Recombinant bacillus Calmette-Guérin (BCG) expressing interferon-alpha 2B enhances human mononuclear cell cytotoxicity against bladder cancer cell lines in vitro
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- Liu, W., O’Donnell, M.A., Chen, X. et al. Cancer Immunol Immunother (2009) 58: 1647. doi:10.1007/s00262-009-0673-z
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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.
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.
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.
Intravesical instillation of BCG has been used to treat superficial transitional cell carcinoma (TCC) of the bladder for three decades [1–3]. 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 [4–7]. 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 [10–15].
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 [16–20]. BAK cells are a CD3−CD8+CD56+ cell population whose cytotoxicity is not major histocompatibility complex (MHC)-restricted [18–20]. BAK cells kill bladder cancer cells through the perforin-mediated lysis pathway  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 , have been suggested to be the major effector cells during intravesical BCG immunotherapy of bladder cancer .
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 . To improve BCG immunotherapy, investigators have combined BCG with Th1-stimulating cytokines including IFN-α to treat bladder cancer [26–29]. 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 . 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) . 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 , 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
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 .
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.
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.
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.
rBCG-IFN-α induces enhanced PBMC cytotoxicity
rBCG-IFN-α exhibits dose-dependent induction of PBMC cytotoxicity
Role of endogenously expressed IFN-γ and IL-2 in induction of PBMC cytotoxicity by rBCG-IFN-α
NK cells and CD8+ T cells contribute to the enhanced PBMC cytotoxicity induced by rBCG-IFN-α
Intravesical BCG immunotherapy has been successfully used for the treatment of bladder TCC for three decades [1–3]. 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 (CD3−CD8+CD56+) are a NK cell subpopulation and are derived from PBMC after BCG stimulation [16–20, 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 [37–39]. 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 . 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, 42–44]. Activated T cells also produce IL-2 and other cytokines . 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 . 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 . 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 [46–48]. 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 , and up-regulation of MHC class I expression on bladder cancer cells . 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.
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.