New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population

Jedema, Inge; Werff, Nicole M. van der; Barge, Renée M. Y.; Willemze, R.; Falkenburg, J.H. Frederik

doi:10.1182/blood-2003-06-2070

Cited by 154 publications

(128 citation statements)

References 18 publications

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“…Results obtained with the CFSE assay have been demonstrated to correlate well with those obtained with the conventional 51 Cr release assay in 4-h incubations. 31 To study the contribution of the death receptor pathway in T-cell-mediated target cell death, we introduced the anti-apoptotic gene FLIP into the target cell via retroviral transduction, and demonstrated that enhanced expression of FLIP caused almost complete inhibition of Fas-induced apoptosis ( Figure 3B). The inhibitory effect of FLIP was variable when target cell death was induced with the different CTL clones.…”

Section: Discussionmentioning

confidence: 99%

“…Cytotoxicity was measured using carboxyfluorescein diacetate succinimidyl ester (CFSE)-based cytotoxicity assays as described by Jedema et al 31 Target cells were labeled with 5 µM CFSE (Molecular Probes Europe, Leiden, The Netherlands), and incubated overnight in a humidified atmosphere of 5% CO2 at 37 °C. For the cytotoxicity assay, 5,000 target cells/well (50 µL) were plated in 96-well microtiter plates (all in triplicate), and 5,000 effector T cells were added in a volume of 100 µL/well.…”

Section: Cfse Cytotoxicity Assaymentioning

confidence: 99%

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Differential activation of the death receptor pathway in human target cells induced by cytotoxic T lymphocytes showing different kinetics of killing

Vries¹,

Borne²,

Luxemburg-Heijs³

et al. 2007

Haematologica

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Section: Discussionmentioning

confidence: 99%

Section: Cfse Cytotoxicity Assaymentioning

confidence: 99%

Differential activation of the death receptor pathway in human target cells induced by cytotoxic T lymphocytes showing different kinetics of killing

Vries¹,

Borne²,

Luxemburg-Heijs³

et al. 2007

Haematologica

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“…A CFSE-based cytotoxicity assay was adapted from Jedema et al (35). Briefly, nonirradiated APCs were labeled with CFSE (2 M), seeded in a 96-well round-bottom plate (1 ϫ 10 4/ well) and pulsed with PPD or M.Tb.-CME.…”

Section: Cfse Cytotoxicity Assaymentioning

confidence: 99%

“…To measure cytolytic activity, we adapted a flow cytometrybased assay that directly quantifies the number of living target cells (35). The assay was validated by comparison to the most widely used 51 Cr-release assay using PBMC-mediated killing of the NK cell target line K562.…”

Section: Functional Analysis Of Isolated Mtb-cme-expanded Cellsmentioning

confidence: 99%

Mycobacterial Lipopeptides Elicit CD4+ CTLs in Mycobacterium tuberculosis-Infected Humans

Bastian¹,

Braun

Bruns³

et al. 2008

The Journal of Immunology

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In searching for immunogenic molecules with the potential to induce protective immune responses against tuberculosis, we developed an ex vivo model to study frequency, phenotype, and effector functions of human T lymphocytes recognizing hydrophobic Ags of Mycobacterium tuberculosis (M.Tb). To obtain unbiased results, we characterized T lymphocytes responding to a crude cell wall extract (chloroform methanol extract of M.Tb (M.Tb-CME)) containing a broad spectrum of mycobacterial glycolipids and lipopeptides. A significant proportion of T lymphocytes recognized M.Tb-CME (290 IFN-γ+ T cells/105 PBMCs) and developed to effector memory cells as determined by the expression of CD45RO and the chemokine receptors CXCR3 and CCR5. Expanded lymphocytes fulfilled all criteria required for an efficient immune response against tuberculosis: 1) release of macrophage-activating Th1 cytokines and chemokines required for the spatial organization of local immune responses, 2) cytolytic activity against Ag-pulsed macrophages, and 3) recognition of infected macrophages and killing of the intracellular bacteria. Phenotypically, M.Tb-CME-expanded cells were CD4+ and MHC class II restricted, challenging current concepts that cytotoxic and antimicrobial effector cells are restricted to the CD8+ T cell subset. Pretreatment of M.Tb-CME with protease or chemical delipidation abrogated the biological activity, suggesting that responses were directed toward mycobacterial lipopeptides. These findings suggest that lipidated peptides are presented by M.Tb-infected macrophages and elicit CD4+ cytolytic and antimicrobial T lymphocytes. Our data support an emerging concept to include hydrophobic microbial Ags in vaccines against tuberculosis.

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“…Flow cytometry-based cytotoxicity studies Flow cytometry-based cytotoxicity assays were performed as previously described with minor adaptations [15,20]. Characteristics of the solid tumor cell lines used as targets are described in Table 1.…”

Section: P2x5 Genotypingmentioning

confidence: 99%

Aberrant expression of the hematopoietic-restricted minor histocompatibility antigen LRH-1 on solid tumors results in efficient cytotoxic T cell-mediated lysis

Overes

Levenga

Vos

et al. 2008

Cancer Immunol Immunother

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CD8 + T cells recognizing minor histocompatibility antigens (MiHA) on solid tumor cells may mediate eVective graft-versus-tumor (GVT) reactivity after allogeneic stem cell transplantation (SCT). Previously, we identiWed LRH-1 as a hematopoietic-restricted MiHA encoded by the P2X5 gene. Here, we report that LRH-1 is aberrantly expressed on solid tumor cells. P2X5 mRNA expression is demonstrated in a signiWcant portion of solid tumor cell lines, including renal cell carcinoma (RCC), melanoma, colorectal carcinoma, brain cancer and breast cancer. Importantly, P2X5 gene expression was also detected in a subset of primary solid tumor specimens derived from RCC, brain cancer and breast cancer patients. Furthermore, P2X5 expressing solid tumor cells can be eVectively targeted by LRH-1-speciWc cytotoxic T lymphocytes under inXammatory conditions. The expression of HLA-B7 and CD54 on tumor cells increases upon cytokine stimulation resulting in improved T cell activation as observed by higher levels of degranulation and enhanced tumor cell lysis. Overall, hematopoietic-restricted MiHA LRH-1 is aberrantly expressed on solid tumor cells and may be used as target in GVT-speciWc immunotherapy after SCT.

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New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population

Cited by 154 publications

References 18 publications

Differential activation of the death receptor pathway in human target cells induced by cytotoxic T lymphocytes showing different kinetics of killing

Differential activation of the death receptor pathway in human target cells induced by cytotoxic T lymphocytes showing different kinetics of killing

Mycobacterial Lipopeptides Elicit CD4+ CTLs in Mycobacterium tuberculosis-Infected Humans

Aberrant expression of the hematopoietic-restricted minor histocompatibility antigen LRH-1 on solid tumors results in efficient cytotoxic T cell-mediated lysis

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