- Email: [email protected]
Workshop Q: Tumor Immunology
Workshop Q Tumor Immunology
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Molecular Tumor Genetics and Immunogenetics, Max-Delbrück-Center for Molecular Medicine, Berlin and 2GSF-National Research Centre for Environment and Health, Neuherberg, Germany
Q. 1 Subcellular localization of latent and early lytic transforming proteins encoded by human herpesvirus 8 J. BENGA1, N. THIRUNARAYANAN1, F. CIFIRE1, E. KREMMER2, and M. LIPP1 All stages of Kaposi’s sarcoma, as well as multifocal Castleman’s disease (MCD) and primary effusion lymphoma (PEL) have been associated with the presence of human Herpesvirus-8 (HHV8), a gammaherpesvirus encoding 81 open reading frames. However, only a small subset has been shown to be transcribed in HHV-8 latently infected PEL-derived cell lines (BCP-1, BCBL-1) and in cells of the KS lesions. Among them, Kaposin, a latent protein has been shown to have transforming potential in Rat-3 cells, and oncogenic potential after injection into nude mice. For the analysis of the subcellular localization of Kaposin, we used monoclonal antibodies which can recognize all three known forms: Kaposin A (small protein of 60 amino acids), Kaposin B (encoding direct repeats only) and Kaposin C (encoding direct repeats and Kaposin A). We noticed by immunofluorescence cytoplasmic localization of Kaposin A in transfected HeLa cells in contrast to different localization of Kaposin proteins in PMA induced BCBL-1 cells. PMA induction of BCBL-1 cells has also shown the significant expression level of an other viral oncogene, the chemokine receptor vGPCR, indicating its early lytic origin. In parallel, cytoplasmic and membrane localization of vGPCR has been shown in retroviral transduced stable NIH3T3 clones and in transiently transfected HeLa cells using specific monoclonal antibody 7D1 by FACS analysis and immunofluorescence. A 45kDa product of vGPCR was detected by Western blot of membrane preparations of transfected HeLa cells. The elucidation of expression profiles and transformation mechanisms of Kaposin and the chemokine receptor vGPCR is important to validate viral proteins as suitable targets for therapy of HHV-8 associated malignancies.
1 Laboratory of Immunobiology, Department of Dermatology and 2Section of Immunobiology, Institute of Zoophysiology, University of Bonn, Bonn, Germany
Q. 2 Activation of MAGE-3 specific T helper cells from healthy donors and melanoma patients by peptide-loaded and Ii/MAGE fusion construct transfected dendritic cells M. BERG1, B. WINZEN2, N. KOCH2, T. BIEBER1, and S. KOCH1 CD4+ T lymphocyte responses are important for anti-tumor immune reactions as has been demonstrated in animal models. Characterization of the CD4+ T cell epitope repertoire on mela-
252 · 33rd Annual Meeting of the German Society of Immunology noma antigens and specific targeting of endogenously synthesized antigens to the class II loading compartment would improve decisively the efficacy of peptide-based immunization protocols in neoplastic patients. MAGE-3 (171–185) peptide has been shown to bind promiscuously to MHC II molecules of all donors tested. Therefore, we used mature DCs as APC to stimulate MAGE-3 specific T cells from healthy donors and melanoma patients. Activation of the T cells was determined using an ELISPOT assay for interferon-g and by measuring T cell proliferation. Using peptide-loaded mature DCs, we could stimulate T cells from all healthy donors tested and from 6 of 11 melanoma patients. MAGE-3 (171–185) peptide specific T cells could be enriched by weekly restimulation. To improve the loading of MHC II molecules and thereby enhance efficacy of antigen presentation, we transfected immature DCs from a healthy donor with a fusion construct consisting of the MHC II associated invariant chain and MAGE-3 (171–185) sequence by means of electroporation. 30% of transfected DCs expressed the transgene and viability after transfection was at 60%. Such transfected DCs were superior to peptideloaded DCs in their capacity to stimulate primary MAGE-3 (171–185) specific T cells, which produced more interferon-g and interleukin-12. This novel antigen delivery system holds promise for a variety of clinical applications including cancer therapy.
Departments of 1Dermatology and 3Pathology, Ludwig-Maximilians University, and Department of Nuclear Medicine, Technical University, Munich, Germany
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Q. 3 Th1 cells inhibit carcinogenesis in transgenic mice H. BRAUMÜLLER1, B.J. PICHLER2, A. SAKRAUSKI1, N. MÜLLER-HERMELINK1, M. KNEILLING1, K. GHORESCHI1, M. SCHWAIGER2, R. HUSS3, W.A. WEBER2, and M. RÖCKEN1 The transgenic mouse model RIP1-Tag2 is a powerful tool to investigate the role of tumor-specific T cells in carcinogenesis. In these mice the SV40 T antigen (Tag) is expressed in insulin-producing beta cells of the pancreas, causing 2% of the islets to develop into adenomas and carcinomas. Subsequently mice die of hypoglycemia at an age of 14–16 weeks. To test, whether Th1 cells can eradicate islet adenomas, we adoptively transferred activated Tag-specific Th1 cells. Th1 cells were generated by stimulating CD4+ cells derived from lymphnodes and spleen of transgenic C3H mice with T antigen, antigen presenting cells and CpG 1668 oligonucleotide. Treatment of RIP1-Tag2 positive mice with Th1 cells resulted in nearly a doubling of the life span. Paraffin-embedded sections revealed strong inhibition of tumor growth in all of the treated animals accompanied by inhibition of angiogenesis. No destruction of the pancreas could be observed in treated mice. IFN-g producing Th1 cells may promote apoptosis of tumor cells or endothelial cells lining the tumor vessels. At an age of 8 weeks, the time when carcinomas start to develop, islet cells and endothelia from untreated mice or Tag-Th1-treated mice still contained similar numbers of apoptotic cells. Alternatively, tumor growth may be controlled by angiogenic inhibitors, namely interferon-inducible protein 10 (IP10) or monokine-induced by IFN-g (Mig). Quantitative PCR of pancreas tissue revealed at least 10-fold higher mRNA levels of IP10 and Mig in Th1-treated mice than in healthy controls or tumor-bearing RIP1-Tag2 mice. Our results show that adoptive transfer of tumor-specific Th1 cells was highly efficient in tumor therapy and did not cause any side effects or any signs of autoimmune diseases. The mechanism of this highly efficient delay of tumor progression seems to base on inhibition of angiogenesis and not direct killing of tumor cells.
33rd Annual Meeting of the German Society of Immunology · 253 1
Department of Gastroenterology, Medizinische Hochschule, Hannover, 3Department of Gastroenterology, University of Ulm, Ulm, Germany, and 2Department of Pharmacology, University of California, La Jolla CA, USA
Q. 4 Immunotherapy of pancreatic cancer in a transgenic mouse model A.I. GARBE1, F. KORANGY1, A. HELLER1, F.R. GRETEN2, R.M. SCHMID3, M.P. MANNS1, and T.F. GRETEN1 Human pancreatic cancer is associated with a poor prognosis and alternative therapies such as immunotherapy are needed. Transgenic mice that develop spontaneously tumors in situ provide a biological more relevant model for the development of vaccination strategies than transplantable tumors. We have begun to analyse TGF-a transgenic mice which overexpress TGF-a in the pancreas (Sandgren, 1990). TGF-a transgenic mice develop fibrosis and ductal pancreatic cancer at the age of one year (Wagner, 1998). Crossbreeding these mice with p53 knockout mice dramatically accelerate tumor development. These mice represent the first model of pancreatic adenocarcinoma with genetic alterations as well as growth characteristics similar to the human disease (Wagner, 2001). Our aim is to establish a potent vaccine against pancreatic cancer in these mice. Preliminary studies showed that a cell line deriving from a tumor of a TGF-a+/–p53+/– mouse on BALB/c¥C57BL/6 background grows in vivo in nu/nu and in BALB/c¥C57BL/6 after s.c. injection and shows the typical growth pattern with cystic and ductal structures, which resemble the primary tumor. Preliminary vaccination experiments show an immunogenicity of this murine pancreatic tumor cell line. For further immunological experiments these mice were bred to a C57BL/6 background. TGF-a+/–p53–/– mice on C57BL/6 background develop ductal pancreatic adenocarcinoma after 80 days. We are presently establishing a pancreatic tumor cell line from these mice to perform further vaccination experiments. DNA vaccination experiments were performed to investigate the role of TGF-a as a potential tumor antigen. Immunization of TGF-a transgenic mice with DNA encoding TGF-a induced a TGF-a specific antibody response in wt littermates but not in TGF-a transgenic mice. A specific cytotoxic T cell response could be detected in all mice which received multiple doses of immunization. Thus, TGF-a seems to represent an antigen capable to elicit a substantial adoptive antitumor immune response.
Department of Surgery, University of Regensburg, Regensburg, Germany
Q. 5 Rapamycin: A double-edged sword capable of preventing allograft rejection, while simultaneously attacking cancer via an antiangiogenic effect M. GUBA, G. KÖHL, M. STEINBAUER, S. FLEGEL, M. ANTHUBER, K.W. JAUCH, and E.K. GEISSLER Background: Cancer is an ominous risk factor for immunosuppressed transplant patients. A potential breakthrough towards addressing this problem comes from our recent finding that immunosuppressive doses of rapamycin (RAPA) destroy tumors in mice. Here, we explored how
254 · 33rd Annual Meeting of the German Society of Immunology RAPA mediates its antitumor effect, focusing on cell proliferation and the proangiogenic cytokine, VEGF. Methods: Proliferation of human umbilical vein endothelial cells (HUVECs), CT26 (murine colon adenocarcinoma) and B16 (murine melanoma) cells in the presence of RAPA (0.01– 1 mg/ml) was determined by BrdU uptake. The effect of RAPA on in vitro VEGF-stimulated angiogenesis was tested in a HUVEC tubular-formation assay. In vitro VEGF protein secretion was measured by ELISA in 24h culture supernatants of RAPA-treated tumor cells. VEGF mRNA levels from tumor cells were quantified by RT-PCR (Light Cycler) 10 h after treatment. Results: Although RAPA, even at the highest dose tested (1 mg/ml), showed only a modest inhibition (<24%) of CT26 and B16 tumor cell proliferation, HUVEC proliferation decreased with RAPA at 0.01, 0.1 and 1 mg/ml (11±7%, 36±8% and 43±12%, respectively). The high sensitivity of HUVECs to RAPA suggested the main antitumor effect of this drug may be via interference of endothelial cell stimulation. Indeed, RAPA decreased recombinant VEGF-stimulated HUVEC proliferation by 76±1%. Furthermore, we could show in vitro tubular formation, stimulated with VEGF, was 100% blocked by RAPA. Besides blocking VEGF stimulation of endothelial cells, RAPA (0.1 mg/ml) inhibited constitutive VEGF production by CT26 and B16 tumor cells (34–37%). RAPA treatment also lowered VEGF mRNA levels 4-fold. Summary: In conclusion, (1) high doses of RAPA modestly inhibit tumor cell proliferation, (2) VEGF-induced endothelial cell activities are blocked by immunosuppressive levels of RAPA, and (3) VEGF production by tumor cells is inhibited by RAPA. Thus, the antiangiogenic effects of RAPA could be useful for the prevention and treatment of tumors in transplant patients.
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany
Q. 6 Nitric oxide differentially regulates expression of proangiogenic IL-8 and VEGF and tumor-suppressive IP-10 in colon carcinoma cells M. HELLMUTH, H. MUHL, J. PAULUKAT, R. NINIC, and J. PFEILSCHIFTER Expression of inducible nitric oxide (NO) synthase and production of NO appears to be a marker of tumor progression in human neoplasia, among them melanoma, head and neck cancer, and colorectal cancer. Since tumor-promoting functions of NO have been associated with increased angiogenesis at the tumor site, we investigated effects of NO on the production of two selected chemokines that are supposed to differentially regulate tumor growth, namely proangiogenic interleukin (IL)-8 and tumorsuppressive interferon-inducible protein-10 (IP-10). Both chemokines were produced by colon carcinoma cells after stimulation with the combination IL-1b/interferon-(IFN)-g. Under these conditions, release of IL-8 was exclusively mediated by IL-1b but not by IFN-g, whereas production of IP-10 was dependent on the presence of both, IL-1b and IFN-g. Effects of NO were analyzed by incubation with the NO-donor DETA-NO which spontaneously releases NO with a characteristically long half-life of 16.5h. NO amplified expression and release of IL-8 from colon carcinoma cells as detected by RNase protection assay and ELISA analysis. In contrast, IL-1b/IFN-g-induced production of IP-10 was suppressed by NO. In addition to IL-8, expression of vascular endothelial growth factor (VEGF), was inducible by NO. The present data are consistent with previous observations that relate NO to enhanced tumor angiogenesis and imply that NO-mediated upregulation of IL-8 and VEGF and downregulation of IP-10 synthesis may contribute to this phenomenon.
33rd Annual Meeting of the German Society of Immunology · 255 Department of Tumor Progression and Immune Defense, German Cancer Research Center, Heidelberg, Germany
Q. 7 Donor T cell and host NK depletion improve the therapeutic efficacy of allogeneic bone marrow cell reconstitution in the non-myeloablatively conditioned tumor-bearing host S. HUMMEL, D. WILMS, M. VITACOLONNA, and M. ZÖLLER Allogeneic bone marrow cell (BMC) reconstitution of the non-myeloablatively conditioned host has the advantage that it can be tolerated in suboptimal health conditions. However, the problem of graft versus host disease (GvHD) remains. Also, graft acceptance may become delicate and HvGD may arise. We report here on advantages/disadvantages of host NK depletion and graft T cell depletion in fully allogeneic healthy and a solid tumor-bearing mice. NK depletion of the healthy host improved the survival rate, whereas graft T cell depletion was disadvantageous. In the tumor-bearing host, graft T cell depletion was beneficial when the host was NK-depleted. Host NK depletion facilitated B lymphopoiesis, repopulation of the thymus, expansion of donor cells and tolerance induction. The disadvantage of graft T cell depletion in the healthy host was due to delayed engraftment. Since in tumor-bearing mice, host, but not graft hematopoiesis was strongly impaired, donor hematopoiesis dominated. Graft T cell depletion reduced GvHD, but hardly interfered with engraftment. Importantly, graft-mediated tumor reactivity appeared late and was unimpaired when the graft was T cell-depleted. Thus, concomitant depletion of host NK and donor T cells is advantageous when approaching therapeutic treatment of solid tumors by allogeneic reconstitution of the non-myeloablatively conditioned host.
Institutes of 1Immunology and 4Pathology, and 2Department of Urology, Medical Faculty Carl Gustav Carus, Technical University of Dresden, Dresden, and 3Department of Immunology, Institute of Cell Biology, University of Tübingen, Tübingen, Germany
Q. 8 Identification of an HLA-A*0201-restricted T cell epitope derived from the prostate cancer-associated protein trp-p8 A. KIESSLING1, S. FÜSSEL2, M. SCHMITZ1, S. STEVANOVIC3, A. MEYE2, B. WEIGLE1, U. KLENK4, M.P. WIRTH2, and E.P. RIEBER1 Immunotherapy of prostate carcinoma (PCa) is advanced by the identification of tumor-associated proteins that are suitable antigens for vaccination strategies. Expression of these proteins should be restricted to prostate and prostate tumors and should ideally be upregulated in the malignant tissue. An additional prerequisite is their capacity to induce an effective immune response in vivo. The number of antigens displaying these characteristics is still limited. Here, we analyzed the newly described (1) PCa-associated protein transient receptor potential-p8 (trp-p8). To determine the abundance of trp-p8 in prostate tumors the expression level of trp-p8 mRNA was quantitatively analyzed in 33 matched samples of malignant and non-malignant prostate tissue. Trp-p8 mRNA was found to be expressed in all prostate tumors and in the corresponding normal prostate tissues. In addition, transcripts were upregulated in the tumorous
256 · 33rd Annual Meeting of the German Society of Immunology samples when compared to the non-tumorous specimens. To identify epitopes recognized by cytotoxic T lymphocytes (CTLs) HLA-A*0201-restricted peptides were selected from the amino acid sequence of the trp-p8 protein using a screening algorithm and tested for their affinity to HLA-A*0201 molecules. Five peptides were loaded on autologous dendritic cells for the in vitro activation of CTLs. One of these peptides (GLTKYIGEV) was found to induce specific CTLs which efficiently lysed HLA-A*0201-positive PCa cells thus confirming the endogenous production and presentation of this peptide by tumor cells. Based on these findings trp-p8 appears as a suitable antigen for the design of vaccines in PCa. (1) Tsavaler et al., Cancer Res. 61:3760 (2001) This work was supported by the Wilhelm-Sander-Stiftung and the Deutsche Forschungsgemeinschaft (SFB510).
Institute für 1Medizinische Mikrobiologie, Immunologie und Hygiene and 2Genetik, Universität zu Köln, Köln, 4Institut für Humangenetik, Universität zu Kiel, Kiel, Deutschland, and 3Department of Biological Sciences, University of Chicago, Chicago IL, USA
Q. 9 Reversal of commitment to the B cell lineage in acute lymphoblastic leukemia harboring a BCR-ABL1 gene rearrangement F. KLEIN1, V.S. BARATH1, N. FELDHAHN2, S. LEE3, G. ZHOU3, S.M. WANG3, S. HARDER4, R. SIEBERT4, M. KRÖNKE1, J.D. ROWLEY3, and M. MÜSCHEN1 Pre-B cells (PBC) have been identified as the precursor of B lineage acute lymphoblastic leukemia (B-ALL) harboring a BCR-ABL1 fusion gene. Normal PBCs are destined to die by apoptosis unless they are rescued by survival signals through the pre-B cell receptor (pBCR). To define growth requirements of such B-ALL clones, we generated and compared genome-wide gene expression profiles of CD34+ hematopoietic progenitor cells (HSC), PBCs and two cases of BALL. To analyze global gene expression profiles of B-ALL cells and their normal precursors, we chose the serial analysis of gene expression (SAGE) technique, which provides quantitative gene expression data at a genome-wide level (including a total of 277,000 SAGE tags). About 80 percent of the genes initially expressed in HSC were silenced in B-ALL cells. Unexpectedly, these genes include virtually all determinants of the B cell lineage. Loss of PBC identity involves constituents of the pBCR (VpreB, l5, IgH Cm, Iga, Igb) and the V(D)J-recombination machinery (RAG1, RAG2, TdT), proximal protein tyrosine kinases such as BTK and BLK and key transcription factors, including E2A, EBF and PAX 5. Given the critical role of pBCR-mediated signals for the survival of PBCs, the question arises, how leukemia cells can establish a highly proliferating clone in the absence of pBCR signaling. Indeed, expression levels of NF-ÍB and other activation-induced nuclear molecules are similar in B-ALL cells as in their normal counterpart. These findings suggest that a transcriptionally silenced pBCR signaling complex in BCR-ABL1+ leukemia cells can be effectively replaced by alternative survival signals, which most likely arise from the oncogenic ABL1 tyrosine kinase. Specific silencing of genes, whose expression was induced during the transition from the HSC to the PBC stage of development demonstrates that BALL cells have phenotypically reversed their commitment to the B cell lineage.
33rd Annual Meeting of the German Society of Immunology · 257 1
Abteilung Genregulation, Gesellschaft für Biotechnologische Forschung (GBF), Braunschweig and 2University Hospital, Freiburg, Germany
Q. 10 Breaking tolerance against hepatocellular carcinoma development by an activatable interferon regulatory factor-1 A. KRÖGER1, M. GEISSLER2, and H. HAUSER1 IRF-1 is a multifunctional transcription factor involved in cell growth regulation, pathogen response and immune activation. Overexpression of IRF-1 leads to the induction of several genes and a significant suppression of cell growth. To elucidate the potential of IRF-1 for cytokine gene therapy of cancer we investigated IRF-1 effects in a mouse model. We expressed an estradiolactivatable fusion protein of IRF-1 in a hepatocellular carcinoma cell line Hepa1–6. Activation of IRF-1 leads to growth inhibition and a decrease of anchorage independent growth. In addition, IRF-1 expressing cells are characterized by enhanced MHC I, MHC II expression and IFN-b secretion. Tumor growth in estradiol-treated syngenic C57L/J mice was completely suppressed, while in estradiol-untreated mice tumors developed normally. The pretreated mice (HepaIRF1hER plus estradiol) were protected against rechallenge with wild type Hepa1–6 cells even in the absense of estradiol, suggesting induction of tumor specific immunity. In fact, significant CTL activity directed against Hepa1–6 and endogenously expressed self antigen a-fetoprotein was detected. However, the antitumoral effects are only partially dependent on CD4+ and CD8+ T cells. In addition, in mice bearing HepaIRF-1hER tumors estradiol treatment resulted in an arrest of tumor expansion. In conclusion, the data demonstrate that IRF-1 suppresses HCC growth through both, a direct antitumor growth effect and enhanced immune cell recognition of the tumor and thus is a promising candidate for gene therapy of HCC.
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Department of Hematology and Oncology, Johannes Gutenberg-University, Mainz, Germany and 2Ludwig Institute of Cancer Research, University of Lausanne, Epalinges, Switzerland
Q. 11 Reprogramming of CD4+ T cells by gene transfer of a class I MHCrestricted hyper-affinity T cell receptor J. KUBALL1, R.-H. VOSS1, F. SCHMITZ1, P. ROMERO2, P. GUILLAUME2, M. JÜLCH1, C. HUBER1, and M. THEOBALD1 T cell receptor (TCR) gene transfer of murine TCR into human T cells allows circumvention of tolerance to tumor associated antigens (TAA). So far, CD8+ T cells could functionally be targeted by class I MHC-restricted TCR gene transfer. To reprogram CD4+ T cells as well as to further improve efficacy of TCR transduced CD8+ T cells, the impact of a CD8-independent TCR was investigated. Cytotoxic T lymphocytes (CTL) specific for p53 (264–272) and MDM2 (81–88) were obtained from A2 and CD8¥A2Kb transgenic (tg) mice, respectively, by peptide immunization. CTLs from A2 tg mice were shown to be CD8-independent as opposed to CTLs obtained from CD8¥A2Kb tg mice. CTLs were cloned, murine TCRs were isolated and expressed by retroviral gene transfer into human T cells. Subpopulations were analysed for tetramer-staining, cytotoxi-
258 · 33rd Annual Meeting of the German Society of Immunology city and cytokine secretion. Gene transfer of an HLA-A2-restricted CD8-independent TCR into human T cells resulted in human CD8+ CTLs that recognized target cells with higher efficacy as compared to the parental murine T cell clone. Reprogramming of CD4+ T cells into class I MHC-restricted antigen-specific effector cells required the expression of a CD8-independent TCR. In summary, efficacy of CD8+ TCR transgenic T cells can be further augmented by a CD8 independent TCR. These hyper-affinity TCRs target CD4+ T cells effectively. This suggests, that class I MHC-restricted TAA-specific CD4+ T cell-mediated help in vivo can be exerted by CD8independent hyper-affinity TCR gene transfer.
Department of Hematology and Oncology, Johannes Gutenberg-University, Mainz, Germany
Q. 12 Universal targeting of multiple myeloma by PRDI-BF1 antigen-specific CTL C. LOTZ, U. LIEWER, C. HUBER, and M. THEOBALD Growing clinical and experimental evidence indicates that multiple myeloma (MM) is susceptible to cytotoxic T lymphocyte (CTL)-based immune interventions. These CTL recognize peptides derived from the endogenous processing of cellular proteins and presented by MHC class I molecules. The PRDI-BF1/Blimp-1 protein is inherently involved in the terminal differentiation of mature B lymphocytes into malignant and non-malignant plasma cells. It likely provides therefore an abundant and universal MM-associated target molecule. HLA-A*0201 (A2.1) transgenic (tg) mice were used to identify A2.1-presented CTL epitopes derived from the PRDI-BF1 protein. CTL with specificity for at least two PRDI-BF1-derived peptides were able to kill various MM cell lines while preserving the integrity of non-transformed cells, such as A2.1+ T and B cells and monocyte-derived dendritic cells (DC). However, recognition by CTL was not limited to malignant plasma cells. Consistent with CTL recognition, protein expression by immunohistochemistry revealed that PRDI-BF1 expression too was not restricted to MM cells only. Substantial protein levels were detected in different solid tumor cell lines, i.e. melanomas, osteosarcomas, breast cancer, and hepatocellular carcinoma. The more abundant expression pattern of PRDI-BF1 suggested that the self-A2.1-restricted human T cell repertoire is affected by PRDI-BF1-specific self-tolerance. To study this hypothesis, naïve CD8+ human T lymphocytes underwent in vitro stimulation with A2.1+ autologous DC pulsed with the relevant PRDI-BF1 peptides. Effector cells were induced at least in one out of two healthy donors. They specifically produced IFN-g but were not able to kill PRDI-BF1 peptide-pulsed target cells. Our results indicate that PRDI-BF1-derived CTL epitopes can serve as MM-associated and, to some extent, as broader tumor antigens. The transduction of PRDI-BF1-specific T cell receptors selected from A2.1-tg mice into human T lymphocytes offers a possibility to turn self-A2.1restricted human T cells into efficient MM-reactive CTL.
33rd Annual Meeting of the German Society of Immunology · 259 Department of Internal Medicine II, Eberhard-Karls-University, Tübingen, Germany
Q. 13 Induction of myeloma specific cytotoxic T cells using dendritic cells transfected with tumor-derived RNA C. MILAZZO, V. REICHARDT, M.R. MÜLLER, F. GRÜNEBACH, and P. BROSSART We investigated myeloma RNA transfection of dendritic cells (DCs) to induce myeloma (MM) specific cytotoxic T cell (CTL) responses in vitro. By this methodology we hope to bypass the need for the identification of MM associated antigens or specific antigens such as idiotype (Id). Monocyte derived DCs from buffy coats, which were matched in the HLA class I haplotype to the myeloma cell lines LP-1 and U266, were used for RNA transfection. DCs were electroporated with total myeloma cell line RNA and were used as antigen presenting cells for the induction of myeloma specific CTL. After a single restimulation with RNA transfected DCs, cytotoxic activity of induced T cells was analyzed in 51Cr release assays. We found that RNA transfected DCs induce CTL that lyse the LP-1 myeloma cells. Cell line specificity was demonstrated by cold target inhibition assay and MHC class I restriction was revealed by antibody blocking studies. Total LP-1 RNA transfected DCs also served as suitable targets in a 51Cr release assay, being equivalent to the respective original tumor cell line. To analyze whether Id specific cytotoxicity contributed to the robust lysis observed, we purified Id protein from LP-1 supernatants and used autologous Id pulsed DCs as targets in 51Cr release assays. Interestingly, LP-1 specific CTL showed no specificity for the idiotype. U266 (MUC1 and HLA-A2 positive) specific CTL were also successfully induced by the same methodology. We furthermore analyzed whether MUC1 specificity added to the lytic activity of U266 specific CTL. As corresponding epitopes we tested the recently described HLA-A2 restricted peptides M1.1 and M1.2 and found a striking fine specificity for M1.2, assuming a possible immunodominance of this peptide. We report here on the successful induction of myeloma specific CTL by RNA transfection of DCs, which could serve as a potential vaccine in vivo.
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Department of Neuropathology, University of Cologne, Cologne, 2Institute of Human Genetics, Christian-Albrechts-University, Kiel, and 3Departments of Neurosurgery and 4 Neuropathology, University of Bonn, Bonn, Germany
Q. 14 Interphase FISH analysis of lymphoma-associated chromosomal breakpoints in primary CNS lymphomas M. MONTESINOS-RONGEN1, R. ZÜHLKE-JENISCH2, S. GESK2, M. FRANITZA1, C. SCHALLER3, D. VAN ROOST3, O.D. WIESTLER4, R. SIEBERT2, and M. DECKERT1 Primary central nervous system lymphomas (PCNSL) are germinal center derived diffuse large B cell lymphomas (DLBCL) arising in and remaining confined to the brain, the pathogenesis of which is poorly understood. We have investigated 13 PCNSL from immunocompetent patients by means of interphase cytogenetics on cryopreserved cells derived from stereotactic biopsies. Interphase fluorescence in situ hybridization (FISH) was performed for the detection of structural alterations for the following loci: IGH (14q32), IGK (2p12), IGL (22q11), BCL6 (3q27),
260 · 33rd Annual Meeting of the German Society of Immunology MYC (8q24), CCND1 (11q13), MLT, and BCL2 (both 18q21). Signal constellations indicating breakpoints within the IGH and IGK locus were detected in five and one PCNSL, respectively. There was no evidence for a t(8;14), t(11;14) or t(14;18) in this series of tumors. Breakpoints in the BCL6 loci were observed in three of the 13 cases. Gains of 18q21 represented the most frequent imbalances present in more than one third of all cases. Interestingly, these gains included the MLT gene. Thus, for the first time this study provides evidence for recurrent chromosomal translocations in PCNSL. While they share similarities with extracerebral DLBCL with respect to the presence of IGH translocations, they appear to differ in the usage of translocation partner genes, which still remain to be identified.
1
Institute of Molecular Immunology, GSF National Research Institute of Environment and Health, München and 2Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany
Q. 15 Understanding tumor-related anergy: In situ and ex vivo studies of leukocytes infiltrating renal cell carcinoma M. ROSMANIT1, I. BERGER2, H.J. GRÖNE2, N. BAUR1, J.J. MYSLIWIETZ1, A. BRANDL1, and E. NÖSSNER1 The observation of lymphocytic infiltrations (TIL) in renal cell carcinoma suggests that immunological processes act in RCC. Despite the consistent demonstration of lymphocytes infiltrating RCC and the identification of TIL-with tumor reactivity in vitro, tumor develops in the patient. To gain insight into the obvious immune anergy TIL-isolated directly from the tumor were analysed by FACS and in parallel TIL-were stained directly at the tumor site (in situ) using immune histology. Morphologically, a capsule of connective tissue separates RCC tumor tissue from surrounding normal tissue. Infiltrating lymphocytes were dominantly found within the capsule and only sparsely distributed throughout the tumor parenchyma. CD8 positive lymphocytes dominated over the CD4 population (contrasting the ratio generally found in PBL). g/d T cells were less than 2,4% of T cells. Of 16 tumors analysed, all had detectable CD3z chain expression. Only upon morphometric comparison of CD3 and CD3z positive cells a reduced number of CD3z positive cells versus CD3 positive cells was detected in about 1/3 of tumor samples. However, lack of CD3z affected less than 10% of T cells. All tumors had very few perforin positive cells. Double staining with CD3 revealed that perforin positive cells were mostly CD3 negative, thus NK cells. CD3 positive T cells were perforin negative. FACS analysis of isolated uncultured TIL-corroborated the histological findings. CD4 positive/CD25 positive regulatory cells were less than 1%. Lack of perforin expression in T cells and sparsity of CD4 T cells are potential explanations why the immune cells do not limit tumor growth.
33rd Annual Meeting of the German Society of Immunology · 261 Departments of 1Internal Medicine II and 2Immunology at the Institute of Cell Biology, Eberhard-Karls-University, Tübingen, Germany
Q. 16 The NKG2D ligand MICA is shed by human tumors H.R. SALIH1, H.G. RAMMENSEE2, and A. STEINLE2 The immunoreceptor NKG2D stimulates tumor immunity through activation of CD8 T cells and NK cells. Its ligand MICA has been shown to be broadly expressed on human tumors of epithelial origin. MICA expression correlates with an enrichment of Vd1 T cells in tumor tissue. We report that human tumor cells spontaneously release a soluble form of MICA encompassing the three extracellular domains, which is present at high levels in sera of patients with gastrointestinal malignancies, but not in healthy donors. Release of MICA from tumor cells is blocked by inhibition of metalloproteinases, concomitantly causing accumulation of MICA on the cell surface. Shedding of MICA by tumor cells may modulate NKG2D mediated tumor immune surveillance. In addition, determination of soluble MICA levels may be implemented as an immunological diagnostic marker in patients with epithelial malignancies.
Department of Gastroenterology, Medizinische Hochschule, Hannover, Germany
Q. 17 Monitoring the effect of Ox-40 on the growth of liver metastasis in mice S.R. SCHEFFER, F. KORANGY, M.P. MANNS, and T.F. GRETEN Different immunotherapy strategies are currently being used for the treatment of cancer. In most studies tumors are injected subcutaneously and tumor growth is monitored using calipers. However, a number of different groups have shown that the site of the tumor influences the efficiency of different cancer vaccines. We therefore decided to inject tumor cells directly into the liver to mimic the development of liver metastasis. Because the size of liver metastasis cannot be measured we used an alternative method: Tumor cells were transfected with a gene expressing the human b-HCG gene. These cells express b-HCG in vivo, which can easily be detected in the urine of mice with a growing tumor. The amount of b-HCG in the urine is shown to be directly proportional to the tumor size. We have already used this system to analyze the effect of the treatment using an antibody against CD134 (Ox-40) for liver metastasis of the murine colon carcinoma CT26 in BALB/c mice. 35 days after injection of 2¥104 CT26 cells directly into the liver 50% of the mice remained tumor free. Moreover, mice receiving anti-Ox40 treatment had a longer delay in tumor development as determined by b-HCG concentration. In conclusion, we can present a new model for the analysis of tumor cell growth in vivo in different organs and the effect of different methods of immunotherapy to treat liver metastasis of colon cancer.
262 · 33rd Annual Meeting of the German Society of Immunology Department of Neurology, Eberhard-Karls-University, Tübingen, Germany
Q. 18 ICOSL expressed on human glioma cells modulates T cell cytokine patterns, but does not confer immunogenicity B. SCHREINER, J. WISCHHUSEN, M. MITSDÖRFFER, M. MEHLING, A. MELMS, E. TOLOSA, M. WELLER, and H. WIENDL Human glioblastoma is a highly lethal tumor that is known for its capability to interfere with effective anti-tumor immune responses. Costimulatory signals are of critical relevance in both inductive and effector phases of immune responses. Inducible costimulator-ligand (ICOSL), a member of the B7 family of costimulatory molecules related to CD80/CD86, has been demonstrated to regulate CD4 as well as CD8 T cell responses via interaction with its receptor, ICOS, on activated T cells. In order to characterize the ICOS/ICOSL costimulatory pathway in human glioblastoma immunobiology, we investigated the expression and functional relevance of ICOSL on glioma cells in vitro. In contrast to CD80 and CD86, ICOSL mRNA and protein expression were detected in six of eleven glioma cell lines. ICOSL expression was upregulated by the inflammatory cytokine, TNF-a, whereas IFN-g had negligible influence on the expression level. Functional studies of the role of ICOSL on glioma cells were conducted using HLA-A2-mismatched alloreactive PBMC. After gene transfer of ICOSL into two ICOSL-negative glioma cell lines (U87MG, LN-229), coculture experiments with alloreactive PBMC revealed enhanced expression of ICOS on activated T cells and increased production of Th1 (IFN-g) as well as Th2 (IL-4, IL-10) cytokines. A neutralizing ICOSL antibody reduced Th1 and Th2 cytokine levels in these cocultures. However, ICOSL gene transfer did not influence primary or secondary alloreactive proliferative responses of the effector cell population. Further, neither primary alloreactive lysis nor recall responses in a secondary alloreactive reaction were modulated by ICOSL. Taken together, we here report that ICOSL is expressed by human glioma cells in the absence of CD80 and CD86. Although ICOSL enhances levels of Th1 and Th2 cytokine production in T cells, expression of this costimulatory ligand does not significantly alter the immunogenicity of human glioma cells in alloassays in vitro.
Abteilungen für 1Urologie und Poliklinik, Chirurgische Klinik, and 2Molekulare Pathologie, Pathologisches Institut, Universitätsklinikum Heidelberg, Heidelberg, Germany
Q. 19 A MSI tumor-specific frameshift mutation in the caspase-5 gene encodes for an HLA-A2.1-restricted CTL-epitope Y. SCHWITALLE1, M. LINNEBACHER2, E. RIPBERGER2, J. GEBERT2, S. WÖRNER2, M. WIESEL1, G. STÄHLER1, and M. VON KNEBEL DÖBERITZ2 Microsatellite instability (MSI) caused by defective DNA mismatch repair (MMR) is a hallmark of hereditary non-polyposis colorectal cancers (HNPCC) but also occurs in about 15% of sporadic tumors. If instability affects microsatellites in coding regions, translational frameshifts lead to truncated proteins often marked by unique frameshift peptide sequences at their C-terminus.
33rd Annual Meeting of the German Society of Immunology · 263 Since MSI tumors show enhanced lymphocytic infiltration and our previous analysis identified numerous coding mononucleotide repeat bearing candidate genes as targets of genetic instability, we examined the role of frameshift peptides in triggering cellular immune responses. Using peptide pulsed autologous CD40-activated B cells (CD40Bs) we have generated cytotoxic T lymphocytes (CTLs) that specifically recognize HLA-A2.1-restricted peptides derived from frameshift sequences. We have previously shown, that peptides derived from (-1) frameshift mutations in genes coding for TGF-bIIR and O-GlcNac transferase, respectively gave rise to CTL capable to lyse MSI cancer cell lines, carrying these frameshift mutations. Here we analysed that two frameshift peptides originating from a (-1) mutation in the Caspase-5 gene, FSP25 (KMFFMVFLI) and FSP26 (FLIIWQNTM) conferred specific lysis of target cells exogenously loaded with cognate peptide. Even more important, CTL specific for peptide FSP26 specifically recognized endogenously Caspase-5(-1)-expressing HCT116, Colo60H and LoVo-HLA-A2.1 colorectal cancer target cells. Presently, we analyse the specific cytotoxic potential of CTL-clones derived by limiting dilution from the above mentioned bulk culture in more detail. Given the huge number of human coding microsatellites and assuming only a fraction being mutated and encoding immunologically relevant peptides in MSI tumors, frameshift protein sequences indeed represent a novel subclass of tumor specific antigens. It is tempting to speculate that a frameshift peptide directed vaccination approach not only could offer new treatment modalities for existing MSI tumors but also might benefit asymptomatic at-risk individuals in HNPCC families by a prophylactic vaccination strategy.
Abteilungen für 1Urologie und Poliklinik, Chirurgische Klinik, and 2Molekulare Pathologie, Pathologisches Institut, Universitätsklinikum Heidelberg, Heidelberg, Germany
Q. 20 Fusions of microsatellite-unstable tumor cells and B cells induce cytotoxic T cells with antitumor potential Y. SCHWITALLE1, M. LINNEBACHER2, C. SUTTER2, M. WIESEL1, G. STÄHLER1, and M. VON KNEBEL DÖBERITZ2 Microsatellite instability (MSI) reflects length variations at short repetitive DNA sequences in diploid tumor cells and is caused by mutational inactivation of different DNA mismatch repair genes. To enhance the immunogenicity of MSI tumors we fused different MSI carcinoma cell lines with CD40-activated B cells (CD40Bs) and examined the ability of generated fusion cell clones for induction of T cell populations with cytotoxic potential. MSI analysis revealed MSI-high (MSI-H) in 2/8 renal cell-, 2/3 prostate-, 1/3 urothelial- and 1/1 colon carcinoma cell lines. MSI-H cell lines were fused with previously generated CD40Bs. To investigate the fusion efficiency, cancer cells were stained with red and Bc with green vital fluorescent dye, respectively. 10–15% of cells detected by flow cytometry exhibited dual fluorescence of fused cells. Fusions from MSI-H colon carcinoma cell line HCT116 and CD40Bs were cloned by limiting dilution. Two selected clones induced outgrow of CD3+ T cells including CD4+ and CD8+ effectors, which produced perforin, granzyme B, IFN-g, IL-2 and TNF-a. Whole CD3+ T cell populations were able to lyse MSI+ tumor cell lines up to 40% in standard chromium release assays. Microsatellite stable tumor cell lines were not lysed and natural killer cell activity could be excluded. Sorting of CD3+ T cells with microbeads resulted in clear CD4+ and CD8+ proliferating T cell populations, respectively. Intracellular cytokine analysis showed
264 · 33rd Annual Meeting of the German Society of Immunology stronger expression of IFN-g, IL-2 and perforin in CD8+ T cells, whereas CD4+ T cells produced more TNF-a and granzyme B. CD8+ effectors revealed high cytotoxic activity with up to 50% lysis of MSI+ target cells. Our results clearly demonstrate the capacity of MSI+ tumor cell/CD40Bs fusions to induce expansion of CD3+ T effector cells with unequivocal cytotoxic potential. Cellular fusion vaccines from tumor cells of MSI patients and autologous B cells may have important clinical implications.
Departments of 1Internal Medicine III and 2Urology, Johannes Gutenberg-University, Mainz, and 3TobLab GmbH and 4Max-Planck-Institute of Biochemistry, Martinsried, Germany
Q. 21 Identification of differentially expressed proteins in renal cell carcinoma by proteome analysis B. SELIGER1, R. LICHTENFELS1, S. MELCHIOR2, W. BRENNER2, A. HARDER3, T. HALDER3, and F. LOTTSPEICH4 Proteomics represents a powerful technology in the identification of proteins and biochemical pathways involved in the development of tumors. Thus, the measurement of protein expression patterns from normal and tumor tissues will lead to the discovery of differentially expressed, cancer related proteins which are potentially useful as diagnostic, prognostic or therapeutic targets. This approach is currently employed to study proteins and pathways involved in renal cell carcinoma (RCC) tumorigenesis. Proteomes of three normal kidney epithelia and of corresponding high grade RCC were analyzed by two-dimensional gel electrophoresis followed by mass spectrometry. More than 30 protein alterations representing either an upregulation or downregulation of protein expression when compared to corresponding normal kidney epithelium were detected in the tumors. Some of these changes were shared among the RCC. The proteins identified as altered during tumorigenesis include proteins associated with stress, cell proliferation/ apoptosis, structure, enzyme metabolism and protein degradation and their significance with regard to tumor biology and immune escape is currently investigated. However, the marked heterogeneity of the observed protein alterations might have an impact on research strategies for profiling analysis of RCC.
33rd Annual Meeting of the German Society of Immunology · 265 1
Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University of Dresden, Dresden and 2Laboratory for Tumor Genetics, Department of Internal Medicine I, University of Cologne, Cologne, Germany
Q. 22 Construction of anti-CD30 chimeric VSV-G-receptors that mediate virus specificity for Hodgkin’s lymphoma cells A. TEMME1, A. HOMBACH2, H. ABKEN2, and E.P. RIEBER1 Retroviral vectors that selectively infect a certain cell type through recognition of specifically expressed surface antigens is a promising approach to target tumor cells. In vitro experiments and clinical trials for gene therapy have adopted retroviral vectors based on the murine leukemia virus system to transfer suicide genes into target cells. For a tumor-specific gene therapy the viral envelope must be engineered in order to reach efficient and selective transduction of cells expressing a tumor-associated antigen. The CD30 antigen is found at high density on Hodgkin’s lymphoma cells and thus represents an appropriate structure for tumor-specific targeting by retroviral vectors. The goal of this study was the development of an experimental therapy based on antiCD30 pseudotyped retroviruses for efficient gene delivery into Hodgkin’s lymphoma cells. The cDNA of the single chain antibody HRS3, which binds specifically the CD30 antigen, was used for the construction of chimeric retroviral receptors. The variable domains of the antibody were fused to a partial sequence of the Vesicular stomatitis virus G-protein (VSV-G). Two different leader sequences, one for immunoglobulins and the other representing the VSV-G leader were included in the construction of the chimeric envelopes. A retroviral vector encoding for an enhanced green fluorescent protein (EGFP) reporter gene was used to visualize transduced cells. To evaluate the target specificity of the anti-CD30 pseudotyped retroviruses CD30 negative cells (HeLa, BL60) and the CD30-expressing Hodgkin’s lymphoma cell line L540 were transduced. All cells were efficiently transduced with retroviruses pseudotyped with the VSV-G wild type envelope. After transduction with particles pseudotyped with the chimeric envelopes EGFP reporter gene expression was restricted to L540 cells suggesting target specificity to the virus pseudotyped with the chimeric receptor. We propose, that the use of retroviral particles genetically engineered to bind tumor antigens is a promising approach for defeating neoplastic growth.
1
Department of Neurosurgery, Martin-Luther-University Halle-Wittenberg, Halle, Germany and 2Department of Neurological Science, University of Liverpool, Liverpool, UK
Q. 23 Identification of tumor-associated antigens in glioma M. VOLKMAR1, K. GANS1, N.G. RAINOV2, and A. SOELING1 Background: Although conventional treatment modalities for gliomas have been improved markedly in the past decades the prognosis for these tumors remains dismal. Therefore, new therapeutic strategies taking into account the biological differences between tumor cells and normal tissue are gaining increasing attention. Our goal was to identify glioma-specific antigens that could serve as potential targets for immunotherapy.
266 · 33rd Annual Meeting of the German Society of Immunology Methods: We performed serological analysis of recombinant cDNA expression libraries (SEREX): A cDNA library from a human gliosarcoma was constructed and screened with pooled allogeneic sera from glioma patients. Expression of candidate antigens in normal and tumor tissue was analysed by semiquantitative RT-PCR. Antigen-specific serum reactivity in brain tumor patients and healthy volunteers was determined by plaque assay. Results: Immunoscreening of 1.2¥106 plaques revealed three tumor-associated antigens: 1) No55, a nuclear autoantigen; 2) the cell cycle-associated protein P1 (a human analogue of yeast Mcm3); 3) an antigen corresponding to the 3’ untranslated region (UTR) of transcription factor NFY-g. Expression levels of the corresponding mRNAs were similar in malignant brain tumors and normal tissues. Sera from 23 healthy donors did not react with any of the antigens while 9 out of 32 (28%) sera from glioma patients reacted with the P1 protein and 1 out of 32 (3%) was immunoreactive with NFY-g. Conclusions: Gliomas, usually considered as being rather weakly immunogenic, can obviously elicit a humoral immune response in the host. Although mRNA expression levels of the 3 detected antigens did not differ substantially between tumor and normal tissues, serological reactivity to these antigens was clearly restricted to cancer patients, thus emphasizing that accessibility to the antigen (e.g. by spontaneous tumor necrosis) may be of greater importance to the immune system than mere overexpression of the antigen in tumor tissues.
Klinik für Innere Medizin I, Universität zu Köln, Köln, Germany
Q. 24 Low cholesterol in Hodgkin’s disease-derived cell line L428 stimulated a converting enzyme-dependent shedding of CD30 TNF-a B. VON TRESKOW, A. ENGERT, and H.P. HANSEN CD30 is selectively expressed on the tumor cells of various malignant lymphomas and is therefore a privileged target for anti-CD30 antibody-based immunotherapy. However, a TACE/ADAM17-dependent CD30 shedding leads to high levels of the soluble ectodomain of CD30 (sCD30) in the serum the patients thus inhibiting successful therapy. From Alzheimer’s disease, there is evidence for a negative correlation between serum cholesterol and the ADAM10dependent non-amyloidogenic pathway of the amyloid precursor protein. In order to improve the tumor therapy we analyzed the influence of cholesterol on CD30 shedding. The release of sCD30 from the Hodgkin’s disease-derived cell line L428 was dramatically increased after treatment with agents known to reduce the cholesterol concentration in cell membranes, such as methyl-b-cyclodextrin or the hydroxymethyl glutaryl-CoA reductase inhibitor lovastatin. A similar effect was achieved by treatment of L428 cells with cholesterol oxidase and the cholesterolbinding polyene antibiotic filipin. This shedding enhancement and a concomitant loss of membrane-anchored CD30 from the tumor cells could be inhibited by the synthetic hydroxamic acidbased metalloproteinase inhibitor BB-3466 as well as TIMP-3 but not by TIMP-1. Accordingly, the CD30 shedding is suggested being ruled by TNF-a converting enzyme in this process as well. In addition, we found that in vitro the cholesterol depleting methyl-b-cyclodextrin inhibited the anti-CD30 antibody internalization whereas enrichment of cholesterol stimulated antibody endocytosis. Since cholesterol is preferentially associated with the lipid ordered (lo) domains of the cell membrane (lipid rafts), CD30 shedding is suggested being localized rather in lipid disordered, CD30 internalization rather in lipid rafts. Since hydroxymethyl glutaryl-CoA reductase
33rd Annual Meeting of the German Society of Immunology · 267 inhibitors are widely used lowering blood cholesterol, they are expected being not indicated during of CD30–targeted immunotherapy.
1
Department of Hematology and Oncology, Johannes Gutenberg-University, Mainz, Germany and 2Ludwig Institute of Cancer Research, University of Lausanne, Epalinges, Switzerland
Q. 25 Phenotype of retrovirally transduced human T cells equipped with tumor-associated antigen-specific high-affinity T cell receptors R.-H. VOSS1, J. KUBALL1, S. THOMAS1, M. JÜLCH1, P. GUILLAUME2, P. ROMERO2, C. HUBER1, and M. THEOBALD1 A promising strategy in the immunotherapy of human malignancies entails the tumor associated antigen (TAA)-specific T cell receptor (TCR) gene delivery into human T cells. Human tolerance to the ubiquitous low level expression of the oncoprotein MDM2 was bypassed by utilizing the HuCD8¥A2Kb transgenic mice for triggering the development of non-tolerant, A2.1-restricted high-affinity T cells. Expression frequency of the exogenous TCRs were increased to almost completeness by appending IRES selection cassettes to the individual TCR transgenes within the LTRs of the retroviral shuttle, facilitating the readout of subtle phenotype changes among the different TCR constructs: following retroviral transfer of the respective TCR genes as for the murine wildtype chains or a diverse set of chimeric TCRs into human T cells, they have been cultured long-term with CD3/CD28 beads and low doses of Il-2 for extensive phenotype studies. Cytotoxicity correlated with both the expression rate on a per cell basis as well as the time-dependent stability of expression as could be quantified by FACS-calibrated vb6-staining. Although some TCR constructs were unable to bind the MDM2(81–88)-specific tetramer to a considerable amount, they turned out to be vastly effective in lysing peptide loaded target cells. CD4+CD8+ subpopulations provided a strong indication that exclusively the transduced CD8+ were inherent the cytotoxic effector function. These results were in line with ELISA/ELISPOT measurements by virtue of IL-2, IFN-g, and IL-5 quantification that proved the TC1-phenotype of CTLs in vitro. Activation status and homing capacity of ex vivo cultured transduced T cells were analysed by a panel of activation markers and the dominant homing receptors CD62L and CCR7. Dissociation kinetics with different TCR constructs demonstrated the usefulness of the «kinetic window» model and to get an estimate for comparative cytolytic efficiencies as a prerequisite for preclinical in vivo studies.
268 · 33rd Annual Meeting of the German Society of Immunology Section of Immunobiology, Institute of Zoophysiology, University of Bonn, Bonn, Germany
Q. 26 Definition of pancreatic tumor antigens for the MHC class I and class II processing pathways B. WINZEN, J. NEUMANN, and N. KOCH Cancer of the pancreas is the fourth frequent cause of death of cancer diseases in the western industrialized countries. Despite advances in chemotherapy and surgical techniques, patients with pancreatic cancer often succumb to local recurrence or metastatic spread. The need for novel advanced therapies for this disease has prompted us to develop a novel strategy for immuno therapy with recombinant pancreatic tumor associated antigens. We utilized the invariant chain (Ii) as a carrier to deliver MHC class I and class II epitopes of the pancreas associated antigen core-2b1–6-N-acetylglucosaminyltransferase (C2GnT) to the MHC class I and class II processing pathways. CD4 and CD8 T cell epitopes were predicted on the basis of motifs, obtained from the SYFPEITHI database. From the C2GnT sequence we received six CD4 T cell epitopes with specificity for DR1, DR3 or DR4. Class I epitopes were specific for HLA-A2. In the recombinant construct, we replaced the MHC class II groove binding segment of Ii by a CD4 T cell epitope, whereas the C-terminal end of Ii contains a CD8 T cell epitope from C2GnT. By addition of a mAb V5-epitope to the C-terminus, the modified invariant chain protein can be monitored in the presence of endogenously expressed Ii. To investigate binding of class II molecules to the recombinant tumor antigens, we transfected COS cells with recombinant Ii and DR cDNAs. Immunoprecipitation of the DR/Ii complex with a mAb directed against DR revealed binding of the recombinant tumor antigen to DR1, DR3 or DR4. The formation of SDS resistant class II molecules demonstrates, that the DR molecules are stably loaded with the CD4 T cell epitopes.
1
Molecular Tumor Genetics and Immunogenetics, Max-Delbrück-Center for Molecular Medicine, Berlin and 2GSF-National Research Centre for Environment and Health, Neuherberg, Germany
Q. 1 Subcellular localization of latent and early lytic transforming proteins encoded by human herpesvirus 8 J. BENGA1, N. THIRUNARAYANAN1, F. CIFIRE1, E. KREMMER2, and M. LIPP1 All stages of Kaposi’s sarcoma, as well as multifocal Castleman’s disease (MCD) and primary effusion lymphoma (PEL) have been associated with the presence of human Herpesvirus-8 (HHV8), a gammaherpesvirus encoding 81 open reading frames. However, only a small subset has been shown to be transcribed in HHV-8 latently infected PEL-derived cell lines (BCP-1, BCBL-1) and in cells of the KS lesions. Among them, Kaposin, a latent protein has been shown to have transforming potential in Rat-3 cells, and oncogenic potential after injection into nude mice. For the analysis of the subcellular localization of Kaposin, we used monoclonal antibodies which can recognize all three known forms: Kaposin A (small protein of 60 amino acids), Kaposin B (encoding direct repeats only) and Kaposin C (encoding direct repeats and Kaposin A). We noticed by immunofluorescence cytoplasmic localization of Kaposin A in transfected HeLa cells in contrast to different localization of Kaposin proteins in PMA induced BCBL-1 cells. PMA induction of BCBL-1 cells has also shown the significant expression level of an other viral oncogene, the chemokine receptor vGPCR, indicating its early lytic origin. In parallel, cytoplasmic and membrane localization of vGPCR has been shown in retroviral transduced stable NIH3T3 clones and in transiently transfected HeLa cells using specific monoclonal antibody 7D1 by FACS analysis and immunofluorescence. A 45kDa product of vGPCR was detected by Western blot of membrane preparations of transfected HeLa cells. The elucidation of expression profiles and transformation mechanisms of Kaposin and the chemokine receptor vGPCR is important to validate viral proteins as suitable targets for therapy of HHV-8 associated malignancies.
1 Laboratory of Immunobiology, Department of Dermatology and 2Section of Immunobiology, Institute of Zoophysiology, University of Bonn, Bonn, Germany
Q. 2 Activation of MAGE-3 specific T helper cells from healthy donors and melanoma patients by peptide-loaded and Ii/MAGE fusion construct transfected dendritic cells M. BERG1, B. WINZEN2, N. KOCH2, T. BIEBER1, and S. KOCH1 CD4+ T lymphocyte responses are important for anti-tumor immune reactions as has been demonstrated in animal models. Characterization of the CD4+ T cell epitope repertoire on mela-
252 · 33rd Annual Meeting of the German Society of Immunology noma antigens and specific targeting of endogenously synthesized antigens to the class II loading compartment would improve decisively the efficacy of peptide-based immunization protocols in neoplastic patients. MAGE-3 (171–185) peptide has been shown to bind promiscuously to MHC II molecules of all donors tested. Therefore, we used mature DCs as APC to stimulate MAGE-3 specific T cells from healthy donors and melanoma patients. Activation of the T cells was determined using an ELISPOT assay for interferon-g and by measuring T cell proliferation. Using peptide-loaded mature DCs, we could stimulate T cells from all healthy donors tested and from 6 of 11 melanoma patients. MAGE-3 (171–185) peptide specific T cells could be enriched by weekly restimulation. To improve the loading of MHC II molecules and thereby enhance efficacy of antigen presentation, we transfected immature DCs from a healthy donor with a fusion construct consisting of the MHC II associated invariant chain and MAGE-3 (171–185) sequence by means of electroporation. 30% of transfected DCs expressed the transgene and viability after transfection was at 60%. Such transfected DCs were superior to peptideloaded DCs in their capacity to stimulate primary MAGE-3 (171–185) specific T cells, which produced more interferon-g and interleukin-12. This novel antigen delivery system holds promise for a variety of clinical applications including cancer therapy.
Departments of 1Dermatology and 3Pathology, Ludwig-Maximilians University, and Department of Nuclear Medicine, Technical University, Munich, Germany
2
Q. 3 Th1 cells inhibit carcinogenesis in transgenic mice H. BRAUMÜLLER1, B.J. PICHLER2, A. SAKRAUSKI1, N. MÜLLER-HERMELINK1, M. KNEILLING1, K. GHORESCHI1, M. SCHWAIGER2, R. HUSS3, W.A. WEBER2, and M. RÖCKEN1 The transgenic mouse model RIP1-Tag2 is a powerful tool to investigate the role of tumor-specific T cells in carcinogenesis. In these mice the SV40 T antigen (Tag) is expressed in insulin-producing beta cells of the pancreas, causing 2% of the islets to develop into adenomas and carcinomas. Subsequently mice die of hypoglycemia at an age of 14–16 weeks. To test, whether Th1 cells can eradicate islet adenomas, we adoptively transferred activated Tag-specific Th1 cells. Th1 cells were generated by stimulating CD4+ cells derived from lymphnodes and spleen of transgenic C3H mice with T antigen, antigen presenting cells and CpG 1668 oligonucleotide. Treatment of RIP1-Tag2 positive mice with Th1 cells resulted in nearly a doubling of the life span. Paraffin-embedded sections revealed strong inhibition of tumor growth in all of the treated animals accompanied by inhibition of angiogenesis. No destruction of the pancreas could be observed in treated mice. IFN-g producing Th1 cells may promote apoptosis of tumor cells or endothelial cells lining the tumor vessels. At an age of 8 weeks, the time when carcinomas start to develop, islet cells and endothelia from untreated mice or Tag-Th1-treated mice still contained similar numbers of apoptotic cells. Alternatively, tumor growth may be controlled by angiogenic inhibitors, namely interferon-inducible protein 10 (IP10) or monokine-induced by IFN-g (Mig). Quantitative PCR of pancreas tissue revealed at least 10-fold higher mRNA levels of IP10 and Mig in Th1-treated mice than in healthy controls or tumor-bearing RIP1-Tag2 mice. Our results show that adoptive transfer of tumor-specific Th1 cells was highly efficient in tumor therapy and did not cause any side effects or any signs of autoimmune diseases. The mechanism of this highly efficient delay of tumor progression seems to base on inhibition of angiogenesis and not direct killing of tumor cells.
33rd Annual Meeting of the German Society of Immunology · 253 1
Department of Gastroenterology, Medizinische Hochschule, Hannover, 3Department of Gastroenterology, University of Ulm, Ulm, Germany, and 2Department of Pharmacology, University of California, La Jolla CA, USA
Q. 4 Immunotherapy of pancreatic cancer in a transgenic mouse model A.I. GARBE1, F. KORANGY1, A. HELLER1, F.R. GRETEN2, R.M. SCHMID3, M.P. MANNS1, and T.F. GRETEN1 Human pancreatic cancer is associated with a poor prognosis and alternative therapies such as immunotherapy are needed. Transgenic mice that develop spontaneously tumors in situ provide a biological more relevant model for the development of vaccination strategies than transplantable tumors. We have begun to analyse TGF-a transgenic mice which overexpress TGF-a in the pancreas (Sandgren, 1990). TGF-a transgenic mice develop fibrosis and ductal pancreatic cancer at the age of one year (Wagner, 1998). Crossbreeding these mice with p53 knockout mice dramatically accelerate tumor development. These mice represent the first model of pancreatic adenocarcinoma with genetic alterations as well as growth characteristics similar to the human disease (Wagner, 2001). Our aim is to establish a potent vaccine against pancreatic cancer in these mice. Preliminary studies showed that a cell line deriving from a tumor of a TGF-a+/–p53+/– mouse on BALB/c¥C57BL/6 background grows in vivo in nu/nu and in BALB/c¥C57BL/6 after s.c. injection and shows the typical growth pattern with cystic and ductal structures, which resemble the primary tumor. Preliminary vaccination experiments show an immunogenicity of this murine pancreatic tumor cell line. For further immunological experiments these mice were bred to a C57BL/6 background. TGF-a+/–p53–/– mice on C57BL/6 background develop ductal pancreatic adenocarcinoma after 80 days. We are presently establishing a pancreatic tumor cell line from these mice to perform further vaccination experiments. DNA vaccination experiments were performed to investigate the role of TGF-a as a potential tumor antigen. Immunization of TGF-a transgenic mice with DNA encoding TGF-a induced a TGF-a specific antibody response in wt littermates but not in TGF-a transgenic mice. A specific cytotoxic T cell response could be detected in all mice which received multiple doses of immunization. Thus, TGF-a seems to represent an antigen capable to elicit a substantial adoptive antitumor immune response.
Department of Surgery, University of Regensburg, Regensburg, Germany
Q. 5 Rapamycin: A double-edged sword capable of preventing allograft rejection, while simultaneously attacking cancer via an antiangiogenic effect M. GUBA, G. KÖHL, M. STEINBAUER, S. FLEGEL, M. ANTHUBER, K.W. JAUCH, and E.K. GEISSLER Background: Cancer is an ominous risk factor for immunosuppressed transplant patients. A potential breakthrough towards addressing this problem comes from our recent finding that immunosuppressive doses of rapamycin (RAPA) destroy tumors in mice. Here, we explored how
254 · 33rd Annual Meeting of the German Society of Immunology RAPA mediates its antitumor effect, focusing on cell proliferation and the proangiogenic cytokine, VEGF. Methods: Proliferation of human umbilical vein endothelial cells (HUVECs), CT26 (murine colon adenocarcinoma) and B16 (murine melanoma) cells in the presence of RAPA (0.01– 1 mg/ml) was determined by BrdU uptake. The effect of RAPA on in vitro VEGF-stimulated angiogenesis was tested in a HUVEC tubular-formation assay. In vitro VEGF protein secretion was measured by ELISA in 24h culture supernatants of RAPA-treated tumor cells. VEGF mRNA levels from tumor cells were quantified by RT-PCR (Light Cycler) 10 h after treatment. Results: Although RAPA, even at the highest dose tested (1 mg/ml), showed only a modest inhibition (<24%) of CT26 and B16 tumor cell proliferation, HUVEC proliferation decreased with RAPA at 0.01, 0.1 and 1 mg/ml (11±7%, 36±8% and 43±12%, respectively). The high sensitivity of HUVECs to RAPA suggested the main antitumor effect of this drug may be via interference of endothelial cell stimulation. Indeed, RAPA decreased recombinant VEGF-stimulated HUVEC proliferation by 76±1%. Furthermore, we could show in vitro tubular formation, stimulated with VEGF, was 100% blocked by RAPA. Besides blocking VEGF stimulation of endothelial cells, RAPA (0.1 mg/ml) inhibited constitutive VEGF production by CT26 and B16 tumor cells (34–37%). RAPA treatment also lowered VEGF mRNA levels 4-fold. Summary: In conclusion, (1) high doses of RAPA modestly inhibit tumor cell proliferation, (2) VEGF-induced endothelial cell activities are blocked by immunosuppressive levels of RAPA, and (3) VEGF production by tumor cells is inhibited by RAPA. Thus, the antiangiogenic effects of RAPA could be useful for the prevention and treatment of tumors in transplant patients.
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany
Q. 6 Nitric oxide differentially regulates expression of proangiogenic IL-8 and VEGF and tumor-suppressive IP-10 in colon carcinoma cells M. HELLMUTH, H. MUHL, J. PAULUKAT, R. NINIC, and J. PFEILSCHIFTER Expression of inducible nitric oxide (NO) synthase and production of NO appears to be a marker of tumor progression in human neoplasia, among them melanoma, head and neck cancer, and colorectal cancer. Since tumor-promoting functions of NO have been associated with increased angiogenesis at the tumor site, we investigated effects of NO on the production of two selected chemokines that are supposed to differentially regulate tumor growth, namely proangiogenic interleukin (IL)-8 and tumorsuppressive interferon-inducible protein-10 (IP-10). Both chemokines were produced by colon carcinoma cells after stimulation with the combination IL-1b/interferon-(IFN)-g. Under these conditions, release of IL-8 was exclusively mediated by IL-1b but not by IFN-g, whereas production of IP-10 was dependent on the presence of both, IL-1b and IFN-g. Effects of NO were analyzed by incubation with the NO-donor DETA-NO which spontaneously releases NO with a characteristically long half-life of 16.5h. NO amplified expression and release of IL-8 from colon carcinoma cells as detected by RNase protection assay and ELISA analysis. In contrast, IL-1b/IFN-g-induced production of IP-10 was suppressed by NO. In addition to IL-8, expression of vascular endothelial growth factor (VEGF), was inducible by NO. The present data are consistent with previous observations that relate NO to enhanced tumor angiogenesis and imply that NO-mediated upregulation of IL-8 and VEGF and downregulation of IP-10 synthesis may contribute to this phenomenon.
33rd Annual Meeting of the German Society of Immunology · 255 Department of Tumor Progression and Immune Defense, German Cancer Research Center, Heidelberg, Germany
Q. 7 Donor T cell and host NK depletion improve the therapeutic efficacy of allogeneic bone marrow cell reconstitution in the non-myeloablatively conditioned tumor-bearing host S. HUMMEL, D. WILMS, M. VITACOLONNA, and M. ZÖLLER Allogeneic bone marrow cell (BMC) reconstitution of the non-myeloablatively conditioned host has the advantage that it can be tolerated in suboptimal health conditions. However, the problem of graft versus host disease (GvHD) remains. Also, graft acceptance may become delicate and HvGD may arise. We report here on advantages/disadvantages of host NK depletion and graft T cell depletion in fully allogeneic healthy and a solid tumor-bearing mice. NK depletion of the healthy host improved the survival rate, whereas graft T cell depletion was disadvantageous. In the tumor-bearing host, graft T cell depletion was beneficial when the host was NK-depleted. Host NK depletion facilitated B lymphopoiesis, repopulation of the thymus, expansion of donor cells and tolerance induction. The disadvantage of graft T cell depletion in the healthy host was due to delayed engraftment. Since in tumor-bearing mice, host, but not graft hematopoiesis was strongly impaired, donor hematopoiesis dominated. Graft T cell depletion reduced GvHD, but hardly interfered with engraftment. Importantly, graft-mediated tumor reactivity appeared late and was unimpaired when the graft was T cell-depleted. Thus, concomitant depletion of host NK and donor T cells is advantageous when approaching therapeutic treatment of solid tumors by allogeneic reconstitution of the non-myeloablatively conditioned host.
Institutes of 1Immunology and 4Pathology, and 2Department of Urology, Medical Faculty Carl Gustav Carus, Technical University of Dresden, Dresden, and 3Department of Immunology, Institute of Cell Biology, University of Tübingen, Tübingen, Germany
Q. 8 Identification of an HLA-A*0201-restricted T cell epitope derived from the prostate cancer-associated protein trp-p8 A. KIESSLING1, S. FÜSSEL2, M. SCHMITZ1, S. STEVANOVIC3, A. MEYE2, B. WEIGLE1, U. KLENK4, M.P. WIRTH2, and E.P. RIEBER1 Immunotherapy of prostate carcinoma (PCa) is advanced by the identification of tumor-associated proteins that are suitable antigens for vaccination strategies. Expression of these proteins should be restricted to prostate and prostate tumors and should ideally be upregulated in the malignant tissue. An additional prerequisite is their capacity to induce an effective immune response in vivo. The number of antigens displaying these characteristics is still limited. Here, we analyzed the newly described (1) PCa-associated protein transient receptor potential-p8 (trp-p8). To determine the abundance of trp-p8 in prostate tumors the expression level of trp-p8 mRNA was quantitatively analyzed in 33 matched samples of malignant and non-malignant prostate tissue. Trp-p8 mRNA was found to be expressed in all prostate tumors and in the corresponding normal prostate tissues. In addition, transcripts were upregulated in the tumorous
256 · 33rd Annual Meeting of the German Society of Immunology samples when compared to the non-tumorous specimens. To identify epitopes recognized by cytotoxic T lymphocytes (CTLs) HLA-A*0201-restricted peptides were selected from the amino acid sequence of the trp-p8 protein using a screening algorithm and tested for their affinity to HLA-A*0201 molecules. Five peptides were loaded on autologous dendritic cells for the in vitro activation of CTLs. One of these peptides (GLTKYIGEV) was found to induce specific CTLs which efficiently lysed HLA-A*0201-positive PCa cells thus confirming the endogenous production and presentation of this peptide by tumor cells. Based on these findings trp-p8 appears as a suitable antigen for the design of vaccines in PCa. (1) Tsavaler et al., Cancer Res. 61:3760 (2001) This work was supported by the Wilhelm-Sander-Stiftung and the Deutsche Forschungsgemeinschaft (SFB510).
Institute für 1Medizinische Mikrobiologie, Immunologie und Hygiene and 2Genetik, Universität zu Köln, Köln, 4Institut für Humangenetik, Universität zu Kiel, Kiel, Deutschland, and 3Department of Biological Sciences, University of Chicago, Chicago IL, USA
Q. 9 Reversal of commitment to the B cell lineage in acute lymphoblastic leukemia harboring a BCR-ABL1 gene rearrangement F. KLEIN1, V.S. BARATH1, N. FELDHAHN2, S. LEE3, G. ZHOU3, S.M. WANG3, S. HARDER4, R. SIEBERT4, M. KRÖNKE1, J.D. ROWLEY3, and M. MÜSCHEN1 Pre-B cells (PBC) have been identified as the precursor of B lineage acute lymphoblastic leukemia (B-ALL) harboring a BCR-ABL1 fusion gene. Normal PBCs are destined to die by apoptosis unless they are rescued by survival signals through the pre-B cell receptor (pBCR). To define growth requirements of such B-ALL clones, we generated and compared genome-wide gene expression profiles of CD34+ hematopoietic progenitor cells (HSC), PBCs and two cases of BALL. To analyze global gene expression profiles of B-ALL cells and their normal precursors, we chose the serial analysis of gene expression (SAGE) technique, which provides quantitative gene expression data at a genome-wide level (including a total of 277,000 SAGE tags). About 80 percent of the genes initially expressed in HSC were silenced in B-ALL cells. Unexpectedly, these genes include virtually all determinants of the B cell lineage. Loss of PBC identity involves constituents of the pBCR (VpreB, l5, IgH Cm, Iga, Igb) and the V(D)J-recombination machinery (RAG1, RAG2, TdT), proximal protein tyrosine kinases such as BTK and BLK and key transcription factors, including E2A, EBF and PAX 5. Given the critical role of pBCR-mediated signals for the survival of PBCs, the question arises, how leukemia cells can establish a highly proliferating clone in the absence of pBCR signaling. Indeed, expression levels of NF-ÍB and other activation-induced nuclear molecules are similar in B-ALL cells as in their normal counterpart. These findings suggest that a transcriptionally silenced pBCR signaling complex in BCR-ABL1+ leukemia cells can be effectively replaced by alternative survival signals, which most likely arise from the oncogenic ABL1 tyrosine kinase. Specific silencing of genes, whose expression was induced during the transition from the HSC to the PBC stage of development demonstrates that BALL cells have phenotypically reversed their commitment to the B cell lineage.
33rd Annual Meeting of the German Society of Immunology · 257 1
Abteilung Genregulation, Gesellschaft für Biotechnologische Forschung (GBF), Braunschweig and 2University Hospital, Freiburg, Germany
Q. 10 Breaking tolerance against hepatocellular carcinoma development by an activatable interferon regulatory factor-1 A. KRÖGER1, M. GEISSLER2, and H. HAUSER1 IRF-1 is a multifunctional transcription factor involved in cell growth regulation, pathogen response and immune activation. Overexpression of IRF-1 leads to the induction of several genes and a significant suppression of cell growth. To elucidate the potential of IRF-1 for cytokine gene therapy of cancer we investigated IRF-1 effects in a mouse model. We expressed an estradiolactivatable fusion protein of IRF-1 in a hepatocellular carcinoma cell line Hepa1–6. Activation of IRF-1 leads to growth inhibition and a decrease of anchorage independent growth. In addition, IRF-1 expressing cells are characterized by enhanced MHC I, MHC II expression and IFN-b secretion. Tumor growth in estradiol-treated syngenic C57L/J mice was completely suppressed, while in estradiol-untreated mice tumors developed normally. The pretreated mice (HepaIRF1hER plus estradiol) were protected against rechallenge with wild type Hepa1–6 cells even in the absense of estradiol, suggesting induction of tumor specific immunity. In fact, significant CTL activity directed against Hepa1–6 and endogenously expressed self antigen a-fetoprotein was detected. However, the antitumoral effects are only partially dependent on CD4+ and CD8+ T cells. In addition, in mice bearing HepaIRF-1hER tumors estradiol treatment resulted in an arrest of tumor expansion. In conclusion, the data demonstrate that IRF-1 suppresses HCC growth through both, a direct antitumor growth effect and enhanced immune cell recognition of the tumor and thus is a promising candidate for gene therapy of HCC.
1
Department of Hematology and Oncology, Johannes Gutenberg-University, Mainz, Germany and 2Ludwig Institute of Cancer Research, University of Lausanne, Epalinges, Switzerland
Q. 11 Reprogramming of CD4+ T cells by gene transfer of a class I MHCrestricted hyper-affinity T cell receptor J. KUBALL1, R.-H. VOSS1, F. SCHMITZ1, P. ROMERO2, P. GUILLAUME2, M. JÜLCH1, C. HUBER1, and M. THEOBALD1 T cell receptor (TCR) gene transfer of murine TCR into human T cells allows circumvention of tolerance to tumor associated antigens (TAA). So far, CD8+ T cells could functionally be targeted by class I MHC-restricted TCR gene transfer. To reprogram CD4+ T cells as well as to further improve efficacy of TCR transduced CD8+ T cells, the impact of a CD8-independent TCR was investigated. Cytotoxic T lymphocytes (CTL) specific for p53 (264–272) and MDM2 (81–88) were obtained from A2 and CD8¥A2Kb transgenic (tg) mice, respectively, by peptide immunization. CTLs from A2 tg mice were shown to be CD8-independent as opposed to CTLs obtained from CD8¥A2Kb tg mice. CTLs were cloned, murine TCRs were isolated and expressed by retroviral gene transfer into human T cells. Subpopulations were analysed for tetramer-staining, cytotoxi-
258 · 33rd Annual Meeting of the German Society of Immunology city and cytokine secretion. Gene transfer of an HLA-A2-restricted CD8-independent TCR into human T cells resulted in human CD8+ CTLs that recognized target cells with higher efficacy as compared to the parental murine T cell clone. Reprogramming of CD4+ T cells into class I MHC-restricted antigen-specific effector cells required the expression of a CD8-independent TCR. In summary, efficacy of CD8+ TCR transgenic T cells can be further augmented by a CD8 independent TCR. These hyper-affinity TCRs target CD4+ T cells effectively. This suggests, that class I MHC-restricted TAA-specific CD4+ T cell-mediated help in vivo can be exerted by CD8independent hyper-affinity TCR gene transfer.
Department of Hematology and Oncology, Johannes Gutenberg-University, Mainz, Germany
Q. 12 Universal targeting of multiple myeloma by PRDI-BF1 antigen-specific CTL C. LOTZ, U. LIEWER, C. HUBER, and M. THEOBALD Growing clinical and experimental evidence indicates that multiple myeloma (MM) is susceptible to cytotoxic T lymphocyte (CTL)-based immune interventions. These CTL recognize peptides derived from the endogenous processing of cellular proteins and presented by MHC class I molecules. The PRDI-BF1/Blimp-1 protein is inherently involved in the terminal differentiation of mature B lymphocytes into malignant and non-malignant plasma cells. It likely provides therefore an abundant and universal MM-associated target molecule. HLA-A*0201 (A2.1) transgenic (tg) mice were used to identify A2.1-presented CTL epitopes derived from the PRDI-BF1 protein. CTL with specificity for at least two PRDI-BF1-derived peptides were able to kill various MM cell lines while preserving the integrity of non-transformed cells, such as A2.1+ T and B cells and monocyte-derived dendritic cells (DC). However, recognition by CTL was not limited to malignant plasma cells. Consistent with CTL recognition, protein expression by immunohistochemistry revealed that PRDI-BF1 expression too was not restricted to MM cells only. Substantial protein levels were detected in different solid tumor cell lines, i.e. melanomas, osteosarcomas, breast cancer, and hepatocellular carcinoma. The more abundant expression pattern of PRDI-BF1 suggested that the self-A2.1-restricted human T cell repertoire is affected by PRDI-BF1-specific self-tolerance. To study this hypothesis, naïve CD8+ human T lymphocytes underwent in vitro stimulation with A2.1+ autologous DC pulsed with the relevant PRDI-BF1 peptides. Effector cells were induced at least in one out of two healthy donors. They specifically produced IFN-g but were not able to kill PRDI-BF1 peptide-pulsed target cells. Our results indicate that PRDI-BF1-derived CTL epitopes can serve as MM-associated and, to some extent, as broader tumor antigens. The transduction of PRDI-BF1-specific T cell receptors selected from A2.1-tg mice into human T lymphocytes offers a possibility to turn self-A2.1restricted human T cells into efficient MM-reactive CTL.
33rd Annual Meeting of the German Society of Immunology · 259 Department of Internal Medicine II, Eberhard-Karls-University, Tübingen, Germany
Q. 13 Induction of myeloma specific cytotoxic T cells using dendritic cells transfected with tumor-derived RNA C. MILAZZO, V. REICHARDT, M.R. MÜLLER, F. GRÜNEBACH, and P. BROSSART We investigated myeloma RNA transfection of dendritic cells (DCs) to induce myeloma (MM) specific cytotoxic T cell (CTL) responses in vitro. By this methodology we hope to bypass the need for the identification of MM associated antigens or specific antigens such as idiotype (Id). Monocyte derived DCs from buffy coats, which were matched in the HLA class I haplotype to the myeloma cell lines LP-1 and U266, were used for RNA transfection. DCs were electroporated with total myeloma cell line RNA and were used as antigen presenting cells for the induction of myeloma specific CTL. After a single restimulation with RNA transfected DCs, cytotoxic activity of induced T cells was analyzed in 51Cr release assays. We found that RNA transfected DCs induce CTL that lyse the LP-1 myeloma cells. Cell line specificity was demonstrated by cold target inhibition assay and MHC class I restriction was revealed by antibody blocking studies. Total LP-1 RNA transfected DCs also served as suitable targets in a 51Cr release assay, being equivalent to the respective original tumor cell line. To analyze whether Id specific cytotoxicity contributed to the robust lysis observed, we purified Id protein from LP-1 supernatants and used autologous Id pulsed DCs as targets in 51Cr release assays. Interestingly, LP-1 specific CTL showed no specificity for the idiotype. U266 (MUC1 and HLA-A2 positive) specific CTL were also successfully induced by the same methodology. We furthermore analyzed whether MUC1 specificity added to the lytic activity of U266 specific CTL. As corresponding epitopes we tested the recently described HLA-A2 restricted peptides M1.1 and M1.2 and found a striking fine specificity for M1.2, assuming a possible immunodominance of this peptide. We report here on the successful induction of myeloma specific CTL by RNA transfection of DCs, which could serve as a potential vaccine in vivo.
1
Department of Neuropathology, University of Cologne, Cologne, 2Institute of Human Genetics, Christian-Albrechts-University, Kiel, and 3Departments of Neurosurgery and 4 Neuropathology, University of Bonn, Bonn, Germany
Q. 14 Interphase FISH analysis of lymphoma-associated chromosomal breakpoints in primary CNS lymphomas M. MONTESINOS-RONGEN1, R. ZÜHLKE-JENISCH2, S. GESK2, M. FRANITZA1, C. SCHALLER3, D. VAN ROOST3, O.D. WIESTLER4, R. SIEBERT2, and M. DECKERT1 Primary central nervous system lymphomas (PCNSL) are germinal center derived diffuse large B cell lymphomas (DLBCL) arising in and remaining confined to the brain, the pathogenesis of which is poorly understood. We have investigated 13 PCNSL from immunocompetent patients by means of interphase cytogenetics on cryopreserved cells derived from stereotactic biopsies. Interphase fluorescence in situ hybridization (FISH) was performed for the detection of structural alterations for the following loci: IGH (14q32), IGK (2p12), IGL (22q11), BCL6 (3q27),
260 · 33rd Annual Meeting of the German Society of Immunology MYC (8q24), CCND1 (11q13), MLT, and BCL2 (both 18q21). Signal constellations indicating breakpoints within the IGH and IGK locus were detected in five and one PCNSL, respectively. There was no evidence for a t(8;14), t(11;14) or t(14;18) in this series of tumors. Breakpoints in the BCL6 loci were observed in three of the 13 cases. Gains of 18q21 represented the most frequent imbalances present in more than one third of all cases. Interestingly, these gains included the MLT gene. Thus, for the first time this study provides evidence for recurrent chromosomal translocations in PCNSL. While they share similarities with extracerebral DLBCL with respect to the presence of IGH translocations, they appear to differ in the usage of translocation partner genes, which still remain to be identified.
1
Institute of Molecular Immunology, GSF National Research Institute of Environment and Health, München and 2Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany
Q. 15 Understanding tumor-related anergy: In situ and ex vivo studies of leukocytes infiltrating renal cell carcinoma M. ROSMANIT1, I. BERGER2, H.J. GRÖNE2, N. BAUR1, J.J. MYSLIWIETZ1, A. BRANDL1, and E. NÖSSNER1 The observation of lymphocytic infiltrations (TIL) in renal cell carcinoma suggests that immunological processes act in RCC. Despite the consistent demonstration of lymphocytes infiltrating RCC and the identification of TIL-with tumor reactivity in vitro, tumor develops in the patient. To gain insight into the obvious immune anergy TIL-isolated directly from the tumor were analysed by FACS and in parallel TIL-were stained directly at the tumor site (in situ) using immune histology. Morphologically, a capsule of connective tissue separates RCC tumor tissue from surrounding normal tissue. Infiltrating lymphocytes were dominantly found within the capsule and only sparsely distributed throughout the tumor parenchyma. CD8 positive lymphocytes dominated over the CD4 population (contrasting the ratio generally found in PBL). g/d T cells were less than 2,4% of T cells. Of 16 tumors analysed, all had detectable CD3z chain expression. Only upon morphometric comparison of CD3 and CD3z positive cells a reduced number of CD3z positive cells versus CD3 positive cells was detected in about 1/3 of tumor samples. However, lack of CD3z affected less than 10% of T cells. All tumors had very few perforin positive cells. Double staining with CD3 revealed that perforin positive cells were mostly CD3 negative, thus NK cells. CD3 positive T cells were perforin negative. FACS analysis of isolated uncultured TIL-corroborated the histological findings. CD4 positive/CD25 positive regulatory cells were less than 1%. Lack of perforin expression in T cells and sparsity of CD4 T cells are potential explanations why the immune cells do not limit tumor growth.
33rd Annual Meeting of the German Society of Immunology · 261 Departments of 1Internal Medicine II and 2Immunology at the Institute of Cell Biology, Eberhard-Karls-University, Tübingen, Germany
Q. 16 The NKG2D ligand MICA is shed by human tumors H.R. SALIH1, H.G. RAMMENSEE2, and A. STEINLE2 The immunoreceptor NKG2D stimulates tumor immunity through activation of CD8 T cells and NK cells. Its ligand MICA has been shown to be broadly expressed on human tumors of epithelial origin. MICA expression correlates with an enrichment of Vd1 T cells in tumor tissue. We report that human tumor cells spontaneously release a soluble form of MICA encompassing the three extracellular domains, which is present at high levels in sera of patients with gastrointestinal malignancies, but not in healthy donors. Release of MICA from tumor cells is blocked by inhibition of metalloproteinases, concomitantly causing accumulation of MICA on the cell surface. Shedding of MICA by tumor cells may modulate NKG2D mediated tumor immune surveillance. In addition, determination of soluble MICA levels may be implemented as an immunological diagnostic marker in patients with epithelial malignancies.
Department of Gastroenterology, Medizinische Hochschule, Hannover, Germany
Q. 17 Monitoring the effect of Ox-40 on the growth of liver metastasis in mice S.R. SCHEFFER, F. KORANGY, M.P. MANNS, and T.F. GRETEN Different immunotherapy strategies are currently being used for the treatment of cancer. In most studies tumors are injected subcutaneously and tumor growth is monitored using calipers. However, a number of different groups have shown that the site of the tumor influences the efficiency of different cancer vaccines. We therefore decided to inject tumor cells directly into the liver to mimic the development of liver metastasis. Because the size of liver metastasis cannot be measured we used an alternative method: Tumor cells were transfected with a gene expressing the human b-HCG gene. These cells express b-HCG in vivo, which can easily be detected in the urine of mice with a growing tumor. The amount of b-HCG in the urine is shown to be directly proportional to the tumor size. We have already used this system to analyze the effect of the treatment using an antibody against CD134 (Ox-40) for liver metastasis of the murine colon carcinoma CT26 in BALB/c mice. 35 days after injection of 2¥104 CT26 cells directly into the liver 50% of the mice remained tumor free. Moreover, mice receiving anti-Ox40 treatment had a longer delay in tumor development as determined by b-HCG concentration. In conclusion, we can present a new model for the analysis of tumor cell growth in vivo in different organs and the effect of different methods of immunotherapy to treat liver metastasis of colon cancer.
262 · 33rd Annual Meeting of the German Society of Immunology Department of Neurology, Eberhard-Karls-University, Tübingen, Germany
Q. 18 ICOSL expressed on human glioma cells modulates T cell cytokine patterns, but does not confer immunogenicity B. SCHREINER, J. WISCHHUSEN, M. MITSDÖRFFER, M. MEHLING, A. MELMS, E. TOLOSA, M. WELLER, and H. WIENDL Human glioblastoma is a highly lethal tumor that is known for its capability to interfere with effective anti-tumor immune responses. Costimulatory signals are of critical relevance in both inductive and effector phases of immune responses. Inducible costimulator-ligand (ICOSL), a member of the B7 family of costimulatory molecules related to CD80/CD86, has been demonstrated to regulate CD4 as well as CD8 T cell responses via interaction with its receptor, ICOS, on activated T cells. In order to characterize the ICOS/ICOSL costimulatory pathway in human glioblastoma immunobiology, we investigated the expression and functional relevance of ICOSL on glioma cells in vitro. In contrast to CD80 and CD86, ICOSL mRNA and protein expression were detected in six of eleven glioma cell lines. ICOSL expression was upregulated by the inflammatory cytokine, TNF-a, whereas IFN-g had negligible influence on the expression level. Functional studies of the role of ICOSL on glioma cells were conducted using HLA-A2-mismatched alloreactive PBMC. After gene transfer of ICOSL into two ICOSL-negative glioma cell lines (U87MG, LN-229), coculture experiments with alloreactive PBMC revealed enhanced expression of ICOS on activated T cells and increased production of Th1 (IFN-g) as well as Th2 (IL-4, IL-10) cytokines. A neutralizing ICOSL antibody reduced Th1 and Th2 cytokine levels in these cocultures. However, ICOSL gene transfer did not influence primary or secondary alloreactive proliferative responses of the effector cell population. Further, neither primary alloreactive lysis nor recall responses in a secondary alloreactive reaction were modulated by ICOSL. Taken together, we here report that ICOSL is expressed by human glioma cells in the absence of CD80 and CD86. Although ICOSL enhances levels of Th1 and Th2 cytokine production in T cells, expression of this costimulatory ligand does not significantly alter the immunogenicity of human glioma cells in alloassays in vitro.
Abteilungen für 1Urologie und Poliklinik, Chirurgische Klinik, and 2Molekulare Pathologie, Pathologisches Institut, Universitätsklinikum Heidelberg, Heidelberg, Germany
Q. 19 A MSI tumor-specific frameshift mutation in the caspase-5 gene encodes for an HLA-A2.1-restricted CTL-epitope Y. SCHWITALLE1, M. LINNEBACHER2, E. RIPBERGER2, J. GEBERT2, S. WÖRNER2, M. WIESEL1, G. STÄHLER1, and M. VON KNEBEL DÖBERITZ2 Microsatellite instability (MSI) caused by defective DNA mismatch repair (MMR) is a hallmark of hereditary non-polyposis colorectal cancers (HNPCC) but also occurs in about 15% of sporadic tumors. If instability affects microsatellites in coding regions, translational frameshifts lead to truncated proteins often marked by unique frameshift peptide sequences at their C-terminus.
33rd Annual Meeting of the German Society of Immunology · 263 Since MSI tumors show enhanced lymphocytic infiltration and our previous analysis identified numerous coding mononucleotide repeat bearing candidate genes as targets of genetic instability, we examined the role of frameshift peptides in triggering cellular immune responses. Using peptide pulsed autologous CD40-activated B cells (CD40Bs) we have generated cytotoxic T lymphocytes (CTLs) that specifically recognize HLA-A2.1-restricted peptides derived from frameshift sequences. We have previously shown, that peptides derived from (-1) frameshift mutations in genes coding for TGF-bIIR and O-GlcNac transferase, respectively gave rise to CTL capable to lyse MSI cancer cell lines, carrying these frameshift mutations. Here we analysed that two frameshift peptides originating from a (-1) mutation in the Caspase-5 gene, FSP25 (KMFFMVFLI) and FSP26 (FLIIWQNTM) conferred specific lysis of target cells exogenously loaded with cognate peptide. Even more important, CTL specific for peptide FSP26 specifically recognized endogenously Caspase-5(-1)-expressing HCT116, Colo60H and LoVo-HLA-A2.1 colorectal cancer target cells. Presently, we analyse the specific cytotoxic potential of CTL-clones derived by limiting dilution from the above mentioned bulk culture in more detail. Given the huge number of human coding microsatellites and assuming only a fraction being mutated and encoding immunologically relevant peptides in MSI tumors, frameshift protein sequences indeed represent a novel subclass of tumor specific antigens. It is tempting to speculate that a frameshift peptide directed vaccination approach not only could offer new treatment modalities for existing MSI tumors but also might benefit asymptomatic at-risk individuals in HNPCC families by a prophylactic vaccination strategy.
Abteilungen für 1Urologie und Poliklinik, Chirurgische Klinik, and 2Molekulare Pathologie, Pathologisches Institut, Universitätsklinikum Heidelberg, Heidelberg, Germany
Q. 20 Fusions of microsatellite-unstable tumor cells and B cells induce cytotoxic T cells with antitumor potential Y. SCHWITALLE1, M. LINNEBACHER2, C. SUTTER2, M. WIESEL1, G. STÄHLER1, and M. VON KNEBEL DÖBERITZ2 Microsatellite instability (MSI) reflects length variations at short repetitive DNA sequences in diploid tumor cells and is caused by mutational inactivation of different DNA mismatch repair genes. To enhance the immunogenicity of MSI tumors we fused different MSI carcinoma cell lines with CD40-activated B cells (CD40Bs) and examined the ability of generated fusion cell clones for induction of T cell populations with cytotoxic potential. MSI analysis revealed MSI-high (MSI-H) in 2/8 renal cell-, 2/3 prostate-, 1/3 urothelial- and 1/1 colon carcinoma cell lines. MSI-H cell lines were fused with previously generated CD40Bs. To investigate the fusion efficiency, cancer cells were stained with red and Bc with green vital fluorescent dye, respectively. 10–15% of cells detected by flow cytometry exhibited dual fluorescence of fused cells. Fusions from MSI-H colon carcinoma cell line HCT116 and CD40Bs were cloned by limiting dilution. Two selected clones induced outgrow of CD3+ T cells including CD4+ and CD8+ effectors, which produced perforin, granzyme B, IFN-g, IL-2 and TNF-a. Whole CD3+ T cell populations were able to lyse MSI+ tumor cell lines up to 40% in standard chromium release assays. Microsatellite stable tumor cell lines were not lysed and natural killer cell activity could be excluded. Sorting of CD3+ T cells with microbeads resulted in clear CD4+ and CD8+ proliferating T cell populations, respectively. Intracellular cytokine analysis showed
264 · 33rd Annual Meeting of the German Society of Immunology stronger expression of IFN-g, IL-2 and perforin in CD8+ T cells, whereas CD4+ T cells produced more TNF-a and granzyme B. CD8+ effectors revealed high cytotoxic activity with up to 50% lysis of MSI+ target cells. Our results clearly demonstrate the capacity of MSI+ tumor cell/CD40Bs fusions to induce expansion of CD3+ T effector cells with unequivocal cytotoxic potential. Cellular fusion vaccines from tumor cells of MSI patients and autologous B cells may have important clinical implications.
Departments of 1Internal Medicine III and 2Urology, Johannes Gutenberg-University, Mainz, and 3TobLab GmbH and 4Max-Planck-Institute of Biochemistry, Martinsried, Germany
Q. 21 Identification of differentially expressed proteins in renal cell carcinoma by proteome analysis B. SELIGER1, R. LICHTENFELS1, S. MELCHIOR2, W. BRENNER2, A. HARDER3, T. HALDER3, and F. LOTTSPEICH4 Proteomics represents a powerful technology in the identification of proteins and biochemical pathways involved in the development of tumors. Thus, the measurement of protein expression patterns from normal and tumor tissues will lead to the discovery of differentially expressed, cancer related proteins which are potentially useful as diagnostic, prognostic or therapeutic targets. This approach is currently employed to study proteins and pathways involved in renal cell carcinoma (RCC) tumorigenesis. Proteomes of three normal kidney epithelia and of corresponding high grade RCC were analyzed by two-dimensional gel electrophoresis followed by mass spectrometry. More than 30 protein alterations representing either an upregulation or downregulation of protein expression when compared to corresponding normal kidney epithelium were detected in the tumors. Some of these changes were shared among the RCC. The proteins identified as altered during tumorigenesis include proteins associated with stress, cell proliferation/ apoptosis, structure, enzyme metabolism and protein degradation and their significance with regard to tumor biology and immune escape is currently investigated. However, the marked heterogeneity of the observed protein alterations might have an impact on research strategies for profiling analysis of RCC.
33rd Annual Meeting of the German Society of Immunology · 265 1
Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University of Dresden, Dresden and 2Laboratory for Tumor Genetics, Department of Internal Medicine I, University of Cologne, Cologne, Germany
Q. 22 Construction of anti-CD30 chimeric VSV-G-receptors that mediate virus specificity for Hodgkin’s lymphoma cells A. TEMME1, A. HOMBACH2, H. ABKEN2, and E.P. RIEBER1 Retroviral vectors that selectively infect a certain cell type through recognition of specifically expressed surface antigens is a promising approach to target tumor cells. In vitro experiments and clinical trials for gene therapy have adopted retroviral vectors based on the murine leukemia virus system to transfer suicide genes into target cells. For a tumor-specific gene therapy the viral envelope must be engineered in order to reach efficient and selective transduction of cells expressing a tumor-associated antigen. The CD30 antigen is found at high density on Hodgkin’s lymphoma cells and thus represents an appropriate structure for tumor-specific targeting by retroviral vectors. The goal of this study was the development of an experimental therapy based on antiCD30 pseudotyped retroviruses for efficient gene delivery into Hodgkin’s lymphoma cells. The cDNA of the single chain antibody HRS3, which binds specifically the CD30 antigen, was used for the construction of chimeric retroviral receptors. The variable domains of the antibody were fused to a partial sequence of the Vesicular stomatitis virus G-protein (VSV-G). Two different leader sequences, one for immunoglobulins and the other representing the VSV-G leader were included in the construction of the chimeric envelopes. A retroviral vector encoding for an enhanced green fluorescent protein (EGFP) reporter gene was used to visualize transduced cells. To evaluate the target specificity of the anti-CD30 pseudotyped retroviruses CD30 negative cells (HeLa, BL60) and the CD30-expressing Hodgkin’s lymphoma cell line L540 were transduced. All cells were efficiently transduced with retroviruses pseudotyped with the VSV-G wild type envelope. After transduction with particles pseudotyped with the chimeric envelopes EGFP reporter gene expression was restricted to L540 cells suggesting target specificity to the virus pseudotyped with the chimeric receptor. We propose, that the use of retroviral particles genetically engineered to bind tumor antigens is a promising approach for defeating neoplastic growth.
1
Department of Neurosurgery, Martin-Luther-University Halle-Wittenberg, Halle, Germany and 2Department of Neurological Science, University of Liverpool, Liverpool, UK
Q. 23 Identification of tumor-associated antigens in glioma M. VOLKMAR1, K. GANS1, N.G. RAINOV2, and A. SOELING1 Background: Although conventional treatment modalities for gliomas have been improved markedly in the past decades the prognosis for these tumors remains dismal. Therefore, new therapeutic strategies taking into account the biological differences between tumor cells and normal tissue are gaining increasing attention. Our goal was to identify glioma-specific antigens that could serve as potential targets for immunotherapy.
266 · 33rd Annual Meeting of the German Society of Immunology Methods: We performed serological analysis of recombinant cDNA expression libraries (SEREX): A cDNA library from a human gliosarcoma was constructed and screened with pooled allogeneic sera from glioma patients. Expression of candidate antigens in normal and tumor tissue was analysed by semiquantitative RT-PCR. Antigen-specific serum reactivity in brain tumor patients and healthy volunteers was determined by plaque assay. Results: Immunoscreening of 1.2¥106 plaques revealed three tumor-associated antigens: 1) No55, a nuclear autoantigen; 2) the cell cycle-associated protein P1 (a human analogue of yeast Mcm3); 3) an antigen corresponding to the 3’ untranslated region (UTR) of transcription factor NFY-g. Expression levels of the corresponding mRNAs were similar in malignant brain tumors and normal tissues. Sera from 23 healthy donors did not react with any of the antigens while 9 out of 32 (28%) sera from glioma patients reacted with the P1 protein and 1 out of 32 (3%) was immunoreactive with NFY-g. Conclusions: Gliomas, usually considered as being rather weakly immunogenic, can obviously elicit a humoral immune response in the host. Although mRNA expression levels of the 3 detected antigens did not differ substantially between tumor and normal tissues, serological reactivity to these antigens was clearly restricted to cancer patients, thus emphasizing that accessibility to the antigen (e.g. by spontaneous tumor necrosis) may be of greater importance to the immune system than mere overexpression of the antigen in tumor tissues.
Klinik für Innere Medizin I, Universität zu Köln, Köln, Germany
Q. 24 Low cholesterol in Hodgkin’s disease-derived cell line L428 stimulated a converting enzyme-dependent shedding of CD30 TNF-a B. VON TRESKOW, A. ENGERT, and H.P. HANSEN CD30 is selectively expressed on the tumor cells of various malignant lymphomas and is therefore a privileged target for anti-CD30 antibody-based immunotherapy. However, a TACE/ADAM17-dependent CD30 shedding leads to high levels of the soluble ectodomain of CD30 (sCD30) in the serum the patients thus inhibiting successful therapy. From Alzheimer’s disease, there is evidence for a negative correlation between serum cholesterol and the ADAM10dependent non-amyloidogenic pathway of the amyloid precursor protein. In order to improve the tumor therapy we analyzed the influence of cholesterol on CD30 shedding. The release of sCD30 from the Hodgkin’s disease-derived cell line L428 was dramatically increased after treatment with agents known to reduce the cholesterol concentration in cell membranes, such as methyl-b-cyclodextrin or the hydroxymethyl glutaryl-CoA reductase inhibitor lovastatin. A similar effect was achieved by treatment of L428 cells with cholesterol oxidase and the cholesterolbinding polyene antibiotic filipin. This shedding enhancement and a concomitant loss of membrane-anchored CD30 from the tumor cells could be inhibited by the synthetic hydroxamic acidbased metalloproteinase inhibitor BB-3466 as well as TIMP-3 but not by TIMP-1. Accordingly, the CD30 shedding is suggested being ruled by TNF-a converting enzyme in this process as well. In addition, we found that in vitro the cholesterol depleting methyl-b-cyclodextrin inhibited the anti-CD30 antibody internalization whereas enrichment of cholesterol stimulated antibody endocytosis. Since cholesterol is preferentially associated with the lipid ordered (lo) domains of the cell membrane (lipid rafts), CD30 shedding is suggested being localized rather in lipid disordered, CD30 internalization rather in lipid rafts. Since hydroxymethyl glutaryl-CoA reductase
33rd Annual Meeting of the German Society of Immunology · 267 inhibitors are widely used lowering blood cholesterol, they are expected being not indicated during of CD30–targeted immunotherapy.
1
Department of Hematology and Oncology, Johannes Gutenberg-University, Mainz, Germany and 2Ludwig Institute of Cancer Research, University of Lausanne, Epalinges, Switzerland
Q. 25 Phenotype of retrovirally transduced human T cells equipped with tumor-associated antigen-specific high-affinity T cell receptors R.-H. VOSS1, J. KUBALL1, S. THOMAS1, M. JÜLCH1, P. GUILLAUME2, P. ROMERO2, C. HUBER1, and M. THEOBALD1 A promising strategy in the immunotherapy of human malignancies entails the tumor associated antigen (TAA)-specific T cell receptor (TCR) gene delivery into human T cells. Human tolerance to the ubiquitous low level expression of the oncoprotein MDM2 was bypassed by utilizing the HuCD8¥A2Kb transgenic mice for triggering the development of non-tolerant, A2.1-restricted high-affinity T cells. Expression frequency of the exogenous TCRs were increased to almost completeness by appending IRES selection cassettes to the individual TCR transgenes within the LTRs of the retroviral shuttle, facilitating the readout of subtle phenotype changes among the different TCR constructs: following retroviral transfer of the respective TCR genes as for the murine wildtype chains or a diverse set of chimeric TCRs into human T cells, they have been cultured long-term with CD3/CD28 beads and low doses of Il-2 for extensive phenotype studies. Cytotoxicity correlated with both the expression rate on a per cell basis as well as the time-dependent stability of expression as could be quantified by FACS-calibrated vb6-staining. Although some TCR constructs were unable to bind the MDM2(81–88)-specific tetramer to a considerable amount, they turned out to be vastly effective in lysing peptide loaded target cells. CD4+CD8+ subpopulations provided a strong indication that exclusively the transduced CD8+ were inherent the cytotoxic effector function. These results were in line with ELISA/ELISPOT measurements by virtue of IL-2, IFN-g, and IL-5 quantification that proved the TC1-phenotype of CTLs in vitro. Activation status and homing capacity of ex vivo cultured transduced T cells were analysed by a panel of activation markers and the dominant homing receptors CD62L and CCR7. Dissociation kinetics with different TCR constructs demonstrated the usefulness of the «kinetic window» model and to get an estimate for comparative cytolytic efficiencies as a prerequisite for preclinical in vivo studies.
268 · 33rd Annual Meeting of the German Society of Immunology Section of Immunobiology, Institute of Zoophysiology, University of Bonn, Bonn, Germany
Q. 26 Definition of pancreatic tumor antigens for the MHC class I and class II processing pathways B. WINZEN, J. NEUMANN, and N. KOCH Cancer of the pancreas is the fourth frequent cause of death of cancer diseases in the western industrialized countries. Despite advances in chemotherapy and surgical techniques, patients with pancreatic cancer often succumb to local recurrence or metastatic spread. The need for novel advanced therapies for this disease has prompted us to develop a novel strategy for immuno therapy with recombinant pancreatic tumor associated antigens. We utilized the invariant chain (Ii) as a carrier to deliver MHC class I and class II epitopes of the pancreas associated antigen core-2b1–6-N-acetylglucosaminyltransferase (C2GnT) to the MHC class I and class II processing pathways. CD4 and CD8 T cell epitopes were predicted on the basis of motifs, obtained from the SYFPEITHI database. From the C2GnT sequence we received six CD4 T cell epitopes with specificity for DR1, DR3 or DR4. Class I epitopes were specific for HLA-A2. In the recombinant construct, we replaced the MHC class II groove binding segment of Ii by a CD4 T cell epitope, whereas the C-terminal end of Ii contains a CD8 T cell epitope from C2GnT. By addition of a mAb V5-epitope to the C-terminus, the modified invariant chain protein can be monitored in the presence of endogenously expressed Ii. To investigate binding of class II molecules to the recombinant tumor antigens, we transfected COS cells with recombinant Ii and DR cDNAs. Immunoprecipitation of the DR/Ii complex with a mAb directed against DR revealed binding of the recombinant tumor antigen to DR1, DR3 or DR4. The formation of SDS resistant class II molecules demonstrates, that the DR molecules are stably loaded with the CD4 T cell epitopes.