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Effect of Anapsos (Polypodium leucotomos extract) on cellular phenotype
T cells in cancer
25 June 1997 - Poster presentations
We wanted to investigate, if the MUCZ molecule comprises HIA-A2-binding epitopes, which are recognized by CTL from the human T-cell repertoire. Matarfal and Methoda: The MUCP-sequence was screened for HlAA2 binding peptldes and ten peptldes wlth predicted high binding affinity were synthesized. The peptides were tested in FACS for binding afffnity and stability to H&A2 with T2-cells, which am A2+. Peptfde-loaded dendritfc cells were used as stimulators for autolcgous T-cells from healthy, A2+ donors. After three rounds of stlmulatfons the T-cell responders were tested for their specificity in a chromium-release cytotoxicity assay. Renuitez Si oeotides were identified which bound to HLA-A2 with an afftnitv comparable to the influenza matnx peptide GILGVFTL. but only four of them showed a stable bindina. which could still be detected after more than 5 h. All six peptides were investigated for their ability to induce cytotoxic T-cells. Two peptides designated 6024 and 6039, which belonged to the group of stable binders, were found to induce specific CTL. These CTL lysed T2 target cells strongly, when T2 were loaded with the MUCP peptides 6024 or 6039, but not when they were loaded with an irrelevant control peptide. The four other MUCP-peptldes did not induce any specific tysis. The lysis of targets loaded with 6039 or 6024 was dependent on the peptide concentration, owning at a half-maximal concentration of 1 &ml. At this time point, the T-cell lines consisted of about 60% CD6* cells and 5% CD4+ cells. Conoluslon: Several peptides detfved from the mucin MUCP were identified as high affinity and stable binders to HLA-W. A2-restricted CTL were established, which recognized MUCPderfved peptides 6024 and 6039. This shows, that the self-antiaen MUCP reoresents a new ootential target antigen for immunotherapy foriatients with mutinous carcinomas. Since-it is not-detectable in normal tissue in breast, ovar and pancreas, but strongly overexpressed in the mutinous tumor cells, it may provide a basis for a tumor&ecific T-cell response in such patients. (Supported by Deutsche Krebshilfs HalllO-1025)
P.5.10.36
Proliferative response and cytokine profile of sinusoidal lymphocytes isolated from humans with liver tumors
B. Lukomska’, M.Winnockz, C. Balabaud2, J. Sarfc3, J. Polanski 4, W.L. Olszewski ’. ’Surg Res Tmnspl Dep. A.&d Res Ctr, pal Acad Sci, Warsaw, Poland, 2GREF; Univetsite de Bordeaux 11,Bordeaux, France, 3Services de Chirurgie Digestive, Hopitat Saint Andre, Bordeaux, France, 4Surg Dep. Med Fat, Med School, Warsaw,Poland
Introduction:Liver sinusoidal lymphocytes (LSL) constitute a distinct phenotypically lymphocyte population, whose function has not been clearfy defined. The unique position of these cells in intimate contact with endothelial cells and in close proximity to environmental antigens suggests that LSL play an important role in immune surveillance. Tumor burden may change the immune cells reactivity. Previously we have shown that LSL adjacent to the tumor expressed lower cytolysis of Raji cells than LSL isdated from the normal tissue. In thle studythe proliferation ability of LSL and cytokfne release was determined in patients undergoing partial hepatectomy for benign and malignant liver tumors. MaterIrIsand Hods: Peripheral blood was collected from 30 patients before operation. In order to obtain LSL-1 population normal liver fragments excised from resected lobes were perfused with Ca”Mg++-free Dulbecco PBS. For LSL-2 collection - perfusion of liver tissue adjacent to the tumor was performed. Proliferating potential of peripheral blood lymphocytes (PBL), LSL-1 and LSL-2 was measured by 3HThy incorporation after phytohemagglutinin (PHA) and wncanavafin (Con A) stimulation. The concentration of cytokines secreted by lymphocytes cultured in the presence of K-562 and IL-2 was determined by enzvrne assav (Quantikine, R&D Svstem). Reaultr: Culture of PBL, LSL-i and LSL-2 with mftogens revealed evident differences. The responsiveness to PHA (16 mg) was 17761 cpm for PBL; 5532 cpm for LSL-1; 11624 cpm for LSL-2 (PBL vs l.AL-1, p CL0.01 j LAL-2 vs LAL-1, p < 0.05). Lymphopmlfferatlve response to Con A (2 mg) was 3969 cpm for PBL; 2916 cpm for LSL-1; 4296 cpm for LSL-2 (LSL-2 vs LSL-1, p < 0.05). Following stlmulatfon by K-562 and IL-2 there was little or no production of IL-l& IFN-G, GM-CSF, TNF-a, TNF-& 11-4,IL-IO, IL-13 and TGF-@l detected in anv of cell culture suoematants. There wsre no diierences between LSL-1 and LSL-2 in the levels of cytokines contributing to T cell activation (156 vs 636 w/ml of IL-ID; 1666 vs 3966 pg/ml of IFN-y; 2197~s 1666 pslml of GM-CSF; 146 vs 163 pslml of TNFar; 211 vs 210 pg/ml of TNF-fl for LSL-1 and LSL-2, respectively) or in the levels of immunosuppressive cytokines (76 vs 75 pglml of IL-4; 34 vs 36 pa/ml of IL-IO; 765 vs 744 @ml of 11-13;1162 vs 1202 &ml of TGF-@l for Lsl_Il and LSL-2, respectively): Concfuelonr: LSL lsolated from tissue adjacent to the tumor revealed higher responsiveness to mitogens than LSL from remote healthy areas however there was no correlation between magnitude of the proliferative response and the amounts of secreted cytokines by LSL-1 and LSM. The level of produced cytoklnes was similar in both cell populations. This indicates that the differences
457
in reactivity of LSL-1 and LSL-2 are not due the autocrtne secretion of cytokines but rather to local factors produced by tumor cells.
P.5.10.37
Effect of Anapsos (Wypodium leucotomos extract) on cellular phenotype
J.M. Sempere I.*, C. Rodrigo2, A. Campos3,*. ’ASACPttarmaceutical International, Spain, 2Division of Immuno/om, University of Alicante, Spain, 3Bbod Bank Centre of Alicante, Spain tntroductfon: Polypodium leucotomos is a fern typical of certain areas of Central America, and its rhizomes are used to treat skin diseases such as psoriasis and atopfc dermatitis. The extract (Anapsos), after being processed into capsules, is given to patients orally. Early studies of this product in vitro, evidenced an antitumoral effect. Further studies also showed an immunomodulating effect in several diseases. Matedat and Metbode: Anapsos was filtered, freeze-dried and granted by ASAC for the present research work. After culturing mononuclear cells from healthy donors with LPS andlor PHA and/or Anapsos for 24 and 46 hours, a phenotypical analysis of different lymphocyte subsets was performed by flow cytometry, according the most appropriate combination of monoclonal antibodies in each case (direct immunofluorescence). Results: Addition of Anapsos to LPS + PHA caused a significant increase in the expression of CD4 (p = 0.017) and CD6 (specially CDS,& antigen (p -Z O.Ol), and a trend towards an increase in the expression of CD3 and CD5. Addtion of Anapsos into the culture, always yielded a significant increase in the percentage of CD4+CD25+, CD4+CD45RO+, CD4+CD36+, CD6+CD25 and CDl6+ cells, vs. cells without Anapsos. Conclusion: Anapsos seems to be able of stimulating proliferation and activation of T and NK lymphocytes, which would indicate a product’s main effect on Cellular Immunity.
P.5.10.38
Evidence for a new T-cell-defined antigen presented by HLA-Al in renal cell carcinoma
N. Brouwenstijn ‘, C. Hoogstraten ‘, C.W. Van der Spek’ , A. Mulder*, J.G.M. Deckers3, P.I. Schrfer ‘. ‘Dept.Clinical oncoloav. Universitv Hospital Lekien, PO Baw9600,230o RC Leiben, The Netherfar& 2Dept. _ lmmunohematology and &cd Bank, University Hospital Leiden, PO Box 9600,230O RC Leiden, The Netherlands, 3Dept. Nephrolog)! Universiry Hospital Leiden, PO Box 9&W, 23W RC Leiden, The Nethedands Introduction: Definition of antigens recognized by tumor-specific CTL is important for understanding the nature of proteins that can serve as T-cell targets and for development of vaccines for anti-cancer immunotherapy. In case of melanoma, several T-cell-defined antigens have been characterized. For renal cell carcinoma (RCC), two Tcelldefined antigens have been identitled at the molecular level thus far: the tumor-specific RAGE-l antigen and a uniquely expressed mutated H&A2 antigen. Employment of these epftopes for immunotherapv, however, does not seem very useful due to the limited distribution of these an&ens in RCC. Therefore other-antigens have to be identified. MaterIdsand Methods:An MLTC using autologous PBL and RCC was performed in the presence of IL2 and 114.Responderlymphocytes were tested for their tumodcidal activity at days 14 and 21 and were cloned by LD. Several dones were tested for MHC class I-restrfction using specific monoclonal antibodies. Reactivity towards both allogeneic RCC cell lines and melanoma cell lines was established. Also primary cultures of proximal tubulus epitfrelium, which are thought to be the cells that RCC originate from, were tested for recognition. Results: Nine CD6+ CTL clones showed MHC class I-restricted lysis of the autologous tumor cells, but not of the EBV-transformed B-cells. Using a panel of MHC class l-typed allogeneic RCC cell lines revealed that 6/9 clones were most likely HLA-Al-restricted. Using a moAb specific for HLA-Al and -A9, lysis by 4/6 different CTL clones could be blocked. CTL 6/55 was studied in more detail. It also recognized 3/4 HLA-Al-positive melanoma cell lines in a TNF assay. Interestingly, HlA-Al-positive-PTEC were efficiently lysed by this CTL clone. Conclusion: We have isolated HlA-Al-restricted CTL from the PBL of a patient with RCC. Interestingly, these clones seem to recognize a widely distributed antigen on both RCC and melanoma cells, which makes it a potential candidate for immunotherapy. Cultures of normal epithelial cells of the kidney, however, were also recognized, stressing the importance of thorough investigation of the nature of this antigen.
25 June 1997 - Poster presentations
We wanted to investigate, if the MUCZ molecule comprises HIA-A2-binding epitopes, which are recognized by CTL from the human T-cell repertoire. Matarfal and Methoda: The MUCP-sequence was screened for HlAA2 binding peptldes and ten peptldes wlth predicted high binding affinity were synthesized. The peptides were tested in FACS for binding afffnity and stability to H&A2 with T2-cells, which am A2+. Peptfde-loaded dendritfc cells were used as stimulators for autolcgous T-cells from healthy, A2+ donors. After three rounds of stlmulatfons the T-cell responders were tested for their specificity in a chromium-release cytotoxicity assay. Renuitez Si oeotides were identified which bound to HLA-A2 with an afftnitv comparable to the influenza matnx peptide GILGVFTL. but only four of them showed a stable bindina. which could still be detected after more than 5 h. All six peptides were investigated for their ability to induce cytotoxic T-cells. Two peptides designated 6024 and 6039, which belonged to the group of stable binders, were found to induce specific CTL. These CTL lysed T2 target cells strongly, when T2 were loaded with the MUCP peptides 6024 or 6039, but not when they were loaded with an irrelevant control peptide. The four other MUCP-peptldes did not induce any specific tysis. The lysis of targets loaded with 6039 or 6024 was dependent on the peptide concentration, owning at a half-maximal concentration of 1 &ml. At this time point, the T-cell lines consisted of about 60% CD6* cells and 5% CD4+ cells. Conoluslon: Several peptides detfved from the mucin MUCP were identified as high affinity and stable binders to HLA-W. A2-restricted CTL were established, which recognized MUCPderfved peptides 6024 and 6039. This shows, that the self-antiaen MUCP reoresents a new ootential target antigen for immunotherapy foriatients with mutinous carcinomas. Since-it is not-detectable in normal tissue in breast, ovar and pancreas, but strongly overexpressed in the mutinous tumor cells, it may provide a basis for a tumor&ecific T-cell response in such patients. (Supported by Deutsche Krebshilfs HalllO-1025)
P.5.10.36
Proliferative response and cytokine profile of sinusoidal lymphocytes isolated from humans with liver tumors
B. Lukomska’, M.Winnockz, C. Balabaud2, J. Sarfc3, J. Polanski 4, W.L. Olszewski ’. ’Surg Res Tmnspl Dep. A.&d Res Ctr, pal Acad Sci, Warsaw, Poland, 2GREF; Univetsite de Bordeaux 11,Bordeaux, France, 3Services de Chirurgie Digestive, Hopitat Saint Andre, Bordeaux, France, 4Surg Dep. Med Fat, Med School, Warsaw,Poland
Introduction:Liver sinusoidal lymphocytes (LSL) constitute a distinct phenotypically lymphocyte population, whose function has not been clearfy defined. The unique position of these cells in intimate contact with endothelial cells and in close proximity to environmental antigens suggests that LSL play an important role in immune surveillance. Tumor burden may change the immune cells reactivity. Previously we have shown that LSL adjacent to the tumor expressed lower cytolysis of Raji cells than LSL isdated from the normal tissue. In thle studythe proliferation ability of LSL and cytokfne release was determined in patients undergoing partial hepatectomy for benign and malignant liver tumors. MaterIrIsand Hods: Peripheral blood was collected from 30 patients before operation. In order to obtain LSL-1 population normal liver fragments excised from resected lobes were perfused with Ca”Mg++-free Dulbecco PBS. For LSL-2 collection - perfusion of liver tissue adjacent to the tumor was performed. Proliferating potential of peripheral blood lymphocytes (PBL), LSL-1 and LSL-2 was measured by 3HThy incorporation after phytohemagglutinin (PHA) and wncanavafin (Con A) stimulation. The concentration of cytokines secreted by lymphocytes cultured in the presence of K-562 and IL-2 was determined by enzvrne assav (Quantikine, R&D Svstem). Reaultr: Culture of PBL, LSL-i and LSL-2 with mftogens revealed evident differences. The responsiveness to PHA (16 mg) was 17761 cpm for PBL; 5532 cpm for LSL-1; 11624 cpm for LSL-2 (PBL vs l.AL-1, p CL0.01 j LAL-2 vs LAL-1, p < 0.05). Lymphopmlfferatlve response to Con A (2 mg) was 3969 cpm for PBL; 2916 cpm for LSL-1; 4296 cpm for LSL-2 (LSL-2 vs LSL-1, p < 0.05). Following stlmulatfon by K-562 and IL-2 there was little or no production of IL-l& IFN-G, GM-CSF, TNF-a, TNF-& 11-4,IL-IO, IL-13 and TGF-@l detected in anv of cell culture suoematants. There wsre no diierences between LSL-1 and LSL-2 in the levels of cytokines contributing to T cell activation (156 vs 636 w/ml of IL-ID; 1666 vs 3966 pg/ml of IFN-y; 2197~s 1666 pslml of GM-CSF; 146 vs 163 pslml of TNFar; 211 vs 210 pg/ml of TNF-fl for LSL-1 and LSL-2, respectively) or in the levels of immunosuppressive cytokines (76 vs 75 pglml of IL-4; 34 vs 36 pa/ml of IL-IO; 765 vs 744 @ml of 11-13;1162 vs 1202 &ml of TGF-@l for Lsl_Il and LSL-2, respectively): Concfuelonr: LSL lsolated from tissue adjacent to the tumor revealed higher responsiveness to mitogens than LSL from remote healthy areas however there was no correlation between magnitude of the proliferative response and the amounts of secreted cytokines by LSL-1 and LSM. The level of produced cytoklnes was similar in both cell populations. This indicates that the differences
457
in reactivity of LSL-1 and LSL-2 are not due the autocrtne secretion of cytokines but rather to local factors produced by tumor cells.
P.5.10.37
Effect of Anapsos (Wypodium leucotomos extract) on cellular phenotype
J.M. Sempere I.*, C. Rodrigo2, A. Campos3,*. ’ASACPttarmaceutical International, Spain, 2Division of Immuno/om, University of Alicante, Spain, 3Bbod Bank Centre of Alicante, Spain tntroductfon: Polypodium leucotomos is a fern typical of certain areas of Central America, and its rhizomes are used to treat skin diseases such as psoriasis and atopfc dermatitis. The extract (Anapsos), after being processed into capsules, is given to patients orally. Early studies of this product in vitro, evidenced an antitumoral effect. Further studies also showed an immunomodulating effect in several diseases. Matedat and Metbode: Anapsos was filtered, freeze-dried and granted by ASAC for the present research work. After culturing mononuclear cells from healthy donors with LPS andlor PHA and/or Anapsos for 24 and 46 hours, a phenotypical analysis of different lymphocyte subsets was performed by flow cytometry, according the most appropriate combination of monoclonal antibodies in each case (direct immunofluorescence). Results: Addition of Anapsos to LPS + PHA caused a significant increase in the expression of CD4 (p = 0.017) and CD6 (specially CDS,& antigen (p -Z O.Ol), and a trend towards an increase in the expression of CD3 and CD5. Addtion of Anapsos into the culture, always yielded a significant increase in the percentage of CD4+CD25+, CD4+CD45RO+, CD4+CD36+, CD6+CD25 and CDl6+ cells, vs. cells without Anapsos. Conclusion: Anapsos seems to be able of stimulating proliferation and activation of T and NK lymphocytes, which would indicate a product’s main effect on Cellular Immunity.
P.5.10.38
Evidence for a new T-cell-defined antigen presented by HLA-Al in renal cell carcinoma
N. Brouwenstijn ‘, C. Hoogstraten ‘, C.W. Van der Spek’ , A. Mulder*, J.G.M. Deckers3, P.I. Schrfer ‘. ‘Dept.Clinical oncoloav. Universitv Hospital Lekien, PO Baw9600,230o RC Leiben, The Netherfar& 2Dept. _ lmmunohematology and &cd Bank, University Hospital Leiden, PO Box 9600,230O RC Leiden, The Netherlands, 3Dept. Nephrolog)! Universiry Hospital Leiden, PO Box 9&W, 23W RC Leiden, The Nethedands Introduction: Definition of antigens recognized by tumor-specific CTL is important for understanding the nature of proteins that can serve as T-cell targets and for development of vaccines for anti-cancer immunotherapy. In case of melanoma, several T-cell-defined antigens have been characterized. For renal cell carcinoma (RCC), two Tcelldefined antigens have been identitled at the molecular level thus far: the tumor-specific RAGE-l antigen and a uniquely expressed mutated H&A2 antigen. Employment of these epftopes for immunotherapv, however, does not seem very useful due to the limited distribution of these an&ens in RCC. Therefore other-antigens have to be identified. MaterIdsand Methods:An MLTC using autologous PBL and RCC was performed in the presence of IL2 and 114.Responderlymphocytes were tested for their tumodcidal activity at days 14 and 21 and were cloned by LD. Several dones were tested for MHC class I-restrfction using specific monoclonal antibodies. Reactivity towards both allogeneic RCC cell lines and melanoma cell lines was established. Also primary cultures of proximal tubulus epitfrelium, which are thought to be the cells that RCC originate from, were tested for recognition. Results: Nine CD6+ CTL clones showed MHC class I-restricted lysis of the autologous tumor cells, but not of the EBV-transformed B-cells. Using a panel of MHC class l-typed allogeneic RCC cell lines revealed that 6/9 clones were most likely HLA-Al-restricted. Using a moAb specific for HLA-Al and -A9, lysis by 4/6 different CTL clones could be blocked. CTL 6/55 was studied in more detail. It also recognized 3/4 HLA-Al-positive melanoma cell lines in a TNF assay. Interestingly, HlA-Al-positive-PTEC were efficiently lysed by this CTL clone. Conclusion: We have isolated HlA-Al-restricted CTL from the PBL of a patient with RCC. Interestingly, these clones seem to recognize a widely distributed antigen on both RCC and melanoma cells, which makes it a potential candidate for immunotherapy. Cultures of normal epithelial cells of the kidney, however, were also recognized, stressing the importance of thorough investigation of the nature of this antigen.