Since the first report of successful treatment by Morales and associates [1], bacillus Calmette-Guérin (BCG) immunotherapy has been the recommended standard treatment for high grade non-muscle-invasive bladder cancer (NMIBC) [2]. The results of recent studies have suggested that not only innate immunity but also acquired immunity plays an important role in this therapy. In contrast, cisplatin-based chemotherapy, e.g., MVAC (methotrexate/vinblastine/doxorubicin/cisplatin), GC (gemcitabine/cisplatin), etc. is a standard systemic therapy for muscle-invasive or metastatic bladder cancer, since urothelial cancer (UC) is chemosensitive. MVAC and GC treatments both result in prolonged survival of up to 14.8 and 13.8 months, respectively, also with long-term followup [3-6]. Several meta-analyses have demonstrated that neoadjuvant cisplatin-containing combination chemotherapy improves overall survival by 5%–7% at 5 years, irrespective of the type of definitive treatment used, whereas neither randomized trials nor meta-analyses have provided sufficient data to support the routine use of adjuvant chemotherapy [7-10]. However, up to 50% of patients are unfit for cisplatin-containing chemotherapy, either due to poor performance status and/or impaired renal function, or to comorbidity that prohibits high-volume hydration [11,12]. Non-platinum combination chemotherapy has produced substantial responses in first- and second-line use, but has not been tested against standard chemotherapy in fit patients or in a purely unfit patient group. There are two main currents for a new paradigm in immunotherapy for UC: (1) enhancement of BCG nonspecific immunotherapy with an adjuvant immunomodulator, e.g., interferon (IFN), granulocyte macrophage colony-stimulating factor (GM-CSF) or specific vaccination for patients with NMIBC, and (2) cancer-specific activation of T cells for patients with muscle-invasive or metastatic UC, e.g., peptide vaccination and dendritic vaccination. In this review, we focus on the progress in immunotherapy for UC and the escape mechanism of cancer cells from the immune system based on data from basic, translational and clinical studies, as well as the down-regulation of major histocompatibility complex (MHC) class I molecules.

Active immunotherapy has the potential to stimulate the immune system, particularly cellular adaptive immunity of the patient to attack the tumor cells. To successfully become recognized as malignant, the response has to be strengthened by vaccination strategies so that target cells can be recognized and eliminated effectively by effector cells such as cytotoxic T lymphocytes (CTLs) [96]. Tumor-infiltrating lymphocytes (TILs) in patients with melanoma, colon cancer and ovarian cancer are associated with favorable clinical outcomes [97]. Several studies of breast cancer indicated that immunological processes, including the development of TILs and disappearance of regulatory T cells, may influence the clinical response to neoadjuvant chemotherapy [98,99]. Lipponen et al. [100] investigated the prognostic value of TILs in a cohort of 514 patients with bladder cancer, and reported that a large density of TILs predicted an unfavorable prognosis in papillary tumors, whereas it was a sign of good prognosis in nodular and T3-T4 tumors. Sharma et al. [101] showed that patients with T2-T4 disease and higher numbers of CD8⁺ TILs had better disease-free survival and overall survival than those with similar-stage bladder cancer and fewer intratumoral CD8⁺ TILs. Furthermore, Marits et al. [102] demonstrated that the sentinel node lymphocytes from patients who underwent radical cystectomy for bladder cancer displayed a retained immunological function upon in vitro restimulation with the tumor antigen, and proposed the use of those lymphocytes as the primary source of immune effector cells for immunotherapy. These data support the idea that active immunotherapy through the augmented T-cell response can have great potential for the treatment of advanced bladder cancer.

Active immunotherapeutic strategies include cancer vaccinations using autologous or allogeneic whole tumor cells or tumor lysates, tumor-derived peptides, gene-modified autologous or allogeneic tumor cells, viral vectors carrying genes encoding for tumor antigens, naked DNA plasmids encoding for tumor antigens, autologous APCs presenting tumor antigens, etc [103]. To date, however, there have been few phase I or II clinical trials of active immunotherapy for bladder cancer published (Table 1).

Sharma et al. evaluated the safety and immunogenicity of a recombinant NY-ESO-1 protein vaccine, which was administered with GM-CSF and BCG as immunologic adjuvants, in a cohort of UC patients whose cancer cells expressed NY-ESO-1, a cancer/testis antigen [104]. Most of the patients exhibited NY-ESO-1-specific antibody and CD4⁺ T cell responses, but CD8⁺ T cell responses were observed in one patient. All patients tolerated the vaccine therapy well and completed all scheduled administrations without any difficulties, although one patient had a grade 2 injection-site reaction consisting of induration and erythema. Honma et al. [105] reported the results of a phase I study of survivin peptide vaccination for advanced UC. HLA-A24-restricted survivin-2B peptide was subcutaneously administered with incomplete Freund's adjuvant. Survivin, a member of the inhibitor of apoptosis protein family, is overexpressed in 88% of bladder cancers, but not in normal adult tissues [106]. This vaccination targeting survivin-expressing cells induced epitope-specific CTLs in five of nine patients, and provided tumor reduction and long-term stable disease in two patients. The vaccination was well tolerated in all patients without severe adverse events (AEs). Sherif et al. [107] harvested T cells from the nearest draining nodes at cystectomy, and reinfused the autologous T-helper cells after in vitro culture and expansion. Tumor-specific-T-helper cells were obtained and reinfused in six of 12 patients without major AEs, but the clinical benefit has not yet been evaluated. Malmström et al. [108] carried out a phase I/II study of intravesical administration of adenoviral vectors expressing a CD40 ligand (AdCD40L). CD40L is a potent Th1 immune stimulator that elicits a robust antitumor response. They reported that gene transfer was detected in biopsies, bladders were heavily infiltrated with T cells, and that therapy was well tolerated with low-grade local pain alone. This study suggests that local AdCD40L therapy is potentially effective for bladder cancer, but there are no data on clinical outcomes. Matsumoto et al. [109] performed a phase I study of personalized peptide vaccination. Cocktail peptides that might induce HLA-A24-restricted or HLA-A2-restricted and tumor-specific CTL activity were administered subcutaneously. Epitope-specific T cell and antibody responses were observed in eight of 10 patients with one complete response, one partial response and two cases of stable disease. The vaccination was well tolerated with no severe AEs.

The results of the phase I and I/II clinical trials suggest that active immunotherapy for UC induces immune and antitumor responses without severe AEs. However, further clinical studies including phases II and III are needed to determine whether such therapies really provide clinical benefits for patients with UC.

The rationale for studies of active immunotherapy is supported by strong cellular immune responses when introducing cancer-specific CTLs from patients. Immune cells must be activated at the tumor site to manifest appropriate effector mechanisms such as direct lysis or cytokine secretion capable of causing tumor destruction, in addition to the need for a sufficient number of the cells. However, there have been many cases in which no clinical response was achieved regardless of good immune responses in immunomonitoring assays. Therefore, tumor cell factors should be evaluated for development of the clinical responses in cancer immunotherapy.

Human leukocyte antigen (HLA), MHC in humans, class I has a critical role in the recognition and lysis of tumor cells by CTLs, and defects in antigen presentation could allow tumors to escape killing by CTLs [110]. Down-regulation of HLA class I in cancer cells is disadvantageous for presentation of a cancer antigen and its peptide to the patient's immune system [111]. HLA class I is down-regulated in approximately 30% of bladder cancers [106,112]. This is a significant prognostic factor in patients undergoing BCG immunotherapy [47] and radical cystectomy for treatment of muscle-invasive disease [112]. It is suggested that tumor cells with down-regulated HLA class I escape from T cell recognition and that, in such cases, BCG immunotherapy is not effective for patients with bladder cancer. In an animal model using chemically induced bladder cancer, the cytotoxic activity of T cells from rats on tumor cells with low MHC class I expression was decreased as compared with tumor cells with high MHC-I expression [113]. Another recent study using surgical specimens of human bladder cancer demonstrated the changes in HLA class I expression during tumor development [114]. This suggests that immunotherapy stimulates an additional strong cycle of immunoselection, eliminating cells with low HLA and/or changes in it, but cancer cells with more profound HLA class I alterations can escape the immune system and develop recurrent tumors. Thus, it is essential not only to analyze the mechanisms of escape from the immune system but also to develop an immunotherapeutic approach targeting the inhibition of the escape, e.g., upregulation of HLA class I on cancer cells [115].

Regulatory T cells (Tregs) are a population of T cells that regulate the activation of both effector/helper T and B cells. Many studies with cancer patients have demonstrated that the prevalence of Tregs is significantly high in cancerous lesions as compared to healthy controls, and the percentage of Tregs among TILs positively correlates with a significantly lower survival rate [116]. In contrast, Winerdal et al. [117] reported that the infiltration of Tregs in bladder cancer was also associated with improved prognosis. Thus the immune escape mechanism through Treg is still controversial in bladder cancer.

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immunosuppressive cells expressing a variety of surface markers such as CD11c⁺, CD11b⁺, CD33⁺, CD34⁺ and CD15⁺. The frequency of these cells also positively correlates with the incidence of recurrence or metastatic disease in patients [116]. Eruslanov et al. [118] have demonstrated that highly activated inflammatory myeloid cells inhibiting in vitro T cell proliferation through induction of Tregs in peripheral blood and tumor tissues from patients with bladder cancer represent a source of multiple chemokines/cytokines and may contribute to inflammation and immune dysfunction in bladder cancer. Further studies are needed to confirm these results, examine the underlying mechanisms in detail, and investigate the prognostic impact on survival.

Various studies have indicated that immunotherapy can eliminate bladder cancer cells, but it is not yet capable of curing bladder cancer, and other therapeutic modalities such as surgery and chemotherapy are still required for this purpose. BCG immunotherapy is effective for NMIBC but does not always control its recurrence and is ineffective for MIBC, indicating that BCG therapy may not induce powerful specific antitumor immunity or immunological memory. However, based on the efficacy and recent advances in understanding of the mechanism of BCG immunotherapy, active immunotherapy has great potential as a weapon against even advanced urothelial carcinoma. Although an HLA class I-restricted peptide vaccine eliciting epitope-specific CTLs seems to be one of the most promising approaches at present, this can also induce CD8⁺ T cell tolerance [119,120], with low response rates in clinical trials. Better understanding of the innate and adaptive immune responses, and of the immune escape mechanisms underlying immunological tolerance, and acknowledgment of the importance of adaptive immunity for control of tumor growth are necessary to develop a more comprehensive immunotherapeutic approach [121]. Alternatively, combination with chemotherapy, molecular-targeting therapy or surgery can be effective ways to apply active immunotherapy to clinical use.