Cancer Immunology, Immunotherapy

, Volume 58, Issue 10, pp 1647–1655

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

Authors

  • Wujiang Liu
    • Department of UrologyUniversity of Iowa
  • Michael A. O’Donnell
    • Department of UrologyUniversity of Iowa
  • Xiaohong Chen
    • Department of UrologyUniversity of Iowa
  • Ruifa Han
    • Tainjin Institute of Urology
    • Department of UrologyUniversity of Iowa
Original Article

DOI: 10.1007/s00262-009-0673-z

Cite this article as:
Liu, W., O’Donnell, M.A., Chen, X. et al. Cancer Immunol Immunother (2009) 58: 1647. doi:10.1007/s00262-009-0673-z

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)-α 2B secreting recombinant (r) BCG (rBCG-IFN-α). We demonstrated that rBCG-IFN-α augmented T helper type 1 (Th1) cytokine IFN-γ production by PBMC. In this study, we further investigated whether rBCG-IFN-α 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-α or control MV261 BCG, and used as effector cells in 51Cr-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-α as well as endogenously expressed IFN-γ and IL-2 in inducing the cytotoxicity, PBMC were stimulated with rBCG-IFN-α in the presence of neutralizing antibodies to IFN-α, IFN-γ 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 51Cr-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-α further increased the PBMC cytotoxicity by up to 2-fold. Elevated production of IFN-γ and IL-2 by PBMC was observed after rBCG-IFN-α stimulation. Blockage of IFN-α, IFN-γ or IL-2 by neutralizing antibodies during rBCG-IFN-α 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-α with the former cell type being more predominant.

Conclusions

rBCG-IFN-α 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.

Keywords

BCGIFN-αBladder cancerCytotoxicity

Introduction

Intravesical instillation of BCG has been used to treat superficial transitional cell carcinoma (TCC) of the bladder for three decades [13]. It has been demonstrated to be more effective than localized chemotherapy and radiotherapy. Although the exact mechanisms by which BCG mediates antitumor immunity remains elusive, induction of localized cellular immunity and inflammation appears to be indispensable for successful BCG immunotherapy of bladder cancer. Following intravesical instillation of BCG, immune cell infiltration in the bladder wall has been observed including T cells, NK cells, and macrophages [47]. In addition, a large number of immune cells in patients’ voided urine have also been reported including neutrophils, T cells, and macrophages [8, 9]. Furthermore, a transient secretion of cytokines and chemokines in patients’ urine after intravesical instillation of BCG has been reported including IL-1, IL-2, IL-6, IL-10, IL-12, IL-18, IFN-γ, TNF-α, GM-CSF, MDC, MCP-1, MIP-1α, IP-10, and Eotaxin [1015].

It has been known that stimulation of human PBMC by viable BCG in vitro leads to the generation of a specialized cell population called BCG-activated killer (BAK) cells [1620]. BAK cells are a CD3CD8+CD56+ cell population whose cytotoxicity is not major histocompatibility complex (MHC)-restricted [1820]. BAK cells kill bladder cancer cells through the perforin-mediated lysis pathway [20] and are effective on lyzing NK cell-resistant bladder cancer cells [18, 19]. Macrophages and CD4+ T cells have been found to be indispensable for the induction of BAK cell killing activity but have no such activity by themselves [17, 19]. Th1 cytokines IFN-γ and IL-2 have also been found to be required for the induction of BAK cell cytotoxicity as neutralizing antibodies to these cytokines could inhibit BCG-induced cytotoxicity [17, 19]. BAK cells, together with lymphokine-activated killer (LAK) cells, a diverse population with NK or T cell phenotypes that are generated by IL-2 [21], have been suggested to be the major effector cells during intravesical BCG immunotherapy of bladder cancer [22].

Even though intravesical instillation of BCG demonstrates a favorable effect on treating bladder cancer, approximately 30% of patients do not respond to BCG and 50% of patients recur after BCG therapy [23, 24]. In addition, intravesical BCG therapy often has common side effects that are occasionally even life threatening [25]. To improve BCG immunotherapy, investigators have combined BCG with Th1-stimulating cytokines including IFN-α to treat bladder cancer [2629]. Previously, we used a reduced BCG dose plus IFN-α 2B to treat bladder cancer and observed a favorable tumor response for patients who had failed previous BCG therapy [29]. We also observed that addition of IFN-α 2B augmented BCG for IFN-γ induction both in vivo (as observed in the urine of bladder cancer patients receiving intravesical BCG) and in vitro (as observed in PBMC culture) [28, 30, 31]. The favorable effects of BCG plus IFN-α 2B have also been observed in others’ clinical trials and laboratory studies [26, 27, 32, 33]. Based on these observations, we developed human IFN-α 2B secreting rBCG (i.e. rBCG-IFN-α) [34]. We observed that rBCG-IFN-α was superior to wild-type BCG for inducing IFN-γ from PBMC in culture. In this study, we further investigated whether rBCG-IFN-α could also enhance cellular cytotoxicity against bladder cancer cells. We have observed that rBCG-IFN-α, due to its expression of rIFN-α, induces more potent PBMC cytotoxicity than control MV261 BCG. We have also observed that PBMC stimulated with rBCG-IFN-α produce significantly increased IFN-γ and IL-2 that are required for the induction of enhanced PBMC cytotoxicity by rBCG-IFN-α. Moreover, we have also observed that CD56+CD8 NK cells and CD8+ T cells both contribute to the enhanced PBMC cytotoxicity induced by rBCG-IFN-α. These results, together with our previous observations [34], suggest that rBCG-IFN-α may serve as an improved BCG agent that allows the use of lower and safer doses of BCG while at the same time preserving or even enhancing its effects on treating bladder cancer.

Materials and methods

BCG

Both rBCG-IFN-α and control MV261 BCG were developed previously from a BCG Pasteur strain [34, 35]. MV261 BCG contains an empty plasmid pMV261 and has been demonstrated to be similar to the commercial lyophilized BCG preparations in immunostimulation. Both rBCG-IFN-α and MV261 BCG contain the kanamycin resistance gene and were routinely cultured at 37°C in Middlebrook 7H9 Bacto broth (Difco, Detroit, MI) supplemented with 10% albumin dextrose catalase (ADC 5% BSA, 2% dextrose, and 0.85% NaCl), 0.05% Tween 80 (Sigma, St. Louis, MO), and 30 μg/ml of kanamycin. One unit of absorbance at 600 nm for the BCG cultures was calculated as 2.5 × 107 colony forming units (CFU)/ml (i.e. 1 OD600 = 2.5 × 107 CFU/ml). rBCG-IFN-α typically secreted approximately 30 IU/ml of rIFN-α in culture under the standardized conditions described previously [34].

Human bladder cancer cell lines

Human bladder cancer cell lines T24, J82, 5637, TCCSUP, and UMUC-3 were obtained from American Type Culture Collection (Rockville, MD). All cell lines were routinely cultured in RPMI 1640 medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS), l-glutamine (2 μM), penicillin-G (100 U/ml), and streptomycin (100 μg/ml) at 37°C in 5% CO2.

Effector cell preparation

In accordance with the approved clinical protocol at our institution, blood samples were collected from healthy donors. All donors gave their informed consent for this study. PBMC were isolated from heparinized blood samples by centrifugation over Ficoll-Paque (Pharmacia, Uppsala, Sweden). Viability of the isolated cells by trypan blue exclusion exceeded 95%. Cells were cultured in RPMI 1640 medium containing 10% FBS, 50 μM of β-mercaptoethnol, and 30 μg/ml of kanamycin at a density of 5 × 106 cells/ml in 6-well plates at 37°C in 5% CO2. To evaluate the effects of BCG on PBMC stimulation, cells were cultured in the absence or presence of indicated doses of MV261 BCG or rBCG-IFN-α. To determine the effect of rIFN-α secreted by rBCG-IFN-α on BCG stimulation of PBMC, a neutralizing antibody for human IFN-α (sheep polyclone) obtained from Schering (Kenilworth, NJ) was added at concentration of 10 μg/ml at the beginning of cell culturing. Since endogenously expressed IFN-γ and IL-2 have been reported to contribute to BCG-induced PBMC cytotoxicity [17, 19], neutralizing antibodies obtained from eBioscience (San Diego, CA) for IFN-γ (clone NIB42, mouse IgG1) and from R&D Systems (Minneapolis, MN) for IL-2 (clone 5334, mouse IgG1) were used at concentration of 20 μg/ml to determine the involvement of these cytokines in the induction of PBMC cytotoxicity by rBCG-IFN-α. Appropriate species and isotype matched control antibodies were included in parallel for comparison (mouse IgG1/clone 107.3 from BD PharMingen, San Diego, CA; sheep IgG from Sigma). After 7-day culturing, viable PBMC were collected via Ficoll-Paque centrifugation and used as effector cells in 51Cr (chromium)-release assays. In some experiments, culture supernatants were collected 3 days after BCG stimulation for analysis of PBMC IFN-γ and IL-2 production using enzyme-linked immunosorbent assay (ELISA). To define the effector cell types responsible for the enhancement of rBCG-IFN-α-mediated PBMC cytotoxicity, NK (CD56+), and CD8+ T cells were isolated from PBMC after BCG stimulation using the MACS negative selection kits (Miltenyi Biotec; Order # 130-092-657 for NK cells and 130-094-156 for CD8+ T cells) and then used as effector cells in 51Cr-release assays.

Cytotoxicity assay

Human bladder cancer cell lines were used as target cells. Cells (2 × 106) were labeled with 200 μCi of Na2[51Cr]O4 (Amersham Bioscience, Pittsburgh, PA) at 37°C in 5% CO2 for 90 min. After three washes, 51Cr-labeled target cells (1 × 104 cells/well) were mixed with effector cells at indicated effector-to-target (E/T) cell ratios in 200 μl of RPMI 1640 complete medium in 96-well U-bottom plates. Plates were centrifuged briefly to initiate cell contact and then incubated at 37°C in 5% CO2 for 20 h. After incubation, plates were centrifuged and the supernatants were collected for radioactive quantitation in a gamma counter. The percent lysis was calculated as the mean of triplicate wells according to the formula: % lysis = [(experimental cpm − spontaneous cpm)/(maximum cpm − spontaneous cpm)] × 100. The maximum 51Cr release was obtained by adding Triton X-100 to target cells (1% final concentration). The spontaneous 51Cr release was obtained from target cells incubated in the absence of effector cells and was, depending on experiments, 12–16% for T24 cells, 9–15% for J82 cells, 12–15% for 5637 cells, 10–16% for TCCSUP cells, and 8–14% for UMUC-3 cells, respectively.

ELISA analysis

Paired monoclonal capture and detecting antibodies were obtained from Endogen (Woburn, MA) for human IFN-γ (clones 2G1 and B133.5) and from BD PharMingen for human IL-2 (clones 5344 and B33-2). A sandwich format ELISA was performed for detecting these cytokines in PBMC culture supernatants according to the manufacturers’ instructions. Cytokine concentration was calculated in standard mass/volume format using standard curve derived from purified rIFN-γ (Endogen) or rIL-2 (Genzyme, Cambridge, MA) and expressed as mean ± standard deviation (SD) of duplicate wells.

Results

rBCG-IFN-α induces enhanced PBMC cytotoxicity

PBMC were prepared from healthy individuals and stimulated with control MV261 BCG or rBCG-IFN-α for 7 days. After stimulation, viable PBMC were collected, mixed with 51Cr-labeled human bladder cancer T24, J82, 5637, TCCSUP, or UMUC-3 cells at E/T ratio of 40:1, and incubated for 20 h. PBMC without BCG stimulation were included for comparison. After incubation, killing of the target cells by PBMC was determined by measuring 51Cr in culture supernatants. Figure 1 represents the cytotoxicity of PBMC from two subjects. Both PBMC preparations showed basal killing on all target cells tested. Stimulation with MV261 BCG increased PBMC cytotoxicity by 1.8- to 3-fold for one subject (Fig. 1, upper panel) and 2.1- to 4.2-fold for the other subject (Fig. 1, bottom panel). Stimulation with rBCG-IFN-α further increased PMBC cytotoxicity by up to 2-fold depending on target cells used (except UMUC-3 cells as targets for the second subject). Such BCG-induced PBMC cytotoxicity and the enhancement of cytotoxicity by rBCG-IFN-α were also observed at E/T ratios of 20:1 and 10:1 (Fig. 2).
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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

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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

rBCG-IFN-α exhibits dose-dependent induction of PBMC cytotoxicity

To determine the effect of BCG dose on inducing PBMC cytotoxicity, PBMC were stimulated with four different BCG doses (0.0001, 0.001, 0.01 or 0.1 OD600/ml) and used as effector cells in a 51Cr-release assay (Fig. 3). PBMC without BCG stimulation were included for comparison. T24, J82, and 5637 cells were used as target cells. Killing of target cells (J82 and 5637) was observed starting at BCG dose of 0.001 OD600/ml, although the killing was weak and showed no clear difference between MV261 BCG and rBCG-IFN-α for cytotoxicity induction. When increased BCG doses (0.01 and 0.1 OD600/ml) were used, enhanced killing of target cells was observed for both MV261 BCG and rBCG-IFN-α with the latter rBCG strain being more potent for cytotoxicity induction. At the BCG dose of 0.01 OD600/ml, T24 cell killing was increased by 3-fold for MV261 BCG and 4.8-fold for rBCG-IFN-α. J82 cell killing was increased by 2.1-fold for MV261 BCG and 4.2-fold for rBCG-IFN-α, and 5637 cell killing was increased by 3.8-fold for MV261 BCG and 4.2-fold for rBCG-IFN-α, respectively. This BCG dose (0.01 OD600/ml) appeared to be optimum for the induction of PBMC cytotoxicity under the experimental conditions, since the use of a high BCG dose (0.1 OD600/ml) led to a reduction of PBMC cytotoxicity toward J82 and 5637 cells. This reduction of PBMC cytotoxicity might result from the inherent toxicity of BCG to PBMC (due to the presence of a high BCG dose). A similar adverse effect of high BCG dose (0.1 OD600/ml) on induction of IFN-γ from PBMC was also observed previously [31].
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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

Role of endogenously expressed IFN-γ and IL-2 in induction of PBMC cytotoxicity by rBCG-IFN-α

It has been reported that IFN-γ and IL-2 play an important role in BCG-induced PBMC cytotoxicity [17, 19]. In this study, we observed that PBMC stimulated with rBCG-IFN-α expressed substantially higher IFN-γ and IL-2 in culture (Fig. 4). Compared with MV261 BCG, rBCG-IFN-α increased PBMC IFN-γ production by 6.8-fold and IL-2 production by 1.9-fold, respectively. The elevated cytokine productions could be reduced or abolished by a neutralizing antibody to IFN-α, indicating that rBCG-IFN-α enhances BCG stimulation of PBMC via its secretion of rIFN-α.
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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

To determine the role of secreted rIFN-α as well as the role of endogenously expressed IFN-γ and IL-2 in cytotoxicity induction, PBMC were stimulated with rBCG-IFN-α in the presence of neutralizing antibodies to IFN-α, IFN-γ or IL-2 (Fig. 5). Compared with non-stimulated PBMC, PBMC stimulated with MV261 BCG showed increased cytotoxicity by 2.9-fold for T24 cells and 2.2-fold for J82 cells. Compared with MV261 BCG, rBCG-IFN-α further enhanced the cytotoxicity by 1.9-fold for T24 cells and 1.7-fold for J82 cells, respectively. Addition of a neutralizing antibody to IFN-α eliminated the effect of rIFN-α on BCG induction of cytotoxicity. Addition of neutralizing antibodies to IFN-γ or IL-2 markedly reduced the cytotoxicity toward the same target cells. Compared with IFN-γ, IL-2 appeared to be more crucial since neutralization of this cytokine nearly abolished all BCG effects on inducing PBMC cytotoxicity.
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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

NK cells and CD8+ T cells contribute to the enhanced PBMC cytotoxicity induced by rBCG-IFN-α

To define the effector cell types responsible for the enhancement of rBCG-IFN-α-mediated PBMC cytotoxicity, we isolated NK and CD8+ T cells from PBMC after BCG stimulation using the MACS negative selection kits. The isolated cells showed a CD56+CD8 phenotype for NK cells and a CD56CD8+ phenotype for CD8+ T cells with 95% purity for both (data not shown). As observed previously, unfractionated PBMC stimulated with rBCG-IFN-α showed enhanced cytotoxicity against bladder cancer J82 cells compared to those stimulated with control MV261 BCG (Figs. 6 and 7, left panels; E/T ratio of 5:1; PBMC from two individuals). Accordingly, compared to NK cells isolated from MV261 BCG-stimulated PBMC, NK cells isolated from rBCG-IFN-α-stimulated PBMC showed increased cytotoxicity by 2.1-fold at E/T ratio of 5:1 (Fig. 6, right panel). Similarly, CD8+ T cells isolated from rBCG-IFN-α-stimulated PBMC also showed an increase (2.2-fold at E/T ratio of 5:1) in cytotoxicity compared to CD8+ T cells isolated from MV261 BCG-stimulated PBMC (Fig. 7, right panel). However, despite these changes, CD8+ T cells appeared to play a minimal or small role relative to NK cells in BCG-induced PBMC cytotoxicity even after stimulation with rBCG-IFN-α.
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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)

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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)

Discussion

Intravesical BCG immunotherapy has been successfully used for the treatment of bladder TCC for three decades [13]. However, the current BCG therapy is empirical and needs to be improved in both its efficacy and side effects. Since a proper induction of localized cellular immunity is considered to be necessary for successful BCG immunotherapy of bladder cancer, investigators have focused on enhancing BCG induction of cellular immune responses. Previously, we developed human IFN-α 2B secreting rBCG (i.e. rBCG-IFN-α) and found that this rBCG strain was superior to wild-type BCG for inducing IFN-γ production by PBMC [31, 34]. In this study, we have further observed that this rBCG strain induces enhanced PBMC cytotoxicity against human bladder cancer cells in vitro. We have also observed that PBMC stimulated with rBCG-IFN-α produce elevated IFN-γ and IL-2, which correlates with and is required for the induction of enhanced PBMC cytotoxicity by this rBCG. Moreover, we have also observed that both CD56+CD8 NK cells and CD8+ T cells contribute to the enhanced PBMC cytotoxicity induced by rBCG-IFN-α. These observations suggest that rBCG-IFN-α may serve as an improved BCG agent for the treatment of bladder cancer.

We have observed that PBMC (non-stimulated) possess basal cytolytic activities on all bladder cancer cell lines tested. This basal killing was largely attributed to their natural NK cell activity, since these PBMC could efficiently kill K-562 cells, a NK cell-susceptible human erythroleukemia cell line, prior to BCG stimulation (data not shown). This basal killing observation was consistent with those reported by others that freshly isolated PBMC naturally acquire NK cell killing activity on human bladder cancer cells [18, 19]. Stimulation of PBMC with MV261 BCG increased the killing of target cells by 1.8- to 4.2-fold. Stimulation with rBCG-IFN-α further increased the PBMC cytotoxicity by up to 2-fold. Although BCG was a potent agent for inducing PBMC cytotoxicity, this induction of cytotoxicity was BCG dose limited since a high BCG dose (0.1 OD600/ml) resulted in a reduction of PBMC cytotoxicity against certain target cells.

BCG induction of PBMC cytotoxicity involves the participation of multiple immune cells and Th1 cytokines. BAK cells (CD3CD8+CD56+) are a NK cell subpopulation and are derived from PBMC after BCG stimulation [1620, 36]. Macrophages and CD4+ T cells have been found to be required for BAK cell induction [17, 19]. Endogenously expressed IFN-γ and IL-2 have also been found to be indispensable for BAK cell induction [17, 19]. In addition to BAK cells, BCG can also act on NK cells and possibly on NKT and γ/δ+ T cells as well to stimulate their cytotoxicity [3739]. Furthermore, IL-2 expression by PBMC after BCG stimulation may lead to the generation of LAK cells that are more potent cytotoxic cells than BAK cells for killing bladder cancer cells [16, 18, 19]. These observations indicate the complexity of the involvement of multiple cellular and cytokine components in determining the overall BCG-induced PBMC cytotoxicity.

Compared with MV261 BCG, rBCG-IFN-α induced up to 2-fold higher PBMC cytotoxicity toward bladder cancer cells. This enhanced cytotoxicity resulted from the expression of rIFN-α by rBCG-IFN-α, since neutralization of this cytokine during rBCG stimulation reduced the cytotoxicity to the levels induced by control MV261 BCG. In addition to its antiviral activity, IFN-α has been reported to mediate a variety of immunoregulatory effects [40]. Based on the current understanding, macrophages serve as a first line of defense in anti-mycobacterial infection and produce IL-12, IFN-α, and other pro-inflammatory cytokines after activation [38, 41, 42]. These macrophage-derived cytokines act as primary initiators of a cellular immune response and cause T cells and NK cells to produce IFN-γ [38, 4244]. Activated T cells also produce IL-2 and other cytokines [45]. Thus, supplemental IFN-α by rBCG-IFN-α could facilitate an early induction of cellular immunity in response to BCG stimulation. These modulatory effects of rBCG-IFN-α may lead to more effective BAK and LAK cell induction as well as NK, NKT and T cell activation, resulting in the observed enhanced PBMC cytotoxicity against bladder cancer cells. Indeed, we have observed that both CD56+CD8 NK cells and CD8+ T cells play a role in the enhancement of rBCG-IFN-α-mediated PBMC cytotoxicity, although the latter cell type appears to be less important in BCG induction of PBMC cytotoxicity as reported previously [18, 19].

Current intravesical BCG immunotherapy of bladder cancer includes weekly instillation of BCG at approximately 1.8 × 108 CFU/dose for at least 6 weeks. This repeated application of high doses of BCG is believed to be a major cause for the commonly seen BCG-related side effects. Previously, we observed that instillation of reduced BCG dose (1/3 or 1/10 of standard BCG dose) plus IFN-α 2B resulted in favorable tumor responses in bladder cancer patients who had failed previous BCG therapy [29]. We also observed that instillation of reduced BCG doses plus IFN-α 2B induced urinary IFN-γ at the levels similar to those induced by standard dose of BCG [28]. Intravesical instillation of IFN-α 2B has been observed to increase bladder infiltration by NK and T cells and be associated with a systemic activation of these cells [4648]. In addition to immune cells, IFN-α 2B also has direct effects on bladder cancer cells including inhibition of bladder cancer cell proliferation [33, 48], inhibition of bladder tumor angiogenesis [49], and up-regulation of MHC class I expression on bladder cancer cells [48]. Thus, rBCG-IFN-α that possesses the BCG and IFN-α 2B dual biological activities may serve as an improved BCG agent that allows the use of lower and safer doses of BCG for the treatment of bladder cancer.

Acknowledgments

This work was supported in part by grants from the National Institutes of Health (RO1DK66079) and Department of Defense (W81XWH-04-1-0070). We would like to thank Mitchell L. Rotman for helping edit this manuscript.

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