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

Shafer‐Weaver, Kimberly; Sayers, Thomas J.; Strobl, Susan; Derby, Eric; Ulderich, Tracy; Baseler, Michael; Malyguine, Anatoli

doi:10.1186/1479-5876-1-14

Cited by 98 publications

(28 citation statements)

References 33 publications

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“…ELISpot granzyme B assays were used since this assay provides a measure of functional CTL activity [33,34]. We observed a significant increase in CTL activity after immunizations with Ad5 [E1-, E2b-]-CEA(6D) indicating that CEA specific CTL activity was induced.…”

Section: Discussionmentioning

confidence: 91%

“…An ELISpot assay for granzyme B-secreting lymphocytes to detect cytolytic T cell activity [33,34] was also performed. Briefly, isolated PBMC (2×10 5 cells/well) from individual patient samples were incubated 36–40 hours with a CEA peptide pool (15mers with 11aa overlap covering full length CEA with the CAP1-6D modification; 0.1 µg/well) to stimulate IFN-γ or granzyme B-secreting cells.…”

Section: Methodsmentioning

confidence: 99%

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Extended evaluation of a phase 1/2 trial on dosing, safety, immunogenicity, and overall survival after immunizations with an advanced-generation Ad5 [E1-, E2b-]-CEA(6D) vaccine in late-stage colorectal cancer

Balint

Gabitzsch

Rice

et al. 2015

Cancer Immunol Immunother

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A phase 1/2 clinical trial evaluating dosing, safety, immunogenicity, and overall survival on metastatic colorectal cancer (mCRC) patients after immunotherapy with an advanced generation Ad5 [E1-, E2b-]-CEA(6D) vaccine was performed. We report our extended observations on long-term overall survival and further immune analyses on a subset of treated patients including assessment of cytolytic T cell responses, T-regulatory (Treg) to T-effector (Teff) cell ratios, flow cytometry on peripheral blood mononuclear cells (PBMC), and determination of HLA-A2 status. An overall survival of 20% (median survival of 11 months) was observed during long-term follow-up and no long-term adverse effects were reported. Cytolytic T cell responses increased after immunizations and cell-mediated immune (CMI) responses were induced whether or not patients were HLA-A2 positive or Ad5 immune. PBMC samples from a small subset of patients were available for follow-up immune analyses. It was observed that the levels of carcinoembryonic antigen (CEA) specific CMI activity decreased from their peak values during follow-up in 5 patients analyzed. Preliminary results revealed that activated CD4+ and CD8+ T cells were detected in a post immunization sample exhibiting high CMI activity. Treg to Teff cell ratios were assessed and samples from 3 of 5 patients exhibited a decrease in Treg to Teff cell ratio during the treatment protocol. Based upon the favorable safety and immunogenicity data obtained, we plan to perform an extensive immunologic and survival analysis on mCRC patients to be enrolled in a randomized/controlled clinical trial that investigates Ad5 [E1-, E2b-]-CEA(6D) as a single agent with booster immunizations.

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Section: Discussionmentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Extended evaluation of a phase 1/2 trial on dosing, safety, immunogenicity, and overall survival after immunizations with an advanced-generation Ad5 [E1-, E2b-]-CEA(6D) vaccine in late-stage colorectal cancer

Balint

Gabitzsch

Rice

et al. 2015

Cancer Immunol Immunother

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“…[32]. Briefly, cells from the HLA-A*02:01-positive T2 cell line were used as target cells and were loaded with pCMV_pp65_A02 and beta-2-microglobulin (Sigma-Aldrich) for 2 hours.…”

Section: Methodsmentioning

confidence: 99%

Impaired Functionality of Antiviral T Cells in G-CSF Mobilized Stem Cell Donors: Implications for the Selection of CTL Donor

et al. 2013

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Adoptive transfer of antiviral T cells enhances immune reconstitution and decreases infectious complications after stem cell transplantation. Information on number and function of antiviral T cells in stem cell grafts is scarce. We investigated (1) immunomodulatory effects of G-CSF on antiviral T cells, (2) the influence of apheresis, and (3) the optimal time point to collect antiviral cells.CMV-, EBV- and ADV-specific T cells were enumerated in 170 G-CSF-mobilized stem cell and 24 non-mobilized platelet donors using 14 HLA-matched multimers. T-cell function was evaluated by IFN-γ ELISpot and granzyme B secretion. Immunophenotyping was performed by multicolor flow cytometry.G-CSF treatment did not significantly influence frequency of antiviral T cells nor their in vitro expansion rate upon antigen restimulation. However, T-cell function was significantly impaired, as expressed by a mean reduction in secretion of IFN-γ (75% in vivo, 40% in vitro) and granzyme B (32% target-independent, 76% target-dependent) as well as CD107a expression (27%). Clinical follow up data indicate that the first CMV-reactivation in patients and with it the need for T-cell transfer occurs while the donor is still under the influence of G-CSF.To overcome these limitations, T-cell banking before mobilization or recruitment of third party donors might be an option to optimize T-cell production.

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“…A strong correlation between Granzyme B and IFN-g ELISPOT assay with cytotoxicity in 51 Cr-release assay (R 2 D 0.95) was reported by Shafer-Weaver et al 39 In the ex vivo ELISPOT assay, which has been previously reported by Patil et al, 40 PBMCs were first incubated with 50 ng/ml of IL-7 and 10 ng/ml of IL-12 in culture medium for 2 h at 37 C. After incubation, the cells were washed with HBSS (Gibco, Life Technologies) and re-suspended at 5 £ 10 5 cells per well with complete culture medium containing RPMI (Gibco, Life Technologies) supplemented with 5% heat-inactivated human AB serum (Gemini Bio-Product) and 1£ penicillin/streptomycin (Gibco, Life Technologies) in a 96-well plate. Then, 50 mg/ml of peptides (appropriately selected depending on patient's HLAtype) was added to the respective wells and the cells were incubated overnight at 37 C and followed by ELISPOT assay as described below.…”

Section: Dendritic Cell Preparation and Stimulation Of T Cellsmentioning

confidence: 55%

Identification of immunogenic MAGED4B peptides for vaccine development in oral cancer immunotherapy

Lim¹,

Chun²,

Gan³

et al. 2014

Human Vaccines & Immunotherapeutics

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The ever-increasing number of tumor-associated antigens has provided a major stimulus for the development of therapeutic peptides vaccines. Tumor-associated peptides can induce high immune response rates and have been developed as vaccines for several types of solid tumors, and many are at various stages of clinical testing. MAGED4B, a melanoma antigen, is overexpressed in oral squamous cell carcinoma (OSCC) and this expression promotes proliferation and cell migration. In this study, we have identified 9 short peptides derived from MAGED4B protein that are restricted in binding to the HLA subtypes common in the Asian population (HLA-A2, A11, and A24). The peptides had good binding affinity with the MHC-Class I molecules and stimulated ex-vivo IFN-gamma and Granzyme-B production in blood samples from OSCC patients, suggesting that they are immunogenic. Further, T cells stimulated with peptide-pulsed dendritic cells showed enhanced T-cell cytotoxic activity against MAGED4B-overexpressing OSCC cell lines. In summary, we have identified MAGED4B peptides that induce anti-tumor immune responses advocating that they could be further developed as vaccine candidates for the treatment of OSCC.

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

Cited by 98 publications

References 33 publications

Extended evaluation of a phase 1/2 trial on dosing, safety, immunogenicity, and overall survival after immunizations with an advanced-generation Ad5 [E1-, E2b-]-CEA(6D) vaccine in late-stage colorectal cancer

Extended evaluation of a phase 1/2 trial on dosing, safety, immunogenicity, and overall survival after immunizations with an advanced-generation Ad5 [E1-, E2b-]-CEA(6D) vaccine in late-stage colorectal cancer

Impaired Functionality of Antiviral T Cells in G-CSF Mobilized Stem Cell Donors: Implications for the Selection of CTL Donor

Identification of immunogenic MAGED4B peptides for vaccine development in oral cancer immunotherapy

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