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. 2003 Dec 29;1(1):14.
doi: 10.1186/1479-5876-1-14.

The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity

Kimberly Shafer-Weaver  1 , Thomas Sayers, Susan Strobl, Eric Derby, Tracy Ulderich, Michael Baseler, Anatoli Malyguine
Affiliations

The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity

Kimberly Shafer-Weaver et al. J Transl Med. .

Abstract

BACKGROUND: The interferon-gamma (IFN-gamma) ELISPOT assay is one of the most useful techniques for immunological monitoring of cancer vaccine trials and has gained increased application as a measure of specific T cell activation. However, it does not assess cell-mediated cytotoxicity directly as IFN-gamma secretion is not limited to only cytolytic cells. Granzyme B (GrB) is a key mediator of target cell death via the granule-mediated pathway. Therefore, the release of GrB by cytolytic lymphocytes upon effector-target interaction may be a more specific indicator of CTL and NK cytotoxic ability than IFN-gamma secretion. METHODS: We assessed whether the GrB ELISPOT assay is a viable alternative to the 51Cr-release and IFN-gamma ELISPOT assays for measuring antigen-specific CTL cytotoxicity. Direct comparisons between the three assays were made using human CTL cell lines (alphaEN-EBV and alphaJY) and an in vitro stimulated anti-Flu matrix peptide (FMP)-specific CTL. RESULTS: When the GrB ELISPOT was directly compared to the IFN-gamma ELISPOT and 51Cr-release assays, excellent cross-correlation between all three assays was shown. However, measurable IFN-gamma secretion in the ELISPOT assay was observed only after 1 hour of incubation and cytotoxicity assessed via the 51Cr-release assay after 4 hours, whereas GrB secretion was detectable within 10 min of effector-target contact with significant secretion observed after 1 h. Titration studies demonstrated a strong correlation between the number of effector cells and GrB spots per well. Irrelevant targets or antigens did not induce significant GrB secretion. Additionally, GrB secretion was abrogated when CTL cultures were depleted of CD8+ cells. CONCLUSION: Our findings demonstrate that the GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-gamma ELISPOT assay, the GrB ELISPOT directly measures the release of a cytotolytic protein. Detection of low frequency tumor-specific CTL and their specific effector functions can provide valuable insight with regards to immunological responses.

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Figures

Figure 1
Figure 1
Granzyme B (GrB) secretion by αEN-EBV CTL cell line in the ELISPOT assay. Effector cells (104, 5 × 103 or 2.5 × 103 cells per well) were run against EN-EBV or K562 (NK control) targets, 5 × 104 cells per well, in a 4 h assay at 37°C. Data is presented as spots per well ± SD and is representative of 3 experiments. Similar results obtained for αJY CTL.
Figure 2
Figure 2
Cytotoxicity of αEN-EBV CTL in the 51Cr-release assay. The αEN-EBV CTL cell lines were utilized as effector cells. Various numbers αEN-EBV CTL cells were tested against 5 × 103 target cells for 4 h at 37°C. Data is representative of 3 experiments. Similar results obtained for αJY CTL.
Figure 3
Figure 3
Specificity of GrB and IFN-γ secretion by αFMP-CTL in the ELISPOT assays. Human αFMP-CTL (7 day culture, 5 × 103 cells per well) were run alone (A) or against various target cells (5 × 104 cells per well): C1R.A2 (B), C1R.A2 pulsed with 5 μg/ml FMP (C) or C1R.A2 pulsed with 3 μM MART-1 (D). Effector and target cells were incubated for 4 h at 37°C assays. Data is presented as spots per well ± SD and is representative of 3 experiments.
Figure 4
Figure 4
Correlation of GrB and IFN-γ secretion in the ELISPOT assay with cytotoxicity in 51Cr-release assay. Human α-FMP-CTL (7 day culture) were used as effector cells. Target cells were C1R.A2 pulsed with FMP, 5 × 103 cells per well. Incubation time was 4 h at 37°C for all three assays. At the 10:1 effector:target ratio the spots in the IFN-γ ELISPOT assay were too numerous to count. Background (CTL alone) was subtracted from the results. Data is representative of 3 experiments.
Figure 5
Figure 5
Effector cells secreting GrB and IFN-γ in the ELISPOT assays. Human αFMP-CTL (7 day culture) or cultures depleted of CD8+ cells were used as effector cells (5 × 103 cells per well). Target cells were C1R.A2 pulsed with FMP (5 × 104 cells per well). CD8+ cells were removed from effector cell cultures using anti-CD8 mAb and magnetic beads. Effector and target cells were incubated for 4 h at 37°C. Data is presented as spots per 105 cells ± SD and is representative of 3 experiments.
Figure 6
Figure 6
Time course of GrB and IFN-γ secretion in the ELISPOT assays and cytotoxicity in 51Cr-release assay. Human αFMP-CTL (7 day culture) were used as effector cells (5 × 103 cells per well). Target cells were C1R.A2 pulsed with FMP (5 × 104 cells per well). Effector and target cells were incubated for the specified times at 37°C. A table containing the number of spots per 105 cells is provided to better clarify the data. Background spots (CTL alone) were subtracted from the results. Data is an average from three experiments.
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References

    1. Keilholz U, Weber J, Finke JH, Gabrilovich DI, Kast WM, Disis ML, Kirkwood JM, Scheibenbogen C, Schlom J, Maino VC, Lyerly HK, Lee PP, Storkus W, Marincola F, Worobec A, Atkins MB. Immunologic monitoring of cancer vaccine therapy: results of a workshop sponsored by the Society for Biological Therapy. J Immunother. 2002;25:97–138. doi: 10.1097/00002371-200203000-00001. - DOI - PubMed
    1. Regner M, Lobigs M, Blanden RV, Mullbacher A. Effector cytolotic function but not IFN-gamma production in cytotoxic T cells triggered by virus-infected target cells in vitro. Scand J Immunol. 2001;54:366–374. doi: 10.1046/j.1365-3083.2001.00955.x. - DOI - PubMed
    1. Lim DG, Bourcier K Bieganowska, Freeman GJ, Hafler DA. Examination of CD8+ T cell function in humans using MHC class I tetramers: similar cytotoxicity but variable proliferation and cytokine production among different clonal CD8+ T cells specific to a single viral epitope. J Immunol. 2000; 165:6214–6220. - PubMed
    1. Slifka MK, Rodriguez F, Whitton JL. Rapid on/off cycling of cytokine production by virus-specific CD8+ T cells. Nature. 1999;401:76–79. doi: 10.1038/43454. - DOI - PubMed
    1. Bachmann MF, Barner M, Viola A, Kopf M. Distinct kinetics of cytokine production and cytolysis in effector and memory T cells after viral infection. Eur J Immunol. 1999;29:291–299. doi: 10.1002/(SICI)1521-4141(199901)29:01<291::AID-IMMU291>3.0.CO;2-K. - DOI - PubMed