Recombinant bacillus Calmette-Guérin (BCG) expressing interferon-alpha 2B enhances human mononuclear cell cytotoxicity against bladder cancer cell lines in vitro

Abstract

Purpose: The proper induction of cellular immunity is required for effective bacillus Calmette-Guérin (BCG) immunotherapy of bladder cancer. It has been known that BCG stimulation of human peripheral blood mononuclear cells (PBMC) leads to the generation of effector cells cytotoxic to bladder cancer cells in vitro. To improve BCG therapy, we previously developed human interferon (IFN)-alpha 2B secreting recombinant (r) BCG (rBCG-IFN-alpha). We demonstrated that rBCG-IFN-alpha augmented T helper type 1 (Th1) cytokine IFN-gamma production by PBMC. In this study, we further investigated whether rBCG-IFN-alpha could also enhance PBMC cytotoxicity toward bladder cancer cells.

Materials and methods: PBMC were prepared from healthy individuals, left alone or stimulated with rBCG-IFN-alpha or control MV261 BCG, and used as effector cells in (51)Cr-release assays. Human bladder cancer cell lines T24, J82, 5637, TCCSUP, and UMUC-3 were used as target cells. To determine the role of secreted rIFN-alpha as well as endogenously expressed IFN-gamma and IL-2 in inducing the cytotoxicity, PBMC were stimulated with rBCG-IFN-alpha in the presence of neutralizing antibodies to IFN-alpha, IFN-gamma or IL-2. To determine the role of natural killer (NK) and CD8(+) T cells in inducing the cytotoxicity, both cell types were isolated after BCG stimulation of PBMC and used as effector cells in (51)Cr-release assays.

Results: Non-stimulated PBMC showed basal levels of cytotoxicity against all target cell lines tested. MV261 BCG increased the PBMC cytotoxicity by 1.8- to 4.2-fold. rBCG-IFN-alpha further increased the PBMC cytotoxicity by up to 2-fold. Elevated production of IFN-gamma and IL-2 by PBMC was observed after rBCG-IFN-alpha stimulation. Blockage of IFN-alpha, IFN-gamma or IL-2 by neutralizing antibodies during rBCG-IFN-alpha stimulation reduced or abolished the induction of PBMC cytotoxicity. Both NK and CD8(+) T cells were found to be responsible for the enhanced PBMC cytotoxicity induced by rBCG-IFN-alpha with the former cell type being more predominant.

Conclusions: rBCG-IFN-alpha is an improved BCG agent that induces enhanced PBMC cytotoxicity against bladder cancer cells in vitro. This rBCG strain may serve as an alternative to BCG for the treatment of superficial bladder cancer.

Fig. 1

rBCG-IFN-α induces enhanced PBMC cytotoxicity against human bladder cancer cells. PBMC were prepared from two healthy individuals, and left alone or stimulated with MV261 BCG (0.01 OD₆₀₀/ml) or rBCG-IFN-α (0.01 OD₆₀₀/ml) for 7 days. ⁵¹Cr-labeled bladder cancer T24, J82, 5637, TCCSUP, and UMUC-3 cells were then incubated with the PBMC at E/T cell ratio of 40:1 for 20 h. The % lysis of target cells by PBMC is presented as the mean of triplicate incubations. Numerical values represent the fold increase in cytotoxicity referring to the basal lysis by non-stimulated PBMC

Fig. 2

PBMC stimulated by rBCG-IFN-α exhibit potent cytotoxicity against human bladder cancer cells. PBMC from a healthy individual were left alone or stimulated with MV261 BCG (0.01 OD₆₀₀/ml) or rBCG-IFN-α (0.01 OD₆₀₀/ml) for 7 days. ⁵¹Cr-labeled bladder cancer T24, J82, and 5637 cells were then incubated with the PBMC at three different E/T cell ratios (40:1, 20:1, and 10:1) for 20 h. The % lysis of target cells by PBMC is presented as the mean of triplicate incubations

Fig. 3

rBCG-IFN-α exhibits dose-dependent induction of PBMC cytotoxicity against human bladder cancer cells. PBMC from a healthy individual were left alone or stimulated with four different doses (0.0001, 0.001, 0.01, and 0.1 OD₆₀₀/ml) of MV261 BCG or rBCG-IFN-α for 7 days. ⁵¹Cr-labeled bladder cancer T24, J82, and 5637 cells were then incubated with the PBMC at E/T cell ratio of 40:1 for 20 h. The % lysis of target cells by PBMC is presented as the mean of triplicate incubations. Numerical values represent the fold increase in cytotoxicity referring to the basal lysis by non-stimulated PBMC

Fig. 4

rBCG-IFN-α induces elevated IFN-γ and IL-2 production by PBMC. PBMC from a healthy individual were left alone or stimulated with MV261 BCG (0.01 OD₆₀₀/ml) or rBCG-IFN-α (0.01 OD₆₀₀/ml) for 3 days. To determine the effect of rIFN-α secreted by rBCG-IFN-α on IFN-γ and IL-2 production, PBMC were also stimulated with rBCG-IFN-α in the presence of a neutralizing antibody to IFN-α. Control sheep IgG was included for comparison. ELISA was performed to evaluate IFN-γ and IL-2 in culture supernatants. Data represents the mean ± SD of duplicate incubations. Numerical values represent the fold changes in cytokine productions referring to those induced by MV261 BCG

Fig. 5

Role of secreted rIFN-α as well as endogenously expressed IFN-γ and IL-2 in induction of PBMC cytotoxicity by rBCG-IFN-α. PBMC from a healthy individual were left alone or stimulated with MV261 BCG (0.01 OD₆₀₀/ml) or rBCG-IFN-α (0.01 OD₆₀₀/ml) for 7 days. To determine the effects of rIFN-α secreted by rBCG-IFN-α as well as endogenously expressed IFN-γ and IL-2 on cytotoxicity induction, PBMC were also stimulated with rBCG-IFN-α in the presence of neutralizing antibodies to IFN-α, IFN-γ or IL-2. Species and isotype matched control antibodies were included for comparison. ⁵¹Cr-labeled bladder cancer T24 and J82 cells were then incubated with the PBMC at E/T cell ratio of 40:1 for 20 h. The % lysis of target cells by PBMC is presented as the mean of triplicate incubations. Numerical values represent the fold increase in cytotoxicity referring to the basal lysis by non-stimulated PBMC

Fig. 6

CD56⁺CD8⁻ NK cells contribute to the enhanced PBMC cytotoxicity induced by rBCG-IFN-α. PBMC from a healthy individual were left alone or stimulated with MV261 BCG (0.01 OD₆₀₀/ml) or rBCG-IFN-α (0.01 OD₆₀₀/ml) for 7 days. Portions of BCG-stimulated PBMC were processed for CD56⁺ NK cell isolation by negative selection. Bladder cancer J82 cells were used as target cells, labeled with ⁵¹Cr, and incubated with unfractionated PBMC (left panel) or the isolated NK cells (right panel) at E/T cell ratio of 5:1 for 20 h. The % lysis of J82 cells is presented as the mean of triplicate incubations. Numerical values represent the fold increase in cytotoxicity referring to the basal lysis by non-stimulated PBMC (for left panel) or the lysis by NK cells isolated from MV261 BCG-stimulated PBMC (for right panel)

Fig. 7

CD8⁺ T cells contribute to the enhanced PBMC cytotoxicity induced by rBCG-IFN-α. PBMC from a healthy individual were left alone or stimulated with MV261 BCG (0.01 OD₆₀₀/ml) or rBCG-IFN-α (0.01 OD₆₀₀/ml) for 7 days. Portions of BCG-stimulated PBMC were processed for CD8⁺ T cell isolation by negative selection. Bladder cancer J82 cells were used as target cells, labeled with ⁵¹Cr, and incubated with unfractionated PBMC (left panel) or the isolated CD8⁺ T cells (right panel) at E/T cell ratio of 5:1 for 20 h. The % lysis of J82 cells is presented as the mean of triplicate incubations. Numerical values represent the fold increase in cytotoxicity referring to the basal lysis by non-stimulated PBMC (for left panel) or the lysis by CD8⁺ T cells isolated from MV261 BCG-stimulated PBMC (for right panel)