Noriko Ikeda PhD, Japan BCG Laboratory, 3-1-5 Matsuyama, Kiyose-city, Tokyo 204-0022, Japan. Email: email@example.com
Abstract Background: The antitumor mechanisms of bacillus Calmette-Guérin (BCG) against bladder cancer is still unclear. We previously reported that BCG was internalized by and survived within murine bladder tumor cells (MBT-2) for at least 40 days. In the present study, we investigated the effect of BCG on the surface antigen expression of bladder tumor cells and the characteristics of these cells as antigen-presenting cells in vitro.
Methods: Surface antigen (major histocompatibility complex (MHC) Class II, CD1, CD80 and intercellular adhesion molecule-1 (ICAM-1)) expression on BCG-treated murine (MBT-2) and human (T-24, J82) bladder tumor cells were analyzed using flow cytometry. The production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) from murine lymphocytes sensitized with BCG or BCG-treated tumor cells were also investigated.
Results: The expressions of MHC Class II, CD1, CD80 and ICAM-1 were augmented in all of the bladder tumor cell lines used; however, they were augmented to varying degrees among the cell lines that were treated with live BCG. Heat-killed BCG had little or no effect. When murine lymph node cells sensitized with BCG or BCG-treated MBT-2 cells were cocultured with BCG-treated MBT-2 cells, significant amounts of IL-2 and IFN-γ were produced in the culture medium.
Conclusions: BCG induced the augmented expression of surface antigens, such as MHC Class II, CD1, CD80 and ICAM-1, of bladder tumor cells. Furthermore, BCG-treated MBT-2 cells could stimulate BCG-sensitized lymphocytes to produce IL-2 and IFN-γ. These results strongly suggest that bladder tumor cells gained the characteristics and functions of antigen-presenting cells (APC).
Intravesical instillation with bacillus Calmette-Guérin (BCG) has been established as the treatment of the first choice for carcinoma in situ (CIS) of the urinary bladder.1 This treatment is also known as the most effective way to prevent the recurrence of superficial bladder cancer after transurethral resection (TUR).1 BCG-instillation is also very effective in treating existing superficial bladder cancer.2 However, the mechanisms of the antitumor effects of BCG have not been completely elucidated, although immunological mechanisms have been assumed.3
Numerous animal experiments and some clinical trials regarding the immunotherapy of various cancers with BCG have shown that the most effective route of administration of BCG is direct intratumoral injection4 and that direct contact between BCG and the tumor cells is an essential initial step in the antitumor activities of BCG.5
In a previous paper, we reported that BCG attached firmly to murine urinary bladder tumor cells (MBT-2) when cocultured in vitro.6 Furthermore, we confirmed by electron microscopy that BCG not only attached to the surface of the tumor cells but also was internalized within the tumor cells and could survive for at least 40 days.7
In addition, we noticed that when bladder tumor cells were cocultured with BCG, tumor cells adhered much more strongly to the wall of the culture vessel than before (unpublished data). This suggests that some changes might occur on the surface molecules of the bladder tumor cells by the direct action of BCG.
From these previous observations, we assumed that bladder tumor cells might take on the characteristics of antigen-presenting cells (APC). To verify such a hypothesis, we studied both the expression of the surface molecules of bladder tumor cells induced by the treatment with BCG in vitro, and the ability of BCG-treated bladder tumor cells to stimulate BCG-sensitized lymphocytes to produce interleukin-2 (IL-2) and interferon-γ (IFN-γ).
C3H/HeN female mice were purchased from Charles River Japan, Inc. (Yokohama City, Japan) and used after at least 1 week of quarantine. Animals were fed with diet and tap water ad libitum.
Urinary bladder tumor cell lines
Three transitional cell carcinoma cell lines were used: MBT-2 of murine origin, and T-24 and J82 of human origin. J82 was kindly donated by Professor K Naito (Department of Urology, Yamaguchi University School of Medicine). These cells were propagated in RPMI-1640 medium (Nissui Pharm. Co. Ltd, Tokyo, Japan) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 0.03% (w/v) L-glutamine at 37°C in 5% CO2. Cells were harvested by treatment with 0.25% trypsin plus 0.02% EDTA in PBS.
Lyophilized preparations of BCG, Tokyo 172 (0.5 or 80 mg wet weight per ampoule [Japan BCG Laboratory, Tokyo, Japan], which contained 37.1 × 106 and 22.9 × 106 colony forming unit (CFU)/0.5 mg wet weight, respectively), were used.
Internalization of BCG by MBT-2 cells
Murine bladder tumor cells (MBT-2) cells were incubated overnight in the above-mentioned medium in a 25 cm2 culture flask (Iwaki Glass Co. Ltd, Tokyo, Japan) at 37°C in 5% CO2. The culture medium was then replaced with l mL RPMI-1640 containing 0.1 mg (wet weight) of BCG. After a 24 h incubation with BCG, the flask was thoroughly washed with PBS to remove the extracellular BCG and the medium was replaced with the same medium containing 100 μg/mL streptomycin to inhibit the extracellular growth of the remaining BCG. After 4 days in the culture, the tumor cells were harvested, washed with PBS and suspended in RPMI-1640 as 5 × 104 cells/mL. MBT-2 cells thus treated are referred to as MBT-2(BCG) hereafter.
Murine lymph-node lymphocytes
1Mice were inoculated with MBT-2(BCG) subcutaneously (1 × 106 cells/mouse). Tumor growth had been suppressed under these conditions. After six weeks, the mice were killed and a single cell suspension was prepared from the draining lymph nodes in the RPMI-1640 medium and culturing them in a plastic dish (90 mm in diameter) for 60 min at 37°C in 5% CO2. Non-adherent cells were collected, washed with PBS and prepared as a single cell suspension of 5 × 106/mL in a RPMI-1640 medium. These lymphocytes are referred to as L(MBT-2(BCG)).
2Mice were inoculated twice with BCG (5 × 106 CFU/mouse) or sheep red blood cells (SRC, 2 × 108/mouse) in both footpads at week 0 and 5. One week after the second injection, non-adherent cell suspensions were prepared from the draining lymph nodes as described above. They are referred to as L(BCG) and L(SRC), respectively.
Expression of the surface antigens on tumor cells
One mL of RPMI-1640 containing 5 × 104 of either MBT-2, T-24 or J82 cells was added into each well of 24-well cell culture plates and incubated at 37°C in 5% CO2. After 24 h, the culture medium was replaced with l mL of RPMI-1640 containing 0.5 mg (wet weight) BCG or heat-killed (100°C, 30 min) BCG. At 0, 40, 60 and 132 h, cells were harvested, washed with PBS and stained with FITC-conjugated antimouse IAk (MHC Class II) mAb, FITC-conjugated antimouse CD80 mAb, FITC-conjugated antimouse CD1d mAb and PE-conjugated antimouse ICAM-1 mAb (PharMingen, Hamburg, Germany) for MBT-2, and FITC-conjugated anti-HLA-DR (part of MHC Class II) mAb (Dako Japan Co. Kyoto, Japan), FITC-conjugated antihuman CD1b mAb, FITC-conjugated antihuman CD80 mAb and PE-conjugated antihuman ICAM-1 mAb (Nippon Becton Dickinson Co. Ltd, Tokyo, Japan) for T-24 and J82, at 4°C in the dark for 30 min. Then the cells were washed with PBS, fixed with 1 ml Lysing Solution ( × 10; Nippon Becton Dickinson Co. Ltd, Tokyo, Japan) at 4°C in the dark for 30 min, washed with PBS four times, suspended in PBS and subjected to flow cytometry using FACSCalibur (Nippon Becton Dickinson Co. Ltd, Tokyo, Japan). Data were analyzed with Cellquest software (Nippon Becton Dickinson Co. Ltd, Tokyo Japan).
Coculture of tumor cells with murine lymphocytes
Murine bladder tumor cells(BCG) were cultured in a 24-well plate (5 × 104/well), together with 5 × 106/well of non-adherent lymphocytes, either L(MBT-2(BCG)), L(BCG) or L(SRC), at 37°C in 5% CO2. The supernatants were collected after 3, 6, 9, 12 and 24 h of culture. As a control, MBT-2 cells, which were not treated with BCG, were cocultured with either L(MBT-2(BCG)), L(BCG) or L(SRC) together with 1 × 104 CFU/well of BCG. The amount of BCG used was equivalent to that internalized by MBT-2(BCG) cells. The supernatants were assayed for IL-2 and IFN-γ.
Anti-IAk mAb pretreatment of MBT-2(BCG) cells
Anti-IAk mAb (50 μL/well; PharMingen, Hamburg, Germany) was added to the MBT-2(BCG) cells (5 × 104/ well). After incubation at 37°C in 5% CO2 for 60 min, the cells were cocultured with L(BCG) (5 × 106/well) for 1, 2, 3 and 4 days and the supernatants were assayed for IL-2 and IFN-γ.
Concentrations of IL-2 and IFN-γ in culture supernatants were determined with ELISA kit (Endogen, Cambridge, USA).
Expressions of MHC Class II, CD1, CD80 and ICAM-1 by BCG-treated tumor cells
MBT-2 cells expressed IAk (MHC Class II), CD1d, CD80 and ICAM-1 at hardly detectable levels before BCG treatment, but their expression was augmented substantially by BCG treatment. Similarly in T-24, expression of HLA-DR (a part of MHC Class ΙΙ), CD1b, CD80 and ICAM-1 were augmented by BCG, although not as strong as MBT-2. J82 was different from these cells in that HLA-DR was clearly expressed and ICAM-1 was slightly expressed before BCG treatment. Their expression was further augmented by BCG, but augmentation of CD1b and CD80 expression was very weak. Killed BCG had little or no effect on these cell lines (Fig. 1).
IL-2 and IFN-γ production by murine lymphocytes
The culture supernatants of L(MBT-2(BCG)) cocultured with MBT-2(BCG) for varying lengths of time were assayed for IL-2 and IFN-γ as described in Methods. As shown in Fig. 2a, IL-2 could be detected at 3 h and its concentration increased with time and reached 60 pg/mL after 24 h. IFN-γ was detected at 9 h and its concentration increased linearly to reach 1000 pg/mL after 24 h. On the other hand, when L(MBT-2(BCG)) was cocultured with MBT-2 cells (not treated with BCG), the level of IL-2 was as low as 15 pg/mL and that of IFN-γ reached a maximum of 180 pg/mL. Neither cytokine was detected in the culture supernatant of lymphocytes alone and lymphocytes plus BCG. When L(BCG) was cocultured with MBT-2(BCG), production of IL-2 and IFN-γ could be detected, but neither cytokine was detectable in the supernatant of L(BCG) alone or of L(BCG) cocultured with MBT-2 or BCG (Fig. 2a). L(SRC) produced neither IL-2 nor IFN-γ when cocultured with MBT-2(BCG) (Fig. 2b).
Influence of anti-IAk mAb pretreatment
When MBT-2(BCG) cells were pretreated with anti-IAk mAb and then cocultured with L(BCG), neither IL-2 nor IFN-γ were detected (Fig. 2c).
In this study, we have focused on bladder-tumor-cell surface antigens (MHC Class II, CD1, CD80 and ICAM-1) that are considered important for antigen presentation. MHC Class II antigens serve as restriction elements for cells that present antigens to CD4+ helper T cells, and it has been reported that intravesical BCG treatment induces the expression of MHC Class II on normal urothelium and bladder tumor cells in patients.8–12
The CD1 family are class I-like antigen presentation molecules, and it is reported that glycolipids of mycobacteria, including BCG, serve as their ligands and activate NKT cells and may have an important role in the antitumor effect.13
Considered most important for T cell activation as a major ligand of CD28 is CD80 (B7-1), which is a major costimulatory molecule.14 ICAM-1 is known to play an important role in the immune response,15,16 and it was reported that either IFN-γ or TNF-α, cytokines induced by BCG, enhanced the expression of ICAM-1 on transitional cell carcinoma.17 An important role of ICAM-1 in the immune response against bladder cancer after intravesical BCG therapy was also suggested.18
In the bladder tumor cell lines used in this study, the expression of all these molecules which characterize APC were augmented by BCG treatment, although augmentations of CD1b and CD80 of J82 were not quite as strong. We report here, for the first time, that the expressions of MHC Class II, CD1, CD80 and ICAM-1 were augmented directly by BCG in vitro, not via the host immune mechanisms. When lymphocytes from BCG-immunized mice (L(BCG)) were cocultured with murine bladder tumor cells treated with BCG (MBT-2(BCG)), IL-2 and IFN-γ were produced in the culture supernatant, but lymphocytes from SRC-immunized mice (L(SRC)) did not produce the cytokines under the same conditions. These cytokine productions were abolished by the pretreatment of MBT-2(BCG) with anti-IAk mAb, thus suggesting that the lymphocytes recognized the BCG antigen presented with MHC Class II on MBT-2(BCG). Taken together, these results indicate that BCG-treated bladder tumor cells, by internalization of BCG, can function as APC. The ineffectiveness of killed BCG seems to imply that a prolonged supply of secretory antigens to the immune system by live BCG is crucial for the expression of antitumor activity, as in the case of protective immunity against tuberculosis. Recently, cells other than professional phagocytes, such as Langerhans cells,19 keratinocytes,20 B cells,21 T cells,22 endothelial cells23 and epithelial cells24,25 were also shown to have APC functions, so it seems natural that this is also the case in MBT-2, T-24 and J82 used in the present study. As for bladder tumor cells, Lattime et al. (1992) observed MHC Class II-restricted, BCG-specific antigen presentation in murine bladder carcinoma cell MB49. However, in their experiment, the expression of MHC Class II(IAb) on the tumor cell surface was induced by the IFN-γ treatment.26 We found that neither IFN-γ nor tumor necrosis factor (TNF)-α were produced when MBT-2 and BCG were cocultured (data not shown), so it is conceivable that the augmentation of surface molecule expression observed in our study is the consequence of the direct effect of (internalized) BCG alone. In vivo, however, this expression may be further augmented by IFN-γ or other cytokines produced by the host immune system. We also investigated the subsets of murine draining lymph node cells immunized with BCG and found that CD4+, CD8+, IAK+, NK1.1+ and NK+ subsets exist, but the production of IL-2 and IFN-γ was not induced by each subset alone when cocultured with MBT-2(BCG) in preliminary experiments (data not shown). Further study about this is required. Recently, it was reported that the transfection of the CD80 gene into tumor cells made the tumor cells recognizable by existing CTL.27 The induction of CD80 expression on the surface of tumor cells by BCG treatment might render the tumor cells more susceptible to the cytotoxicity by CTL and this might be a part of the mechanism of the antitumor effect of BCG. The elucidation of the nature of putative effector cell needs further investigation.
The authors wish to thank Dr K Naito for the kind donation of J82 and for useful discussions.