[Uro-oncological research from the laboratory and clinical practice]. / Uroonkologische Forschung aus Labor und Klinik.

This report presents current work and results of projects in the uro-oncological field from the Cantonal Hospital of St. Gallen. The first part deals with dendritic cell-based immunotherapy of hormone refractory prostate cancer. In the second part, some recent results of clinical and laboratory work for non-muscle invasive bladder cancer are highlighted.

[Adaptive immunotherapy of the advanced prostate cancer - cancer testis antigen (CTA) as possible target antigens]. / Adaptive Immuntherapie des fortgeschrittenen Prostatakarzinoms - Cancer Testis Antigene (CTA) als mögliche Zielantigene.

Prostate cancer (PCa) like other tumors expresses antigens that may serve as target for specific immunotherapy. Special antigen-presenting cells (e. g., dendritic cells) are capable of generating tumor-specific immunity. Cytotoxic T-cells (killer cells) are very effective against antigens and, consequently, against the respective tissue or tumor. Cancer testis antigens (CTA) are expressed in various human cancers but, aside from the testicles, not in normal tissue. Therefore, they are suitable for a specific tumor immunotherapy. We looked at different CTA (LAGE-1, PRAME, MAGE-C2, NY-ESO-1, SSX-2 and PAGE4) and their occurrence in prostatic cancer. Expression of CTA in various PCa cell lines and PCa material from patients was very heterogeneous. Only PAGE4 was expressed in primary PCa and in LnCaP cells as well as in hormone-dependent and hormone-refractory PCa probes. We conclude that PAGE4 should be further evaluated as a potential target for immunotherapy of PCa.

Dendritic cell vaccination and viral infection--animal models.

Dendritic cells (DCs) play a pivotal role in the initiation and maintenance of immune responses against viruses and other microbial pathogens. Adoptively transferred, in vitro manipulated DCs presenting antigen derived from different viruses have been shown to elicit cytotoxic T cell (CTL) and T helper (Th) cell responses and to induce protective antiviral immunity. Furthermore, DC-based adoptive immunotherapies have the potential to specifically (re)activate antiviral immunity in chronic viral diseases such as HIV or hepatitis virus infections. Cellular dendritic cell vaccines, however, are not suitable for large-scale prophylactic immunization. Strategies for vaccine development should therefore aim at the specific delivery of microbial antigens to DCs in situ. Furthermore, appropriate mobilization and activation of DCs by the vaccine is important for the generation of optimal antimicrobial immune responses. Here, we discuss recent data on induction of antiviral immunity with various DC-vaccination approaches and outline future directions for the development of specific antigen targeting to DCs.

Dendritic cells in autoimmune diseases.

Subclinical autoimmune responses can be frequently detected in healthy individuals. Sustained activation of autoreactive lymphocytes is, however, required for the development of autoimmune diseases associated with ongoing tissue destruction either in single organs or generalized with multiple manifestations. Clinical and experimental evidence suggests that prolonged presentation of self antigens by dendritic cells is crucial for the development of destructive autoimmune disease. We discuss here a simplified threshold model where the key parameters for the magnitude of the autoimmune response are the amount of previously ignored self peptides presented by dendritic cells and the duration of the antigen presentation in secondary lymphoid organs. Multiple factors influence the threshold for the conversion of an autoimmune response to overt autoimmune disease. Frequent or persistent viral infections of the target organ may favor autoimmune disease by increasing the amounts of released self antigens, generating cytokine-mediated bystander activation of self-reactive lymphocytes and/or sustaining a chronic response via neoformation of lymphoid structures in the target organ.

Perforin-independent regulation of dendritic cell homeostasis by CD8(+) T cells in vivo: implications for adaptive immunotherapy.

We investigated here the effects of perforin on CTL responses during interaction of dendritic cells (DC) with cytotoxic T lymphocytes in vivo. Using MHC class I tetramers complexed with the immunodominant CTL epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP33), we followed the kinetics of DC-induced CTL responses. GP33-presenting DC induced rapid primary expansion of both perforin-competent and -deficient CTL with similar kinetics. Secondary CTL responses in perforin-deficient and normal control mice after DC-booster immunization were more rapid than the primary responses, but never reached the high initial levels, suggesting that reactivated memory CTL eliminated the antigen-presenting DC and thereby limited the booster effect. Whereas killingof DC in vitro was strictly dependent on perforin, elimination of GP33-presenting DC by CTL in vivo was largely independent of perforin and Fas. Taken together, these results suggest that control of DC homeostasis by CTL, i. e. elimination of DC by the effector cells they had elicited, is controlled via multiple and probably redundant signals and represents an important fail-safe mechanism to avoid exaggerated CTL responses.

Rapid peptide turnover and inefficient presentation of exogenous antigen critically limit the activation of self-reactive CTL by dendritic cells.

This study evaluated to what extent presentation of exogenously acquired self-Ags via MHC class I molecules on DC might contribute to the activation of self-reactive CTL and subsequent development of autoimmune disease. We show here by using the rat insulin promotor lymphocytic choriomeningitis virus glycoprotein model of autoimmune diabetes that the activation of self-reactive CTL by DC after uptake of exogenous Ag is very limited, first by the short half-life of MHC class I-associated peptides on DC in vitro and in vivo, and second by the rather inefficient MHC class I presentation of cell-associated self-Ags by DC. These two mechanisms are probably crucial in establishing high thresholds for the induction of self-reactive CTL that prevent autoimmune sequelae after release of sequestered and previously immunologically ignored tissue Ags.

Hypercholesterolemia exacerbates virus-induced immunopathologic liver disease via suppression of antiviral cytotoxic T cell responses.

The immune system has to be optimally balanced to be highly effective against infections with cytopathic microbial pathogens and must guarantee efficient destruction of cells infected with noncytopathic agents while leaving the integrity of noninfected cells largely unaltered. We describe here the effects of genetically induced hypercholesterolemia on cellular immunity in apolipoprotein E (ApoE(-/-)) and low density lipoprotein receptor-deficient (LDLR(-/-)) mice during infection with the hepatotropic lymphocytic choriomeningitis virus WE strain. In both ApoE(-/-) and LDLR(-/-) mice hypercholesterolemia aggravated virus-induced immunopathologic liver disease. ApoE(-/-) mice exhibited a higher susceptibility to virus-induced immunopathology than LDLR(-/-) mice and usually succumbed to immunopathologic disease when infected with high doses of virus. Initial virus spread was not influenced by the hypercholesterolemia, whereas clearance of the virus from spleen and nonlymphoid organs, including liver, was delayed. Activation of antiviral CTL, measured by ex vivo cytotoxicity and IFN-gamma production, and recruitment of specific CTL into blood and liver were impaired in hypercholesterolemic mice, indicating that hypercholesterolemia had a significant suppressive effect on cellular immunity. Taken together, these data provide evidence that hypercholesterolemia suppresses antiviral immune responses, thereby changing the host-virus balance, and can increase susceptibility to acute or chronic and potentially lethal virus-induced immunopathologic disease. These findings impinge on our understanding of hypercholesterolemia as a disease parameter and may explain aspects of the frequent association of persistent pathogens with hypercholesterolemia-induced diseases, such as atherosclerosis.

Linking immune-mediated arterial inflammation and cholesterol-induced atherosclerosis in a transgenic mouse model.

Arterial inflammatory responses are thought to be a significant component of atherosclerotic disease. We describe here, using a transgenic approach, the mutual perpetuation of immune-mediated arterial inflammation and cholesterol-induced atherosclerosis. Mice expressing the bacterial transgene beta-galactosidase exclusively in cardiomyocytes and in smooth muscle cells in lung arteries and the aorta (SM-LacZ), and hypercholesterolemic apolipoprotein E-deficient SM-LacZ mice (SM-LacZ/apoE(-/-)) developed myocarditis and arteritis after immunization with dendritic cells presenting a beta-galactosidase-derived immunogenic peptide. Hypercholesterolemia amplified acute arteritis and perpetuated chronic arterial inflammation in SM-LacZ/apoE(-/-) mice, but had no major impact on acute myocarditis or the subsequent development of dilated cardiomyopathy. Conversely, arteritis significantly accelerated cholesterol-induced atherosclerosis. Taken together, these data demonstrate that the linkage of immune-mediated arteritis and hypercholesterolemia favors initiation and maintenance of atherosclerotic lesion formation. Therapeutic strategies to prevent or disrupt such self-perpetuating vicious circles may be crucial for the successful treatment of atherosclerosis.

In vivo antigen loading and activation of dendritic cells via a liposomal peptide vaccine mediates protective antiviral and anti-tumour immunity.

Initiation of antiviral and anti-tumour T cell responses is probably achieved mainly by dendritic cells (DC) transporting antigen from the periphery into organised lymphoid tissues. To develop T cell vaccines it is, therefore, important to understand the accessibility of the antigen to DC in vivo and whether DC are activated by vaccination. Here we have evaluated the immunogenicity of a liposomal vaccine formulation with antigenic peptides derived from the glycoprotein of the lymphocytic choriomeningitis virus. Liposome-encapsulated peptides were highly immunogenic when administered intradermally and elicited protective antiviral immunity. After intradermal injection, liposomes formed antigen depots which facilitated long-lasting in vivo antigen loading of dendritic cells almost exclusively in the local draining lymph nodes. The immunogenicity of the liposomal peptide vaccine was further enhanced by incorporation of immunostimulatory oligonucleotides leading to activation of DC. This optimised liposomal peptide vaccine elicited also anti-tumour immunity and induced CTL responses comparable to adoptively transferred, peptide-presenting DC. Thus, our data show that liposomal formulations of peptide vaccines are highly effective at direct in vivo antigen loading and activation of DC leading to protective antiviral and anti-tumour immune responses.

Immunotherapy with dendritic cells directed against tumor antigens shared with normal host cells results in severe autoimmune disease.

Vaccination with dendritic cells (DCs) presenting tumor antigens induces primary immune response or amplifies existing cytotoxic antitumor T cell responses. This study documents that antitumor treatment with DCs may cause severe autoimmune disease when the tumor antigens are not tumor-specific but are also expressed in peripheral nonlymphoid organs. Growing tumors with such shared tumor antigens that were, at least initially, strictly located outside of secondary lymphoid organs were successfully controlled by specific DC vaccination. However, antitumor treatment was accompanied by fatal autoimmune disease, i.e., autoimmune diabetes in transgenic mice expressing the tumor antigen also in pancreatic beta islet cells or by severe arteritis, myocarditis, and eventually dilated cardiomyopathy when arterial smooth muscle cells and cardiomyocytes expressed the transgenic tumor antigen. These results reveal the delicate balance between tumor immunity and autoimmunity and therefore point out important limitations for the use of not strictly tumor-specific antigens in antitumor vaccination with DCs.

Dendritic cells and differential usage of the MHC class II transactivator promoters in the central nervous system in experimental autoimmune encephalitis.

In the normal central nervous system (CNS) expression of MHC class II is minimal, but has been found to be highly up-regulated on microglia cells in experimental autoimmune encephalitis (EAE). Here we used the EAE model to examine the regulation of expression of the class II transactivator (CIITA), which is required for activation of MHC class II genes. EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55. CIITA mRNA form I (specific for dendritic cells) and form IV (IFN-gamma inducible) but not form III (B cell specific) were detected in brain and spinal cord of mice with acute EAE. In unimmunized or mock-immunized mice, none of the three CIITA forms was found to be induced. Dendritic cells (DC) were identified by immunostainings for CD11c in perivascular and meningeal cell infiltrates in EAE spinal cord and brain. Time-course analysis showed (1) the appearance of DC in the CNS shortly before onset of disease, (2) the recruitment of CD11b cells occuring much earlier and (3) the absence of CIITA and MHC class II expression in these CD11b+ cells at preclinical stages.

Induction, binding specificity and function of human ICOS.

Recently, we have identified the inducible co-stimulator (ICOS), an activation-dependent, T cell-specific cell surface molecule related to CD28 and CTLA-4. Detailed analysis of human ICOS presented here shows that it is a 55-60-kDa homodimer with differently N-glycosylated subunits of 27 and 29 kDa. ICOS requires both phorbol 12-myristate 13-acetate and ionomycin for full induction, and is sensitive to Cyclosporin A. ICOS is up-regulated early on all T cells, including the CD28- subset, and continues to be expressed into later phases of T cell activation. On stimulation of T cells by antigen-presenting cells, the CD28/B7, but not the CD40 ligand/CD40 pathway is critically involved in the induction of ICOS. ICOS does not bind to B7-1 or B7-2, and CD28 does not bind to ICOS ligand; thus the CD28 and ICOS pathways do not cross-interact on the cell surface. In vivo, ICOS is expressed in the medulla of the fetal and newborn thymus, in the T cell zones of tonsils and lymph nodes, and in the apical light zones of germinal centers (predominant expression). Functionally, ICOS co-induces a variety of cytokines including IL-4, IL-5, IL-6, IFN-gamma, TNF-alpha, GM-CSF, but not IL-2, and superinduces IL-10. Furthermore, ICOS co-stimulation prevents the apoptosis of pre-activated T cells. The human ICOS gene maps to chromosome 2q33 - 34.

Transgenic animal models for virus-induced autoimmune diseases.

In the pathogenesis of autoimmune diseases, tolerance against self-determinants is lost and autoreactive lymphocytes are activated leading to pathological damage of single or multiple organs. Viral and other microbial infections have been implicated in these processes. Viruses may induce immunopathological damage by maintaining a chronic immune response against the locally persisting infectious agent. Alternatively, viruses may help to initiate anti-self immunoreactivity, e.g. by induction of an inflammatory milieu needed to overcome tolerance against self-antigens. Presentation of viral antigens and/or previously immunologically ignored self-antigens in secondary lymphoid organs is most probably the key event in the initiation of autoimmune diseases. Translocation of antigens to secondary lymphoid organs and primary induction of T cell responses is primarily mediated by dendritic cells (DCs). We discuss here two transgenic models of autoimmune diseases where DC-mediated antigen transport initiated autoimmune responses against microbial neoself antigens. In the first model, dose and timing of antigen delivery by DCs and turnover of antigenic peptides presented by DCs are the main parameters regulating the outcome of autoimmune diabetes. In the second model, chronic stimulation of organ-specific immune responses via DCs leads to severe cardiovascular immunopathology with arteritis, myocarditis and eventually dilated cardiomyopathy. Taken together, transgenic mouse models are valuable tools for delineating basic pathogenic mechanisms and evaluating therapeutic strategies to interfere with early detrimental processes that lead to manifest autoimmune diseases.

Induction of optimal anti-viral neutralizing B cell responses by dendritic cells requires transport and release of virus particles in secondary lymphoid organs.

Dendritic cells (DC) are sentinels of the immune system, transporting antigens from the periphery to secondary lymphoid organs. This study investigates the interactions of DC with B cells for the induction of anti-viral neutralizing antibody responses. Using the vesicular stomatitis virus glycoprotein (VSV-G) as a model antigen, we show that DC contain infection with cytopathic VSV in the presence of a functional IFN system, facilitating transport and release of low levels of live virus in secondary lymphoid organs. DC exposed to live virus induced efficient neutralizing anti-viral B cell responses. In contrast, DC transporting UV-inactivated viral antigens were poor activators of anti-viral B cells, although they were capable of very efficiently inducing virus-specific Th cells. Transgenic DC expressing a membrane-bound form of VSV-G induced neutralizing B cell responses; however, this DC-induced, Th-dependent B cell response was significantly slower than the anti-viral B cell response induced by DC infected with live VSV, and was strongly dependent on concomitant priming of T help. These results suggest that DC may play a double role during infection with cytopathic virus: they transport and release live virus in secondary lymphoid tissues for optimal direct B cell induction and offer MHC class II-associated determinants for induction of T help.

Role of dendritic cells in the induction and maintenance of autoimmune diseases.

Autoimmune diseases are characterised by the loss of tolerance against self-determinants, activation of autoreactive lymphocytes and pathological damage to single or multiple organs. The mechanisms by which autoimmune responses are triggered and activation of autoreactive lymphocytes is initiated and maintained are not yet fully understood. Translocation of previously immunologically ignored antigens from the periphery to secondary lymphoid organs is probably a key step in the initiation of autoimmunity. Antigen transport and primary sensitisation of T lymphocytes is mainly mediated by dendritic cells which reside in peripheral non-lymphoid tissues and maintain a continuous gradient of antigens towards secondary lymphoid tissues. In the transgenic rat insulin promoter-glycoprotein model of autoimmune diabetes, dendritic cell (DC)-mediated antigen transport initiates an autoimmune response against a pancreatic neoself-antigen. Dose and timing of antigen delivery by DC and turnover of antigenic peptides presented by DC are the main parameters regulating the outcome of autoimmune diabetes in this model system. An important sequel of continued antigenic stimulation via DC is the formation of lymphoid structures in the pancreas. Thus, appropriate and repeated activation of cytotoxic T lymphocytes by DC, in concert with local inflammatory processes leading to formation of organised lymphoid tissue in the target organ, is likely to be crucial in the development of destructive autoimmunity. Therapeutic intervention to selectively manipulate antigen transport by dendritic cells or to influence antigen presentation may prove beneficial for the treatment of autoimmune diseases. Furthermore, the capacity of DC to induce potent antiself responses might have implications for the use of DC presenting self-antigens in treatment of established tumours.

Protective antiviral cytotoxic T cell memory is most efficiently maintained by restimulation via dendritic cells.

Dendritic cells (DC) play a key role in the initiation of T cell-mediated immune responses and may therefore be successfully used in antiviral and antitumor vaccination strategies. Because both strength and duration of an immune response determines the outcome of a vaccination protocol, we evaluated the life span of DC-induced antiviral CTL memory against systemic and peripheral challenge infections with lymphocytic choriomeningitis virus (LCMV). We found that expansion and activation of CTL by DC was transient. Protection against systemic LCMV infection after DC immunization was relatively long-lived (>60 days), whereas complete protection against peripheral infection via intracerebral infection or infection into the footpad with LCMV, where rapid recruitment of effector T cells to the site of infection and elimination of viral pathogen plays a major role, was short-lived (<30 days). Protective immunity was most efficiently restored by administration of antigenic peptides via DC, rather than in combination with IFA or in liposomes. These results suggest that Ag presentation by DC may be crucial for both initiation and maintenance of protective CTL-mediated immunity against viruses infecting solid organs or against peripheral mesenchymal or epithelial tumors.

Immune surveillance against a solid tumor fails because of immunological ignorance.

Many peripheral solid tumors such as sarcomas and carcinomas express tumor-specific antigens that can serve as targets for immune effector T cells. Nevertheless, overall immune surveillance against such tumors seems relatively inefficient. We studied immune surveillance against a s.c. sarcoma expressing a characterized viral tumor antigen. Surprisingly, the tumor cells were capable of inducing a protective cytotoxic T cell response if transferred as a single-cell suspension. However, if they were transplanted as small tumor pieces, tumors readily grew. Tumor growth correlated strictly with (i) failure of tumor cells to reach the draining lymph nodes and (ii) absence of primed cytotoxic T cells. Cytotoxic T cells were not tolerant or deleted because a tumor antigen-specific cytotoxic T cell response was readily induced in lymphoid tissue by immunization with virus or with tumor cells even in the presence of large tumors. Established tumors were rejected by vaccine-induced effector T cells if effector T cells were maintained by prolonged or repetitive vaccination, but not by single-dose vaccination. Thus, in addition to several other tumor-promoting parameters, some antigenic peripheral sarcomas-and probably carcinomas-may grow not because they anergize or tolerize tumor-specific T cells, but because such tumors are immunologically dealt with as if they were in a so-called immunologically privileged site and are ignored for too long.

Qualitative and quantitative requirements for CD4+ T cell-mediated antiviral protection.

CD4+ Th cells deliver the cognate and cytokine signals that promote the production of protective virus-neutralizing IgG by specific B cells and are also able to mediate direct antiviral effector functions. To quantitatively and qualitatively analyze the antiviral functions of CD4+ Th cells, we generated transgenic mice (tg7) expressing an MHC class II (I-Ab)-restricted TCR specific for a peptide derived from the glycoprotein (G) of vesicular stomatitis virus (VSV). The elevated precursor frequency of naive VSV-specific Th cells in tg7 mice led to a markedly accelerated and enhanced class switching to virus-neutralizing IgG after immunization with inactivated VSV. Furthermore, in contrast to nontransgenic controls, tg7 mice rapidly cleared a recombinant vaccinia virus expressing the VSV-G (Vacc-IND-G) from peripheral organs. By adoptive transfer of naive tg7 CD4+ T cells into T cell-deficient recipients, we found that 105 transferred CD4+ T cells were sufficient to induce isotype switching after challenge with a suboptimal dose of inactivated VSV. In contrast, naive transgenic CD4+ T cells were unable to adoptively confer protection against peripheral infection with Vacc-IND-G. However, tg7 CD4+ T cells that had been primed in vitro with VSV-G peptide were able to adoptively transfer protection against Vacc-IND-G. These results demonstrate that the antiviral properties of CD4+ T cells are governed by the differentiation status of the CD4+ T cell and by the type of effector response required for virus elimination.

Dendritic cells induce autoimmune diabetes and maintain disease via de novo formation of local lymphoid tissue.

Activation of autoreactive T cells can lead to autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM). The initiation and maintenance of IDDM by dendritic cells (DC), the most potent professional antigen-presenting cells, were investigated in transgenic mice expressing the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) under the control of the rat insulin promoter (RIP-GP mice). We show that after adoptive transfer of DC constitutively expressing the immunodominant cytotoxic T lymphocyte (CTL) epitope of the LCMV-GP, RIP-GP mice developed autoimmune diabetes. Kinetic and functional studies of DC-activated CTL revealed that development of IDDM was dependent on dose and timing of antigenic stimulation. Strikingly, repeated CTL activation by DC led to severe destructive mononuclear infiltration of the pancreatic islets but also to de novo formation of islet-associated organized lymphoid structures in the pancreatic parenchyma. In addition, repetitive DC immunization induced IDDM with lymphoid neogenesis also in perforin-deficient RIP-GP mice, illustrating that CD8(+) T cell-dependent inflammatory mechanisms independent of perforin could induce IDDM. Thus, DC presenting self-antigens not only are potent inducers of autoreactive T cells, but also help to maintain a peripheral immune response locally; therefore, the induction of autoimmunity against previously ignored autoantigens represents a potential hazard, particularly in DC-based antitumor therapies.