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Is Fas ligand (CD95L) expression by astrocytoma cells a major mechanism of inactivation of tumour infiltrating lymphocytes?
25 June 1997 - Poster presentations P4 1..
Evasion of the immune system
. 1Identification of amino acid residues within the
09 05
EWl9K protein of adenovirus type 2 critically involved In HLA binding M. Sester, K. Koebemib, B. Men& H.-G. Burged. Max-van Pettenkofer-Institute, Dep. of W&?g~
GenZentNf77,
Miinchen, Germany
Introduction: The E3/19K protein of human adenovirus type 2 (Ad2) is a resident transmembrane glycoprotein of the endoplasmic reticulum. Its ability to associate with class I histccompatibilii (MHC) antigens, impairs the transport and antigen presentation function of MHC ant&fens. At present, it is unclear which structure of the E3/19K protein mediates binding to MHC molecules. Surprisingly, E3/19K molecules from different adenovirus subgroups (B, C and D) share little homology. Only 25 of +- 150 amino acids are strictly conserved. We hypothesize that these conserved residues are important for the function of E3l19K. MeterlaIr and Methode: Using alanine scanning mutagenesis single amino acid substitutions were introduced at conserved positions in the E3/19K protein of Ad2. After expression in 293 cells, interaction of these mutant proteins with HfA was examined by coimmunoprecipitation, pulse-chase experiments and FACS analysis. Structural changes introduced by the mutations were monitored by a panel of monoclonal antibodies. Reeuk First, we show that E3/19K contains two intra-molecular disulfide bonds, which are absolutely critical for the interaction with human MHC antigens. Several other amino acids were identified whose substitution completely abrogated E3/19K function. Interestingly, for some of these mutants only minor structural changes are detected. Other exchanges exert intenediate or no effects. Condusions: The results suggest that a conformational determinant based on two disutfide bonds and other conserved amino acids is crucial for the function of the E3/19K molecule. Together with antibody binding data a refined structural model of E3/19K will be presented.
I..P 4
. 1Is
09 06
Fas ligand (CD95L) expression by astrocytoma cells a major mechanism of inactivation of tumour infiltrating lymphocytes?
,
P. Sass' P.R. Walker’, M. Hahne2, A.-L. Quiquerez’, V. Schnuriger ‘, G. Penin’, L. French3, E.G. Van Meir4, N. de Tnbolet4, J. Tschopp*, P.-Y.Dietrich ’ ’ Divisionof Onoology,UniversityHospital Geneva, Switzerland, *Institute of Biochemistry Epalinges, Switzedand, 3Division of Dermatolw Geneva, Switzerland, 4Laboratory of Tumor Biology and Genetics, CHlJV Lausanne, Switzerland Introduction: Astrocytomas are among the most common brain tumours that are usually fatal in their malignant form. They appear to progress without significant impedance from the immune system, despite the presence of intratumoral T cell infiltration. To date, this has been thought to be the result of T cell immunosuppression induced by asbccytoma derived cytokines. Here, we propose that a cell contact mediated event (i.e., functional FasL expression by astrocytoma cells) may also play a role in inhibiting T infiltratfng lymphocytes (TIL) function. Meterlals and Methods: FasL expression was studied by RT-PCR, Western blot, flow cytometry and immunohistochemistry.The function of FasL expressed by tumour cell lines (primary and established) and tumour ex-vivo was measured by 20 hr chromium release assay or/and by DNA fragmentation assay (the JAM test) using parental P815 or Fas transfectsd P815 cells as targets. An autologous TIL line was also used as a target. Specificity was confined using anti-human FasL mAb NOK-1. Reeults~ FasL mRNA expression was observed in most human astrocytoma cell lines and tumour biopsy samples. These results were extended using immunoblotting: a 40-kD protein corresponding to the membrane bound form was identified in the cell lysate from 1Wll tumour biopsies and 6/6 cell lines tested. lmmunohistochemistry on tumour sections confirmed that certain astrocytoma cells express FasL in vivo. Non-tumoral control tissue (human temporal cortex) showed no FasL expression by normal astrocytes in contrast to the clear staining of neurons. Despite modest (but constitutive) FasL cell surface expression (flow cytometry), rodent and human astrocytoma lines efficiently killed Fas transfected P815 targets at modest E!T ratios, but not the parental P815 cells. FasL mediated function is not only a feature of established cell lines, since astrocytoma cells directly obtained after enzymatic dissociation of tumour biopsy and early passage cell lines also specifically killed Fas positive targets, with DNA fragmentation totaky inhibited by NOK-1. Furthermore, in one case tested, astrocytoma cells kill both autdogous CD4 and CD8 TIL lines by FasL-Fas interaction. Conclusion: Our findings demonstrated that functional FasL is widely expressed by astrccytoma. This could profoundly influence the efficacy of the immune response against this tumour within the central nervous system. This important question has to be addressed in future in vivo experiments. FasL expression by astrocytoma may extend the processes that are postulated to occur in normal brain to maintain immune privilege, since we also observed FasL expression by neurons.
P.4.09.07
435
The CDla molecule from antigen presenting cells is involved In superantigen-induced activation
M.-T. Zilber, D. Charron, C. Gelin. INSERM WW, lnstitutd’H6matofogie, Hbpital Saint Louis, Paris, France The CDla molecules are highly expressed on human cortical thymocytes, and like MHC class II molecules, on antigen presenting cells. In the present study, we investigated the role of the CD1 molecules in T cell responses to bacterial superantigen. Firstly, CDla molecules were detected on all CD14 positive cells of certain heafthy donors (5/30 tested). The expression of the CD1a molecules on APC, was correlated with the presence of CD25 positive cells wfthff the peripheral blood lymphocytes of these donors, suggesting an activation state of these populations. The proliferative response of those peripheral blood cells to the bacterial toxins SEA and TSST-I was clearly inhibited by the addition of monoclonal antibodies directed against the CDla molecule (50 to 70% inhibition). The ability of the CDla molecule to transmit activation signals in CD14 positive cells was next examined. An intracellular calcium flux was initiated via CDla on CD14 positive cells; this calcium flux was inhibited by pre-incubation of these cells with the toxin TSST-I. These data demonstrate for the first tkns that a signal transduction pathway, invofving an intracellular calcium flux can be mediated via the CDla molecule expressed on the surface of CD14 positive cells and that this pathway can be instrumental in superantigen induced activation of peripheral blood T cells.
P.4.09.06
Expression of Fas-ligand (CtML) on rat epfthelial tumor cell lines
T. Ritter, M. Seifert, A. Krfngel, K. Vogt, H.-D. Volk. lnsfitute of Medical Immunolog)! HumbokMJniversity, Charftb, Berlin, Germany Introduction: Tumor cells escape immune recognition by various mechanisms including down-regulation of MHC-molecules and production of immunosuppressive agents. Recently the Fas Ligand (CD95L)ffas Receptor (CD95) pathway has been suggested to be involved in these processes too. The interaction of Fas Ugand with its receptor FasR is known to be very important for home&asis and regulation of immune development. Ligation of Fas with FasL induces apoptosis in Far+expressing cells. Matetlalsand Methods: Three different rat epithelial tumor cell lines derived from different organs have been investigated for expression of Fas Ltgand by RT-PCR with specific oligonucleotides and by immunofluorescence staining using an &D95L-antibody. Furthermore these tumor cell lines have been cocultured with activated rat T-cells and the capacity of apoptosis induction has been determined by demonstration of DNA-fragmentation and the TUNEL method. Reeulte: We have shown by immunocytology that Fas Ligand is expressed on rat epithelial cell lines of different origin. This interesting finding has been confirmed by detection of Fas Ligand mRNA with specific primers after RT-PCRanalysis. Moreover, the functional expression of Fas Ligand could be determined by the induction of apoptosis on rat T-cells when cocultured with these tumor cells. Conclusion: We have been shown that expression of Fas Ligand on rat epithelial cell lines induces apoptosis on activated T-cells. As the expression of Fas Ligand on human colon carcinoma and mouse melanoma cells has been proven recently, this could be a more general strategy of tumor cells to protect themselves against immune recognition. Further experiments are in progress to investigate induction of apoptosis by tumor cells.
LP.4.09.09 1 Modulation of in I&U response to interleukin-1 (IL-l) by immunotherapy with superantigens in autoimmune diseases V. Cristea’, M. Cd$an2, C. Stugren2, 0. Sort@*. ‘UniveMyof Medicine and Pharmacy “Mu Hefieganu”, Cluj-Napoca, Romania, 2Oncobgical Institute “1. Chiricu/a” Cluj-Napoca,Romania Introduction: We found that in vitro incubation of lymphocytes, isolated from patients with some autoimmune diseases in evofution, with intedeukin-1 (IL-I) induced an increase of active E-rosette forming cells (from 16.75 f 15.79% to 25.88 f 23.311%, p < 0.001; %A = +101.82). Inversely. in normal controls it was observed an inhibition of active E-rosettes at 37‘C by IL-1 preincubatfon (from 20.23 f 11.457% to 9.34 f 5.870%, p i 0.091; %A = -52.81). Material and Methods: The objective of the study was to evaluate the clinical and immunological responses in 33 patients to immunotherapy with superantigens (Staphylococcus exotoxins, two i.d. injection weekly with increasing doses). We treated 33 patients with dtfferent autoimmune diseases (anterior uveitis, Sj6gren syndrome, rheumatoid polyarthrttis, ankilosing spondilitis): 19 with evoluttve diseases, and 14 with stable diseases. Reeub After 5 weeks of immunotherapy in the ffrst group clinical improvement were noticed. In acute anterior uveitis (17 cases) were obtained suppression of: ocular pain, photophobia, blurring of the vision, pericomeal injection. hyperemia and edema of the ins, cellular exudation and aqueos precipitates.
Evasion of the immune system
. 1Identification of amino acid residues within the
09 05
EWl9K protein of adenovirus type 2 critically involved In HLA binding M. Sester, K. Koebemib, B. Men& H.-G. Burged. Max-van Pettenkofer-Institute, Dep. of W&?g~
GenZentNf77,
Miinchen, Germany
Introduction: The E3/19K protein of human adenovirus type 2 (Ad2) is a resident transmembrane glycoprotein of the endoplasmic reticulum. Its ability to associate with class I histccompatibilii (MHC) antigens, impairs the transport and antigen presentation function of MHC ant&fens. At present, it is unclear which structure of the E3/19K protein mediates binding to MHC molecules. Surprisingly, E3/19K molecules from different adenovirus subgroups (B, C and D) share little homology. Only 25 of +- 150 amino acids are strictly conserved. We hypothesize that these conserved residues are important for the function of E3l19K. MeterlaIr and Methode: Using alanine scanning mutagenesis single amino acid substitutions were introduced at conserved positions in the E3/19K protein of Ad2. After expression in 293 cells, interaction of these mutant proteins with HfA was examined by coimmunoprecipitation, pulse-chase experiments and FACS analysis. Structural changes introduced by the mutations were monitored by a panel of monoclonal antibodies. Reeuk First, we show that E3/19K contains two intra-molecular disulfide bonds, which are absolutely critical for the interaction with human MHC antigens. Several other amino acids were identified whose substitution completely abrogated E3/19K function. Interestingly, for some of these mutants only minor structural changes are detected. Other exchanges exert intenediate or no effects. Condusions: The results suggest that a conformational determinant based on two disutfide bonds and other conserved amino acids is crucial for the function of the E3/19K molecule. Together with antibody binding data a refined structural model of E3/19K will be presented.
I..P 4
. 1Is
09 06
Fas ligand (CD95L) expression by astrocytoma cells a major mechanism of inactivation of tumour infiltrating lymphocytes?
,
P. Sass' P.R. Walker’, M. Hahne2, A.-L. Quiquerez’, V. Schnuriger ‘, G. Penin’, L. French3, E.G. Van Meir4, N. de Tnbolet4, J. Tschopp*, P.-Y.Dietrich ’ ’ Divisionof Onoology,UniversityHospital Geneva, Switzerland, *Institute of Biochemistry Epalinges, Switzedand, 3Division of Dermatolw Geneva, Switzerland, 4Laboratory of Tumor Biology and Genetics, CHlJV Lausanne, Switzerland Introduction: Astrocytomas are among the most common brain tumours that are usually fatal in their malignant form. They appear to progress without significant impedance from the immune system, despite the presence of intratumoral T cell infiltration. To date, this has been thought to be the result of T cell immunosuppression induced by asbccytoma derived cytokines. Here, we propose that a cell contact mediated event (i.e., functional FasL expression by astrocytoma cells) may also play a role in inhibiting T infiltratfng lymphocytes (TIL) function. Meterlals and Methods: FasL expression was studied by RT-PCR, Western blot, flow cytometry and immunohistochemistry.The function of FasL expressed by tumour cell lines (primary and established) and tumour ex-vivo was measured by 20 hr chromium release assay or/and by DNA fragmentation assay (the JAM test) using parental P815 or Fas transfectsd P815 cells as targets. An autologous TIL line was also used as a target. Specificity was confined using anti-human FasL mAb NOK-1. Reeults~ FasL mRNA expression was observed in most human astrocytoma cell lines and tumour biopsy samples. These results were extended using immunoblotting: a 40-kD protein corresponding to the membrane bound form was identified in the cell lysate from 1Wll tumour biopsies and 6/6 cell lines tested. lmmunohistochemistry on tumour sections confirmed that certain astrocytoma cells express FasL in vivo. Non-tumoral control tissue (human temporal cortex) showed no FasL expression by normal astrocytes in contrast to the clear staining of neurons. Despite modest (but constitutive) FasL cell surface expression (flow cytometry), rodent and human astrocytoma lines efficiently killed Fas transfected P815 targets at modest E!T ratios, but not the parental P815 cells. FasL mediated function is not only a feature of established cell lines, since astrocytoma cells directly obtained after enzymatic dissociation of tumour biopsy and early passage cell lines also specifically killed Fas positive targets, with DNA fragmentation totaky inhibited by NOK-1. Furthermore, in one case tested, astrocytoma cells kill both autdogous CD4 and CD8 TIL lines by FasL-Fas interaction. Conclusion: Our findings demonstrated that functional FasL is widely expressed by astrccytoma. This could profoundly influence the efficacy of the immune response against this tumour within the central nervous system. This important question has to be addressed in future in vivo experiments. FasL expression by astrocytoma may extend the processes that are postulated to occur in normal brain to maintain immune privilege, since we also observed FasL expression by neurons.
P.4.09.07
435
The CDla molecule from antigen presenting cells is involved In superantigen-induced activation
M.-T. Zilber, D. Charron, C. Gelin. INSERM WW, lnstitutd’H6matofogie, Hbpital Saint Louis, Paris, France The CDla molecules are highly expressed on human cortical thymocytes, and like MHC class II molecules, on antigen presenting cells. In the present study, we investigated the role of the CD1 molecules in T cell responses to bacterial superantigen. Firstly, CDla molecules were detected on all CD14 positive cells of certain heafthy donors (5/30 tested). The expression of the CD1a molecules on APC, was correlated with the presence of CD25 positive cells wfthff the peripheral blood lymphocytes of these donors, suggesting an activation state of these populations. The proliferative response of those peripheral blood cells to the bacterial toxins SEA and TSST-I was clearly inhibited by the addition of monoclonal antibodies directed against the CDla molecule (50 to 70% inhibition). The ability of the CDla molecule to transmit activation signals in CD14 positive cells was next examined. An intracellular calcium flux was initiated via CDla on CD14 positive cells; this calcium flux was inhibited by pre-incubation of these cells with the toxin TSST-I. These data demonstrate for the first tkns that a signal transduction pathway, invofving an intracellular calcium flux can be mediated via the CDla molecule expressed on the surface of CD14 positive cells and that this pathway can be instrumental in superantigen induced activation of peripheral blood T cells.
P.4.09.06
Expression of Fas-ligand (CtML) on rat epfthelial tumor cell lines
T. Ritter, M. Seifert, A. Krfngel, K. Vogt, H.-D. Volk. lnsfitute of Medical Immunolog)! HumbokMJniversity, Charftb, Berlin, Germany Introduction: Tumor cells escape immune recognition by various mechanisms including down-regulation of MHC-molecules and production of immunosuppressive agents. Recently the Fas Ligand (CD95L)ffas Receptor (CD95) pathway has been suggested to be involved in these processes too. The interaction of Fas Ugand with its receptor FasR is known to be very important for home&asis and regulation of immune development. Ligation of Fas with FasL induces apoptosis in Far+expressing cells. Matetlalsand Methods: Three different rat epithelial tumor cell lines derived from different organs have been investigated for expression of Fas Ltgand by RT-PCR with specific oligonucleotides and by immunofluorescence staining using an &D95L-antibody. Furthermore these tumor cell lines have been cocultured with activated rat T-cells and the capacity of apoptosis induction has been determined by demonstration of DNA-fragmentation and the TUNEL method. Reeulte: We have shown by immunocytology that Fas Ligand is expressed on rat epithelial cell lines of different origin. This interesting finding has been confirmed by detection of Fas Ligand mRNA with specific primers after RT-PCRanalysis. Moreover, the functional expression of Fas Ligand could be determined by the induction of apoptosis on rat T-cells when cocultured with these tumor cells. Conclusion: We have been shown that expression of Fas Ligand on rat epithelial cell lines induces apoptosis on activated T-cells. As the expression of Fas Ligand on human colon carcinoma and mouse melanoma cells has been proven recently, this could be a more general strategy of tumor cells to protect themselves against immune recognition. Further experiments are in progress to investigate induction of apoptosis by tumor cells.
LP.4.09.09 1 Modulation of in I&U response to interleukin-1 (IL-l) by immunotherapy with superantigens in autoimmune diseases V. Cristea’, M. Cd$an2, C. Stugren2, 0. Sort@*. ‘UniveMyof Medicine and Pharmacy “Mu Hefieganu”, Cluj-Napoca, Romania, 2Oncobgical Institute “1. Chiricu/a” Cluj-Napoca,Romania Introduction: We found that in vitro incubation of lymphocytes, isolated from patients with some autoimmune diseases in evofution, with intedeukin-1 (IL-I) induced an increase of active E-rosette forming cells (from 16.75 f 15.79% to 25.88 f 23.311%, p < 0.001; %A = +101.82). Inversely. in normal controls it was observed an inhibition of active E-rosettes at 37‘C by IL-1 preincubatfon (from 20.23 f 11.457% to 9.34 f 5.870%, p i 0.091; %A = -52.81). Material and Methods: The objective of the study was to evaluate the clinical and immunological responses in 33 patients to immunotherapy with superantigens (Staphylococcus exotoxins, two i.d. injection weekly with increasing doses). We treated 33 patients with dtfferent autoimmune diseases (anterior uveitis, Sj6gren syndrome, rheumatoid polyarthrttis, ankilosing spondilitis): 19 with evoluttve diseases, and 14 with stable diseases. Reeub After 5 weeks of immunotherapy in the ffrst group clinical improvement were noticed. In acute anterior uveitis (17 cases) were obtained suppression of: ocular pain, photophobia, blurring of the vision, pericomeal injection. hyperemia and edema of the ins, cellular exudation and aqueos precipitates.