Gerald Willimsky

Sporadic immunogenic tumours avoid destruction by inducing T-cell tolerance

The recognition and elimination of tumours by T cells, a process termed cancer immunosurveillance, is effective against certain virus-associated cancers. Spontaneous tumours often induce a specific immune response and are therefore also immunogenic. However, it is not clear whether they can be controlled by T cells. The immunosurveillance hypothesis postulates that tumours, if they eventually grow, escaped T-cell recognition by losing immunogenicity. Here we show, by generating a mouse model of sporadic cancer based on rare spontaneous activation of a dormant oncogene, that immunogenic tumours do not escape their recognition but induce tolerance. In this model, tumours derive from single cells and express a tumour-specific transplantation rejection antigen. Whereas vaccinated mice remain tumour-free throughout their lifetime, naive mice always develop a progressively growing tumour. We also show that despite specific recognition by T cells, the tumours do not lose their intrinsic immunogenicity and are rejected after transplantation in T-cell-competent recipients. Furthermore, in the primary host tumour-induced tolerance is associated with the expansion of non-functional T cells. Together, our data argue against immunosurveillance of spontaneous cancer.

Immunosuppressive plasma cells impede T cell-dependent immunogenic chemotherapy

Cancer-associated genetic alterations induce expression of tumour antigens that can activate CD8+ cytotoxic T cells (CTLs), but the microenvironment of established tumours promotes immune tolerance through poorly understood mechanisms. Recently developed therapeutics that overcome tolerogenic mechanisms activate tumour-directed CTLs and are effective in some human cancers. Immune mechanisms also affect treatment outcome, and certain chemotherapeutic drugs stimulate cancer-specific immune responses by inducing immunogenic cell death and other effector mechanisms. Our previous studies revealed that B cells recruited by the chemokine CXCL13 into prostate cancer tumours promote the progression of castrate-resistant prostate cancer by producing lymphotoxin, which activates an IκB kinase α (IKKα)-BMI1 module in prostate cancer stem cells. Because castrate-resistant prostate cancer is refractory to most therapies, we examined B cell involvement in the acquisition of chemotherapy resistance. Here we focus on oxaliplatin, an immunogenic chemotherapeutic agent that is effective in aggressive prostate cancer. We show that mouse B cells modulate the response to low-dose oxaliplatin, which promotes tumour-directed CTL activation by inducing immunogenic cell death. Three different mouse prostate cancer models were refractory to oxaliplatin unless genetically or pharmacologically depleted of B cells. The crucial immunosuppressive B cells are plasmocytes that express IgA, interleukin (IL)-10 and programmed death ligand 1 (PD-L1), the appearance of which depends on TGFβ receptor signalling. Elimination of these cells, which also infiltrate human-therapy-resistant prostate cancer, allows CTL-dependent eradication of oxaliplatin-treated tumours.

Immunogenicity of premalignant lesions is the primary cause of general cytotoxic T lymphocyte unresponsiveness.

Cancer is sporadic in nature, characterized by an initial clonal oncogenic event and usually a long latency. When and how it subverts the immune system is unknown. We show, in a model of sporadic immunogenic cancer, that tumor-specific tolerance closely coincides with the first tumor antigen recognition by B cells. During the subsequent latency period until tumors progress, the mice acquire general cytotoxic T lymphocyte (CTL) unresponsiveness, which is associated with high transforming growth factor (TGF) β1 levels and expansion of immature myeloid cells (iMCs). In mice with large nonimmunogenic tumors, iMCs expand but TGF-β1 serum levels are normal, and unrelated CTL responses are undiminished. We conclude that (a) tolerance to the tumor antigen occurs at the premalignant stage, (b) tumor latency is unlikely caused by CTL control, and (c) a persistent immunogenic tumor antigen causes general CTL unresponsiveness but tumor burden and iMCs per se do not.

The adaptive immune response to sporadic cancer

Summary: Most of the current experimental cancer models do not reflect the pathophysiology of real‐life cancer. Cancer usually occurs sporadically and is clonal in origin. Between tumor initiation and progression, clinically unapparent pre‐malignant cells may persist for years or decades in humans. Recently, mouse models of sporadic cancer have been developed. The mouse germ‐line can be engineered with high precision so that defined genes can be switched on and off in the adult organism in a targeted manner. Analysis of the immune response against sporadic tumors requires the knowledge of a tumor antigen. Ideally, a silent oncogene, for which the mice are not tolerant, is stochastically activated in individual cells. This approach offers the opportunity to analyze the adaptive immune response throughout the long process of malignant transformation and most closely resembles cancer in humans. In such a model with the highly immunogenic SV40 large T antigen as a dormant oncogene, we discovered that sporadic cancer is recognized by the adaptive immune system at the pre‐malignant stage, concomitant with the induction of tumor antigen‐specific tolerance. These results demonstrated that even highly immunogenic sporadic tumors are unable to induce functional cytotoxic T lymphocytes. Based on this model, we conclude that immunosurveillance plays little or no role against sporadic cancer and that tumors must not escape immune recognition or destruction.

Cell-based vaccines for renal cell carcinoma: genetically-engineered tumor cells and monocyte-derived dendritic cells

Initial vaccine developments for renal cell carcinoma (RCC) have concentrated on cell-based approaches in which tumor cells themselves provide mixtures of unknown tumor-associated antigens as immunizing agents. Antigens derived from autologous tumors can direct responses to molecular composites characteristic of individual tumors, whereas antigens derived from allogeneic tumor cells must be commonly shared by RCC. Three types of cell-based vaccine for RCC have been investigated: isolated tumor cell suspensions, gene modified tumor cells and dendritic cells (DCs) expressing RCC-associated antigens. Approaches using genetic modification of autologous RCC have included ex vivo modification of tumor cells or modification of tumors in vivo. We have used gene-modification of allogeneic tumor cell lines to create generic RCC vaccines. More recently, emphasis has shifted to the use of DCs as cell-based vaccines for RCC. DCs have moved to a position of central interest because of their excellent stimulatory capacity, combined with their ability to process and present antigens to both naive CD4 and CD8 cells. The long impasse in identifying molecular targets for specific immunotherapy of RCC is now rapidly being overcome through the use of tools and information emerging from human genome research. Identification of candidate molecules expressed by RCC using cDNA arrays, combined with protein arrays and identification of peptides presented by MHC molecules, allow specific vaccines to be tailored to the antigenic profile of individual tumors, providing the basis for development of patient-specific vaccines.

Virus-induced hepatocellular carcinomas cause antigen-specific local tolerance

T cell surveillance is often effective against virus-associated tumors because of their high immunogenicity. It is not clear why surveillance occasionally fails, particularly against hepatitis B virus- or hepatitis C virus-associated hepatocellular carcinoma (HCC). We established a transgenic murine model of virus-induced HCC by hepatocyte-specific adenovirus-induced activation of the oncogenic SV40 large T antigen (TAg). Adenovirus infection induced cytotoxic T lymphocytes (CTLs) targeted against the virus and TAg, leading to clearance of the infected cells. Despite the presence of functional, antigen-specific T cells, a few virus-infected cells escaped immune clearance and progressed to HCC. These cells expressed TAg at levels similar to HCC isolated from neonatal TAg-tolerant mice, suggesting that CTL clearance does not select for cells with low immunogenicity. Virus-infected mice revealed significantly greater T cell infiltration in early-stage HCC compared with that in late-stage HCC, demonstrating progressive local immune suppression through inefficient T cell infiltration. Programmed cell death protein-1 (PD-1) and its ligand PD-L1 were expressed in all TAg-specific CD8+ T cells and HCC, respectively, which contributed to local tumor-antigen-specific tolerance. Thus, we have developed a model of virus-induced HCC that may allow for a better understanding of human HCC.

The Sorting Receptor Sortilin Exhibits a Dual Function in Exocytic Trafficking of Interferon-γ and Granzyme A in T Cells

Immunological control of infections or tumors depends on the release of effector cytokines and polarized secretion of cytotoxic granules from T cells and natural killer cells. Here we show that the sorting receptor Sortilin controlled both processes. In murine Sortilin-deficient cytotoxic T lymphocytes, regulated secretion of granzyme A and cytotoxic killing was enhanced and correlated with increased vesicle-associated membrane protein 7 availability. In contrast, loss of Sortilin reduced the release of interferon-γ upon infections and in autoimmune colitis. Exit of interferon-γ from the Golgi apparatus required the presence of Sortilin. Furthermore, we tracked the transport route of interferon-γ beyond this Sortilin-dependent Golgi to early endosome step. In wild-type T cells, trafficking of interferon-γ from the endosomal sorting platform to the plasma membrane proceeded independently of recycling endosomes, and interferon-γ remained excluded from late endosomes. Our results suggest that Sortilin modulates systemic immune responses through exocytic sorting of immunological effector molecules.

Influence of CD80, Interleukin-2, and Interleukin-7 Expression in Human Renal Cell Carcinoma on the Expansion, Function, and Survival of Tumor-Specific CTLs

Purpose: A renal cell carcinoma (RCC) line, RCC-26, has been identified as a suitable candidate for development of an allogeneic tumor cell vaccine based on its expression of a variety of tumor-associated antigens (TAA). To improve immunogenicity, RCC-26 cells were genetically engineered to express CD80 alone or in combination with interleukin (IL)-2 or IL-7. The effect of these modifications on proliferation, function, and survival of autologous and allogeneic tumor-specific CTLs was assessed. Experimental Design: RCC-26 sublines expressing different transgenes were tested for their capacity to reactivate cytokine secretion and cytotoxicity in autologous tumor-infiltrating lymphocytes, to improve proliferation and survival of tumor-associated T cells present in autologous peripheral blood, and to induce tumor-associated responses in naive allogeneic lymphocytes. The expression of several common TAA was quantitated in the RCC-26 sublines using reverse transcription-PCR to identify surrogate markers for immune monitoring in clinical trials. Results: Gene-modified RCC-26 cells showed enhanced immunogenicity. CD80 expression was necessary to induce RCC-associated CTL in blood of healthy allogeneic donors. It also improved proliferation of autologous effector-memory T cells. Further enhancement was achieved with IL-2 through induction of the antiapoptosis protein Bcl-xL. The candidate vaccine lines overexpressed several common TAA that are suitable markers for immune monitoring. Conclusions: RCC-26 cells coexpressing CD80 and cytokine transgenes display improved immunogenic characteristics, supporting their use as allogeneic tumor cell vaccines for HLA-A2-matched patients with metastatic RCC.

Tumor-induced antibodies resemble the response to tissue damage

Tumor‐associated antibodies are frequently detected in cancer patients. To ask whether the recognized antigens are rejection antigens, we screened a cDNA expression library of the mouse TS/A tumor with TS/A‐immune serum and isolated 8 IgG‐reactive clones, representing self‐antigens that were expressed in normal tissues and other tumor lines. Three of the antigens had previously been identified in the human system by this cloning strategy. None of the antigens revealed to be a rejection antigen in normal mice demonstrated by an otherwise effective plasmid immunization. For one of the identified antigens, α‐catenin, it is shown that the induction of IgG antibodies by protein immunization does not correlate with tumor rejection. For another antigen, vimentin, it is shown that vimentin‐deficient but not vimentin‐competent mice reject vimentin‐expressing tumors indicating T ‐cell tolerance despite the fact that tumor cell immunization induces antivimentin IgG antibodies. Tissue damage induced by adenovirus infection induced an antibody response similar to tumor cell immunization, exemplified with 2 of the antigens. We conclude that the tumor‐induced antibodies mirror tissue damage and that the antibody‐inducing antigens can serve as rejection antigens if they are recognized as foreign.

Fas expression by tumor stroma is required for cancer eradication

The contribution of molecules such as perforin, IFN-γ (IFNγ), and particularly Fas ligand (FasL) by transferred CD8+ effector T (TE) cells to rejection of large, established tumors is incompletely understood. Efficient attack against large tumors carrying a surrogate tumor antigen (mimicking a “passenger” mutation) by TE cells requires action of IFNγ on tumor stroma cells to avoid selection of antigen-loss variants. Because “cancer-driving” antigens (CDAs) are rarely counterselected, IFNγ may be expected to be dispensable in elimination of cancers by targeting a CDA. Here, initial regression of large, established tumors required neither IFNγ, FasL, nor perforin by transferred CD8+ TE cells targeting Simian Virus (SV) 40 large T as CDA. However, cytotoxic TE cells lacking IFNγ or FasL could not prevent relapse despite retention of the rejection antigen by the cancer cells. Complete tumor rejection required IFNγ-regulated Fas by the tumor stroma. Therefore, TE cells lacking IFNγ or FasL cannot prevent progression of antigenic cancer because the tumor stroma escapes destruction if its Fas expression is down-regulated.