Ex vivo activation of peripheral blood stem cells (PBSC) using
interleukin-2 (IL-2) results in cytotoxic effector cells that may possess beneficial in vivo effects. We proposed to evaluate ex vivo stimulation of PBSC using various
cytokines alone or in combination to optimize their function.
Cytokine-activated PBSC were analyzed for
tumor-directed cytotoxicity and their ability to remove
tumor cells from long-term clonogenic assays. Mononuclear cells were obtained from the
apheresis products of normal donors and cultured with
IL-2 (1000 U/ml),
interferon-alpha (IFN-alpha) (1000 U/ml), or
IL-12 (50 U/ml) either alone or in combinations at 37 degrees C and 5% CO(2) for 24 h. Colony-forming unit-
tumor (CFUT) assays were initiated using
cytokine-activated PBSC with varying concentrations of MCF-7 or SKBR-3 human
breast cancer cells. Standard 4-h (51)Cr-release assays were performed with
cytokine-activated PBSC using MCF-7 or SKBR-3 cells as targets. Activation of PBSC with
IL-2, IFN-alpha, or
IL-12 resulted in enhanced cytotoxicity against the two
breast cancer cell lines when compared to controls. PBSC activated with
IL-2 and IFN-alpha or
IL-2 and
IL-12 were more cytotoxic than PBSC activated with single
cytokines (p = 0.0004 for MCF-7 cells and p < 0.001 for SKBR-3 cells). Using clonogenic assays, IL-2-activated PBSC reduced the number of CFU-T to a greater extent than did
IL-12 or IFN-alpha-activated PBSC (p = 0.0006). However, PBSC activated with a combination of
IL-2 and IFN-alpha or
IL-2 and
IL-12 demonstrated 95% and 90% reductions, respectively, compared to 79% reduction using IL-2-activated PBSC (p < 0.0001). The greatest reduction in cytotoxicity occurred in the cell populations depleted of CD56(+) cells (p = 0.016) and CD8(+) CD56(+) cells (p = 0.002), suggesting that the effector cell population includes a combination of cytotoxic CD8(+) T cells and CD56(+) natural killer cells. These results demonstrate that the ex vivo activation of PBSC with
cytokines, either alone or in combination, enhances cytotoxicity against, and removal of two human
breast cancer cells. The combinations of
IL-2 with IFN-alpha or
IL-12 are most beneficial in cytotoxicity and purging assays. These results could play an important role in designing adoptive cellular immunotherapy clinical trials in the autologous hematopoietic stem cell transplant setting.