The International Symposium on Tumour Escape and its Determinants was held in Salzburg, Austria, October 10–13, 2004, under the joint sponsorship of the International Union Against Cancer (UICC, Geneva), the Fritz-Bender-Foundation (Munich), the Roswell Park Cancer Institute, Buffalo, USA, the Institute of Immunology, University Witten/Herdecke, Germany, the Department of Cell Biology and Genetics, Faculty for Basic Sciences, Paris Lodron University, Salzburg, Austria.
The presentations concerned five areas of the immunobiology of cancer: (1) evidence of immune responses to tumour, chaired by H. zur Hausen and F. Entschladen, (2) escape mechanisms: tumour cell mechanisms, chaired by Kurt S. Zänker and Suzanne Ostrand-Rosenberg, (3) escape mechanisms: tumour-induced suppression, chaired by Soldano Ferrone and Graham Pawelec, (4) escape counteraction: immunological mechanisms, chaired by Rolf Kiessling and Yasmin Thanavala, and (5) escape modification: angiogenesis and stroma, chaired by Peter Eckl and Enrico Mihich.
In her keynote address, Eva Klein, Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden, spoke about EBV infection in man. Viral maintenance relies on lack of immunogenicity when inserted into the genome of the cell and on survival factors that are present in the in vivo microenvironment; on the other hand, immunosurveillance eliminates EBV-driven potentially malignant cells. During the long history of human coexistence with EBV, both the virus and the host evolved a balanced interaction, favouring the persistence of the virus and survival of the host. EBV engages in a variety of non-lytic strategies. The alternative viral gene expression determines the fate of the infected cell. Depending on the expression of virally induced proteins, Type I, Type IIa, Type IIb and Type III patterns have been defined. EBV has been detected in cultured Burkitt lymphoma; these tumours express only the EBNA-1 protein in vivo (Type I program). Burkitt lymphoma patients are immunocompetent. B cells with Type I and Type III EBV expression pattern can be detected in lymph nodes and tonsils of infectious mononucleosis (IM) patients and in tissues of EBV-positive immunoblastomas, arising in immunosuppressed patients. EBNA-1- and LMP-1-positive Type IIa and EBNA-1–6-positive but LMP-1-negative Type IIb B cells are also found in these tissues. In EBV-positive Hodgkin’s lymphomas H/RS cells express the Type IIa pattern; Type IIb malignancies do not occur. The H/RS cells are surrounded by T lymphocytes; however, the genesis of Hodgkin’s lymphomas is not well understood. Crosstalk between the malignant and stroma cells, involving antiapoptotic, immunostimulatory and immunosuppressive (IL-10) cytokines seems to be important. The fate of Type IIa and Type IIb cells in IM patients is also unknown. The incidence of Hodgkin’s lymphoma is higher in individuals with a history of IM, therefore, Type IIa cells may occasionally give rise to Hodgkin’s lymphomas. EBV infection in man is a prominent example of successful immune surveillance. Although the virus has a powerful potential to transform B lymphocytes, humans have built up equally powerful mechanisms that safeguard the host and malignancies occur only by cytogenetic or immunosuppressive accidents.
Session I
Kai Masur, Institute of Immunology, University Witten/Herdecke, Germany, spoke on a novel role of CD26 in immune function and tumour progression. The multifunctional protein CD26 contains an endopeptidase function and is characterized as a DiPeptidyl Peptidase (DPP IV), the key enzyme to degrade and inactivate glucagon-like-peptide (GLP) −1 and −2. The inhibition of CD26 (synonym DPP IV) activity increases the locomotion and activation of T lymphocytes as assessed by CD25 expression. Concomitantly, the sibling GLP-2, which is secreted concurrently with GLP-1 and which is also a substrate for CD26, causes increased crypt cell proliferation in the gut, improves nutrient uptake and stimulates gastric and gut motility. GLP-2 induces cell proliferation and motility in colon cancer cell lines. The application of DPP IV inhibitors as suggested for the treatment of Type 2 Diabetes mellitus increases the risk of misbalancing immune competence and, even more fatally, the risk of tumour progression by sustaining colon cancer cell proliferation and migration.
Zhihai Qin, Institute of Immunology, Charite Campus Benjamin Franklin, Berlin, Germany, discussed diverse immunological mechanisms against transplanted and chemical carcinogen-induced tumours. In most tumour transplantation models, tumour rejection requires INFγR expression by the host but not by tumour cells. IFNγ produced by either CD4- or CD8-postive T cells acts on non-haematopoietic tumour stroma cells and induces angiostasis. This prevents rapid tumour burden build-up and allows residual tumour cells to be eliminated by immunological mechanisms. Furthermore, chemical carcinogenesis (methylcholanthrene, MCA) is inhibited by an IFNγ-receptor-dependent foreign body reaction. Locally produced IFNγ induces the formation of a fibrotic capsule. Encapsulated MCA can persist virtually life-long in mice without inducing tumours. The foreign body reaction against MCA prevents malignant transformation, probably by reducing DNA damage.
Magnus von Knebel-Doeberitz, Institute of Molecular Pathology, University of Heidelberg, Heidelberg, Germany, focused his talk on predictable tumour antigens in microsatellite unstable cancers: towards a vaccine for familial forms of colon cancer. About 15% of all human colorectal, gastric, and endometrial tumours, and the majority of tumours in patients suffering from hereditary non-polyposis colorectal cancer syndrome, are caused by the loss of DNA mismatch repair functions. Mutations in coding microsatellites (cMS) cause translational frameshifts that may destroy gene function. These frameshifts could also cause the translation of immunogenic neopeptides at the carboxy terminus. He described the immunogenic properties of five additional HLA-A0201-restricted frameshift-induced neopeptides derived from mutations in three cMS-containing genes (Caspase-5, TAF-1b and HT001). One Caspase-5 derived frameshift peptide (FSP 26) was identified as a novel HLA-A0201-restricted CTL epitope. FSP 26-specific CTLs efficiently lyse colon carcinoma cells expressing HLA-A0201 and the underlying (−1) mutation. Thus, newly identified CTL epitopes may serve in a multivalent vaccine against cancers with microsatellite instability.
Session II
Andrea Anichini, Unit of Human Tumour Immunotherapy and Immunobiology, Instituto Nazionale Tumori, Milan, Italy spoke about attack and escape in T lymphocyte-tumour cell interaction. Tumour cells may express antigens which are recognized as HLA/peptide complexes by T cells. The majority of metastatic melanoma patients can recognize differentiation antigens (e.g. Melan-A/MART-1), whereas such a recognition is scanty in the early phase of the disease and in healthy subjects. Despite the presence of melanoma-specific T cells infiltrating tumour lesions, tumour rejection rarely occurs. The inefficient anti-tumour response might be due to an anti-T cell counterattack mediated by FasL-positive tumour cells; FasL is located in the cytoplasm of melanoma cells and is transported into the tumour microenvironment through the release of melanosomes. Furthermore, inducible nitric oxide syntheses might play an important role in determining the inhibition of apoptosis in melanoma cells, which make such cells resistant to T cell attack.
Soldano Ferrone, Department of Immunology, Roswell Park Cancer Institute, Buffalo, USA, discussed the role of HLA class I antigen-peptide complexes in T cell recognition of malignant cells. Convincing evidence has shown that defects in the antigen processing machinery (APM) and HLA class I antigen expression are frequently found in malignant cells. This phenomenon appears to play a major role in the clinical course of the disease, since APM components and/or HLA class I antigen downregulation show a statistically significant, although not absolute, association with reduced disease-free intervals. It is assumed that these clinical findings reflect the escape of tumour cells from T cell recognition. Therefore, this paradigm emphasizes the need to monitor the level of HLA class I antigen–peptide complexes on tumour cells when utilizing CTLs as effector cells to attack tumour cells.
Peter Krammer, Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany, addressed the issue: No life without death: the role of apoptosis in cancer. Many tumour cells express CD95L (CD178, FasL, APO-1L) and may thus kill tumour-infiltrating lymphocytes, a phenomenon called tumour counterattack. The receptor and its ligand are important for apoptosis of peripheral T cells, for downregulation of an immune response, and, at least in part, also for peripheral T cell tolerance. In Aids, apoptosis mediated by this system might contribute to the depletion of T helper lymphocytes; likewise, in disease in which liver cells are destroyed the CD95 system might play a major role. In hepatitis cytotoxic T lymphocytes might use the CD95 system to kill infected hepatocytes. In Morbus Wilson, copper overload leads to upregulation of the CD95 ligand that may finally contribute to acute liver failure. Within an in vitro TCR transgenic T cell model including a model tumour antigen, CD95L(+) tumour cells killed activated T cells and inhibited the expansion of cytotoxic antitumour T cells in mixed lymphocyte-tumour reactions. It is most likely that CD95L mediates tumour counterattack.
Graham Pawelec, Center for Medical Research, University of Tübingen Medical School, Tübingen, Germany talked about the tumour milieu as a hostile environment for immune cells. Tumours are regularly found to be infiltrated by tumour antigen specific T cells and other immunocytes. Why, then, are tumours not usually successfully rejected by the host? The explanation is that tumours are Darwinian paragons commonly winning the battle against the forces of natural immune selection. The escape mechanisms may be classified into at least six major groups: (1) alteration of MHC class I and tumour antigen expression, (2) dysregulated expression of adhesion/accessory molecules by tumour and/or antigen presenting cells, (3) tumour utilization of products of stimulated leukocytes, i.e. immunostimulation of cancer, (4) secretion of immunosuppressive soluble factors either by tumour cells or infiltrating T cells or both, (5) induction of immune non-responsiveness via anergy or clonal deletion of responding T cells or induction of suppressor cells, and (6) changes in T cell signal transduction molecules. In addition to these considerations, which in most cases focus on the cancer cells themselves rather than the whole tumour, it will almost certainly be necessary to target other components, particularly blood vessel endothelial cells and additional stromal elements. Tumour escape from immunosurveillance represents the last series of hurdles to be overcome in formulating truly effective cancer immunotherapy, but given the immense plasticity of the tumour cells, and the complex balance between pro- and anti-tumour activity of the very same effector pathways, this remains a major challenge for the 21st century.
Session III
Jay Berzofsky, Vaccine Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MA, USA, spoke on the role of CD4+ NK T cells, IL-13, myeloid cells, and TGF-ß in the regulation of tumour immunosurveillance. He has characterized a novel immunoregulatory circuit in which CD1d-restricted NK T cells inhibit tumour immunosurveillance against a regressor fibrosarcoma tumour which grew, regressed and then recurred. CD4+ NK T cells were induced by the tumour to secrete IL-13, which led to inhibition of CD8+ T cell-mediated regression of the tumour, allowing late tumour recurrence. The NK T cells and IL-13 are necessary to induce a non-lymphoid cell, defined as a myeloid cell, to secrete TGF-ß, which can directly suppress CTL induction. Blockade or elimination of any of these four components: CD4+ NK T cells, Il-13, Gr-1 positive myeloid cells, or TGF-ß could prevent tumour recurrence.
Suzanne Ostrand-Rosenberg, University of Maryland, Baltimore, USA, talked about tumour immunity: a balancing act between T cell activation and suppression. Immune suppression, which occurs in many cancer patients, is a major impediment for developing successful cancer immunotherapy. Although there are numerous types of immune suppression, tumour-induced myeloid suppressor cells (MSC), also known as immature myeloid cells, are found in many patients and in animals with transplanted and spontaneous tumours. She has recently identified a gene, the Signal Transducer and Activator of Transcription 6, (STAT6) gene, that when deleted in the host, confers resistance to established metastatic disease, if the primary 4T1 tumour is surgically removed. More than 60% of STAT6-deficient mice are resistant and survive indefinitely as compared to less than 5% of STAT6-competent BALB/C mice. The STAT6 gene appears to play a pivotal role in enhanced immunity via two pathways. The STAT6 transcription factor is thought to transmit intracellular signals initiated by cytokines that mediate the accumulation of MSC. The macrophages of STAT6-deficient mice are tumouricidal because they produce iNOS and do not produce arginase. Since STAT6 proteins are essential for transmitting signals that induce arginase biosynthesis, in the absence of STAT6 protein, macrophages are polarized towards iNOS production (M1 response).
Barbara Seliger, Institute of Medical Immunology, University Halle, Halle/Saale, Germany, talked about the hide and present in tumours—the role for HLA antigens in mediating immune privilege. Proper HLA class I antigen processing and presentation is a prerequisite for the recognition of tumour cells by cytotoxic T cells. In murine in vitro models of oncogenic transformation, an association between ras-mediated transformation and down-regulation of different components of the MHC class I antigen processing machinery (APM) has been described. To date there exists only limited information on the expression of components of the HLA class I APM in human tumours compared to normal tissue. In colorectal cancer, APM component deficiencies (e.g. TAP1, LMP2, and tapasin) occur more frequently in Ki-ras-mutated colorectal carcinoma lesions. In primary and metastatic renal cell carcinoma a high frequency of a total lack of heterogeneous TAP1, LMP2 and LMP7 expression could be found. In Ki-ras mutated colorectal carcinomas the APM abnormalities appear to be associated with the disease stage, but in renal cell carcinomas the APM component deficiencies are associated with tumour subtypes but not with tumour grading and staging.
Theresa Whiteside, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA, spoke on apoptosis of T cells in the circulation of patients with cancer and its consequences. The fate of CD8+ effector T cells in cancer patients is of concern, because data are showing that tumour infiltrating as well as circulating T lymphocytes are susceptible to spontaneous apoptosis. Dysfunction of T cells and decreased absolute T cell subset numbers are common findings in patients with head and neck, breast ovarian carcinomas and in melanomas. Elimination of CD8+ T cells via a novel mechanism involving circulating membranous vesicles (MV) in patients with cancer has been observed and depends on expression of the membrane form of FasL on MV. Pretreatment of T cells obtained from cancer patients with cytokines known to regulate T cell homeostasis (IL-2, Il-7, IL-12, and IL-15) on molecular targets engaged in apoptosis can rescue T cells from apoptosis.
Session IV
Olivera Finn, Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, discussed the good and bad interactions between antigen presenting cells and tumour antigens. Shared tumour antigens that are immunogenic in humans are important tools to develop tumour vaccines. One such antigen is MUC1 mucin. The antigen is expressed on normal epithelial cells as a highly O-glycosylated molecule, composed of 20 aa tandem repeats; soluble MUC1 produced by tumour cells, however, cannot be processed and presented by the patient’s antigen processing cells, resulting in the lack of MUC1-specific T helper cells, low frequency of cytotoxic T cells and low titers of IgM. A shorter, synthetic form of MUC1 (100 mer peptide) is processed by dendritic cells and elicits MUC-specific helper T cells, a higher frequency of cytotoxic cells and multiple IgG antibody isotypes resulting in tumour rejection in MUC1 transgenic mice. In spite of the high level of immunogenicity observed in animal models with a MUC1 vaccine (peptide plus SB-AS2 adjuvant), the immunogenicity of this vaccine was very low in pancreatic carcinoma patients. MUC1 may attract immature dendritic cells to the tumour through chemotaxis and then negatively influence their ability to stimulate effective helper T cell response; this may be an explanation for the failure of patients to generate better anti-MUC1 responses during the growth of the tumour, or to process and present this antigen when delivered as a vaccine. Another trial is ongoing testing a vaccine of MUC1 antigen-loaded and matured dendritic cells injected subcutaneously in the upper arm once every 3 weeks for a total of three doses; a booster dose is given 6 months later. Twelve patients are enrolled to date and dendritic cell generation from peripheral blood has been successful. Two patients have shown isotype switching after the second injection, suggesting activation of helper T cells.
Licia Rivoltini, Unit of Immunotherapy of Human Tumours, National Tumour Institute of Milan, Milan, Italy, addressed the role of tumour-released microvesicles in the down-modulation of T cell-mediated immune responses. During the process of oncogenic transformation, tumour cells escape from normal growth control and tissue homeostasis mechanisms, and acquire the ability to invade surrounding tissues and establish metastases. Cancer cells must be capable of eluding recognition by the immune system, despite their expression of antigenic determinants that might be detected by specific T cells. Melanoma cells were found to release massive amounts of melanosome-derived microvesicles (MV) bearing melanoma antigens and active FasL, which induce Fas-mediated apoptosis in activated T lymphocytes and thereby promote potential tumour immune escape; melanoma cells are not killed because they are resistant to Fas-induced apoptosis. Melanoma-released MV share several phenotypical features with another type of organelles, the so-called ”exosomes”; these are vesicles of 50–100 nm diameter, secreted through vesicular transport and exocytosis by different cells under normal as well as pathological conditions, and play a role in receptor discharge, tissue-specific functions, and intercellular crosstalk. Secretion, which is amplified in tumour cells, can thus represent a powerful tool for modulating host environment and creating favourable conditions for neoplastic progression. Monocytes, for example, which are resistant to the pro-apoptotic activity of tumour-derived MV, are impaired in their ability to differentiate into iDC or macrophages when co-incubated with tumour-released MV. MV release might represent a new mechanism of tumour immune escape that might have important prognostic and therapeutic implications.
Rolf Kiessling, Cancer Center Karolinska Institute, Stockholm, Sweden, spoke on the great escape: similarities and differences by which tumour cells evade NK cells and T cells. While loss of MHC class I expression may lead to less efficient rejection of a tumour by CD8+ T cells, it primes such tumour variants to enhance rejection by NK cells due to abrogation of signalling through inhibitory NK receptors. Therefore, the consequences of exposing tumour cells to factors such as IL-10 and IFNγ result in their divergent susceptibility to NK-mediated versus T cell-meditated anti-tumour mechanisms. Data gathered from mouse models as well as observations with the uncommon malignancy, uveal melanoma, have demonstrated that high MHC class I expression for certain tumours can be a poor prognostic sign. These data contrast the current paradigm that low expression of MHC class I favours escape from T cell-mediated immunity. As CD8+ T cells often express NK inhibitory receptors like CD94/NKG2A which bind to the non-classical HLA-E molecule, upregulation of this NK ligand by IFNγ may also protect rather than enhance tumour sensitivity to T cell-mediated cytotoxicity. Oxidative stress is also a critical factor responsible for tumour-induced immune suppression, which affects T cells as well as NK cells.
Nikolaus Bresgen, Department of Cell Biology, University of Salzburg, Austria, spoke on the potential role of isoferritins in apoptosis. Conditioned media (CM) from primary rat hepatocyte cultures contain compounds which are able to induce apoptosis as well as prominent changes of cell morphology in a time-dependent manner in primary rat hepatocytes. Furthermore, purified CM is able to significantly induce apoptosis in human hepatoma cells. Purification of the active principle of CM showed that they contained acidic isoferritins, for which, in addition to iron storage function, immunomodulatory and immunosuppressive properties have been demonstrated. Placental isoferritin participates in the suppression of the maternal immune response to fetal antigens and a novel isoferritin subunit (placental immunomodulatory factor) has been identified containing a specific domain at the C-terminus (c48 domain) which appears to be the immunoregulatory unit. It is possible that Fe2+ released from the isoferritin molecule may lead to conditions of enhanced oxidative stress, driven by the free radical-generating Fenton reaction and lipid peroxidation, thereby inducing apoptosis.
Session V
Karin de Visser spoke for Lisa Coussens, Cancer Research Institute, Department of Pathology and the UCSF Comprehensive Cancer Center, University of California, San Francisco, USA about the innate and adaptive immune interactions which regulate cancer development. Accumulating clinical and experimental evidence suggest that chronic inflammation contributes to cancer development. Using a transgenic mouse model of multi-stage squamous carcinogenesis, K14-HPV 16 mice, they have shown that mast cells potentiate early epithelial carcinogenesis through their release of growth factors and specific serine and metalloproteases. Since genetic elimination of either CD4+ or CD8+ T cells alone does not result in reduced leukocyte infiltration or altered characteristics of premalignant progression, they hypothesized that initiation of the tumour-promoting innate immune response is mediated by a B cell-dependent process. Indeed, adoptively transferred B cells from HPV16 mice to HPV16 mice lacking the complete adaptive immune system restores the recruitment of innate immune cells into neoplastic skin as well as enhancing the characteristic features of premalignant progression, e.g. proliferation and angiogenesis. Such findings suggest that the adaptive immune system is required for innate immune cell-mediated potentiation of tumourigenesis.
Kerstin Lang, Institute of Immunology, University Witten/Herdecke, Germany, reconsidered the seed and soil hypothesis with particular reference to new molecules involved in the metastatic process. The ”seed and soil” hypothesis was originally postulated by Paget (1889), where both ”seed” (the cancer cell) and ”soil” (factors in the organ environment) contribute to the organ specificity of metastasis formation. The development of metastases is a multi-step cascade for which the metastasizing tumour cell needs, among other things, to acquire the capability of active migration. It is well known for immune competent cells that ligands to G protein-coupled receptors (GPCRs), such as chemokines, regulate the migratory activity and the homing of leukocytes. Recently, it could be shown that chemokines, especially neurotransmitters like norepinephrine, dopamine and substance P are strong cues for the initiation of the locomotory activity of breast and colon cancer cells. The neurotransmitter-induced development of a metastatogenic phenotype is accompanied by a significant activation of cAMP-response element binding protein (CREB), caused by down-regulation of the two actin-binding proteins gelsolin and MARCKS as well as by up-regulation of the collagen receptor α2integrin. The neurotransmitter signals, which switch the locomotory machinery from an idle to an active state can be abrogated by inhibitory neurotransmitters like GABA and anandamide or blocked by clinically established receptor agonists and antagonists. The functional interplay between the nervous system, the immune system and the tumour cells demonstrates a further dimension of complexity of metastasis formation, yet concomitantly opening new pathways for specific intervention.
Kurt S. Zaenker, University Witten/Herdecke, Germany, concluded with a summary of this symposium and expressed special thanks to the officers of the Fritz-Bender-Foundation (Munich, Germany) for supporting this 10th very successful conference in this respected series of symposia organized annually together with the UICC, Geneva, Switzerland, and held around the world.