International Journal of Cell Cloning, Vol 9, 65-77, Copyright © 1991 by AlphaMed Press
Gamma-irradiated peripheral blood mononuclear cells can express LAK activity
AS Chong, DE Bier, WJ Grimes and EM Hersh
Department of General Surgery, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois.
Peripheral blood mononuclear cells (PBMC) irradiated with high dose
gamma-radiation (1000-5000 rad) are commonly used as feeder cells during
the cloning of T lymphocytes, natural killer (NK) and lymphokine activated
killer (LAK) cells. We report here that such gamma-irradiated PBMC can be
stimulated with interleukin 2 (IL-2) to express the ability to lyse a
variety of tumor cell targets. The non-major histocompatibility complex
(MHC) restricted cytotoxicity demonstrated by irradiated PBMC is, however,
lower than that expressed by their non- irradiated counterparts. The
numbers of viable, gamma-irradiated LAK cells are significantly increased
by the addition of the mitogen, phytohemagglutinin (PHA). Purification of
the gamma-irradiated cells expressing cytotoxic activity by flow cytometry
determined that the effector cells were predominantly CD3- cells, although
some CD3+ cells also expressed moderate LAK activity. The ability of
gamma-irradiated cells to proliferate in the presence of PHA alone, or with
IL-2 + PHA, was maximal at day 4-5; but proliferation, as detected by
3H-thymidine uptake, was not detectable beyond 12-15 days of in vitro
culture. Because many of the LAK, T cell and NK cell cloning procedures
require the presence of feeder layers, growth factors (usually IL-2) and
mitogens, the presence of residual feeder cells expressing cytotoxic
activity may affect the specificity of such clones. Thus, efforts should be
made to ensure that such gamma-radiation-resistant cells capable of
expressing cytotoxic activity are completely eliminated before the cloned
cells are used for further experiments.
