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Activation of human CD4+ T cell clones through CD40 ligand
56
Co-stimulation
LDL from APC to T-cell, evidence for DNA synthesis induced by oxidised LDL is involved a co-stimulatory process. Oxidised LDL can stimulate expression of CD80 on monocytes and B cells and expression of CD26 on the T cells and oxidised LDL also can stimulate expression of ICAM- on monocytes and LFA-1 on the T cells. These surface markers on cells were determined bv FACAcan with an immunofluorescence method. Blocking CD80 signal by CTiA4-lg may completely inhibit T cells DNA synthesis induced by oxidised LDL. Anti-ICAMantibodies may pattially inhibit 7 cells proliferation induced by oxidised LDL. Oxidised LDL as immunogenecily is related to development of atherosclerosis. Studying the stimulatory mechanism of T-cell synthesis by oxldisad LDL may contribute to regulation of immune responses thus further affect atherosclerotic process.
( P.l .01.231 Activation of human CD4+ T cell clones through CD40 llgand Nathalie Foumier, Etic Garcia, lsabelle Durand, Claude Gaillard, Dominique Blanchard, Jacques Banchereau, Pierre Garrone. Schering-Plough, Laboratory for lmmunobgical Research, Datdilly; France
Introduction: The CD40 ligand (CD4OL),a member of the tumor necrosis factor (TNF) family, is rapidly induced on activated CD4+ T calls. CD4OL binds to its counter-receptor, CD40, a membrane glycoprotein expressed on the surface of APC (B cells, dendtitic cells...). While CD4GCD4OL interactions are clearly beneficial to B cells (B cell growth, isotype switching and Ig synthesis), several studies have shown that a reciprocal costimulation of T cells can also occur. To examine this issue, we studied the effects of CD4OL engagement on activation of human CD4+ T cell clones. Materials and Methods: The CD4+ T cell done MT9 (Thl) was delived from a healthy donor, FS35 (Th2) from an atopic donor and KD20 (Thl) from a patient suffering from X-linked hyper-IgM syndrome and expressing a truncated CD4OL. Clones were weekly stimulated with irradiated feeder cells, PHA and IL-2 for 3 days and then cultured with IL-2 for4 days. T cell clones (resting) were used 7 days after the last stimulation with feeder cells and were cultured with immobilized anti-CD3 mAb (OKT3), IL-2 or an antibody raised against human CD4OL (mAb Ll2). Their pmliieration, pattern of cytokine secretion (by ELISA) and viability (Ttypan blue or propidium iodide) were studied. Results: The expression of the CMOL molecule on resting MT9 and FS35 cells was strongly up-regulated by activation with immobilized anti-CD3 mAb or to a lesser extend with 11-2.Addition of anti-CD4OL mAb LL2 enhanced proliferation of MT9 and FS35 cells in the presence of 11-2,but did not costimulate with anti-CD3 mAb. As obtained with anti-CD3 activation, en0agement of CD4OL bv mAb Ll2 increased the secretion of IFN-y by MT9 &&and of IL-lo, IL-5 a& IL-4 by FS35 cells cultured with IL-2. Activation of MT9 cells with anti-CD4OL and 11-2resulted in a rapid decrease of the viable cell number, as also obtained with anti-CD3 plus 11-2. While proliferation of CD40L- or CD9activated MT9 cells was strongly increased bv addition of the antaoonistic anti-Fas mAb 284 (that blocks FadFasL interactibn), this antibody fal6d to prevent the death of MT9 cells cultured with anti-CD4OL or anti-CD3 mAb. In contrast, FS35 cell viability was only weakly affected by CD4OL or CD3 activation, and addition of mAb 284 neither modified FS35 cell proliferation nor viability. As expected, mAb Ll2 did not bind to anti-CD3 and IL-2 activated CD4OL-deficient KD20 ceils, and did not modify their proliferation and cytoklne secretion. Moreover, KD20 cells rapidly died when cultured with anti-CD3 mAb but not with anti-CD4OL mAb Ll2, thus demonstrating the overall effects of mAb LL2 were mediated by engagement of CD4OL. Conclurlon: These results indicate that engagement of the CD4OL in the presence of IL-2 can induce proliferation, cytokine secretion and death of CD4+ T cell clones, that do not require CD3 costlmulation.
23 June 1997 - Poster presentations Sequence analysis of the largest cDNA clone showed a insert of 850 bp containing an open reading frame of 595 bp. Not surprisingly, the predicted protein is a type II transmembrane protein with a molecular weight of 21 kDa, and 3 potential N-linked glycceylatlon sites. Comparison to human CD70 shows an overall homology of 55%. The highest homology is found within the extracellular parl of the protein, which is similar to what is found for other memben of the TNF-family. Remarkablv, sequence homoloaVbetween the intracellular domains is restritiid to the first 5 N-terminal amino &ids. Knowing the cDNA sequence of mutine CD70, PCR based strategies were used to identify a Pl plasmid containing the murine CD70 gene (Ginome Systems, INC), &d to &ermine its structure. The murlne CD70 gene compromises 3 exons and 2 intmns spread over 4 kb of genomic DNA. The first exon contains the 5’ untranslated region, the intracellular region, the transmembrane region and a small part oftheextracellular region. The second exons is very small and only contains 36 nucleotides. The third and last exon contains the largest part of the extracellular region and the 3’ untranslated region, which is characteristic for members of theTNF family. Currently we are transfecting hamster fibroblasts with the murlne CD70 cDNA. These transfectants will be used to immunise hamsters and subsequently obtain monoclonal antibodies directed against mutine CD70. Wii the previous identification of murine CD27 and the production of anti-mouse CD27 r&b, all the necessary tools are then available to study the role of the CD27-CD70 interactions in vivo.
1P.l .01.25 1 CD40 and CD70 co-stimulate a potent in vlvo anti-tumor T cell response Yvo F. Graus, Barbara L. Kremers, John D. Nieland, Ada M. Kruisbeek. Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CXAmsierdam, The Netherlands Genetically modified tumor cell vaccines have been widely explored for their ability to induce systemic long-lasting anti-tumor speclflc T cell immunity. This appears to be a consequence of augmented tumor antigen presentation, either by the tumor itself or by host APC. In several studies, the co-stlmulatoty molecule CD80. when transfected into tumor cells, induced systemic CD8 antltumor T cell responses. However, expression of CD80 by tumors not always insures generation of a T cell mediated anti-tumor response. We addressed the efficacy of other co-stlmulatory ligands, in particular CD40 end CD70, for their ability to co-stimulate anti-tumor responses. CD40 and CD70 transfected P815 wei first tested for their capacity h co-stimulate T cells in vitro. P815-CD70 co-stimulated both CD4 and CD8 T cells whereas P815-CD40 preferentiallv co-stimulated CD4 T cells. Subsequently it was investigated ðer the & tifm co-stlmulatory capacity of P8156D40 and CD70 transfectants correlated with the in vivo induction of an anti-tumor response. Despite the fact that, in vitro, CD40 predominantly cc-stimulates CD4 T cells it was demonstrated that MHC class II negative P815-CD40 tumor induce strong anti-tumor immunity and induce a long-lasting memory tumor specific CTL response against both transfected and wild type (wt) tumor. A large fraction of mice rejecting the transfected P815 were even protected against a challenge with wl P815. Similar tumor rejection was observed with P815CD70. Co-stimulation of PElCCD40 in vitro was found to be dependent on CD80 whereas CD70 appeared to co-stimulate tumor-specific CTL responses directly or via other cc-stimulatory molecules than CDBO/CD86. CD40 and CD70 thus emerge as powerful alternatives to CD80 for improving tumor immunogenicity in viw. The mechanisms by which they do so are currently being defined in TCR-transgenic mouse models. These findings suggest additional strategies for Immunotherapy. Supported by grants from the Dutch Cancer Foundation.
P.l .01.26 1P.l .01.241 Murlne CD70: cDNA cloning and gene structure K. Tesselaar, L. Gravestein I, J. Borst l, R.A.W. van tier. Central Laboratory of the Red Cross Blood TransfusionService and Labomtory for Experimental and Clinical lmmunolow; University of Amstetdam, The Netherlands, ’ Division of Immunology the Netherlands Cancer Institute, Amsterdam, The Netherlands Interactions between members of the tumor necrosis receptor family and tumor necrosis family are essential in the regulation of immune responses. Ligation of CD27, a member of the TNF-R family, by its ligand CD70, provides a strong co-stimulatoty signal for growth and differentiation of activated T and B ceils. To further investigate the role of the interaction between CD27 and CD70 in vivo, we set out to characterize murlne CD70. A murine CD70 expressing cell line (B771) was identified, using a murine CD27-Fc construct. Further confirmation of CD70 expression was obtained via immunoprecipitation. The CD27-Fc construct specifically precipitated a protein of approximately 29 kDa, which is in agreement with the MW of human CD70. Of the B771 cell line a cDNA library was prepared in lambda gtll and screening of the library with a human CD70 PCR probe revealed eight strongly hybrldizlng clones.
;h~~~~;lYLA4
durlng the acthmtion of resting
M.C. Brunner l, F. Chan ‘, C. Hinkel 2, J. Hanke2, A. Winoto ‘, J.P. Allison’. ’ Department of Molecular and Ceil Bidogy; -_ UC Berkelw CA 94720, USA, 2Pti& Inc., Groton, C7; USA
Introduction:CD28 costlmulation enhances, while CTLA-4 engagement inhibits, IL-2 production and proliferation of T-cells upon T-cell activation. The inhibition meditated by CT&4 is not a result of induction of cell death, but rather due to inhibition of cell cycle progression. We analyzed the mechanisms of CTLA-4 inhibition early during activation of resting CD4+ T-cells. Materials and Methods: CD4+ LN T-cells were stimulated with &D3, aCD28, and/or aCTlA-4 antibodies bound to microspheres. IL-2 production was measured by ELISA. IL-2 mRNA was quantltated by competitive PCR. Transcription initiation of IL-2 was addressed using transgenic mice bearing a reporter gene with the IL-2 promoter. Nuclear extracts were analyzed by gel shift assays. Furthermore, Gl-kinases were examined by Western blotting. Reeuh Stimulation of T-cells with &D3 and nCD28 resulted in IL-2 mRNA accumulation as early as 4 hrs which is prevented by coligatlon of CTLA4. CTLA-4 crosslinking had no effect on the CD28-mediated stabilization of IL-2 mRNA. Using transgenic mice, CTLA4 engagement inhibits initiation of IL-2 tran-
Co-stimulation
LDL from APC to T-cell, evidence for DNA synthesis induced by oxidised LDL is involved a co-stimulatory process. Oxidised LDL can stimulate expression of CD80 on monocytes and B cells and expression of CD26 on the T cells and oxidised LDL also can stimulate expression of ICAM- on monocytes and LFA-1 on the T cells. These surface markers on cells were determined bv FACAcan with an immunofluorescence method. Blocking CD80 signal by CTiA4-lg may completely inhibit T cells DNA synthesis induced by oxidised LDL. Anti-ICAMantibodies may pattially inhibit 7 cells proliferation induced by oxidised LDL. Oxidised LDL as immunogenecily is related to development of atherosclerosis. Studying the stimulatory mechanism of T-cell synthesis by oxldisad LDL may contribute to regulation of immune responses thus further affect atherosclerotic process.
( P.l .01.231 Activation of human CD4+ T cell clones through CD40 llgand Nathalie Foumier, Etic Garcia, lsabelle Durand, Claude Gaillard, Dominique Blanchard, Jacques Banchereau, Pierre Garrone. Schering-Plough, Laboratory for lmmunobgical Research, Datdilly; France
Introduction: The CD40 ligand (CD4OL),a member of the tumor necrosis factor (TNF) family, is rapidly induced on activated CD4+ T calls. CD4OL binds to its counter-receptor, CD40, a membrane glycoprotein expressed on the surface of APC (B cells, dendtitic cells...). While CD4GCD4OL interactions are clearly beneficial to B cells (B cell growth, isotype switching and Ig synthesis), several studies have shown that a reciprocal costimulation of T cells can also occur. To examine this issue, we studied the effects of CD4OL engagement on activation of human CD4+ T cell clones. Materials and Methods: The CD4+ T cell done MT9 (Thl) was delived from a healthy donor, FS35 (Th2) from an atopic donor and KD20 (Thl) from a patient suffering from X-linked hyper-IgM syndrome and expressing a truncated CD4OL. Clones were weekly stimulated with irradiated feeder cells, PHA and IL-2 for 3 days and then cultured with IL-2 for4 days. T cell clones (resting) were used 7 days after the last stimulation with feeder cells and were cultured with immobilized anti-CD3 mAb (OKT3), IL-2 or an antibody raised against human CD4OL (mAb Ll2). Their pmliieration, pattern of cytokine secretion (by ELISA) and viability (Ttypan blue or propidium iodide) were studied. Results: The expression of the CMOL molecule on resting MT9 and FS35 cells was strongly up-regulated by activation with immobilized anti-CD3 mAb or to a lesser extend with 11-2.Addition of anti-CD4OL mAb LL2 enhanced proliferation of MT9 and FS35 cells in the presence of 11-2,but did not costimulate with anti-CD3 mAb. As obtained with anti-CD3 activation, en0agement of CD4OL bv mAb Ll2 increased the secretion of IFN-y by MT9 &&and of IL-lo, IL-5 a& IL-4 by FS35 cells cultured with IL-2. Activation of MT9 cells with anti-CD4OL and 11-2resulted in a rapid decrease of the viable cell number, as also obtained with anti-CD3 plus 11-2. While proliferation of CD40L- or CD9activated MT9 cells was strongly increased bv addition of the antaoonistic anti-Fas mAb 284 (that blocks FadFasL interactibn), this antibody fal6d to prevent the death of MT9 cells cultured with anti-CD4OL or anti-CD3 mAb. In contrast, FS35 cell viability was only weakly affected by CD4OL or CD3 activation, and addition of mAb 284 neither modified FS35 cell proliferation nor viability. As expected, mAb Ll2 did not bind to anti-CD3 and IL-2 activated CD4OL-deficient KD20 ceils, and did not modify their proliferation and cytoklne secretion. Moreover, KD20 cells rapidly died when cultured with anti-CD3 mAb but not with anti-CD4OL mAb Ll2, thus demonstrating the overall effects of mAb LL2 were mediated by engagement of CD4OL. Conclurlon: These results indicate that engagement of the CD4OL in the presence of IL-2 can induce proliferation, cytokine secretion and death of CD4+ T cell clones, that do not require CD3 costlmulation.
23 June 1997 - Poster presentations Sequence analysis of the largest cDNA clone showed a insert of 850 bp containing an open reading frame of 595 bp. Not surprisingly, the predicted protein is a type II transmembrane protein with a molecular weight of 21 kDa, and 3 potential N-linked glycceylatlon sites. Comparison to human CD70 shows an overall homology of 55%. The highest homology is found within the extracellular parl of the protein, which is similar to what is found for other memben of the TNF-family. Remarkablv, sequence homoloaVbetween the intracellular domains is restritiid to the first 5 N-terminal amino &ids. Knowing the cDNA sequence of mutine CD70, PCR based strategies were used to identify a Pl plasmid containing the murine CD70 gene (Ginome Systems, INC), &d to &ermine its structure. The murlne CD70 gene compromises 3 exons and 2 intmns spread over 4 kb of genomic DNA. The first exon contains the 5’ untranslated region, the intracellular region, the transmembrane region and a small part oftheextracellular region. The second exons is very small and only contains 36 nucleotides. The third and last exon contains the largest part of the extracellular region and the 3’ untranslated region, which is characteristic for members of theTNF family. Currently we are transfecting hamster fibroblasts with the murlne CD70 cDNA. These transfectants will be used to immunise hamsters and subsequently obtain monoclonal antibodies directed against mutine CD70. Wii the previous identification of murine CD27 and the production of anti-mouse CD27 r&b, all the necessary tools are then available to study the role of the CD27-CD70 interactions in vivo.
1P.l .01.25 1 CD40 and CD70 co-stimulate a potent in vlvo anti-tumor T cell response Yvo F. Graus, Barbara L. Kremers, John D. Nieland, Ada M. Kruisbeek. Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CXAmsierdam, The Netherlands Genetically modified tumor cell vaccines have been widely explored for their ability to induce systemic long-lasting anti-tumor speclflc T cell immunity. This appears to be a consequence of augmented tumor antigen presentation, either by the tumor itself or by host APC. In several studies, the co-stlmulatoty molecule CD80. when transfected into tumor cells, induced systemic CD8 antltumor T cell responses. However, expression of CD80 by tumors not always insures generation of a T cell mediated anti-tumor response. We addressed the efficacy of other co-stlmulatory ligands, in particular CD40 end CD70, for their ability to co-stimulate anti-tumor responses. CD40 and CD70 transfected P815 wei first tested for their capacity h co-stimulate T cells in vitro. P815-CD70 co-stimulated both CD4 and CD8 T cells whereas P815-CD40 preferentiallv co-stimulated CD4 T cells. Subsequently it was investigated ðer the & tifm co-stlmulatory capacity of P8156D40 and CD70 transfectants correlated with the in vivo induction of an anti-tumor response. Despite the fact that, in vitro, CD40 predominantly cc-stimulates CD4 T cells it was demonstrated that MHC class II negative P815-CD40 tumor induce strong anti-tumor immunity and induce a long-lasting memory tumor specific CTL response against both transfected and wild type (wt) tumor. A large fraction of mice rejecting the transfected P815 were even protected against a challenge with wl P815. Similar tumor rejection was observed with P815CD70. Co-stimulation of PElCCD40 in vitro was found to be dependent on CD80 whereas CD70 appeared to co-stimulate tumor-specific CTL responses directly or via other cc-stimulatory molecules than CDBO/CD86. CD40 and CD70 thus emerge as powerful alternatives to CD80 for improving tumor immunogenicity in viw. The mechanisms by which they do so are currently being defined in TCR-transgenic mouse models. These findings suggest additional strategies for Immunotherapy. Supported by grants from the Dutch Cancer Foundation.
P.l .01.26 1P.l .01.241 Murlne CD70: cDNA cloning and gene structure K. Tesselaar, L. Gravestein I, J. Borst l, R.A.W. van tier. Central Laboratory of the Red Cross Blood TransfusionService and Labomtory for Experimental and Clinical lmmunolow; University of Amstetdam, The Netherlands, ’ Division of Immunology the Netherlands Cancer Institute, Amsterdam, The Netherlands Interactions between members of the tumor necrosis receptor family and tumor necrosis family are essential in the regulation of immune responses. Ligation of CD27, a member of the TNF-R family, by its ligand CD70, provides a strong co-stimulatoty signal for growth and differentiation of activated T and B ceils. To further investigate the role of the interaction between CD27 and CD70 in vivo, we set out to characterize murlne CD70. A murine CD70 expressing cell line (B771) was identified, using a murine CD27-Fc construct. Further confirmation of CD70 expression was obtained via immunoprecipitation. The CD27-Fc construct specifically precipitated a protein of approximately 29 kDa, which is in agreement with the MW of human CD70. Of the B771 cell line a cDNA library was prepared in lambda gtll and screening of the library with a human CD70 PCR probe revealed eight strongly hybrldizlng clones.
;h~~~~;lYLA4
durlng the acthmtion of resting
M.C. Brunner l, F. Chan ‘, C. Hinkel 2, J. Hanke2, A. Winoto ‘, J.P. Allison’. ’ Department of Molecular and Ceil Bidogy; -_ UC Berkelw CA 94720, USA, 2Pti& Inc., Groton, C7; USA
Introduction:CD28 costlmulation enhances, while CTLA-4 engagement inhibits, IL-2 production and proliferation of T-cells upon T-cell activation. The inhibition meditated by CT&4 is not a result of induction of cell death, but rather due to inhibition of cell cycle progression. We analyzed the mechanisms of CTLA-4 inhibition early during activation of resting CD4+ T-cells. Materials and Methods: CD4+ LN T-cells were stimulated with &D3, aCD28, and/or aCTlA-4 antibodies bound to microspheres. IL-2 production was measured by ELISA. IL-2 mRNA was quantltated by competitive PCR. Transcription initiation of IL-2 was addressed using transgenic mice bearing a reporter gene with the IL-2 promoter. Nuclear extracts were analyzed by gel shift assays. Furthermore, Gl-kinases were examined by Western blotting. Reeuh Stimulation of T-cells with &D3 and nCD28 resulted in IL-2 mRNA accumulation as early as 4 hrs which is prevented by coligatlon of CTLA4. CTLA-4 crosslinking had no effect on the CD28-mediated stabilization of IL-2 mRNA. Using transgenic mice, CTLA4 engagement inhibits initiation of IL-2 tran-