- Email: [email protected]
Workshop K: Tumor Immunology
WORKSHOPK
Tumor Immunology
Institute for Medical Microbiology and Hygiene, TU Munich, Munich, Germany
K.1 Identification of a Cll recognized epitope in the E7 protein of human papillomavirus type 16 S. BAUER, K. HEEG, H. WAGNER, and G. LIPFORD Human papillomavirus (HPV) is responsible for the pathogenesis of warts and several squamous lesions. Some types of HPV, mainly type 16 and 18, are associated with cervical carcinoma. The early oncoproteins E7 and E6 are necessary for the transforming and immortalizing activity of the virus. It has previously been shown, that mouse melanoma cells transfected with the E6 or the E7 genes are specifically rejected by mice immunized with syngenic fibroblasts carrying the same genes. This rejection could be blocked by anti-CD8 antibodies, implying, that CTL are involved. To find the recognized epitope, we screened the amino acid sequence of E6 and E7 for possible peptides fitting into the Kb binding motif. Four sequences from E6 and two from E7 were selected, the peptides synthesized and tested for Kb-binding and Kb-stabilization. The Kb binding studies were performed by titrating the peptides in a competition assay using a 1251-labeled reporter peptide. The ability of the peptides to stabilize Kh molecules was investigated in a RMA-S MHC class I stabilization assay. Out of six peptides two peptides, E6 50-57 and E7 21-28 were able to bind and stabilize Kb. For the peptide E7 21-28 we could induce a primary CTL response by inoculating mice with peptide entrapped within Quil A containing liposomes. We have shown that the peptide E7 21-28 binds and stabilizes Kb and was also immunogenic in vivo. These observations should allow for the development of a mouse model for peptide vaccination against tumor cells expressing E7. Further work on the peptide E6 50-57 is in progress.
GSF-Institut fur Klinische Hamatologie, Munchen, Germany
K.2 Cell surface localization of a 72 KD heat shock protein (HSP72) on human malignant cells after sublethal heat treatment C. BOTZLER, G. MULTHOH', M. WIESNET, M. ESSLER, and R. D. ISSELS To study immunological effects of hyperthermia treatment upon human malignant cells, we compared cell surface markers of either heat-treated or untreated cells. Using indirect immunofluorescence, we detected cell surface expression of heat shock protein 72 (HSP72) on human Ewings's sarcoma (ES) and on osteosarcoma (HOS58) cells after a single sublethal heat dose (41.8 0C). Biochemically, this HSP72 localization was also demonstrated by selective cell surface radioiodination. Even without heat treatment,
152 . 24th Meeting of the Society of Immunology HSP72 cell surface expression was detectable on PBL of 'patients suffering from acute leucemia and on HEK 293 cells, transformed with the EIA sequence of adenovirus. In contrast, PBL and fibroblasts derived from healthy human individuals did not express HSP72 on the cell surface under any heating conditions. This indicates that «affected" cells may have different HSP regulation mechanisms compared to normal cells. This possibly results in cytoplasmic HSP72 overexpression followed by cell surface expression. Kinetic studies showed that HSP72 surface expression on ES and HOS cells is heating time dependent. Furthermore, a pre-heat incubation of ES cells with the proteinsynthesis inhibitor cycloheximide totally blocks cell surface expression of HSP72. Therefore, we conclude that only newly synthesized HSP72 molecules are expressed on the cell surface. Regarding the long-term surface localization of HSP72 (for at least 4 days) after a single heat treatment, we speculate that HSP72 could playa possible role as antigenic determinant on heat-treated tumor cells.
Institute of Immunological Reproduction, Magdeburg, Germany
K.3 Activity of granulocytes during chemotherapy H. DONAT and G. SKIBA It is known that during the chemotherapy the number of peripheral blood leucocytes is decreased because of the myelo-depression. The aim of the present study was to measure the activity of granulocytes by luminol and lucigenin chemiluminescence. 15 patients included in this study were operated because of ovarian cancer and treated with carboplatin and cyclophosphamide over 6 months. One day before and one day after each cytostatic treatment heparinized blood was obtained from the patients and the white blood cells were separated by gradient centrifugation. For the measurement we used the luminometer 1250 of LKB. The results have shown that one day after the infusion with cytostatics the activity of granulocytes is markedly increased. During the following time we observed a moderate decrease of the activity to the 4th cycle and then a significant decrease to the 6th cycle. It is concluded that the short time high activity of the granulocytes is caused by the stress situation during the infusion of cytostatics. The following decrease of the activity is due to the suppressive influence of the antineoplastic drugs to the bone marrow.
lDept. of Biological Chemistry and 2Dept. of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main, Germany
K.4 Phenotyping and functional analysis of peripheral blood lymphocytes from patients with ovarian cancer after immunoscintigraphy B. DONNERSTAGl, R. P. BAUM 2,J. B. OLTROGGEl, K. HENZEL 1, and G. HOR2 The development of human anti-mouse antibodies (HAMA) is well known for patients with ovarian cancer after immunoscintigraphy. Longer survival rates have been previously reported for patients developing high amounts of HAMA, which has been
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connected with the induction of the idiotypic network. Due to these observations we have examined phenotypes and function of peripheral blood lymphocytes (PBL) from the patients before and during the development of HAMA. These immunological parameters were compared with the clinical course. For phenotyping of lymphocytes, HAM A which can interfere with flow cytometry, were eliminated with protein G affinity chromatography. The following lymphocytic antigens were determined: CD4, CDS, CD3, CD19, CD16, CD56, CD2, CD25, CD45 and HLA-DR. For measurement of cytotoxic activity NK-cell and non-MHC restricted T-cell activity was determined with a fluorescence-based assay. PBL of 35 patients injected 1 to 6 times with 1 mg In-Ill OC 125 F(ab')2 (CIS, France) or 2 mg intact Tc-99m B43.13 (Biomira, Canada) antibody were tested for their immunological status at monthly intervals. In comparison, the PBL of ten healthy controls, not previously exposed to murine immunoglobulins, served as controls. Our results demonstrate that high HAMA titers (1 x 103-5 x 10 5 ng/ml) are often associated with normal phenotypes and cytotoxic activity of peripheral blood lymphocytes. Whereas PBL of patients with HAMA-titers below 1000 ng/ml show cytotoxic activity, which is decreased to as much as 20 % of the normal values. Concerning the functionality of non-MHC restricted cytotoxic T-lymphocytes we noticed an increase of more than 100 % indicating a decrease in specificity concerning T-cellular activity. These results enable us to define a correlation between immunological parameters and the progression of tumor growth. These immunologial parameters obtained by long-term phenotyping and functional analysis of peripheral blood lymphocytes compared to the clinical course will give a new insight into the role of anti-idiotypic antibodies in the immune response.
lZentrum der Biologischen Chemie and 2Klinik fur Allgemeinchirurgie, J.- W.-Goethe Universitat, Frankfurt/Main, Germany
K.S Relevance of immunological parameters in patients with colorectal carcinoma B. DONNERSTAG l , K. HENZELl, E. STAlil-SELBER2,J. B. OLTROGGEl, and M. LORENZ 2 In addition to the proliferation rate tumor growth is influenced by functionality of the immune system. From 26 patients with colorectalliver metastases the clinical course was compared with their immunological functions. For investigating the influence of tumor and/or chemotherapy we analyzed the following groups: A: no chemotherapy, B: patients with adjuvant therapy, C: patients with palliative regional arterial or systemic therapy of liver metastases, or D: patients with lung and liver metastases and cytostatic treatment. In comparison we analyzed phenotypes and function of peripheral blood lymphocytes (PBL) from 10 healthy controls. Phenotyping was done with flow cytometry by investigating the following parameters: CD4, CDS, CD19, CD3, CD16, CD56, CD2, CD25 and HLA-DR. For evaluation of cytotoxicity PBL stimulated with Interleukin-2 (nIL-2) were used as effector cells. Target cells were NK-sensitive (K562) or resistant (Raji). Percent lysis was calculated by fluorescence analysis. The results show a good -correlation between phenotyping and cytotoxic activity in the groups. Only little influence could be evaluated after application of 5-fluorouracil and folinic acid. Furthermore it was possible to define immunological parameters correlated with the progression of tumor growth.
154 . 24th Meeting of the Society of Immunology GSF-Institut fur Klinische Hamatologie, Munchen, Germany
K.6 An autologous in vitro model to study in vivo effects of regional hyperthermia/chemotherapy M. ESSLER, G. MULTHOFF, C. BOTZLER, M. WIESNET, and R. D. ISSELS A 72kD heat shock protein (HSP72) is expressed on the cell surface of a Ewing's sarcoma (ES) cell-line after sublethal heat exposure (41.8 Qq, whereas cell lines derived from normal tissues did not show this effect. The expression of HSP72 on the surface of sarcoma cells can be correlated with sensitivity to NK-like lysis in an allogeneic in vitro system. For better understanding of in vivo mechanisms after a combined regional hyperthermia/cytostatica therapy of sarcomas we want to generate an autologous system using PBL and tumor biopsy material derived from the same patient. For this purpose vital tumor specimens (liposarcoma) were minced mechanically and digested enzymatically in order to generate single cell suspensions. A permanent monolayer cell line was obtained, which showed similar growth characteristics (regarding plating efficiency and doubling time) to that of ES cells which were used in previous studies. To separate malignant cells from fibroblasts we performed softagar cloning experiments. After establishing of malignant, clonal cell-lines we first want to analyze if HSP cell surface expression is already apparent on in vivo heated tumor cells, or if heat treatment can induce surface expression. Furthermore, we want to study if autologous PBL derived from the patient either untreated or stimulated with recombinant IL2 are capable to lyse these autologous, clonal tumor cells in vitro.
Chirurgische Klinik GroGhadern, Munchen, Germany
K.7 Aggregate formation of micrometastatic cells in epithelial primary tumors (breast/stomach) S. FRIES, K. W.]AUCH, M. M. HEISS, U. GROTZNER, F. W. SCHILDBERG, and I. FUNKE
Introduction: Nowadays the immunocytochemical identification of epithelial tumor cells in bone marrow (micro metastases) allows to diagnose early tumor cell dissemination. Several groups even demonstrated a prognostic significance of this finding at the time of primary diagnosis and therapy. An astoundingly high frequency of micrometastatic cells was not only found in breast cancer, but also in gastrointestinal cancer. Results: Starting from the observation that micrometastases are not only detectable as single cells, but also as small cell aggregates, we compared two tumor types with different patterns in organ manifestation of solid metastases (i.e. breast and gastric cancer) according to the quantitative aspect. Aggregates were found in 21/45 (47%) of the micrometastases positive breast cancer patients (45/132) compared 18/45 (40%) of the positive gastric cancer patients (45/102). Analyses of aggregate size (grouped in A: 2-4; B: 5-9; C: > 10 cells/aggregate) revealed that 33 % of the aggregate forming cells in breast cancer belonged to group A versus 61 % in gastric cancer. Samples belonging to group C were found exclusively in breast cancer patients (10/45). The differences in the size of cell aggregates between these two tumor types are statistically significant (p = 0.009; chi-square).
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Conclusion: We hypothesize that micrometastases are indicative for the general disseminative capability of an individual tumor. Considering the differences in the formation of cell aggregates the expression of cell adhesion molecules may play an important role in this process. Cell surface molecule mediated interactions with the cells of their new microenvironment enable them to proliferate or stay «dormant». First double staining analyses revealed the expression of the epithelial cell adhesion molecule E-cadherin in 15/21 micrometastases positive breast cancer patients.
Institute for Immunology, University of Kiel, Kiel, Germany
K.8 Leukemia treatment with allogeneic T cells. 1: limiting dilution assays for donor selection
c. HERWARTZ,]. STEINMANN, T. GASKA, M. SALOMO, and W. MOLLER-RuCHHOLTZ Both, clinical data from allogeneic bone marrow transplantation and in vitro investigations provide evidence, that allogeneic T cells are principally capable to discriminate leukemia cells from nonmalignant cells. Here we demonstrate limiting dilution assays for cytotoxic T cell precursors (CTL-p) and interleukin-2 producing T helper cells (THl). Peripheral blood cells were stimulated with leukemia cells and tested for cytotoxicity and IL-2 production with leukemia cells and Iymphoblastoid cells of a patient with acute B lymphocyte leukemia (HLA A2S.30 BlS.57 Cw5.6 DR3.7). Whereas leukemia specific T cells were not detectable in the blood of the patient (in remission), of his syngeneic brother and of several allogeneic blood donors, one allogeneic donor (HLA A2.23 B7.53 Cw7.- DR2.7) showed a high frequency of CTL-p (20S per million) which differentiate in leukemia-reactive CTL that do not lyse the lymphoblastoid cell line. We suggest that the limiting dilution assay with split well analysis is a promising method for selecting the blood donors, if one plans T cell therapy of leukemia with allogeneic T cells.
1Pharmaceutical Research, E. Merck, Darmstadt; 2 Applied Immunology, D KFZ, INF 2S0, Heidelberg, Germany
K.9 Bispecific antibody for targeted cellular cytotoxicity C. S.JAGGU:I, S. NICKI, N. BAHLI, G. KREysCHI, D. ROTHI, D. MOLLER-POMPALLAI, S. c. MEUER2 , and W. STRITTMATTER 1 The potential of immune enhancing substances (especially cytokines) for the treatment of various types of cancer has been recognized. Anti-T cell antibodies are among the most potent and most specific immune enhancers known. Our aim was to combine the immune enhancing power of anti-T cell antibodies with the targeting properties of a tumor-selective anti-epidermal growth factor-receptor (EGF-R) antibody. Bispecific antibodies (BAb) have both properties. We have used a biochemical coupling procedure (based on Ellman's reagent) in order to generate bispecific antibodies. As a tumor selective principle we have used anti-EGF-R antibody (EMD 55900). Novel anti-CD2 antibodies have been developed (see M. WILD et al. this meeting) and used for triggering
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and targeting of effector cells. Previous studies suggested that cytotoxic T cells and natural killer cells are effectors involved in the control of tumor. CD2 based bispecific antibody activates both types of effector cells. In addition we have compared BAb induced lytic activity in a system based on the patients own lymphocytes and tumor cells with the antitumor activity induced with lymphocytes isolated from healthy donors. The two systems turned out to be comparable. BAb triggered cytolysis was greatly EGF-R specific and showed only weak bystander lysis. In summary, our results demonstrate that anti-CD2 x anti-EGF-R BAbs are able to retarget and activate autologous TIL's and PBMC's leading to active cytolysis of tumor cells. The further potential of BAbs for therapeutic application is under investigation.
Institute of Immunology, University of Munich, Munich, Germany
K.l0 Tcell receptor repertoire of tumor-infiltrating lymphocytes (TI L) in renal cell carcinoma (RCC) P. 1ANTZER, O. G. SEGURADO, and D. J. SCHENDEL Solid tumors such as RCC often show rich cellular infiltrates indicating a host anti-tumor immune response. In vitro cultivation of TIL led to the expansion of populations of highly specific MHC-restricted, CDS-positive cytotoxic T lymphocytes (CTL). Characterization at the molecular level revealed a preferential use of a limited number of V-genesegments with the repertoire of these TIL. These results are now reinforced by a comparative in situ analysis of primary tumor, normal kidney and metastatic tissue versus in vitro expanded TIL and PBL. In at least one patient we could see a stronger expression of unique V-gene-segments in primary RCC in relation to PBL or normal kidney. This might be an indication of an oligoclonal proliferation of T cells within the tumor which are specific for peptides derived from tumor-associated antigens (TAA). Some of these Vgene-segment-families are also detectable in the in vitro cultivated TIL which gives evidence for their functional relevance. These results and findings regarding the restriction elements involved in presenting immunodominant pep tides open the possibility to design new immunotherapies for patients suffering from RCC and to monitor the effectiveness of these therapies.
Medizinische Klinik I, Universitatskliniken des Saarlandes, Homburg/Saar, Germany
K.ll Expression of the activation antigen C030, its extracellular domain and immunoglobulin fusion proteins in different systems W. lUNG, H. GINER, and M. PFREUNDSCHUH Expression of the human activation antigen CD30 is found on activated or virus transformed lymphoid cells of either T or B origin, Hodgkin and Reed-Sternberg cells of Hodgkin's disease and cells of anaplastic large cell lymphoma. cDNA cloning has revealed that the CD30 antigen is a member of the growing family of cell surface receptors sharing homology with the nerve growth factor receptor. We have amplified
24th Meeting of the Society of Immunology . 157 the complete open reading frame (ORF) of the CD30 antigen from the Hodgkin derived cell line L540 by RT-PCR and cloned it into pTZ19. The translation product of one of the established clones was identical to the published data as confirmed by dideoxysequencing. Using PCR and standard cloning techniques on this cDNA we produced constructs encoding for the extracellular domain and two different immunoglobulin fusion proteins (CD30FPs). These DNAs and DNA encoding for the complete ORF were subcloned into vectors suitable for expression in COS-7-cells and CHO-cells, respectively, and were used to generate recombinant baculoviruses for expression in Sf9 cells. Purification protocols for the various recombinant proteins are being established in order to obtain pure recombinant proteins for functional tests and biochemical characterization.
Institute of Immunology, University of Munich, Munich, Germany
K.12 HLA-A2 restricted recognition of autologous and allogeneic human renal cell carcinomas by tumor-infiltrating lymphocytes S. KRESSENSTEIN, K. SEEBART, A. STEINLE, B. MAGET, and D. J. SCHENDEL Tumor-infiltrating lymphocytes isolated from a primary renal cell carcinoma (RCC) using a low concentration of recombinant IL-2 consist predominantly of MHCrestricted CD3 and CDS positive CTL. These CTL lysed autologous tumor cells but did not lyse normal kidney cells, a lymphokine activated killer sensitive cell line (Daudi), or a natural killer sensitive cell line (K562). Some allogeneic tumors of the same histological type were also recognized when shared HLA-A2 antigens were present. Blocking studies revealed that the MHC class I molecule, HLA-A2 serves as one restriction element for the presentation of a tumor-derived peptide to CTL within the TIL population. Some but not all allogeneic HLA-A2 positive RCC tumors were recognized by the CTL. Sequencing of exons 2 and 3 of HLA-A2 of autologous tumor cells and two allogeneic lines that were lysed showed that they had N:'0201 alleles. Only two of the five lines which were not lysed had N:'0201 alleles; peptide variability of longterm culture could be responsible for their lack of recognition. These results demonstrate that MHC-restricted CTL can recognize antigenic determinants on RCC tumors. Identification of specific CTL and definition of the molecular epitopes they recognize could lead to new approaches for immunotherapy for metastatic RCC.
Medizinische Klinik I, Universitatskliniken des Saarlandes, Homburg/Saar, Germany
K.13 cDNA sequence comparison of the CD30 antigen expressed on activated T-cells and a Hodgkin derived cell line L540 S. KRUGER, W. JUNG, L. TROMPER, and M. PFREUNDSCHUH The Hodgkin's disease associated activation antigen CD30 is a member of the growing family of cell surface receptors showing homology with the nerve growth factor receptor. In the hemopoietic cell lineage, expression of the CD30 antigen is predominantly found
158 . 24th Meeting of the Society of Immunology on Hodgkin and Reed-Sternberg cells (H&RS-cells), cells of anaplastic large celllymphoma and activated or virus transformed lymphoid cells of either T or B origin. In order to investigate whether structural differences of the CD30 antigen between normal T-cells and neoplastic H&RS-cells are involved in the growth regulation of H&RS-cells we have compared the open reading frames (ORF) of the CD30 antigen expressed on normal activated T -cells and a Hodgkin derived cell line sharing features of an activated T-cell, L540. The ORFs were cloned by PCR and independent clones were sequenced. Comparison with the published cDNA sequence of the CD30 antigen from the HTLV-I transformed T-cell line HUT-102 revealed a silent mutation at position 771 of the ORF in both cell types (A --> G). Besides this no significant mutations could be detected. We conclude that the primary amino acid sequence of the CD30 antigen expressed on the Hodgkin derived cell L540 is identical to that expressed on activated T -cells.
Institute of Clinical Immunology, !Dept. of Anaesthesiology and Intensive Care, and 2Dept. of Pediatric Surgery, Leipzig University, Leipzig, Germany
K.14 Whole body hyperthermic treatment in children alters the lymphocyte population in peripheral blood 1. LEHMANN, H. SCHADLICH, U. SACK, G. METZNER, U. BURKHARDT!, and R. B. TROBS 2 Because tumor cells react more sensitively to the action of heat than normal cells a combined application of hyperthermia with cytostatic drugs could lead to better treatment results. Since radiation and chemotherapy are known to cause a long term depression of immune response we observed the immune system of patients during controlled hyperthermia. Peripheral blood MNC of children having advanced malignant diseases treated by whole body hyperthermia (41,8 °e/2 hours) in combination with cytostatic drugs (Vepesid, Dactinomycin, Ifosfamid) was analyzed. Blood samples were taken before hyperthermia, in the period of temperature increase, at the time of highest body temperature and after hyperthermia. Induction of hyperthermia alters the lymphocyte populations rapidly. Among T lymphocytes we found a shift toward the CD8+ cells. A high percentage of this CD8+ cells showed an expression of HLA-DR antigen. Additionally an increasing of the natural killer cell population was observed. Although IFN gamma is involved in generation of cytotoxic T lymphocytes, in activation of natural killer cells and induction of HLA antigens on the surface no important changes were found in serum IFN gamma levels. During hyperthermia the IL2 receptor expression on CD4+ cells was reduced but reconstituted after reaching normal body temperature. The soluble IL2 receptor increased during hyperthermic treatment. It seems that hyperthermia in combination with cytostatic therapy does not suppress the immune system but in contrary activate the cytotoxic cells which could potentiate the anticancer activity.
24th Meeting of the Society of Immunology . 159 Klinikum GroBhadern, University of Munich, Munich, Germany
K.1S Loss of HLA A locus products in primary gastric cancer C. LORENZ, 1. FUNKE, K. W. JAUCH, J. P. JOHNSON, and B. MAYER
Down-regulation of HLA class I and HLA class II antigens is postulated to be a mechanism used by tumor cells to escape immunological recognition. Although many human tumors have been investigated for changes in class I and II antigen expression, the reagents employed have generally been unable to distinguish between the products of different loci or of different alleles. In the present study, the expression of HLA ABC and HLA D region molecules on gastric carcinomas was investigated immunohistochemically using monoclonal antibodies directed to the individual locus products and to several individual allelic specificities. 48 primary gastric carcinomas and autologous normal mucosae were examined as were metastatic lesions derived from various locations. HLA class II expression by normal mucosae was correlated with the presence of infiltrating leukocytes and inflammation. While the majority of primary tumors expressed HLA-DR molecules, the expression of HLA-DP and DQ products was less common. The frequency of HLA class I antigen expression as estimated with the antibody W6132, did not significantly differ between the tumors and the normal tissue. However, examination of locus and allelic specific products revealed a loss of particular HLA A locus products in more than 60 % of the primary tumors. The expression of HLA molecules by the primary tumors was correlated with a variety of prognostic factors such as tumor diameter, differentiation state, grading, and lymphatic vessel invasion and with tumor recurrence and survival (mean follow up: 23 months).
GSF-Institut fiir Klinische Hamatologie, Miinchen, Germany
K.16 A 72 kD heat shock protein acts as a target structure for NK cells G. MULTHOFF, M. WI ESNET, C. BOTZLER, M. ESSLER, and R. D. ISSELS Earlier we showed by indirect immunofluorescence technique and selective cell surface radioiodination that cell surface expression of HSP72 can be induced by sublethal heat shock on several human malignant cells, i.e. Ewing's sarcoma (ES) cells, osteosarcoma cells (HOS58) and transformed HEK293 cells. Regarding these results we addressed the question if HSP72 that is selectively expressed on affected cells, might elicit an antitumor immune response. Cytotoxic effector cells were derived from autologous stimulation of different peripheral blood lymphocytes (PBL) with EBV-transformed B-LCL, which were preincubated with heat treated tumor cell lysates. By magnetic beads separation method we separated CDr NK-like effector cells from CD3+ T-lymphocytes. Using the NK-enriched effector cell population in a cell mediated lympholysis assay (CML), we demonstrated that heat treated ES and K562 cells (either untreated or heattreated) were lysed significantly better compared to untreated ES cells or allogeneic BLCL. This NK-mediated lysis of heat stressed ES cells could be blocked using HSP72 moAb, whereas blocking with MHC class I specific antibody (W6/32) had no influence on the lysis pattern. Furthermore, lysis of untreated K562 cells was slightly reduced by HSP72 moAb. Cold target inhibition CML experiments using either heat treated ES or
160 . 24th Meeting of the Society of Immunology K562 cells as competitors clearly demonstrate that heat treated ES cells and K562 cells share one recognition structure for NK cells. Regarding our results of antibody blocking studies we conclude that HSP72 possibly is this common recognition structure for NK cells with anti-tumor activity.
Institute for Immunology, University of Munich, Munich; Medical Clinic II, Zentralklinikum, Augsburg, Germany; Sandoz Research Institute, Vienna, Austria
K.17 Decrease of individual metastatic tumor cells in bone marrow of breast cancer patients treated with monoclonal antibody K. PANTEL, G. SCHLIMOK, H. LOIBNER, and G. RIETHMULLER Limited accessibility and antigenic heterogeneity of cells incorporated in epithelial parenchyma hamper severely the efficacy of monoclonal antibody therapy in patients with advanced solid tumors. In contrast, individual epithelial tumor cells dispersed in mesenchymal compartments during the early phase of metastasis appear more appropriate targets for i.v. injected antibodies. Since such cells can now be detected immunocytochemically by cytokeratin (CK) antibodies in bone marrow, the question arose whether cytotoxic activity of murine monoclonal IgG3 antibody ABL364 can be directly demonstrated by monitoring cytokeratin-positive tumor cells in bone marrow. The used antibody is directed against the Lewis Y carbohydrate antigen, widely expressed on human epithelial tumors; it exhibits remarkable cytotoxicity against human tumor cells in vitro via activation of human complement and effector cells. Ten patients with breast cancer presenting with high numbers of epithelial cells in bone marrow (> 20/4 x 10 5 nucleated marrow cells) were treated in a randomized fashion either with 6 x 100 mg of the monoclonal Lewis Y antibody during two weeks or with human serum albumin (HSA) as a placebo. Epithelial cytokeratin-positive tumor cells in marrow were monitored on day 15 and 60 after commencement of treatment. Of the seven evaluable patients treated with antibody, five showed a distinct reduction or eradication of CK-positivel Lewis Y-positive cells of at least one log kill (96-100 %) while two patients, presenting only with CK-positive/Lewis Y-negative cells, showed no response. Similarly, in the three patients receiving HSA no significant tumor cell reduction was observed. Thus, the presented data may open a new approach towards a more rational selection of antibodies for adjuvant studies in minimal residual disease. Because of the marked cytotoxicity the antibody ABL364 exhibits in ex vivo experiments with serum of treated patients we postulate that the observed disappearance of tumor cells from bone marrow can be reduced to the action of the injected antibody.
24th Meeting of the Society of Immunology . 161 Beilinson Medical Center, Tel Aviv University Petah Tiqva, Israel; Max Planck Institute of Experimental Medicine, Gottingen, Germany
K.la Primary structure/amyloidogenicity relationship of AL-Bence Jones proteins POL, EZI and DIA A. I. PICK, H. KRATZIN, H. KLAfKI, and N. HILSCHMANN We have shown that the soluble urinary Bence Jones protein DIA (B.].) is a dimer of monoclonal L-chains with a primary structure identical to that of the myloid L-chain DIA and thus represents the amyloid precursor protein. The primary structure of Al protein POL is typical for the lambda 1.1 subclass. It has fifteen AA exchanges and shows a homology of 93 % with protein VOR and an increase in hydrophilic rather than hydrophobic groups. It shows a lack of homology with amyloid associated lambda-I and VI subclass B.]. (average 56.07 %) and a marked degree of homology with non-amyloid lambda-I subclass B.J. VOR (93 %) and KOL (77.78 %). The N-terminal H. is similar to the V -lambda-II B.]. protein VIL. In B.J. POL, the replacement of T by A in position 20 is unique. AL B.J. EZI is V lambda 1.2, it has seven AA replacements and a slight increase in hydrophilic groups. A comparison with 2t AL proteins shows an average homology of 54.23 %, the highest being with AL proteins EPS and HAR, 83.33 and 75 % respectively. The location of A in position 68, and of G in position 89 in the beta pleated sheet, remains unknown. A in position 68 and 97 is unique, the replacement of L by P in position 39 also occurred in AL protein POL. AL B.J. proteins could be the result of a subtle sequence alteration similar to that described in Alzheimer's disease.
'Institute of Clinical Immunology, Faculty of Medicine, and 2Dept. of Analysis, Faculty of Chemistry, University Leipzig, Leipzig, Germany; 3Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
K.19 Genotoxicity assessment of waste products with the SOS chromotest and the salmonella/microsome test F. RAABE', S. ]ANZ1,3, and R. HERZSCHUH 2 We evaluated 36 characteristic waste products from the plasma etching of aluminum and 7 waste gas condensates of municipal incinerators. The majority of the samples showed genotoxic activity in tester strain Escherichia coli PQ37 without metabolic activation using S9 mix. In the presence of S9, a deactivation of the samples of plasma etching and also of the municipal incinerators was regularly observed. Comparable studies with the Salmonella/microsome (Ames) test using tester strains Salmonella typhimurium TA98 and TAtOO indicated actual mutagenicity of the waste products. Gas chromatograms of the organic constituents of the samples from the plasma etching were performed in parallel with the genotoxic assays. In contrast to the similarity of the peak patterns of all chromatograms, the biological effects of the individual waste products showed large differences. The results of a representative sample from the plasma etching and also the influences of sample preparation are shown. The results of prokaryotic test systems alone are not sufficient to predict the potential danger to exposed people, they suggest consideration of those waste products as harmful and carcinogenic to man, and warrant further studies.
162 . 24th Meeting of the Society of Immunology Institute of Medical Immunology and IDept. of Urology, MLU, Halle, Germany
K.20 Aminopeptidase N/C013 on tumor-infiltrating T lymphocytes D. RIEMANN, B. GOHRINGl, A. KEHLEN, and]. LANGNER Aminopeptidase N/CD13 is a new activation-associated molecule on human T cells. CD13 cannot be demonstrated on PB lymphocytes - but on cells of inflammatory body effusions (1, 2). Functionally the enzyme could be involved in the metabolism of biologically active pep tides (3) or in antigen processing (4). We studied the immunophenotype of tumor-infiltrating T lymphocytes derived from human renal carcinoma and lung cancer. Fresh tumor specimens of 20 patients were minced and enzymatically dissociated. After separation on Ficoll-Hypaque, the immunophenotype of mononuclear cells was determined by two-color flow cytometry. CD3+ T cells expressed high amounts of CD45RO (94±3%), CD69 (84± 10'/"0), HLA-DR (53 ± 15 %) and CD25 (21 ± 8 % of CD3 + cells). Whereas in most cases only a small part of T cells expressed CD13 (13 ± 6 %), 3 tumor specimens contained 55-80 % CD13+ T cells. After cultivation of T cells of 1 patient in RPMI + 10 % FCS and 1000 U/ml IL2 for 30 days, only 28 % of CD3+ T cells expressed CD13 yet (initially 80 %). The ratio CD4:CD8 was 2.2 before and after cultivation. Using Ala-pNA as substrate, aminopeptidase activity of this pure T cell population could be estimated to be 18.2 pkat/l0 6 cells. Actinonine (10-4M) inhibited enzymatic activity by 80 %. By use of intron spanning PCR, cultivated T cells were found to produce mRNA for CD13 as well as ILIa, IL4, ILIO and IFNy. A possible function of CD13 on tumor-infiltrating T cells will be discussed. 1. 2. 3. 4.
RIEMANN, D. et al.: Immunobiol. 187,24-35 (1993). RIEMANN, D. et al.: Clin. Rheumatol. 12,31 (1993). MATSAS, R. et al.: FEBS Lett. 175, 124 (1984). MOURITSEN, S. et al.:]. Immunol. 149, 1987-1993 (1992).
This work was supported by the BMFT -Project Oncology 01 ZZ 9105, part XIII.
Innere Medizin I, Universitatsklinik Homburg, Homburg/Saar, Germany
K.2l Single Hodgkin and Reed-Sternberg cells lack rearrangements of the immunoglobulin and T cell receptor genes ]. ROTH, H. DAUS, L. TRUMPER, A. GAUSE, and M. PFREUNDSCHUH We tested the hypothesis that Hodgkin- and Reed-Sternberg cells (H&RS cells) have a lymphatic origin by analyzing the rearrangement status of single H&RS cells isolated from biopsy tissue by micromanipulation. In order to detect rearrangements of the immunoglobulin heavy chain (IgH) and T-cell receptor-y (TCR) genes, primers were constructed corresponding to the variable and joining regions of the IgH and TCR-y genes. These primers were shown to amplify a PCR-product of ca. 350 bp in length from PBLs, various cell lines and patients with non-Hogkin's lymphomas at a single cell level. The specificity of the PCR products was proven by Southern blotting and sequencing. Single H&RS cells isolated from biopsy material of nine patients were tested for IgH and TCR gene rearrangements. Rearrangements were not detected in the H&RS cells. As a
24th Meeting of the Society of Immunology . 163 positive control, sequences of the B-actin gene were amplified from single H&RS cells of all patients in a parallel reaction. From these results we conclude, that H&RS cells do not seem to be related to lymphoid cells.
Med. Universitatsklinik und Poliklinik, Abt. II, Tubingen, Germany
K.22 Differential regulation of HLA-class I genes by interferon H. SCHMIDT, I. STEIERT, E. KELLERMANN- KEGREISS, J. W ALZ, R. ZINSER, and C. A.MuLLER Expression of HLA class I antigens can be modulated in vitro and in vivo by interferon (IFN) a but also by IFN y. A strong induction of HLA-class I antigens was found on both hematopoietic progenitors and normal peripheral blood mononuclear cells after one month of IFN treatment in 18 patients with myeloproliferative syndrome. By daily injections of IFN in the first month of therapy stimulation was continuously increasing suggesting a major effect of IFN a on hematopoietic progenitors with sustained enhanced expression of HLA-class I antigens during differentiation of myelomonocytic cells. Differential in vivo regulation of HLA-class I antigens by IFN was demonstrated by comparison of HLA-A2 with HLA-B antigen expression. In vitro expression of the HLA-B7 and -Bw64 genes was significantly more inducible by IFN than the genes coding for the HLA-B27, HLA-B51, HLA-B38, HLA-B39, HLA-Cw3 and HLA-A2 antigens after transfection into mouse L cells. Modification of the 5' ends of the HLA-B7 and HLA-B27 genes before transfection in mouse L cells revealed the presence of enhancer sequences responding to interferon treatment in the 5' untranslated region of the HLA-B7, but not of the HLA-B27 gene and suggested further independently acting enhancer elements down-stream of the transcription initiation site. When different fragments of HLA B7 or B38, including introns, 3' and 5' untranslated regions, were cloned in front of CAT genes IFN responding enhancers were only detected at the 5' end of the HLA-B7 gene. Further IFN independent enhancers could be detected within introns and at the 3' end of HLA class I genes. These findings may indicate specific regulatory mechanisms of HLA class I antigen expression possibly influencing T-cell recognition in immune response.
I. Medizinische Klinik und Poliklinik, Johannes-Gutenberg-U niversitat, Mainz; 1Max-Planck -Institut fur Biologie, Abtl. Immungenetik, Tubingen, Germany
K.23 Recognition of human melanoma cells by autologous cytotoxic T lymphocytes (eTL): detection of target peptide antigens after elution from HLA-A2.1
J. SCHNEIDER, K. FALKt, O. ROTZSCHKEt, T. WOLFEL, K.-H. MEYERZUMBOsCHENfELDE, and H.-G. RAMMENSEE 1
In the melanoma model A V a panel of tumor-reactive CTL clones were derived from autologous peripheral blood lymphocytes. By immunoselection it was found that they were directed against four different antigens (A a, Ab, B, C) and were all restricted by
164 . 24th Meeting of the Society of Immunology HLA-A2.1. HLA-A2.1 molecules were purified by affinity chromatography from batches of each 5 x 109 AV melanoma cells. Pep tides bound to HLA-A2.1 were eluted with trifluoroacetic acid and were further separated by ultrafiltration and high-performance liquid chromatography (HPLC). Various targets were preincubated with these HPLC fractions and tested for CTL recognition in s'Cr-release assays. When antigennegative AV tumor cell variants, A2-positive EBV-transformed cells or A2-transfected P815 cells were used as target cells, none of the fractions caused lysis. Using the A2positive transport deletion mutant T2 as a target, we detected positive peptide fractions with CTL anti-Aa, but not with CTL against other AV antigens. However, after pretreatment of T2 cells with the anti-HLA-A2 antibody MA2.1 known to enhance peptide association with HLA-A2.1 (BODMER et aI., Nature 342: 443, 1989), we detected naturally processed peptide antigens Aa, Ab, Band C in different fractions of the HLAA2.1 peptide pool. The results were reproducible with different HPLC preparations. Improving the sensitivity of peptide detection assays, as described herein, is a prerequisite for biochemical characterization of CTL-defined peptide antigens which so far has been impossible for non-viral endogeneous peptides.
GSF-Institut fur Klinische Hamatologie, Munchen, Germany
K.24 Modulation of cell surface markers during combined hyperthermia/chemotherapy M. WIESNET, G. MULTHOFF, C. BOTZLER, M. ESSLER, and R. D. ISSELS Patients suffering from local advanced sarcomas were treated in a clinical phase-II study with systemic chemotherapy combined with regional hyperthermia in repeated cycles. The parameters of this study were defined in a RHT-91 protocol (R. D. ISSELs). To examine the effects of this therapy in regard to the immune system, we analyzed cell surface markers of freshly isolated peripheral blood lymphocytes (PBL) of eight sarcoma patients (cycle 1 to 6) within one therapeutic cycle. Additionally, these PBL were stimulated with 100 U of human recombinant IL2 for 7 days and tested again using the following mAb in a FACScan-analysis: CD3, CD4, CD8, CDI6/CD56. After one treatment cycle a significant reduction of all tested cell surface markers in five out of eight PBL of patients was observed. This effect did not show any correlation with the number of treatment cycle. PBL of three patients did not show changes in the tested cell surface markers. Interestingly, reconstitution of the immune status to normal levels was achieved by incubation of PBL of patients with IL2 in vitro. The function of this IL2 expanded PBL of four patients was defined in a cell mediated lympholysis assay (CML) using either untreated (37°C) or heat treated (41.8 0c) Ewing's sarcoma cells (ES) as target cells. Our results show that IL2 expanded PBL derived after treatment show better lysis of heat treated ES cells compared to untreated ES cells. There was no difference in the lysis pattern of either untreated or heat treated ES cells when PBL were derived before treatment. These results lead us to the hypothesis that regional hyperthermia in combination with chemotherapy in vivo can induce a selection of cytotoxic effector cells with specificity for heat treated sarcoma cells.
24th Meeting of the Society of Immunology . 165 !Applied Immunology, D KFZ, INF 280, Heidelberg; 2Pharmaceutical Research, E. Merck, Darmstadt, Germany
K.2S T cell targeting by anti-C02 X anti-EGF-R bispecific antibodies M. WILD!, B. SCHRAVEN!, H. KIRCHGESSNER!, D. C. ELKINS!, W. STRITTMATTER2 , S. MATZKU 1 , and S. C. MEUER 1 Bispecific antibodies (BsAbs) can be employed to redirect effector cells to tumour cells independent of specific target recognition by the TCR. In addition, antibodies can provide triggering signals for effector cell activation. Among the lymphocyte receptors considered for T cell targeting strategies CD2 appears to be favorable: 1) CD2 is strongly expressed by both T cells and natural killer cells. 2) Both effector cell populations can be triggered to express their functional programs following stimulation via CD2. 3) Efficient lymphocyte triggering through CD2 requires the simultaneous binding of two different CD2 mAbs. Therefore, a two step activation protocol can be established using one CD2 mAb as a CD2 x anti-tumor BsAb for targeting and the second one for activation of targeted cells thus likely avoiding unwanted systemic side effects. We have produced two novel anti-CD2 mAbs, AICD2.M1 (M1) and AICD2.M2 (M2), which in combination are mitogenic for human T cells. These mAbs do not interfere with CDS8/LFA-3 binding to CD2 nor do they individually inhibit activation of T cells via the TCR unlike many other CD2 mAbs. We used a chemical conjugation method to couple anti-EGFreceptor Fab' to M1- or M2-Fab' fragments. The combinations of M1 x anti-EGF-R BsAb plus M2 F(ab'h or alternatively M2 x anti-EGF-R BsAb plus M1 F(ab')2 were mitogenic for PBMC. Lysis of EGF-R(+) A 431 cells by PBMC was induced when effector cells were preincubated with the above mentioned Ab-combinations. As expected cytotoxicity was partly mediated by NK activity. Targeting with the BsAbs was proven in experiments with a CD8(+) cytotoxic human T cell clone which lysed A 431 but not EGF-R(-) cells in the presence of BsAb. Moreover, BsAb-induced lysis of A 431 cells by the T cell clone was blocked by an excess of EGF-R mAb. Our in vitro studies demonstrate that CD2 x anti-tumour antigen BsAbs in combination with a second CD2 mAb are able to mediate activation and tumor antigen specific targeting of effector cells and subsequent specific lysis of tumor cells. In vivo studies in hCD2 transgenic mice are In progress.
Tumor Immunology
Institute for Medical Microbiology and Hygiene, TU Munich, Munich, Germany
K.1 Identification of a Cll recognized epitope in the E7 protein of human papillomavirus type 16 S. BAUER, K. HEEG, H. WAGNER, and G. LIPFORD Human papillomavirus (HPV) is responsible for the pathogenesis of warts and several squamous lesions. Some types of HPV, mainly type 16 and 18, are associated with cervical carcinoma. The early oncoproteins E7 and E6 are necessary for the transforming and immortalizing activity of the virus. It has previously been shown, that mouse melanoma cells transfected with the E6 or the E7 genes are specifically rejected by mice immunized with syngenic fibroblasts carrying the same genes. This rejection could be blocked by anti-CD8 antibodies, implying, that CTL are involved. To find the recognized epitope, we screened the amino acid sequence of E6 and E7 for possible peptides fitting into the Kb binding motif. Four sequences from E6 and two from E7 were selected, the peptides synthesized and tested for Kb-binding and Kb-stabilization. The Kb binding studies were performed by titrating the peptides in a competition assay using a 1251-labeled reporter peptide. The ability of the peptides to stabilize Kh molecules was investigated in a RMA-S MHC class I stabilization assay. Out of six peptides two peptides, E6 50-57 and E7 21-28 were able to bind and stabilize Kb. For the peptide E7 21-28 we could induce a primary CTL response by inoculating mice with peptide entrapped within Quil A containing liposomes. We have shown that the peptide E7 21-28 binds and stabilizes Kb and was also immunogenic in vivo. These observations should allow for the development of a mouse model for peptide vaccination against tumor cells expressing E7. Further work on the peptide E6 50-57 is in progress.
GSF-Institut fur Klinische Hamatologie, Munchen, Germany
K.2 Cell surface localization of a 72 KD heat shock protein (HSP72) on human malignant cells after sublethal heat treatment C. BOTZLER, G. MULTHOH', M. WIESNET, M. ESSLER, and R. D. ISSELS To study immunological effects of hyperthermia treatment upon human malignant cells, we compared cell surface markers of either heat-treated or untreated cells. Using indirect immunofluorescence, we detected cell surface expression of heat shock protein 72 (HSP72) on human Ewings's sarcoma (ES) and on osteosarcoma (HOS58) cells after a single sublethal heat dose (41.8 0C). Biochemically, this HSP72 localization was also demonstrated by selective cell surface radioiodination. Even without heat treatment,
152 . 24th Meeting of the Society of Immunology HSP72 cell surface expression was detectable on PBL of 'patients suffering from acute leucemia and on HEK 293 cells, transformed with the EIA sequence of adenovirus. In contrast, PBL and fibroblasts derived from healthy human individuals did not express HSP72 on the cell surface under any heating conditions. This indicates that «affected" cells may have different HSP regulation mechanisms compared to normal cells. This possibly results in cytoplasmic HSP72 overexpression followed by cell surface expression. Kinetic studies showed that HSP72 surface expression on ES and HOS cells is heating time dependent. Furthermore, a pre-heat incubation of ES cells with the proteinsynthesis inhibitor cycloheximide totally blocks cell surface expression of HSP72. Therefore, we conclude that only newly synthesized HSP72 molecules are expressed on the cell surface. Regarding the long-term surface localization of HSP72 (for at least 4 days) after a single heat treatment, we speculate that HSP72 could playa possible role as antigenic determinant on heat-treated tumor cells.
Institute of Immunological Reproduction, Magdeburg, Germany
K.3 Activity of granulocytes during chemotherapy H. DONAT and G. SKIBA It is known that during the chemotherapy the number of peripheral blood leucocytes is decreased because of the myelo-depression. The aim of the present study was to measure the activity of granulocytes by luminol and lucigenin chemiluminescence. 15 patients included in this study were operated because of ovarian cancer and treated with carboplatin and cyclophosphamide over 6 months. One day before and one day after each cytostatic treatment heparinized blood was obtained from the patients and the white blood cells were separated by gradient centrifugation. For the measurement we used the luminometer 1250 of LKB. The results have shown that one day after the infusion with cytostatics the activity of granulocytes is markedly increased. During the following time we observed a moderate decrease of the activity to the 4th cycle and then a significant decrease to the 6th cycle. It is concluded that the short time high activity of the granulocytes is caused by the stress situation during the infusion of cytostatics. The following decrease of the activity is due to the suppressive influence of the antineoplastic drugs to the bone marrow.
lDept. of Biological Chemistry and 2Dept. of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main, Germany
K.4 Phenotyping and functional analysis of peripheral blood lymphocytes from patients with ovarian cancer after immunoscintigraphy B. DONNERSTAGl, R. P. BAUM 2,J. B. OLTROGGEl, K. HENZEL 1, and G. HOR2 The development of human anti-mouse antibodies (HAMA) is well known for patients with ovarian cancer after immunoscintigraphy. Longer survival rates have been previously reported for patients developing high amounts of HAMA, which has been
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connected with the induction of the idiotypic network. Due to these observations we have examined phenotypes and function of peripheral blood lymphocytes (PBL) from the patients before and during the development of HAMA. These immunological parameters were compared with the clinical course. For phenotyping of lymphocytes, HAM A which can interfere with flow cytometry, were eliminated with protein G affinity chromatography. The following lymphocytic antigens were determined: CD4, CDS, CD3, CD19, CD16, CD56, CD2, CD25, CD45 and HLA-DR. For measurement of cytotoxic activity NK-cell and non-MHC restricted T-cell activity was determined with a fluorescence-based assay. PBL of 35 patients injected 1 to 6 times with 1 mg In-Ill OC 125 F(ab')2 (CIS, France) or 2 mg intact Tc-99m B43.13 (Biomira, Canada) antibody were tested for their immunological status at monthly intervals. In comparison, the PBL of ten healthy controls, not previously exposed to murine immunoglobulins, served as controls. Our results demonstrate that high HAMA titers (1 x 103-5 x 10 5 ng/ml) are often associated with normal phenotypes and cytotoxic activity of peripheral blood lymphocytes. Whereas PBL of patients with HAMA-titers below 1000 ng/ml show cytotoxic activity, which is decreased to as much as 20 % of the normal values. Concerning the functionality of non-MHC restricted cytotoxic T-lymphocytes we noticed an increase of more than 100 % indicating a decrease in specificity concerning T-cellular activity. These results enable us to define a correlation between immunological parameters and the progression of tumor growth. These immunologial parameters obtained by long-term phenotyping and functional analysis of peripheral blood lymphocytes compared to the clinical course will give a new insight into the role of anti-idiotypic antibodies in the immune response.
lZentrum der Biologischen Chemie and 2Klinik fur Allgemeinchirurgie, J.- W.-Goethe Universitat, Frankfurt/Main, Germany
K.S Relevance of immunological parameters in patients with colorectal carcinoma B. DONNERSTAG l , K. HENZELl, E. STAlil-SELBER2,J. B. OLTROGGEl, and M. LORENZ 2 In addition to the proliferation rate tumor growth is influenced by functionality of the immune system. From 26 patients with colorectalliver metastases the clinical course was compared with their immunological functions. For investigating the influence of tumor and/or chemotherapy we analyzed the following groups: A: no chemotherapy, B: patients with adjuvant therapy, C: patients with palliative regional arterial or systemic therapy of liver metastases, or D: patients with lung and liver metastases and cytostatic treatment. In comparison we analyzed phenotypes and function of peripheral blood lymphocytes (PBL) from 10 healthy controls. Phenotyping was done with flow cytometry by investigating the following parameters: CD4, CDS, CD19, CD3, CD16, CD56, CD2, CD25 and HLA-DR. For evaluation of cytotoxicity PBL stimulated with Interleukin-2 (nIL-2) were used as effector cells. Target cells were NK-sensitive (K562) or resistant (Raji). Percent lysis was calculated by fluorescence analysis. The results show a good -correlation between phenotyping and cytotoxic activity in the groups. Only little influence could be evaluated after application of 5-fluorouracil and folinic acid. Furthermore it was possible to define immunological parameters correlated with the progression of tumor growth.
154 . 24th Meeting of the Society of Immunology GSF-Institut fur Klinische Hamatologie, Munchen, Germany
K.6 An autologous in vitro model to study in vivo effects of regional hyperthermia/chemotherapy M. ESSLER, G. MULTHOFF, C. BOTZLER, M. WIESNET, and R. D. ISSELS A 72kD heat shock protein (HSP72) is expressed on the cell surface of a Ewing's sarcoma (ES) cell-line after sublethal heat exposure (41.8 Qq, whereas cell lines derived from normal tissues did not show this effect. The expression of HSP72 on the surface of sarcoma cells can be correlated with sensitivity to NK-like lysis in an allogeneic in vitro system. For better understanding of in vivo mechanisms after a combined regional hyperthermia/cytostatica therapy of sarcomas we want to generate an autologous system using PBL and tumor biopsy material derived from the same patient. For this purpose vital tumor specimens (liposarcoma) were minced mechanically and digested enzymatically in order to generate single cell suspensions. A permanent monolayer cell line was obtained, which showed similar growth characteristics (regarding plating efficiency and doubling time) to that of ES cells which were used in previous studies. To separate malignant cells from fibroblasts we performed softagar cloning experiments. After establishing of malignant, clonal cell-lines we first want to analyze if HSP cell surface expression is already apparent on in vivo heated tumor cells, or if heat treatment can induce surface expression. Furthermore, we want to study if autologous PBL derived from the patient either untreated or stimulated with recombinant IL2 are capable to lyse these autologous, clonal tumor cells in vitro.
Chirurgische Klinik GroGhadern, Munchen, Germany
K.7 Aggregate formation of micrometastatic cells in epithelial primary tumors (breast/stomach) S. FRIES, K. W.]AUCH, M. M. HEISS, U. GROTZNER, F. W. SCHILDBERG, and I. FUNKE
Introduction: Nowadays the immunocytochemical identification of epithelial tumor cells in bone marrow (micro metastases) allows to diagnose early tumor cell dissemination. Several groups even demonstrated a prognostic significance of this finding at the time of primary diagnosis and therapy. An astoundingly high frequency of micrometastatic cells was not only found in breast cancer, but also in gastrointestinal cancer. Results: Starting from the observation that micrometastases are not only detectable as single cells, but also as small cell aggregates, we compared two tumor types with different patterns in organ manifestation of solid metastases (i.e. breast and gastric cancer) according to the quantitative aspect. Aggregates were found in 21/45 (47%) of the micrometastases positive breast cancer patients (45/132) compared 18/45 (40%) of the positive gastric cancer patients (45/102). Analyses of aggregate size (grouped in A: 2-4; B: 5-9; C: > 10 cells/aggregate) revealed that 33 % of the aggregate forming cells in breast cancer belonged to group A versus 61 % in gastric cancer. Samples belonging to group C were found exclusively in breast cancer patients (10/45). The differences in the size of cell aggregates between these two tumor types are statistically significant (p = 0.009; chi-square).
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Conclusion: We hypothesize that micrometastases are indicative for the general disseminative capability of an individual tumor. Considering the differences in the formation of cell aggregates the expression of cell adhesion molecules may play an important role in this process. Cell surface molecule mediated interactions with the cells of their new microenvironment enable them to proliferate or stay «dormant». First double staining analyses revealed the expression of the epithelial cell adhesion molecule E-cadherin in 15/21 micrometastases positive breast cancer patients.
Institute for Immunology, University of Kiel, Kiel, Germany
K.8 Leukemia treatment with allogeneic T cells. 1: limiting dilution assays for donor selection
c. HERWARTZ,]. STEINMANN, T. GASKA, M. SALOMO, and W. MOLLER-RuCHHOLTZ Both, clinical data from allogeneic bone marrow transplantation and in vitro investigations provide evidence, that allogeneic T cells are principally capable to discriminate leukemia cells from nonmalignant cells. Here we demonstrate limiting dilution assays for cytotoxic T cell precursors (CTL-p) and interleukin-2 producing T helper cells (THl). Peripheral blood cells were stimulated with leukemia cells and tested for cytotoxicity and IL-2 production with leukemia cells and Iymphoblastoid cells of a patient with acute B lymphocyte leukemia (HLA A2S.30 BlS.57 Cw5.6 DR3.7). Whereas leukemia specific T cells were not detectable in the blood of the patient (in remission), of his syngeneic brother and of several allogeneic blood donors, one allogeneic donor (HLA A2.23 B7.53 Cw7.- DR2.7) showed a high frequency of CTL-p (20S per million) which differentiate in leukemia-reactive CTL that do not lyse the lymphoblastoid cell line. We suggest that the limiting dilution assay with split well analysis is a promising method for selecting the blood donors, if one plans T cell therapy of leukemia with allogeneic T cells.
1Pharmaceutical Research, E. Merck, Darmstadt; 2 Applied Immunology, D KFZ, INF 2S0, Heidelberg, Germany
K.9 Bispecific antibody for targeted cellular cytotoxicity C. S.JAGGU:I, S. NICKI, N. BAHLI, G. KREysCHI, D. ROTHI, D. MOLLER-POMPALLAI, S. c. MEUER2 , and W. STRITTMATTER 1 The potential of immune enhancing substances (especially cytokines) for the treatment of various types of cancer has been recognized. Anti-T cell antibodies are among the most potent and most specific immune enhancers known. Our aim was to combine the immune enhancing power of anti-T cell antibodies with the targeting properties of a tumor-selective anti-epidermal growth factor-receptor (EGF-R) antibody. Bispecific antibodies (BAb) have both properties. We have used a biochemical coupling procedure (based on Ellman's reagent) in order to generate bispecific antibodies. As a tumor selective principle we have used anti-EGF-R antibody (EMD 55900). Novel anti-CD2 antibodies have been developed (see M. WILD et al. this meeting) and used for triggering
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and targeting of effector cells. Previous studies suggested that cytotoxic T cells and natural killer cells are effectors involved in the control of tumor. CD2 based bispecific antibody activates both types of effector cells. In addition we have compared BAb induced lytic activity in a system based on the patients own lymphocytes and tumor cells with the antitumor activity induced with lymphocytes isolated from healthy donors. The two systems turned out to be comparable. BAb triggered cytolysis was greatly EGF-R specific and showed only weak bystander lysis. In summary, our results demonstrate that anti-CD2 x anti-EGF-R BAbs are able to retarget and activate autologous TIL's and PBMC's leading to active cytolysis of tumor cells. The further potential of BAbs for therapeutic application is under investigation.
Institute of Immunology, University of Munich, Munich, Germany
K.l0 Tcell receptor repertoire of tumor-infiltrating lymphocytes (TI L) in renal cell carcinoma (RCC) P. 1ANTZER, O. G. SEGURADO, and D. J. SCHENDEL Solid tumors such as RCC often show rich cellular infiltrates indicating a host anti-tumor immune response. In vitro cultivation of TIL led to the expansion of populations of highly specific MHC-restricted, CDS-positive cytotoxic T lymphocytes (CTL). Characterization at the molecular level revealed a preferential use of a limited number of V-genesegments with the repertoire of these TIL. These results are now reinforced by a comparative in situ analysis of primary tumor, normal kidney and metastatic tissue versus in vitro expanded TIL and PBL. In at least one patient we could see a stronger expression of unique V-gene-segments in primary RCC in relation to PBL or normal kidney. This might be an indication of an oligoclonal proliferation of T cells within the tumor which are specific for peptides derived from tumor-associated antigens (TAA). Some of these Vgene-segment-families are also detectable in the in vitro cultivated TIL which gives evidence for their functional relevance. These results and findings regarding the restriction elements involved in presenting immunodominant pep tides open the possibility to design new immunotherapies for patients suffering from RCC and to monitor the effectiveness of these therapies.
Medizinische Klinik I, Universitatskliniken des Saarlandes, Homburg/Saar, Germany
K.ll Expression of the activation antigen C030, its extracellular domain and immunoglobulin fusion proteins in different systems W. lUNG, H. GINER, and M. PFREUNDSCHUH Expression of the human activation antigen CD30 is found on activated or virus transformed lymphoid cells of either T or B origin, Hodgkin and Reed-Sternberg cells of Hodgkin's disease and cells of anaplastic large cell lymphoma. cDNA cloning has revealed that the CD30 antigen is a member of the growing family of cell surface receptors sharing homology with the nerve growth factor receptor. We have amplified
24th Meeting of the Society of Immunology . 157 the complete open reading frame (ORF) of the CD30 antigen from the Hodgkin derived cell line L540 by RT-PCR and cloned it into pTZ19. The translation product of one of the established clones was identical to the published data as confirmed by dideoxysequencing. Using PCR and standard cloning techniques on this cDNA we produced constructs encoding for the extracellular domain and two different immunoglobulin fusion proteins (CD30FPs). These DNAs and DNA encoding for the complete ORF were subcloned into vectors suitable for expression in COS-7-cells and CHO-cells, respectively, and were used to generate recombinant baculoviruses for expression in Sf9 cells. Purification protocols for the various recombinant proteins are being established in order to obtain pure recombinant proteins for functional tests and biochemical characterization.
Institute of Immunology, University of Munich, Munich, Germany
K.12 HLA-A2 restricted recognition of autologous and allogeneic human renal cell carcinomas by tumor-infiltrating lymphocytes S. KRESSENSTEIN, K. SEEBART, A. STEINLE, B. MAGET, and D. J. SCHENDEL Tumor-infiltrating lymphocytes isolated from a primary renal cell carcinoma (RCC) using a low concentration of recombinant IL-2 consist predominantly of MHCrestricted CD3 and CDS positive CTL. These CTL lysed autologous tumor cells but did not lyse normal kidney cells, a lymphokine activated killer sensitive cell line (Daudi), or a natural killer sensitive cell line (K562). Some allogeneic tumors of the same histological type were also recognized when shared HLA-A2 antigens were present. Blocking studies revealed that the MHC class I molecule, HLA-A2 serves as one restriction element for the presentation of a tumor-derived peptide to CTL within the TIL population. Some but not all allogeneic HLA-A2 positive RCC tumors were recognized by the CTL. Sequencing of exons 2 and 3 of HLA-A2 of autologous tumor cells and two allogeneic lines that were lysed showed that they had N:'0201 alleles. Only two of the five lines which were not lysed had N:'0201 alleles; peptide variability of longterm culture could be responsible for their lack of recognition. These results demonstrate that MHC-restricted CTL can recognize antigenic determinants on RCC tumors. Identification of specific CTL and definition of the molecular epitopes they recognize could lead to new approaches for immunotherapy for metastatic RCC.
Medizinische Klinik I, Universitatskliniken des Saarlandes, Homburg/Saar, Germany
K.13 cDNA sequence comparison of the CD30 antigen expressed on activated T-cells and a Hodgkin derived cell line L540 S. KRUGER, W. JUNG, L. TROMPER, and M. PFREUNDSCHUH The Hodgkin's disease associated activation antigen CD30 is a member of the growing family of cell surface receptors showing homology with the nerve growth factor receptor. In the hemopoietic cell lineage, expression of the CD30 antigen is predominantly found
158 . 24th Meeting of the Society of Immunology on Hodgkin and Reed-Sternberg cells (H&RS-cells), cells of anaplastic large celllymphoma and activated or virus transformed lymphoid cells of either T or B origin. In order to investigate whether structural differences of the CD30 antigen between normal T-cells and neoplastic H&RS-cells are involved in the growth regulation of H&RS-cells we have compared the open reading frames (ORF) of the CD30 antigen expressed on normal activated T -cells and a Hodgkin derived cell line sharing features of an activated T-cell, L540. The ORFs were cloned by PCR and independent clones were sequenced. Comparison with the published cDNA sequence of the CD30 antigen from the HTLV-I transformed T-cell line HUT-102 revealed a silent mutation at position 771 of the ORF in both cell types (A --> G). Besides this no significant mutations could be detected. We conclude that the primary amino acid sequence of the CD30 antigen expressed on the Hodgkin derived cell L540 is identical to that expressed on activated T -cells.
Institute of Clinical Immunology, !Dept. of Anaesthesiology and Intensive Care, and 2Dept. of Pediatric Surgery, Leipzig University, Leipzig, Germany
K.14 Whole body hyperthermic treatment in children alters the lymphocyte population in peripheral blood 1. LEHMANN, H. SCHADLICH, U. SACK, G. METZNER, U. BURKHARDT!, and R. B. TROBS 2 Because tumor cells react more sensitively to the action of heat than normal cells a combined application of hyperthermia with cytostatic drugs could lead to better treatment results. Since radiation and chemotherapy are known to cause a long term depression of immune response we observed the immune system of patients during controlled hyperthermia. Peripheral blood MNC of children having advanced malignant diseases treated by whole body hyperthermia (41,8 °e/2 hours) in combination with cytostatic drugs (Vepesid, Dactinomycin, Ifosfamid) was analyzed. Blood samples were taken before hyperthermia, in the period of temperature increase, at the time of highest body temperature and after hyperthermia. Induction of hyperthermia alters the lymphocyte populations rapidly. Among T lymphocytes we found a shift toward the CD8+ cells. A high percentage of this CD8+ cells showed an expression of HLA-DR antigen. Additionally an increasing of the natural killer cell population was observed. Although IFN gamma is involved in generation of cytotoxic T lymphocytes, in activation of natural killer cells and induction of HLA antigens on the surface no important changes were found in serum IFN gamma levels. During hyperthermia the IL2 receptor expression on CD4+ cells was reduced but reconstituted after reaching normal body temperature. The soluble IL2 receptor increased during hyperthermic treatment. It seems that hyperthermia in combination with cytostatic therapy does not suppress the immune system but in contrary activate the cytotoxic cells which could potentiate the anticancer activity.
24th Meeting of the Society of Immunology . 159 Klinikum GroBhadern, University of Munich, Munich, Germany
K.1S Loss of HLA A locus products in primary gastric cancer C. LORENZ, 1. FUNKE, K. W. JAUCH, J. P. JOHNSON, and B. MAYER
Down-regulation of HLA class I and HLA class II antigens is postulated to be a mechanism used by tumor cells to escape immunological recognition. Although many human tumors have been investigated for changes in class I and II antigen expression, the reagents employed have generally been unable to distinguish between the products of different loci or of different alleles. In the present study, the expression of HLA ABC and HLA D region molecules on gastric carcinomas was investigated immunohistochemically using monoclonal antibodies directed to the individual locus products and to several individual allelic specificities. 48 primary gastric carcinomas and autologous normal mucosae were examined as were metastatic lesions derived from various locations. HLA class II expression by normal mucosae was correlated with the presence of infiltrating leukocytes and inflammation. While the majority of primary tumors expressed HLA-DR molecules, the expression of HLA-DP and DQ products was less common. The frequency of HLA class I antigen expression as estimated with the antibody W6132, did not significantly differ between the tumors and the normal tissue. However, examination of locus and allelic specific products revealed a loss of particular HLA A locus products in more than 60 % of the primary tumors. The expression of HLA molecules by the primary tumors was correlated with a variety of prognostic factors such as tumor diameter, differentiation state, grading, and lymphatic vessel invasion and with tumor recurrence and survival (mean follow up: 23 months).
GSF-Institut fiir Klinische Hamatologie, Miinchen, Germany
K.16 A 72 kD heat shock protein acts as a target structure for NK cells G. MULTHOFF, M. WI ESNET, C. BOTZLER, M. ESSLER, and R. D. ISSELS Earlier we showed by indirect immunofluorescence technique and selective cell surface radioiodination that cell surface expression of HSP72 can be induced by sublethal heat shock on several human malignant cells, i.e. Ewing's sarcoma (ES) cells, osteosarcoma cells (HOS58) and transformed HEK293 cells. Regarding these results we addressed the question if HSP72 that is selectively expressed on affected cells, might elicit an antitumor immune response. Cytotoxic effector cells were derived from autologous stimulation of different peripheral blood lymphocytes (PBL) with EBV-transformed B-LCL, which were preincubated with heat treated tumor cell lysates. By magnetic beads separation method we separated CDr NK-like effector cells from CD3+ T-lymphocytes. Using the NK-enriched effector cell population in a cell mediated lympholysis assay (CML), we demonstrated that heat treated ES and K562 cells (either untreated or heattreated) were lysed significantly better compared to untreated ES cells or allogeneic BLCL. This NK-mediated lysis of heat stressed ES cells could be blocked using HSP72 moAb, whereas blocking with MHC class I specific antibody (W6/32) had no influence on the lysis pattern. Furthermore, lysis of untreated K562 cells was slightly reduced by HSP72 moAb. Cold target inhibition CML experiments using either heat treated ES or
160 . 24th Meeting of the Society of Immunology K562 cells as competitors clearly demonstrate that heat treated ES cells and K562 cells share one recognition structure for NK cells. Regarding our results of antibody blocking studies we conclude that HSP72 possibly is this common recognition structure for NK cells with anti-tumor activity.
Institute for Immunology, University of Munich, Munich; Medical Clinic II, Zentralklinikum, Augsburg, Germany; Sandoz Research Institute, Vienna, Austria
K.17 Decrease of individual metastatic tumor cells in bone marrow of breast cancer patients treated with monoclonal antibody K. PANTEL, G. SCHLIMOK, H. LOIBNER, and G. RIETHMULLER Limited accessibility and antigenic heterogeneity of cells incorporated in epithelial parenchyma hamper severely the efficacy of monoclonal antibody therapy in patients with advanced solid tumors. In contrast, individual epithelial tumor cells dispersed in mesenchymal compartments during the early phase of metastasis appear more appropriate targets for i.v. injected antibodies. Since such cells can now be detected immunocytochemically by cytokeratin (CK) antibodies in bone marrow, the question arose whether cytotoxic activity of murine monoclonal IgG3 antibody ABL364 can be directly demonstrated by monitoring cytokeratin-positive tumor cells in bone marrow. The used antibody is directed against the Lewis Y carbohydrate antigen, widely expressed on human epithelial tumors; it exhibits remarkable cytotoxicity against human tumor cells in vitro via activation of human complement and effector cells. Ten patients with breast cancer presenting with high numbers of epithelial cells in bone marrow (> 20/4 x 10 5 nucleated marrow cells) were treated in a randomized fashion either with 6 x 100 mg of the monoclonal Lewis Y antibody during two weeks or with human serum albumin (HSA) as a placebo. Epithelial cytokeratin-positive tumor cells in marrow were monitored on day 15 and 60 after commencement of treatment. Of the seven evaluable patients treated with antibody, five showed a distinct reduction or eradication of CK-positivel Lewis Y-positive cells of at least one log kill (96-100 %) while two patients, presenting only with CK-positive/Lewis Y-negative cells, showed no response. Similarly, in the three patients receiving HSA no significant tumor cell reduction was observed. Thus, the presented data may open a new approach towards a more rational selection of antibodies for adjuvant studies in minimal residual disease. Because of the marked cytotoxicity the antibody ABL364 exhibits in ex vivo experiments with serum of treated patients we postulate that the observed disappearance of tumor cells from bone marrow can be reduced to the action of the injected antibody.
24th Meeting of the Society of Immunology . 161 Beilinson Medical Center, Tel Aviv University Petah Tiqva, Israel; Max Planck Institute of Experimental Medicine, Gottingen, Germany
K.la Primary structure/amyloidogenicity relationship of AL-Bence Jones proteins POL, EZI and DIA A. I. PICK, H. KRATZIN, H. KLAfKI, and N. HILSCHMANN We have shown that the soluble urinary Bence Jones protein DIA (B.].) is a dimer of monoclonal L-chains with a primary structure identical to that of the myloid L-chain DIA and thus represents the amyloid precursor protein. The primary structure of Al protein POL is typical for the lambda 1.1 subclass. It has fifteen AA exchanges and shows a homology of 93 % with protein VOR and an increase in hydrophilic rather than hydrophobic groups. It shows a lack of homology with amyloid associated lambda-I and VI subclass B.]. (average 56.07 %) and a marked degree of homology with non-amyloid lambda-I subclass B.J. VOR (93 %) and KOL (77.78 %). The N-terminal H. is similar to the V -lambda-II B.]. protein VIL. In B.J. POL, the replacement of T by A in position 20 is unique. AL B.J. EZI is V lambda 1.2, it has seven AA replacements and a slight increase in hydrophilic groups. A comparison with 2t AL proteins shows an average homology of 54.23 %, the highest being with AL proteins EPS and HAR, 83.33 and 75 % respectively. The location of A in position 68, and of G in position 89 in the beta pleated sheet, remains unknown. A in position 68 and 97 is unique, the replacement of L by P in position 39 also occurred in AL protein POL. AL B.J. proteins could be the result of a subtle sequence alteration similar to that described in Alzheimer's disease.
'Institute of Clinical Immunology, Faculty of Medicine, and 2Dept. of Analysis, Faculty of Chemistry, University Leipzig, Leipzig, Germany; 3Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
K.19 Genotoxicity assessment of waste products with the SOS chromotest and the salmonella/microsome test F. RAABE', S. ]ANZ1,3, and R. HERZSCHUH 2 We evaluated 36 characteristic waste products from the plasma etching of aluminum and 7 waste gas condensates of municipal incinerators. The majority of the samples showed genotoxic activity in tester strain Escherichia coli PQ37 without metabolic activation using S9 mix. In the presence of S9, a deactivation of the samples of plasma etching and also of the municipal incinerators was regularly observed. Comparable studies with the Salmonella/microsome (Ames) test using tester strains Salmonella typhimurium TA98 and TAtOO indicated actual mutagenicity of the waste products. Gas chromatograms of the organic constituents of the samples from the plasma etching were performed in parallel with the genotoxic assays. In contrast to the similarity of the peak patterns of all chromatograms, the biological effects of the individual waste products showed large differences. The results of a representative sample from the plasma etching and also the influences of sample preparation are shown. The results of prokaryotic test systems alone are not sufficient to predict the potential danger to exposed people, they suggest consideration of those waste products as harmful and carcinogenic to man, and warrant further studies.
162 . 24th Meeting of the Society of Immunology Institute of Medical Immunology and IDept. of Urology, MLU, Halle, Germany
K.20 Aminopeptidase N/C013 on tumor-infiltrating T lymphocytes D. RIEMANN, B. GOHRINGl, A. KEHLEN, and]. LANGNER Aminopeptidase N/CD13 is a new activation-associated molecule on human T cells. CD13 cannot be demonstrated on PB lymphocytes - but on cells of inflammatory body effusions (1, 2). Functionally the enzyme could be involved in the metabolism of biologically active pep tides (3) or in antigen processing (4). We studied the immunophenotype of tumor-infiltrating T lymphocytes derived from human renal carcinoma and lung cancer. Fresh tumor specimens of 20 patients were minced and enzymatically dissociated. After separation on Ficoll-Hypaque, the immunophenotype of mononuclear cells was determined by two-color flow cytometry. CD3+ T cells expressed high amounts of CD45RO (94±3%), CD69 (84± 10'/"0), HLA-DR (53 ± 15 %) and CD25 (21 ± 8 % of CD3 + cells). Whereas in most cases only a small part of T cells expressed CD13 (13 ± 6 %), 3 tumor specimens contained 55-80 % CD13+ T cells. After cultivation of T cells of 1 patient in RPMI + 10 % FCS and 1000 U/ml IL2 for 30 days, only 28 % of CD3+ T cells expressed CD13 yet (initially 80 %). The ratio CD4:CD8 was 2.2 before and after cultivation. Using Ala-pNA as substrate, aminopeptidase activity of this pure T cell population could be estimated to be 18.2 pkat/l0 6 cells. Actinonine (10-4M) inhibited enzymatic activity by 80 %. By use of intron spanning PCR, cultivated T cells were found to produce mRNA for CD13 as well as ILIa, IL4, ILIO and IFNy. A possible function of CD13 on tumor-infiltrating T cells will be discussed. 1. 2. 3. 4.
RIEMANN, D. et al.: Immunobiol. 187,24-35 (1993). RIEMANN, D. et al.: Clin. Rheumatol. 12,31 (1993). MATSAS, R. et al.: FEBS Lett. 175, 124 (1984). MOURITSEN, S. et al.:]. Immunol. 149, 1987-1993 (1992).
This work was supported by the BMFT -Project Oncology 01 ZZ 9105, part XIII.
Innere Medizin I, Universitatsklinik Homburg, Homburg/Saar, Germany
K.2l Single Hodgkin and Reed-Sternberg cells lack rearrangements of the immunoglobulin and T cell receptor genes ]. ROTH, H. DAUS, L. TRUMPER, A. GAUSE, and M. PFREUNDSCHUH We tested the hypothesis that Hodgkin- and Reed-Sternberg cells (H&RS cells) have a lymphatic origin by analyzing the rearrangement status of single H&RS cells isolated from biopsy tissue by micromanipulation. In order to detect rearrangements of the immunoglobulin heavy chain (IgH) and T-cell receptor-y (TCR) genes, primers were constructed corresponding to the variable and joining regions of the IgH and TCR-y genes. These primers were shown to amplify a PCR-product of ca. 350 bp in length from PBLs, various cell lines and patients with non-Hogkin's lymphomas at a single cell level. The specificity of the PCR products was proven by Southern blotting and sequencing. Single H&RS cells isolated from biopsy material of nine patients were tested for IgH and TCR gene rearrangements. Rearrangements were not detected in the H&RS cells. As a
24th Meeting of the Society of Immunology . 163 positive control, sequences of the B-actin gene were amplified from single H&RS cells of all patients in a parallel reaction. From these results we conclude, that H&RS cells do not seem to be related to lymphoid cells.
Med. Universitatsklinik und Poliklinik, Abt. II, Tubingen, Germany
K.22 Differential regulation of HLA-class I genes by interferon H. SCHMIDT, I. STEIERT, E. KELLERMANN- KEGREISS, J. W ALZ, R. ZINSER, and C. A.MuLLER Expression of HLA class I antigens can be modulated in vitro and in vivo by interferon (IFN) a but also by IFN y. A strong induction of HLA-class I antigens was found on both hematopoietic progenitors and normal peripheral blood mononuclear cells after one month of IFN treatment in 18 patients with myeloproliferative syndrome. By daily injections of IFN in the first month of therapy stimulation was continuously increasing suggesting a major effect of IFN a on hematopoietic progenitors with sustained enhanced expression of HLA-class I antigens during differentiation of myelomonocytic cells. Differential in vivo regulation of HLA-class I antigens by IFN was demonstrated by comparison of HLA-A2 with HLA-B antigen expression. In vitro expression of the HLA-B7 and -Bw64 genes was significantly more inducible by IFN than the genes coding for the HLA-B27, HLA-B51, HLA-B38, HLA-B39, HLA-Cw3 and HLA-A2 antigens after transfection into mouse L cells. Modification of the 5' ends of the HLA-B7 and HLA-B27 genes before transfection in mouse L cells revealed the presence of enhancer sequences responding to interferon treatment in the 5' untranslated region of the HLA-B7, but not of the HLA-B27 gene and suggested further independently acting enhancer elements down-stream of the transcription initiation site. When different fragments of HLA B7 or B38, including introns, 3' and 5' untranslated regions, were cloned in front of CAT genes IFN responding enhancers were only detected at the 5' end of the HLA-B7 gene. Further IFN independent enhancers could be detected within introns and at the 3' end of HLA class I genes. These findings may indicate specific regulatory mechanisms of HLA class I antigen expression possibly influencing T-cell recognition in immune response.
I. Medizinische Klinik und Poliklinik, Johannes-Gutenberg-U niversitat, Mainz; 1Max-Planck -Institut fur Biologie, Abtl. Immungenetik, Tubingen, Germany
K.23 Recognition of human melanoma cells by autologous cytotoxic T lymphocytes (eTL): detection of target peptide antigens after elution from HLA-A2.1
J. SCHNEIDER, K. FALKt, O. ROTZSCHKEt, T. WOLFEL, K.-H. MEYERZUMBOsCHENfELDE, and H.-G. RAMMENSEE 1
In the melanoma model A V a panel of tumor-reactive CTL clones were derived from autologous peripheral blood lymphocytes. By immunoselection it was found that they were directed against four different antigens (A a, Ab, B, C) and were all restricted by
164 . 24th Meeting of the Society of Immunology HLA-A2.1. HLA-A2.1 molecules were purified by affinity chromatography from batches of each 5 x 109 AV melanoma cells. Pep tides bound to HLA-A2.1 were eluted with trifluoroacetic acid and were further separated by ultrafiltration and high-performance liquid chromatography (HPLC). Various targets were preincubated with these HPLC fractions and tested for CTL recognition in s'Cr-release assays. When antigennegative AV tumor cell variants, A2-positive EBV-transformed cells or A2-transfected P815 cells were used as target cells, none of the fractions caused lysis. Using the A2positive transport deletion mutant T2 as a target, we detected positive peptide fractions with CTL anti-Aa, but not with CTL against other AV antigens. However, after pretreatment of T2 cells with the anti-HLA-A2 antibody MA2.1 known to enhance peptide association with HLA-A2.1 (BODMER et aI., Nature 342: 443, 1989), we detected naturally processed peptide antigens Aa, Ab, Band C in different fractions of the HLAA2.1 peptide pool. The results were reproducible with different HPLC preparations. Improving the sensitivity of peptide detection assays, as described herein, is a prerequisite for biochemical characterization of CTL-defined peptide antigens which so far has been impossible for non-viral endogeneous peptides.
GSF-Institut fur Klinische Hamatologie, Munchen, Germany
K.24 Modulation of cell surface markers during combined hyperthermia/chemotherapy M. WIESNET, G. MULTHOFF, C. BOTZLER, M. ESSLER, and R. D. ISSELS Patients suffering from local advanced sarcomas were treated in a clinical phase-II study with systemic chemotherapy combined with regional hyperthermia in repeated cycles. The parameters of this study were defined in a RHT-91 protocol (R. D. ISSELs). To examine the effects of this therapy in regard to the immune system, we analyzed cell surface markers of freshly isolated peripheral blood lymphocytes (PBL) of eight sarcoma patients (cycle 1 to 6) within one therapeutic cycle. Additionally, these PBL were stimulated with 100 U of human recombinant IL2 for 7 days and tested again using the following mAb in a FACScan-analysis: CD3, CD4, CD8, CDI6/CD56. After one treatment cycle a significant reduction of all tested cell surface markers in five out of eight PBL of patients was observed. This effect did not show any correlation with the number of treatment cycle. PBL of three patients did not show changes in the tested cell surface markers. Interestingly, reconstitution of the immune status to normal levels was achieved by incubation of PBL of patients with IL2 in vitro. The function of this IL2 expanded PBL of four patients was defined in a cell mediated lympholysis assay (CML) using either untreated (37°C) or heat treated (41.8 0c) Ewing's sarcoma cells (ES) as target cells. Our results show that IL2 expanded PBL derived after treatment show better lysis of heat treated ES cells compared to untreated ES cells. There was no difference in the lysis pattern of either untreated or heat treated ES cells when PBL were derived before treatment. These results lead us to the hypothesis that regional hyperthermia in combination with chemotherapy in vivo can induce a selection of cytotoxic effector cells with specificity for heat treated sarcoma cells.
24th Meeting of the Society of Immunology . 165 !Applied Immunology, D KFZ, INF 280, Heidelberg; 2Pharmaceutical Research, E. Merck, Darmstadt, Germany
K.2S T cell targeting by anti-C02 X anti-EGF-R bispecific antibodies M. WILD!, B. SCHRAVEN!, H. KIRCHGESSNER!, D. C. ELKINS!, W. STRITTMATTER2 , S. MATZKU 1 , and S. C. MEUER 1 Bispecific antibodies (BsAbs) can be employed to redirect effector cells to tumour cells independent of specific target recognition by the TCR. In addition, antibodies can provide triggering signals for effector cell activation. Among the lymphocyte receptors considered for T cell targeting strategies CD2 appears to be favorable: 1) CD2 is strongly expressed by both T cells and natural killer cells. 2) Both effector cell populations can be triggered to express their functional programs following stimulation via CD2. 3) Efficient lymphocyte triggering through CD2 requires the simultaneous binding of two different CD2 mAbs. Therefore, a two step activation protocol can be established using one CD2 mAb as a CD2 x anti-tumor BsAb for targeting and the second one for activation of targeted cells thus likely avoiding unwanted systemic side effects. We have produced two novel anti-CD2 mAbs, AICD2.M1 (M1) and AICD2.M2 (M2), which in combination are mitogenic for human T cells. These mAbs do not interfere with CDS8/LFA-3 binding to CD2 nor do they individually inhibit activation of T cells via the TCR unlike many other CD2 mAbs. We used a chemical conjugation method to couple anti-EGFreceptor Fab' to M1- or M2-Fab' fragments. The combinations of M1 x anti-EGF-R BsAb plus M2 F(ab'h or alternatively M2 x anti-EGF-R BsAb plus M1 F(ab')2 were mitogenic for PBMC. Lysis of EGF-R(+) A 431 cells by PBMC was induced when effector cells were preincubated with the above mentioned Ab-combinations. As expected cytotoxicity was partly mediated by NK activity. Targeting with the BsAbs was proven in experiments with a CD8(+) cytotoxic human T cell clone which lysed A 431 but not EGF-R(-) cells in the presence of BsAb. Moreover, BsAb-induced lysis of A 431 cells by the T cell clone was blocked by an excess of EGF-R mAb. Our in vitro studies demonstrate that CD2 x anti-tumour antigen BsAbs in combination with a second CD2 mAb are able to mediate activation and tumor antigen specific targeting of effector cells and subsequent specific lysis of tumor cells. In vivo studies in hCD2 transgenic mice are In progress.