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

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