Abstract
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.
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References
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 8:130
Fox BA, Schendel DJ, Butterfield LH et al (2011) Defining the critical hurdles in cancer immunotherapy. J Transl Med 9:214
Butterfield LH, Palucka AK, Britten CM et al (2011) Recommendations from the iSBTc-SITC/FDA/NCI workshop on immunotherapy biomarkers. Clin Cancer Res 17:3064–3076
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 65:109–121
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 7:145–154
Cox JH, Ferrari G, Janetzki S (2006) Measurement of cytokine release at the single cell level using the ELISPOT assay. Methods 38:274–282
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 9:231–235
Cui Y, Chang L-J (1997) Computer-assisted, quantitative cytokine enzyme-linked immunospot analysis of human immune effector cell function. Biotechniques 22:1146–1149
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 203:141–152
Moodie Z, Price L, Gouttefangeas C et al (2010) Response definition criteria for ELISPOT assays revisited. Cancer Immunol Immunother 59:1489–1501
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 9:641–649
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 9:9
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 27:298–308
Maecker HT, Moon J, Bhatia S et al (2005) Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT. BMC Immunol 6:17
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 322:57–69
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 6:61
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 45:20–27
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 6:227–234
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 57:303–315
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 59:609–618
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 43:88–100
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 261:145–156
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 9:998–1008
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: 11895–11899
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 22:4474–4485
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 11:7692–7699
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 15:1443–1451
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
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Butterfield, L.H., Buffo, M.J. (2014). Immunologic Monitoring of Cancer Vaccine Trials Using the ELISPOT Assay. In: Thurin, M., Marincola, F. (eds) Molecular Diagnostics for Melanoma. Methods in Molecular Biology, vol 1102. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-727-3_5
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DOI: https://doi.org/10.1007/978-1-62703-727-3_5
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