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In the 1980s, subgroups of patients with T1G3 bladder tumours (BTs) and/or carcinoma in situ had a significantly higher progression rate to carcinoma invading the bladder muscle and a poorer long-term survival, which led to the concept of ‘high-risk’ non-muscle-invasive BT. 
In the past, patients with high-risk BTs were candidates for undergoing total cystectomy.  However, recently, the accepted standard treatment for high-risk BTs consists of transurethral resection, removing all visible lesions, followed by intravesical therapy, in particular, intravesical instillation of BCG, now recognized as the best treatment for high-risk BT [3,4]. Although BCG was employed to treat many kinds of solid tumours in the 1970s, its usefulness has proven to be limited in most tumours [5,6]. BCG instillation therapy was first introduced for BT by Morales in 1976 , and its usefulness was confirmed in numerous reports [3,4]. Thus, BT is one of the few tumours for which immunotherapy is effective.
Two novel IL-12-related cytokines, IL-23 and IL-27, were recently identified [7,8]. IL-23 is composed of p19 and the p40 subunit of IL-12. It is mainly secreted by activated dendritic cells and monocytes/macrophages. IL-23 induces the proliferation of memory Th1 CD4+ T cells and the production of interferon-γ (IFN-γ) from activated T cells. IL-23 and IL-12 play an essential role in linking innate and adaptive immunity. Because IL-12 is known to have strong antitumour effects in vivo, IL-23 may also possess anti-tumour activity [9–12].
In urology, systemic immunotherapy using IL-2 and IFN-γ is applied for RCC. In particular, high-dose IL-2 therapy can result in long-term complete remission in a minority of metastatic RCC patients. However, severe side effects such as fatal vascular leak syndrome limit the application of this therapy. To overcome this problem, immunogene therapy is a possible strategy. Introducing a cytokine gene into tumour cells can maintain a high concentration of cytokine at the tumour site, while keeping systemic levels low, thus diminishing the systemic side effects. We previously reported antitumour activities using different cytokines, such as IL-2 [13,14], IL-12, IL-18 [15–17] and IL-21 [18,19].
In the present study, we evaluated the antitumour effects of IL-23 gene transfer into a mouse bladder carcinoma (MBT2) cell line. We also investigated the mechanisms mediating this antitumour effect and the possibility of developing a cancer vaccine for future clinical use.
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IL-23 is a member of the IL-12 family of heterodimeric cytokines, composed of the p40 subunit shared with IL-12 and the IL-23-specific p19 subunit. IL-23 is normally secreted by activated macrophages and dendritic cells and stimulates proliferation of and IFN-γ production by Th1 CD4+ T cells. IL-23 plays important roles in Th1 immunity similar to IL-12. We have previously established that antitumour activities occur by using IL-12 gene transduction into RCC cell lines. Cells transduced with the IL-12 gene were completely rejected in syngeneic mice but not in athymic nude mice. Cells secreting IL-12 can also act as tumour vaccines. CD8+ T cells and NK cells were essential for this tumour vaccine effect. In the present study, we introduced IL-23 gene, which has a heterodimeric structure similar to that of IL-12, into a murine BT cell line and evaluated the antitumour activities of IL-23 in tumour immunity.
In previous experiments using IL-12, we transfected two expression vectors encoding p40 and p35 simultaneously into RCC cell lines. In the present study using IL-23, we also first applied the same cotransfection methods using expression vectors encoding p40 and p19. However, we were unable to create transfectants expressing the heterodimeric form of IL-23 using this approach. Therefore, we used an expression vector encoding single chain IL-23 composed of the p40 chain, a (Gly4Ser)3 linker, and the p19 chain [20,21]. By using this system, we succeeded in constructing transfectants which secreted the heterodimeric form of IL-23, as detected by sandwich ELISA.
The proliferation rate of clones of MBT2/IL-23 in vitro was not different from that of the parental cells (data not shown). However, MBT2/IL-23 tumours were completely rejected in vivo. In a previous study, we introduced IL-23 and IL-27 genes into a poorly immunogenic melanoma (B16F10) using the same expression vector and evaluated their antitumour activities . Interestingly, IL-23-transfected B16F10 (B16/IL-23) tumours exhibited almost the same growth curve as B16F10 parental tumour until about 20 days after tumour injection, but then showed growth inhibition or even regression. This phenomenon had been observed previously in studies by other investigators [10,12]. In contrast, the MBT2/IL-23 tumour exhibited significant retardation of growth from the early stage. In fact, some authors have reported that expression of IL-23 gene in other tumour cells could cause tumour regression from the early stage in animal tumour models [9,11].
Additionally, the syngeneic C3H mice that had rejected MBT2/IL-23 rejected a subsequent challenge with parental MBT2 cells, suggesting that systemic immunity was conferred by inoculation of MBT2/IL-23. However, with regard to clinical applications, live tumour cell vaccines will not be acceptable. Therefore, we investigated the feasibility of tumour vaccine therapy using MBT2/IL-23 treated with MMC. Treatment with MMC completely inhibits proliferation of tumour cells, while preserving cytokine secretion of MBT2/IL-23 (data not shown). We showed that MMC-treated MBT2/IL-23 cell injection could inhibit the growth of parental MBT2 distant sites, suggesting that these cells could be used as a tumour vaccine. In a previous study, we reported that B16/IL-23-vaccinated mice showed significant protective immunity against B16F10 parental tumour cells. Because the tumour vaccine effect shown in this study was also observed in previous investigations , we suggest that this may be a useful approach to vaccine therapy in clinical applications.
We investigated the prophylactic vaccine effect combined with anti-CD25 mAb for depletion of regulatory T cells (Tregs). Interestingly, in this setting, the protective immunity against MBT2 parental tumour endowed by MBT2/IL-23 vaccination was markedly enhanced. The immune system has established an elaborate network of central and peripheral tolerance mechanisms to discriminate between self and non-self. An important component of this network are the Tregs, which mediate self-tolerance and immune homeostasis by acting in a dominant cell-extrinsic manner to regulate immune functions[23–25]. Two sets of observations also implicate Tregs in suppression of tumour immunity. First, Tregs accumulation at tumour sites in patients with cancer correlated with disease progression . Second, elimination of Tregs in mice by treatment with a CD25 antibody enhances the immune-mediated rejection of tumours [27,28] and synergizes with vaccination protocols [29,30]. Therefore we analysed the distribution of the CD4+ CD25+ population and the CD4+ Foxp3+ population of regional lymph nodes in a tumour vaccine model. As expected, the CD4+CD25+ and CD4+ Foxp3 population is notably smaller in the mice that had been treated with anti CD25+ mAb. We also expected that the CD4+ CD25+ and CD4+ Foxp3 population would be larger in the mice bearing MBT2, but smaller in the mice vaccinated with MBT2/IL23. However, the populations were similar to those in non-treated mice. In a recent clinical trial, depletion of Tregs in patients with RCC using an IL-2/diphtheria toxin fusion product (ONTAK) led to enhanced vaccine-induced antitumour immune responses . Thus, depletion of Tregs could represent an important addition to cancer immunotherapy.
For clinical applications, these results suggest that IL-23 has great potency in a cytokine-based tumour vaccine and that anti-CD25 treatment could act as an efficient adjuvant in an IL-23-based tumour vaccination.
Mechanisms underlying the antitumour effects observed were investigated in syngeneic mice by depleting CD8 or CD4 T cells, or NK cells using appropriate antibodies. The antitumour effect of MBT2/IL-23 was partially inhibited in mice depleted of CD4 T cells and NK cells at the initial phase, but was almost completely abrogated in mice depleted of CD8 T cells. Using immunohistochemistory, we confirmed that CD8 T cells infiltrated the tumour.
A similar antitumour response has been reported in studies using other tumour types. Wang et al. . reported that the expression of IL-23 in CT26 murine colon carcinoma cells resulted in antitumour effects that were mediated through CD8+T cells secreting IFN-γ. Lo et al.  also reported that antitumour activity was mediated through CD8+ T cells but not CD4+ T cells or NK cells. In fact, it seems to be hard to define the mechanism of tumour shrinkage engineered to secrete cytokines. In terms of this complication, Musiani et al.  suggested that continual crosstalk between leukocytes and lymphocytes plays a much important role than the single effector population which has been assumed to be stimulated by secreted cytokine. They reconstructed the cell events that take place at the site of the challenge with tumour cells engineered with many kinds of cytokine genes as determined by sequential histological, immunocytochemical and ultrastructural observations. They found that granulocytes induced by T cells at the tumour site play a crucial role in the rejection mechanism. This unexpected and major role for granulocytes seems to be key to understanding the mechanism.
In conclusion, we observed that CD8+ T cells are required for the IL-23-mediated antitumour effects described here. Moreover, MMC-treated IL-23-secreting MBT2 cells acted as a tumour vaccine for parental MBT2 rejection. We found that this vaccine effect was enhanced by combining it with CD25 antibody treatment. We will continue our vaccination studies in preparation for future clinical application.