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Intravesical Mycobacterium bovis BCG therapy is effective against carcinoma in situ and as a prophylaxis against the recurrence of bladder cancer [1–5]. In addition to the direct anti-tumour effect, it is widely recognized that intravesical BCG therapy is more potent in preventing tumour recurrence than intravesical chemotherapy . Although intravesical BCG therapy is effective, it is not free from serious side effects (e.g. high fever, granulomatous prostatitis, pneumonitis, hepatitis, and BCG sepsis) . To avoid such unfavourable events, it is necessary to develop a more active and less toxic immunotherapeutic agent.
Although the BCG-cell wall skeleton (CWS) has long been investigated for this purpose, its clinical use is very limited because of difficulties relating to solubility and stability. To overcome these unfavourable physicochemical properties of the BCG-CWS preparation, we have applied octaarginine-modified liposomes (R8-liposomes) as a vector to transport BCG-CWS into the cytoplasm effectively. R8-liposomes were initially developed to transfer highly negatively charged DNA molecules into the cytoplasm by macropinocytosis [8–10]. R8-liposomes resemble an envelope-type virus and their surface are modified by anchored R8, a characteristic and efficient cell-penetrating peptide .
We have previously reported that R8-liposome-incorporating mycobacterial cell walls (R8-liposome-BCGCW) successfully attached to the surface of MBT-2 cells and were efficiently internalized into the cytoplasm within 1 h of co-incubation . Internalized BCG-CW was then distributed to the lysosome of the MBT-2 cells. Furthermore, R8-liposome-BCGCW has been shown to completely inhibit the growth of MBT-2 tumours in vivo. However, the mechanisms of the anti-tumour effect remain to be clarified, especially for the immune effector cells involved in R8-liposome-BCGCW-induced immunity.
In BCG immunotherapy, both the anti-tumour activity mediated by cytotoxic T lymphocytes (CTLs) and the anti-tumour activity mediated by an innate immune response in natural killer (NK) cells have a direct anti-tumour effect, as well as a prophylactic effect. The role of NK cells in this process was initially unclear , although more recent observations have shown that NK cells play a central role in the immune response that eradicates bladder cancer after intravesical instillation of BCG. In an in vivo mouse orthotopic bladder cancer model, Brandau et al.  noted that BCG-activated killer (BAK) cells are essential for a positive response to BCG. Furthermore, Suttmann et al.  reported the molecular mechanisms of BCG-immunotherapy involved in the process of cell-mediated cytotoxicity of both BAK cells and lymphokine-activated killer (LAK) cells against bladder cancer cells. NK cells were the major effector cell population of both BAK cells and LAK cells.
More recently, the natural killer group 2, member D (NKG2D) has been shown to be an important activating receptor present on the surface of NK cells. The NKG2D serves as a primary activation receptor, which is able to trigger cytotoxicity by itself. Previous studies have established that the expression of NKG2D ligands such as MHC class I-related chain A (MICA), MHC class I-related chain B (MICB) and a structurally distinct family of UL-16-binding protein (ULBP) proteins on tumours renders them susceptible to killing by NK cells [15–17]. However, the role of NKG2D and its ligands in BCG immunotherapy has not yet been investigated.
In the present study, we investigated whether BCG or R8-liposome-BCG-CWS treatment could induce the up-regulation of NKG2D ligands in human bladder cancer cell lines. In addition, we examined the susceptibility to LAK cells of cancer cells with or without R8-liposome-BCG-CWS treatment. The findings obtained show that the non-live bacterial agent, R8-liposome-BCG-CWS, can directly enhance the susceptibility of bladder cancer cells to LAK cells.
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We previously reported that R8-liposome-BCG-CW completely inhibited the growth of MBT-2 tumours in C3H/HeN mice, whereas BCG-CW alone did not. Animals vaccinated with a mixture of MBT-2 cells and R8-liposome-BCG-CW showed a significant inhibition of the growth of R8-liposome-BCG-CW pretreated MBT-2 cells . This suggests that, under conditions of immune tolerance from the host, bladder cancer cells usually can be recognized by the presence of BCG-related molecules in cancer cells.
Both the antigen-specific activity of CTLs and innate immune activity of NK cells are considered to be involved in BCG-induced anti-tumour immunity. Recent studies have reported that NK cells are essential for effective BCG immunotherapy. In addition, there is increasing evidence to support a significant role of NKG2D and its ligands in NK cell cytotoxicity. For example, human macrophages infected with either influenza or Sendai virus are known to have up-regulated MICB expression, which stimulates NKG2D-dependent interferon-γ release by NK cells .
The present study aimed to investigate the immune mechanism activated by a non-live bacterial agent, R8-liposome-BCG-CWS, using human bladder cancer cell lines and allogenic LAK cells. The results obtained clearly showed that the R8-liposome-BCG-CWS can directly enhance the susceptibility of bladder cancer cells to LAK cells, possibly through the up-regulation of NKG2D ligands on cancer cells. In the present study, we decided to use LAK cells rather than BAK cells as NK cell dominant effector cells in an attempt to establish a well known, non-live bacterial model. Generally, LAK cells represent a composite of CD3- NK cells and CD3+ T cells, and have the capacity to kill a variety of tumour cells and MHC class I-negative target cells. However, most importantly, activated NK cells, but not T cells, play a major role in LAK cell activity [29,30].
One of the cell lines used in the present study, T24, which is a well known line of human bladder cancer , expresses a markedly lower level of MHC class I molecules compared to normal cells. Hence, the T24 cells may be regulated by cells in a class I MHC molecule-unrelated manner, rather than by the class I MHC molecule-restricted CTLs.
In the present study, the induction of the surface NKG2D ligands MICB by R8-liposome-BCG-CWS treatment rendered T24 cells more susceptible to LAK cells. Higuch et al.  reported that cytotoxicity against T24 cells by live BCG-treated PBMCs containing mostly activated NKT cells, as well as some γδT and NK cells, was markedly inhibited by an anti-MICA/MICA specific antibody . Therefore, MICA/MICB molecules on cancer cells appear to be possible tumour cell ligands for BCG-activated innate immune cell recognition. MICB expression was increased on the T24 cells when they were cultured with R8-liposome-BCG-CWS, and ULBP1 expression was also increased on the RT-112 cells in the present study. MICB and ULBP1 expression were also increased on T24 and RT-112 co-cultured with live BCG. The reasons for the different expression of MICB and ULBP1 on these cells treated with R8-liposome-BCG-CWS remain to be clarified.
MICA and MICB expression is restricted or absent on normal tissues but is induced in response to various stresses and pathological conditions, including epithelial-derived tumours [33,34]. Additionally, the MICA/MICB protein has been detected in intestinal epithelial cells infected with Mycobacterium tuberculosis or Escherichia coli, and MICA has been shown on the surface of cytomegalovirus-infected fibroblasts [36,37]. ULBP ligands are expressed at the mRNA level in many tissues and cell lines, including the lung, heart, liver, testis, brain and colon, but cell surface expression by normal cells has not been detected [26,38]. These findings suggest that transcriptional regulation of individual NKG2D ligands may differ substantially between normal tissues and tumours.
Unfortunately, little is known about the regulation and expression of these molecules, except that they all share the common property of being inducible by cellular distress. Nevertheless, the available data clearly suggest that they are differentially expressed in normal tissues and in tumours from different origins. MICA and MICB proteins are frequently overexpressed in epithelial tumours of multiple origins but are less frequently expressed in haematopoietical malignancies [33,39–41]. This variable pattern of expression of NKG2D in cancer could be part of the immunoediting process , although it is more likely related to the fact that the expression of NKG2D ligands is controlled by different activation pathways. Up-regulation of MICA expression is considered to be mediated by the heat shock elements identified in the promoters of genes for MIC . By contrast, ULBP family members lack these motifs . Taken together, this suggest that NKG2D ligand diversity not only reflects the redundant expression of molecules with the same function, but also may indicate that these ligands have evolved to be differentially regulated in diverse physiological or pathological situations.
In conclusion, the results obtained in the present study show that R8-liposome-BCG-CWS up-regulated the expression of the ligand of NKG2D on bladder cancer cells. Accordingly, LAK cells recognized the bladder cancer cells and induced cytolysis of the cells. Therefore, LAK cells are crucial for the BCG-induced control of bladder tumour growth. In the future, the development of this non-live bacterial agent may provide a more active and less toxic tool as a substitute for live BCG in immunotherapy against bladder cancer.