Quantification of T-cell-mediated apoptosis in heterogeneous leukemia populations using four-color multiparameter flow cytometry.
BACKGROUND
The unique capacity of dendritic cells to present antigens to naive T cells is being increasingly utilized in cancer therapy. The efficacy of cell-based immunotherapy can be analyzed by determination of cytotoxic activity of T cells toward tumor cells in vitro. This study supplies a flow cytometric method to analyze T-cell-mediated cytotoxic activity toward heterogeneous leukemic cell populations at a single-cell level.


METHODS
The fluorescent probe SYTO16 and the dead-cell dye 7-aminoactinomycine-D (7-AAD) were used to identify early and late stages of apoptosis in combination with leukemia cell-identifying markers. Determination of viable, apoptotic, and necrotic cells was performed by inclusion of fluorescent beads.


RESULTS
In nine acute myeloid leukemia samples and three leukemic cell lines the use of SYTO16 next to the dead-cell marker 7-AAD significantly increased (P = 0.001) the sensitivity of the cytotoxicity assay as compared with single use of 7-AAD. Analysis of several effector-to-target ratios revealed the ability to determine dose-response effects. Enumeration of absolute numbers resulted in coefficients of variation of 4.1% and 8.4% for cell lines and leukemic samples, respectively.


CONCLUSIONS
The presented flow cytometric cytotoxicity assay enables the study of T-cell-mediated apoptosis in a heterogenous leukemia population.
Westers
Theresia M
TM
Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands.
Houtenbos
Ilse
I
Schuurhuis
Gerrit Jan
GJ
Ossenkoppele
Gert J
GJ
van de Loosdrecht
Arjan A
AA
D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000032:analysis; D018106:CD28 Antigens / Q000276:immunology; D016923:Cell Death; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic; D003609:Dactinomycin / Q000031:analogs & derivatives; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D015470:Leukemia, Myeloid, Acute / Q000276:immunology / Q000473:pathology*; D013601:T-Lymphocytes / Q000276:immunology*; D020298:U937 Cells
D014408:Biomarkers, Tumor; D018106:CD28 Antigens; D005456:Fluorescent Dyes; D003609:Dactinomycin; C025942:7-aminoactinomycin D