Scholar
  11. 11.
    Whiteside TL, Zhao Y, Tsukishiro T et al (2003) Enzyme-linked immunospot, cytokine flow cytometry, and tetramers in the detection of T-cell responses to a dendritic cell-based multipeptide vaccine in patients with melanoma. Clin Cancer Res 9:641–649PubMedGoogle Scholar
  12. 12.
    Maecker HT, Hassler J, Payne JK et al (2008) Precision and linearity targets for validation of an IFN-γ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides. BMC Immunol 9:9CrossRefPubMedGoogle Scholar
  13. 13.
    Speiser DE, Pittet MJ, Guillaume P et al (2004) Ex vivo analysis of human antigen-specific CD8+ T-cell responses: quality assessment of fluorescent HLA-A2 multimer and interferon-γ ELISPOT assays for patient immune monitoring. J Immunother 27:298–308CrossRefPubMedGoogle Scholar
  14. 14.
    Maecker HT, Moon J, Bhatia S et al (2005) Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT. BMC Immunol 6:17CrossRefPubMedGoogle Scholar
  15. 15.
    Bull M, Lee D, Stucky J et al (2007) Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials. J Immunol Methods 322:57–69CrossRefPubMedGoogle Scholar
  16. 16.
    Xu Y, Theobald V, Sung C et al (2008) Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic responses to gp100 and MelanA/MART-1 in melanoma patients. J Transl Med 6:61CrossRefPubMedGoogle Scholar
  17. 17.
    Dubey S, Clair J, Fu T-M et al (2007) Detection of HIV vaccine-induced cell-mediated immunity in HIV-seronegative clinical trial participants using an optimized and validated enzyme-linked immunospot assay. J Acquir Immune Defic Syndr 45:20–27CrossRefPubMedGoogle Scholar
  18. 18.
    Zhang W, Caspell R, Karulin AY et al (2009) ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring-even in hands of ELISPOT-inexperienced investigators. J Immunotoxicol 6:227–234CrossRefPubMedGoogle Scholar
  19. 19.
    Janetzki S, Panageas KS, Ben-Porat L et al (2007) Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol Immunother 57:303–315CrossRefPubMedGoogle Scholar
  20. 20.
    Janetzki S, Price L, Britten CM et al (2010) Performance of serum-supplemented and serum-free media in IFNγ Elispot assays for human T cells. Cancer Immunol Immunother 59:609–618CrossRefPubMedGoogle Scholar
  21. 21.
    Meidenbauer N, Harris DT, Spitler LE et al (2000) Generation of PSA-reactive effector cells after vaccination with a PSA-based vaccine in patients with prostate cancer. Prostate 43:88–100CrossRefPubMedGoogle Scholar
  22. 22.
    Bennouna J, Hildesheim A, Chikamatsu K et al (2002) Measurements of helper type T-cell responses in humans using ELISPOT assays for IL-5. J Immunol Methods 261:145–156CrossRefPubMedGoogle Scholar
  23. 23.
    Butterfield LH, Ribas A, Dissette VB et al (2003) Determinant spreading associated with clinical response in dendritic cell-based immunotherapy for malignant melanoma. Clin Cancer Res 9:998–1008PubMedGoogle Scholar
  24. 24.
    Welters MJ, Kenter GG, de Vos van Steenwijk PJ et al (2010) Success or failure of vaccination for HPV16-positive vulvar lesions correlates with kinetics and phenotype of induced T-cell responses. Proc Natl Acad Sci U S A 107: 11895–11899CrossRefPubMedGoogle Scholar
  25. 25.
    Slingluff CL, Petroni GR, Yamshchikov GV et al (2004) Immunologic and clinical outcomes of vaccination with a multiepitope melanoma peptide vaccine plus low-dose interleukin-2 administered either concurrently or on a delayed schedule. J Clin Oncol 22:4474–4485CrossRefPubMedGoogle Scholar
  26. 26.
    Linette GP, Zhang D, Hodi S et al (2005) Immunization using autologous dendritic cells pulsed with the melanoma-associated antigen gp100-derived G280-9V peptide elicits CD8+ immunity. Clin Cancer Res 11:7692–7699CrossRefPubMedGoogle Scholar
  27. 27.
    Kirkwood JM, Lee S, Moschos SJ et al (2009) Immunogenicity and antitumor effects of vaccination with peptide vaccine +/- granulocyte-monocyte colony-stimulating factor and/or IFN-alpha2b in advanced metastatic melanoma: eastern cooperative oncology group phase II Trial E1696. Clin Cancer Res 15:1443–1451CrossRefPubMedGoogle Scholar
  28. 28.
    Schaefer C, Butterfield LH, Lee S et al (2012) Function but not phenotype of melanoma peptide-specific CD8+ T cells correlate with survival in a multi-epitope peptide vaccine trial (ECOG 1696). Int J Cancer 131:874–884. doi: 10.1002/ijc.26481 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Lisa H. Butterfield
    • 1
  • Mary Jo Buffo
    • 1
  1. 1.University of Pittsburgh Cancer Institute, University of Pittsburgh School of MedicinePittsburghUSA

Personalised recommendations