CTL ELISPOT Assay
Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8+ CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells.
The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method described herein would like to offer helpful and clear protocols for researchers that apply Elispot. IFN-γ and perforin Elispot assays are described.
Key wordsElispot T cell Cytokines Cancer
This work was supported by Progetto Strategico Regione Puglia grant (E.R., 2008), Ministero dell’Istruzione, dell’Università e della Ricerca (MiUR) FIRB, CAROMICS grant (E.R., 2011), and a Postdoctoral Fellowship by MiUR, PROGRAMMA OPERATIVO NAZIONALE (PON) RICERCA E COMPETITIVITÀ 2007–2013 PONa3_00395 “BIOSCIENCES AND HEALTH” (B&H) (M.G., 2013). The authors declare no conflicts of interest.
- 2.Zhang W, Caspell R, Karulin AY, Ahmad M, Haicheur N, Abdelsalam A, Johannesen K, Vignard V, Dudzik P, Georgakopoulou K, Mihaylova A, Silina K, Aptsiauri N, Adams V, Lehmann PV, McArdle S (2009) ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring even in hands of ELISPOT-inexperienced investigators. J Immunotoxicol 6:227–234PubMedCrossRefGoogle Scholar
- 6.Kuerten S, Kleen T, Assad RJ, Lehmann PV*, Tary-Lehmann M* (*contributed equally) (2007) Dissociated production of perforin, granzyme B and IFN-γ by HIV-specific CD8+ cells in HIV infection. AIDS Res Hum Retroviruses 24:62–71Google Scholar
- 8.Butterfield LH, Palucka AK, Britten CM, Dhodapkar MV, Hakansson L, Janetzki S, Kawakami Y, Kleen TO, Lee PP, Maccalli C, Maecker HT, Maino VC, Maio M, Malyguine A, Masucci G, Pawelec G, Potter DM, Rivoltini L, Salazar LG, Schenedel DJ, Slingluff CL, Song W, Stroncek DF, Tahara H, Thurin M, Trinchieri G, van Der Burg SH, Whiteside TL, Wigginton JM, Marincola F, Khleif S, Fox BA, Disis ML (2011) Recommendations from the iSBTc-SITC/FDA/NCI Workshop on Immunotherapy BioMarkers. Clin Cancer Res 17:3064–3076PubMedCentralPubMedCrossRefGoogle Scholar