New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population.
For the clinical evaluation of the efficacy of cellular immunotherapy it is necessary to analyze the effector functions of T cells against primary leukemic target cell populations which are usually considerably heterogeneous caused by differential maturation stages of the leukemic cells. An appropriate assay should not only allow the quantitative analysis of rapid cell death induction as measured by the conventional 51Cr release assay but also of the more slowly executing pathways of T-cell-induced apoptosis occurring within days instead of hours which cannot be measured using this method. Furthermore, it should dissect the differential susceptibility to T-cell-induced cell death of various target cell subpopulations and characterize the malignant precursor cells capable of producing malignant progeny. To fulfill these requirements we developed a new assay based on carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the target cell population combined with antibody staining of specific cell populations and addition of fluorescent microbeads to quantitatively monitor target cell death occurring within a longer time frame up to at least 5 days. This new assay facilitates the analysis of differential recognition of distinct cell types within a heterogeneous target cell population and allows simultaneously evaluation of the proliferative status of surviving target cells in response to relevant cytokines.
Jedema
Inge
I
Department of Hematology, Leiden University Medical Center, The Netherlands. i.jedema@lumc.nl
van der Werff
Nicole M
NM
Barge
Renée M Y
RM
Willemze
Roel
R
Falkenburg
J H Frederik
JH
D002455:Cell Division; D002999:Clone Cells; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006681:HLA-D Antigens / Q000276:immunology; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology / Q000473:pathology; D015470:Leukemia, Myeloid, Acute / Q000276:immunology* / Q000473:pathology; D011289:Preleukemia / Q000276:immunology* / Q000473:pathology; D013388:Succinimides; D013601:T-Lymphocytes / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006681:HLA-D Antigens; D015395:Histocompatibility Antigens Class I; D013388:Succinimides