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Evaluation of antigen-specific lymphocytes anti-tumour effect with use of non-radioactive cytotoxicity test


Authors: L. Zahradová 1,2;  D. Očadlíková 1;  L. Kovářová 1,3;  R. Hájek 1,2;  A. Buliková 3;  M. Penka 3;  J. Michálek 1,4
Authors‘ workplace: Laboratoř experimentální hematologie a buněčné imunoterapie (LEHABI), FN Brno, Bohunice, 2Interní hematoonkologická 1;  klinika, FN Brno - Bohunice, 3Oddělení klinické hematologie, FN Brno - Bohunice, I. dětská interní klinika, FN Brno, Dětská nemocnice 4
Published in: Transfuze Hematol. dnes,12, 2006, No. 4, p. 244-248.
Category: Comprehensive Reports, Original Papers, Case Reports

Overview

Objectives:
Cytotoxicity test using radioactive chromium 51Cr has been standardly used to evaluate specific cytolytic activity of T lymphocytes. This test has some significant disadvantages because of the high non-specific background caused by spontaneous release of 51Cr from target cells, low sensitivity, and health risks associated with the use of a radioactive isotope. To avoid these limitations we have introduced a cytotoxicity test using 5-(6-) karboxyfluorescein diacetate succinimidyl ester (CFSE) and propidium iodide (PI).

Aim:
The aim of this study was to evaluate cytotoxic effect of antigen-specific T lymphocyte using a non-radioactive test with CFSE and PI staining.

Methods:
The model of cytotoxic T lymphocytes specific to multiple myeloma cell line ARH 77 was used. Activated myeloma-specific T cells that produce interferon-γ were isolated using immunomagnetic beads and further expanded in vitro. T lymphocyte labeled with CFSE were co-cultured with ARH77 target cells. To identify target cells in late apoptosis or necrosis, PI was used and measured by flow cytometry.

Results:
The optimal staining concentration of CFSE has been found (1μM). Cytotoxicity has been evaluated with effector cells:target cells ratio 2:1, 10:1 and 50:1 after 4, 24 and 48 hours of in vitro cultivation. Tumor specificity has been verified by 2 sets of controls: third-party PBMC as negative control target cells and expanded CFSE-labeled interferon gamma negative fraction of T cells as negative control effector cells.

Conclusion:
The newly established cytotoxicity test is a valuable instrument to evaluate effector part of the immune system and efficacy of anticancer vaccines.

Key words:
cytotoxicity test, CFSE, cytotoxic T lymphocytes


Labels
Haematology Internal medicine Clinical oncology

Article was published in

Transfusion and Haematology Today

Issue 4

2006 Issue 4

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