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Melanoma cells express Fas ligand: Implications for tumor immune escape
343
WEDNESDAY, 25 JUNE 1997
Oral Presentations
lO:oo-12:oo
0.1.08
10.1.08.1
Room B
Regulation of apoptosis 1 Apoptosis and T cell renewal during antlretroviral therapy in HIV-l-Infected children
Th. Bbhler‘, J. Walcher ‘, M.Geiss *, P. Schnitzler’, I. Herra, K.-M. Debatin 1.3.’ Hematology/Oncolo~, Children’s Hospital, University of Heidelberg, Heidelberg, Germany, 2institute of Medical virology; University of Heidelberg,Heidelberg, German)! 3Departmenr of Molecular Oncology German Cancer Research Center, Heidelberg. Germany
Introduction:Increased activation-induced programmed cell death (apoptosis, AICD) of T ceils is thought to be involved in progressive T cell depletion in HIV-l infected individuals. The CD95 (APO-l/Fas) receptor/ligand (L) system, a key regulator of apcptosis in normal and malignant T cells, is profoundly deregulated in peripheral blood T cells during HIV-l infection (Blood 1994; 83: 3101; Blood 1996; 88: 1741). It is not known, however, how T cell apoptosis and deregulation of the CD95/CD95L svstem are related to HIV-l olasma viral load and T cell homeostasis during a&etroviral therapy. Materlalsand Methods:Peripheral venous blood from 30 HIV-l+ children was obtained by venipuncture. In i0 patients sequential samples were analysed durfng antiretroviral treatment using combinations of two HIV-I-RT-inhibitors with or without an inhibitor of the HIV-l -protease. Expression of CD95, CD45RA, CD45RO and CD62L on peripheral blood T cells was studied by immunophenotyping using a whole blood lysis method and flow cytometry. Sensitivity of isolated peripheral blood T cells towards apoptosis induced by immobilized anti-CD3 or anti-CD95 monoclonal antibodies was assessed by flow cytometry after 24 hours short term culture in vitro. CD95L expression in freshly isolated peripheral T cells was measured using quantitative FlT-PCRand HIV-1 plasma viral load was measured by branched DNA determination (Quantiplex bDNA 2.0, Chiron Diagnostics). Results: CD95expression (%CD95highcells) in peripheral blood T cells increased with disease progression as judged by decreasing CD4+ T cell counts and decreasing percentages of naive (CD45FtA+ CD62Lhigh)T cells. AICD of CD4+ T cells also increased significantly with disease progression. The expression of CD95L mRNA in peripheral blood T cells was found to be heavily increased in symptomatic patients without AIDS, whereas asymptomatic patients and patients with advanced disease showed lower expression levels. No correlation was found between HIV-l plasma viral load and any of these parameters. However, as early as 8 weeks after starting antiretroviral combination therapy a statistically significant reduction of CD95L levels in freshly isolated peripheral blood T cells was seen in those patients where HIV-l plasma viral load dropped by at least 1.Olog. Furthermore, the percentage of CD95hQhcells T cells (which are mostly CD4+) and CD4+ T cell apoptosis in vitro decreased early dutfng antiretroviral treatment. A decrease in the percentage of primed/memory (CD45RA- CDRO+) together with an increase in naive (CD45RA+ CD62Lhigh)T cells was not observed initially but occurred during later treatment phases (>12 weeks). Conclusion: Increased AICD and deregulation of the CD95 receptorfiigand system in T cells from HIV-l-infected children increase with advanced CD4+ T &II loss. CD4+ T cell apoptosis in HIV-l-infection correlates with disease stage but not with viral load. The effect of antiretroviral combination therapy on disease progression and T cell renewal may be partly explained by reduced expression of CD95 receptor/ligand in T cells and reduced AICD of CD4+ T cells during therapy. 10.1.08.2
1 Role of FADD and Caspase family members in TNF-induced apoptosis in a T ceil hybridoma
G. Denecker, W. Declercq, G. Van Loo, 8. Depuyt, M. Van de Craen, W. Fiers, P. Vandenabeele. Laboratory of Molecular Biology. V/B and University of Gent, K.L. Ledeganckstraar 35, Ghent, Balgium Introductfon: Although accumulating data have been presented resolving the TNF-R55 post-receptor events leading to cell death, the mechanism involved in TNF-R75-induced cell killing is not yet known. We investigated the role of FADD
and the CED3XE (caspase) family of cysteine proteases in TNF-R75-induced apoptosis. Materialsand Methods: The rat/mouse T cell hybidoma PC60 was transfected with both human TNF-R55 and TNF-R75: PC60 RXVR75. These cells were either stably or transiently transfected with respectively, CrmA or a FADD dominant negative deletion mutant (FADD-DN). Induction of apoptosis in PC60 R55/R75: 2.104 cells/well were stimulated with hTNF or its TNF-R-specific muteins, this in the presence or absence of peptide caspase inhibitors. After 20 h incubation, the number of apoptotic cells was measured by the PI exclusion method. Caspase enzyme activity was determined in cell lysates by standard procedures making use of the fluorooenic substrates AC-YVAD-amcor Ac-DEVD-amt. Results:-ln PC60 R55/R75, a rat/mouse T-cell hybtfdoma transfected with both human TNF-R, either TNF-R55 and TNF-R75 can mediate TNF-induced apoptosis. Here, we show that apoptosis elicited by TNF-R55 as well as by TNF-R75, share the same signal transducing protein, namely FADD. FADD is a cvtoplasmic protein that mediates Fas/Aool and TNF-R55 death-inducina ac&iti&. Transient overexpression of FAD&DN completely inhibited apoptc sis induced by either TNF-R55 or TNF-R75. In addition, expression of CrmA. a viral inhibitor of the caspases, blocked the TNF-depenbent apoptosis by both TNF-Rs. This observation was further substantiated by the use of peptide caspase inhibitors. TNF-R55- as well as TNF-R75-induced apoptosis in PC60 R55/R75, can be completely inhibited by Z-VAD-fmk, this in contrast to Ac-DEVD-fmk and Ac-YVADcmk inhibitors. By using fluorescent tetrapeptide substrates we could detect a late activation of CPP32-like cysteine proteases, but not of the ICE;-like proteases. Moreover both TNF-receptors could induce this CPP32-like enzyme activity. Conclusion: These results indicate that both TNF-R55 and TNF-R75 use the same receptor associated molecule (FADD). Also, TNF-R75 makes use of the same cysteine protease cascade as TNF-R55.
0.1.08.3
Melanoma ceils express Fas iigand: impiications for tumor immune escape
M. Hahne, D. Rimoldi, M. SchrOter,P. Romera, L. French, J.-C. Cerottini, J. Tschopp. BIL Research Center Lausanne, CH-1066 Epalinges, Bwitzerland Malignant melanoma account for most of the growing mortality from skin cancer. Melanoma cells were found to express Fas ligand (FasL). In metastatic lesions, Fas-expressing T cell infiltrates were proximal to FasL+ tumor cells. In vitro, apoptosis of Fas-sensitive target cells occurred upon incubation with melanoma tumor cells, and in viva, injection of FasL-positive mouse melanoma cells in mice led to rapid tumor formation. In contrast, tumorigenesis was delayed in Fas-deficient lpr mutant mice in which immune effector cells cannot be killed by FasL. Thus, FasL may contribute to the immune privilege of tumors. We are presently investigating the role of the Fas system in other diseases.
0.1.08.4
identification of pathways involved in Bceii receptor-induced and FASinduced B ceil death
S.M.A. Lens’, B.F.A. den Drijver’, K. Tesselaar’, M.H.J. van Oers*, R.A.W. van tier’. ’ C.L.B. & Lab. Exp. C/in. Immunofog~ University of Amsterdam, The Netherlands, ’ Department of Hematology A.M.C., Amsterdam, The Netherlands Our previous findings have shown that selection of mature B cells can occur at two stages. First cross-linking of the B-cell receptor (BCR) can result in cell death unless T-cell dependent signals such as CD40-ligand or TNFarare present. However, these latter signals can make B-cells sensitive to Fas-mediated apoptosis, suggesting the need for additional stimuli which overcome Fas-induced apoptosis and allow positive selection (S.M.A. Lens et al., J. Immunol. 156: 507, 1996). To dissect pathways involved in antigen-receptor mediated and Fas-mediated B cell death, clones were selected from the Burkitt lymphoma cell line Ramos that differred in their sensitivity to Fas-induced and BCR-induced apoptosis. Enhanced sensitivity to Fas-induced apoptosis coincided with increased susceptibility for BCR-mediated death. In contrast, clones which were resistant to Fas-induced cell death, could still undergo apoptosis in response to BCR cross-linking. Although both CD40 mAb and TNFu enhanced Fas expression to levels observed in the wild-type Ramos cell line, these agents did not make Fas-resistant clones susceptible for Fas-mediated death. Based on these find-
WEDNESDAY, 25 JUNE 1997
Oral Presentations
lO:oo-12:oo
0.1.08
10.1.08.1
Room B
Regulation of apoptosis 1 Apoptosis and T cell renewal during antlretroviral therapy in HIV-l-Infected children
Th. Bbhler‘, J. Walcher ‘, M.Geiss *, P. Schnitzler’, I. Herra, K.-M. Debatin 1.3.’ Hematology/Oncolo~, Children’s Hospital, University of Heidelberg, Heidelberg, Germany, 2institute of Medical virology; University of Heidelberg,Heidelberg, German)! 3Departmenr of Molecular Oncology German Cancer Research Center, Heidelberg. Germany
Introduction:Increased activation-induced programmed cell death (apoptosis, AICD) of T ceils is thought to be involved in progressive T cell depletion in HIV-l infected individuals. The CD95 (APO-l/Fas) receptor/ligand (L) system, a key regulator of apcptosis in normal and malignant T cells, is profoundly deregulated in peripheral blood T cells during HIV-l infection (Blood 1994; 83: 3101; Blood 1996; 88: 1741). It is not known, however, how T cell apoptosis and deregulation of the CD95/CD95L svstem are related to HIV-l olasma viral load and T cell homeostasis during a&etroviral therapy. Materlalsand Methods:Peripheral venous blood from 30 HIV-l+ children was obtained by venipuncture. In i0 patients sequential samples were analysed durfng antiretroviral treatment using combinations of two HIV-I-RT-inhibitors with or without an inhibitor of the HIV-l -protease. Expression of CD95, CD45RA, CD45RO and CD62L on peripheral blood T cells was studied by immunophenotyping using a whole blood lysis method and flow cytometry. Sensitivity of isolated peripheral blood T cells towards apoptosis induced by immobilized anti-CD3 or anti-CD95 monoclonal antibodies was assessed by flow cytometry after 24 hours short term culture in vitro. CD95L expression in freshly isolated peripheral T cells was measured using quantitative FlT-PCRand HIV-1 plasma viral load was measured by branched DNA determination (Quantiplex bDNA 2.0, Chiron Diagnostics). Results: CD95expression (%CD95highcells) in peripheral blood T cells increased with disease progression as judged by decreasing CD4+ T cell counts and decreasing percentages of naive (CD45FtA+ CD62Lhigh)T cells. AICD of CD4+ T cells also increased significantly with disease progression. The expression of CD95L mRNA in peripheral blood T cells was found to be heavily increased in symptomatic patients without AIDS, whereas asymptomatic patients and patients with advanced disease showed lower expression levels. No correlation was found between HIV-l plasma viral load and any of these parameters. However, as early as 8 weeks after starting antiretroviral combination therapy a statistically significant reduction of CD95L levels in freshly isolated peripheral blood T cells was seen in those patients where HIV-l plasma viral load dropped by at least 1.Olog. Furthermore, the percentage of CD95hQhcells T cells (which are mostly CD4+) and CD4+ T cell apoptosis in vitro decreased early dutfng antiretroviral treatment. A decrease in the percentage of primed/memory (CD45RA- CDRO+) together with an increase in naive (CD45RA+ CD62Lhigh)T cells was not observed initially but occurred during later treatment phases (>12 weeks). Conclusion: Increased AICD and deregulation of the CD95 receptorfiigand system in T cells from HIV-l-infected children increase with advanced CD4+ T &II loss. CD4+ T cell apoptosis in HIV-l-infection correlates with disease stage but not with viral load. The effect of antiretroviral combination therapy on disease progression and T cell renewal may be partly explained by reduced expression of CD95 receptor/ligand in T cells and reduced AICD of CD4+ T cells during therapy. 10.1.08.2
1 Role of FADD and Caspase family members in TNF-induced apoptosis in a T ceil hybridoma
G. Denecker, W. Declercq, G. Van Loo, 8. Depuyt, M. Van de Craen, W. Fiers, P. Vandenabeele. Laboratory of Molecular Biology. V/B and University of Gent, K.L. Ledeganckstraar 35, Ghent, Balgium Introductfon: Although accumulating data have been presented resolving the TNF-R55 post-receptor events leading to cell death, the mechanism involved in TNF-R75-induced cell killing is not yet known. We investigated the role of FADD
and the CED3XE (caspase) family of cysteine proteases in TNF-R75-induced apoptosis. Materialsand Methods: The rat/mouse T cell hybidoma PC60 was transfected with both human TNF-R55 and TNF-R75: PC60 RXVR75. These cells were either stably or transiently transfected with respectively, CrmA or a FADD dominant negative deletion mutant (FADD-DN). Induction of apoptosis in PC60 R55/R75: 2.104 cells/well were stimulated with hTNF or its TNF-R-specific muteins, this in the presence or absence of peptide caspase inhibitors. After 20 h incubation, the number of apoptotic cells was measured by the PI exclusion method. Caspase enzyme activity was determined in cell lysates by standard procedures making use of the fluorooenic substrates AC-YVAD-amcor Ac-DEVD-amt. Results:-ln PC60 R55/R75, a rat/mouse T-cell hybtfdoma transfected with both human TNF-R, either TNF-R55 and TNF-R75 can mediate TNF-induced apoptosis. Here, we show that apoptosis elicited by TNF-R55 as well as by TNF-R75, share the same signal transducing protein, namely FADD. FADD is a cvtoplasmic protein that mediates Fas/Aool and TNF-R55 death-inducina ac&iti&. Transient overexpression of FAD&DN completely inhibited apoptc sis induced by either TNF-R55 or TNF-R75. In addition, expression of CrmA. a viral inhibitor of the caspases, blocked the TNF-depenbent apoptosis by both TNF-Rs. This observation was further substantiated by the use of peptide caspase inhibitors. TNF-R55- as well as TNF-R75-induced apoptosis in PC60 R55/R75, can be completely inhibited by Z-VAD-fmk, this in contrast to Ac-DEVD-fmk and Ac-YVADcmk inhibitors. By using fluorescent tetrapeptide substrates we could detect a late activation of CPP32-like cysteine proteases, but not of the ICE;-like proteases. Moreover both TNF-receptors could induce this CPP32-like enzyme activity. Conclusion: These results indicate that both TNF-R55 and TNF-R75 use the same receptor associated molecule (FADD). Also, TNF-R75 makes use of the same cysteine protease cascade as TNF-R55.
0.1.08.3
Melanoma ceils express Fas iigand: impiications for tumor immune escape
M. Hahne, D. Rimoldi, M. SchrOter,P. Romera, L. French, J.-C. Cerottini, J. Tschopp. BIL Research Center Lausanne, CH-1066 Epalinges, Bwitzerland Malignant melanoma account for most of the growing mortality from skin cancer. Melanoma cells were found to express Fas ligand (FasL). In metastatic lesions, Fas-expressing T cell infiltrates were proximal to FasL+ tumor cells. In vitro, apoptosis of Fas-sensitive target cells occurred upon incubation with melanoma tumor cells, and in viva, injection of FasL-positive mouse melanoma cells in mice led to rapid tumor formation. In contrast, tumorigenesis was delayed in Fas-deficient lpr mutant mice in which immune effector cells cannot be killed by FasL. Thus, FasL may contribute to the immune privilege of tumors. We are presently investigating the role of the Fas system in other diseases.
0.1.08.4
identification of pathways involved in Bceii receptor-induced and FASinduced B ceil death
S.M.A. Lens’, B.F.A. den Drijver’, K. Tesselaar’, M.H.J. van Oers*, R.A.W. van tier’. ’ C.L.B. & Lab. Exp. C/in. Immunofog~ University of Amsterdam, The Netherlands, ’ Department of Hematology A.M.C., Amsterdam, The Netherlands Our previous findings have shown that selection of mature B cells can occur at two stages. First cross-linking of the B-cell receptor (BCR) can result in cell death unless T-cell dependent signals such as CD40-ligand or TNFarare present. However, these latter signals can make B-cells sensitive to Fas-mediated apoptosis, suggesting the need for additional stimuli which overcome Fas-induced apoptosis and allow positive selection (S.M.A. Lens et al., J. Immunol. 156: 507, 1996). To dissect pathways involved in antigen-receptor mediated and Fas-mediated B cell death, clones were selected from the Burkitt lymphoma cell line Ramos that differred in their sensitivity to Fas-induced and BCR-induced apoptosis. Enhanced sensitivity to Fas-induced apoptosis coincided with increased susceptibility for BCR-mediated death. In contrast, clones which were resistant to Fas-induced cell death, could still undergo apoptosis in response to BCR cross-linking. Although both CD40 mAb and TNFu enhanced Fas expression to levels observed in the wild-type Ramos cell line, these agents did not make Fas-resistant clones susceptible for Fas-mediated death. Based on these find-