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Cyclosporin-A enhances IFN-γ, IL-5 and IL-13 production by T cells costimulated through CD28
23 June 1997 - Poster presentations
blocking agents, T cell proliferation was measured by thymidine incorporation, cytokine productions were measured with ELISA, and generation of cytotoxic T cell activity was measured with standard chromium release assay. Reaultr: Anti-CD86 mAb or e&CD40 mAb alone added during the primary MLR induced hyporesponsiveness. The combination of anti-CD86 and CsA induced nonresponslveness as published previously. When stimulated with allogeneic PBMC in the presence of a combination of blocking anti-CD46 mAb and anti-CD86 rnAb, T cell activation was completely blocked and T cells entered a state of nonresponsiveness as evidenced upon allogeneic restimulation. Addition of anti-CD86 mAb did not significantly after the results, and the combination of anti-CD80 and an&CD86 were also insufficient to induce nortresponslveness. Conclusion: The cooperative effect of blocking the CD46-CD46L interaction with antiCD66, is probably based upon prevention of expression of CD60 on APC. Other costimulatory APC function might also be blocked as evidenced by the fact that anti-CD86 + anti-CD46 was more effllient than anti-CD66 + anti-CD86 The combination of anti-CD40 mAb and anti-CD66 mAb offers a new strategy for immunosuppression and induction of nonresponsiveness.
P.l.Ol.18 Cyclosporin-A enhances IFN-y, IL-5 and IL-13 production by T cells costimulated through CD28 Khadija Rafiq, Stefaan W. Van Gool, Ahmad Kasran, Xiaohui Peng, Jan L. Ceuppens. Laborafory of Experimental /mmuno/cgK Catholic Universify of Leuven, Leuven, Belgium Introduction: The CD80/CDB&CD28 interaction provides a costimulatory signal for cyclospotinA (C&)-resistant IL-2 production and generation of cytotoxic T cell activity. We studied whether CsA differentially affects lhl- and Th2-type cytokine secretion by T cells when costimulated through CD28. Msterlalsand Methods:Anti-CD3 mAb was cross-linked on culture plates or on FcR-expressing P815 cells. Astimulating anti-CD28 mAb or CD86 (CD86), transfected into P815 cells, provided the costimulatory signal. IL-2, 11-4, IL-5 IL-lo, IL-13 and IFN-y were measured by ELISA. Raau~ Similar to previous reports, IL-2 production was hardly affected by CsA in these stimulating conditions. The productions of IL4 and (to a lesser extent) of IL-10 were significantly blocked. On the contrary, the production of IFN-y, IL-5 and IL-13 was markedly enhanced by CsA. By using combinations of PMA with anti-CD3 andlor anti-CD28 costlmulation, we could show that the effect of CsA on IL-5 IL-13 and IFNy production only occurred when both CD3 and CD28 were also triggered. Moreover, 11-5,IL-13 and IFN-y production could be inhibited by blocking IL-2 and its receptor. The CD4(+) subpopulation was responsible for the phenomena observed. Conclusion:CsA facilitates IFN-)I, IL-5 and IL-13 production by CD4(+) T cells. This suggests the existence of a negative regulatory signal which is CD3-dependent and calcium-dependent. It also indicates diierent regulatory pathways for two groups of Th2 cytokines: IL-4 and IL-10 on the one hand, and IL-13 and IL-5 on the other hand. This finding has major implications for immunosuppressive strategies based upon the use of CsA.
P.l.Ol.19 Clonlng of bovine CD28, CTLA4 and their ligands K.R. Parsons, C.J. Howard. lnsfifufe forAnimalHealth, Compton, UK When processed antigen is recognized in association with major histocompatibility antigen complex molecules on an antigen presenting cell (APC) by the T cell receptor (TCR) secondary costimulatory signals are required to initiate cytokine synthesis and T cell proliferation. Without the signal, from this secondary accessory molecule, stimulation via the TCR can render T cells anergic. CD80 and CD66, present on APC, interact with Tcells providing a costtmulatory signal that initiates an immune response through CD28 or terminates the response via CTLA4. The ability to enhance or suppress immune responses by manipulation of this costimulatory pathway makes these molecules a target for immune intervention. To enable manipulation of immune responses in cattle the objective of this report was to clone, sequence and express bovine CD28, CTLA-4 and their ligands. CD28 and CTLA-4 genes have been cloned by reverse-transcription polymerase chain reaction (RT-PCR) from a concanavalin-A activated peripheral blood leucocyte cDNA library, using primers based on the human sequences. Human CD86 has been shown to bind to both bovine molecules when expressed in transfected COS-7 cells. The homology between CD86 and CD86, and their analogues in other species is low and attempts to clone the genes encoding these molecules by RT-PCR has been unsuccessful. However, chimeric fusion proteins of mouse and human CTLA4 cross-react with the corresponding ligands in the other species. Mouse CTLA4-Ig has been used to identify cDNA clones encoding bovine CD60 from libraries constructed in pCDM6 and expressed in COS-7 cells. All three bovine cDNAs have been sequenced and chime&. fusion proteins have been produced of bovine CTLA-4 and CD86, which bind to their ligands. Studies of the distribution and function compared with analogues in other species using PCR, chimeric fusion proteins and transfected cell lines will be presented.
Co-stimulation
55
P.l.01.20 CD3KD28 actlvatlon of human naive neonatal T n cells S. Kilpinen, M. Hurme. Depattmenf of Microbiology and fmmunof~, University of Tampere Me&al School, POB 607, F/N-33101, Tampwe, Finland
Introduction:Optimal activation of T cells results in increased production of IL-2, enhanced cell proliferation and an effective immune response. To become optimally activated T cells require costimulatory signals in addition to the signal provided via the CD3/TCR for antigen. CD26 appears to be an important costimulatory signal receptor on T cells. The biochemical events foflowtng this CD28 ligation are still poorly known but there is some evidence that the CD28 costimulatory pathway could induce the production of reactive oxygen intermediates (ROls). We wanted to analyse IL-2 production, possible ROls formation and activation of transcription factors after CD3/CD28 stimulation in neonatal and adult T cells. Neonatal T cells may be considered as naive on the basis of their origin, their phenotype and their functional properties. Msterlalsand Methods:Cord blood samples were obtained from healthy, full-term newborns after normal vaginal delivery and leucocyte-rich huffy coats were obtained from healthy adult blood donors. The levels of IL-2 were determined using a commercial ELISA IL-2 kit. Intracellular reacttve oxygen intermediates (ROls) were measured using fluorescent DCHF, which could be detected by flow cytometer. Activation of transcription factors were analysed using electrophoretic mobility shift assay (EMSA). Result% CD3/CD28 stimulation increased IL-2 production in adult and in neonatal T cells, but neonatal T cells produced much lower amounts of IL-2. However, the adult naive T cells (CD45RA) and the adult memoryleffector T cells (CD45RO) produced equal amounts of IL-2 after CD3JCD28 stimulation. CD3/CD28 costimulatfon induced the production of ROls in neonatal and adult T cells, but the formation of ROls was stronger in adult T cells. Also CDtiCD28 increased activation of AP-1 and NF-kB in both groups, but the levels of transcription factors were lower in newborn cells compared to those of adult cells. Conclusions:Although CD3/CD28 stimulation enhanced IL-2 production, ROI formation and activation of NF-kB and AP-1 in neonatal T cells, levels remained lower. Results suggest that neonatal T cells are at different stags of development and differentiation, and also their activation requirements may differ.
P.l.01.21 CD28 positive avlan y6 T cells K. Koskela, P. Arstila, 0. Lassila. Turku Immunobgy Centre and Department of Medical Microbiollog)! Turku University, Turku, Finland
Introduction:Activation of y8 T cells is poorly understood. Major part of our knowledge in T cell activation is based on UP T cell studies. In the chicken yS T cell activation is shown to be dependent on CD4+ a/l T cells and cytokines produced by them. Avian CD28 is expressed and capable of giving costimulatory signal to (YBT cells. We have studied the function of CD28 molecule in yS T cells. Mefariels and Methods:Phenotype of avian peripheral mononuclear cells was determined by triple immunofluorescence and flowcytometry. Enriched yS T cell subset was obtained by depletion of a,9 T cells with magnetic beads. Further CD28 positive cells were depleted. These cells were stimulated with PMA and anti-CD28 mAb. In antigen specific stimulations PBLs of CFA primed chickens were stimulated with either MT (mycobacterium tuberculosis sonicate) or PPD (mycobacterial purified protein derivative). Reeuftaz Most avian peripheral y6 Tcells do not express CD28 costimulatory molecule. We have found a small CD28+ yS T cell subset that expressed CD8 and CD5 at higher level than CD28 negative cells. yS T cells responded to PMA and anti-CD28 costimulation but when CD28+ cells were removed all responsiveness disappears. This emphasises the importance of the CD28+ yS T cells. We tested whether this CD26+ y6 T cell subpcpulation becomes independent from up T cells during antigen specific activation. Our results revealed that the activation of both CD28+ and CD28- yS T cells was entirely dependent on the presence of UP T cells. Conclusion:Although avian CD26+ yS T cells can receive costimulatory signal through CD28 the results indicate a central role for CD4+ @J T cells in the physiological function of all y6 T cell subsets. P.l.01.22 Co-etlmulatory effect In T cell prollferatlon Induced by oxldleed LDL Ruihua Wu, Ann Kari Lefvert, Johan Frostegard. Medicine of Dept. Karolinska Hospifal, Stockhofm, Sweden LDL may undergo oxidatively modified in vtvo. Oxidised LDL is immunogenic and correlated closely to development of atherosclerosis. Oxidised LDL can stimulate human peripheral T cells to increased DNA synthesis. This process depended on antigen presenting cells (APC) and is restricted by MHC class II molecule. In this study, we have measured signal transduction of oxidised
blocking agents, T cell proliferation was measured by thymidine incorporation, cytokine productions were measured with ELISA, and generation of cytotoxic T cell activity was measured with standard chromium release assay. Reaultr: Anti-CD86 mAb or e&CD40 mAb alone added during the primary MLR induced hyporesponsiveness. The combination of anti-CD86 and CsA induced nonresponslveness as published previously. When stimulated with allogeneic PBMC in the presence of a combination of blocking anti-CD46 mAb and anti-CD86 rnAb, T cell activation was completely blocked and T cells entered a state of nonresponsiveness as evidenced upon allogeneic restimulation. Addition of anti-CD86 mAb did not significantly after the results, and the combination of anti-CD80 and an&CD86 were also insufficient to induce nortresponslveness. Conclusion: The cooperative effect of blocking the CD46-CD46L interaction with antiCD66, is probably based upon prevention of expression of CD60 on APC. Other costimulatory APC function might also be blocked as evidenced by the fact that anti-CD86 + anti-CD46 was more effllient than anti-CD66 + anti-CD86 The combination of anti-CD40 mAb and anti-CD66 mAb offers a new strategy for immunosuppression and induction of nonresponsiveness.
P.l.Ol.18 Cyclosporin-A enhances IFN-y, IL-5 and IL-13 production by T cells costimulated through CD28 Khadija Rafiq, Stefaan W. Van Gool, Ahmad Kasran, Xiaohui Peng, Jan L. Ceuppens. Laborafory of Experimental /mmuno/cgK Catholic Universify of Leuven, Leuven, Belgium Introduction: The CD80/CDB&CD28 interaction provides a costimulatory signal for cyclospotinA (C&)-resistant IL-2 production and generation of cytotoxic T cell activity. We studied whether CsA differentially affects lhl- and Th2-type cytokine secretion by T cells when costimulated through CD28. Msterlalsand Methods:Anti-CD3 mAb was cross-linked on culture plates or on FcR-expressing P815 cells. Astimulating anti-CD28 mAb or CD86 (CD86), transfected into P815 cells, provided the costimulatory signal. IL-2, 11-4, IL-5 IL-lo, IL-13 and IFN-y were measured by ELISA. Raau~ Similar to previous reports, IL-2 production was hardly affected by CsA in these stimulating conditions. The productions of IL4 and (to a lesser extent) of IL-10 were significantly blocked. On the contrary, the production of IFN-y, IL-5 and IL-13 was markedly enhanced by CsA. By using combinations of PMA with anti-CD3 andlor anti-CD28 costlmulation, we could show that the effect of CsA on IL-5 IL-13 and IFNy production only occurred when both CD3 and CD28 were also triggered. Moreover, 11-5,IL-13 and IFN-y production could be inhibited by blocking IL-2 and its receptor. The CD4(+) subpopulation was responsible for the phenomena observed. Conclusion:CsA facilitates IFN-)I, IL-5 and IL-13 production by CD4(+) T cells. This suggests the existence of a negative regulatory signal which is CD3-dependent and calcium-dependent. It also indicates diierent regulatory pathways for two groups of Th2 cytokines: IL-4 and IL-10 on the one hand, and IL-13 and IL-5 on the other hand. This finding has major implications for immunosuppressive strategies based upon the use of CsA.
P.l.Ol.19 Clonlng of bovine CD28, CTLA4 and their ligands K.R. Parsons, C.J. Howard. lnsfifufe forAnimalHealth, Compton, UK When processed antigen is recognized in association with major histocompatibility antigen complex molecules on an antigen presenting cell (APC) by the T cell receptor (TCR) secondary costimulatory signals are required to initiate cytokine synthesis and T cell proliferation. Without the signal, from this secondary accessory molecule, stimulation via the TCR can render T cells anergic. CD80 and CD66, present on APC, interact with Tcells providing a costtmulatory signal that initiates an immune response through CD28 or terminates the response via CTLA4. The ability to enhance or suppress immune responses by manipulation of this costimulatory pathway makes these molecules a target for immune intervention. To enable manipulation of immune responses in cattle the objective of this report was to clone, sequence and express bovine CD28, CTLA-4 and their ligands. CD28 and CTLA-4 genes have been cloned by reverse-transcription polymerase chain reaction (RT-PCR) from a concanavalin-A activated peripheral blood leucocyte cDNA library, using primers based on the human sequences. Human CD86 has been shown to bind to both bovine molecules when expressed in transfected COS-7 cells. The homology between CD86 and CD86, and their analogues in other species is low and attempts to clone the genes encoding these molecules by RT-PCR has been unsuccessful. However, chimeric fusion proteins of mouse and human CTLA4 cross-react with the corresponding ligands in the other species. Mouse CTLA4-Ig has been used to identify cDNA clones encoding bovine CD60 from libraries constructed in pCDM6 and expressed in COS-7 cells. All three bovine cDNAs have been sequenced and chime&. fusion proteins have been produced of bovine CTLA-4 and CD86, which bind to their ligands. Studies of the distribution and function compared with analogues in other species using PCR, chimeric fusion proteins and transfected cell lines will be presented.
Co-stimulation
55
P.l.01.20 CD3KD28 actlvatlon of human naive neonatal T n cells S. Kilpinen, M. Hurme. Depattmenf of Microbiology and fmmunof~, University of Tampere Me&al School, POB 607, F/N-33101, Tampwe, Finland
Introduction:Optimal activation of T cells results in increased production of IL-2, enhanced cell proliferation and an effective immune response. To become optimally activated T cells require costimulatory signals in addition to the signal provided via the CD3/TCR for antigen. CD26 appears to be an important costimulatory signal receptor on T cells. The biochemical events foflowtng this CD28 ligation are still poorly known but there is some evidence that the CD28 costimulatory pathway could induce the production of reactive oxygen intermediates (ROls). We wanted to analyse IL-2 production, possible ROls formation and activation of transcription factors after CD3/CD28 stimulation in neonatal and adult T cells. Neonatal T cells may be considered as naive on the basis of their origin, their phenotype and their functional properties. Msterlalsand Methods:Cord blood samples were obtained from healthy, full-term newborns after normal vaginal delivery and leucocyte-rich huffy coats were obtained from healthy adult blood donors. The levels of IL-2 were determined using a commercial ELISA IL-2 kit. Intracellular reacttve oxygen intermediates (ROls) were measured using fluorescent DCHF, which could be detected by flow cytometer. Activation of transcription factors were analysed using electrophoretic mobility shift assay (EMSA). Result% CD3/CD28 stimulation increased IL-2 production in adult and in neonatal T cells, but neonatal T cells produced much lower amounts of IL-2. However, the adult naive T cells (CD45RA) and the adult memoryleffector T cells (CD45RO) produced equal amounts of IL-2 after CD3JCD28 stimulation. CD3/CD28 costimulatfon induced the production of ROls in neonatal and adult T cells, but the formation of ROls was stronger in adult T cells. Also CDtiCD28 increased activation of AP-1 and NF-kB in both groups, but the levels of transcription factors were lower in newborn cells compared to those of adult cells. Conclusions:Although CD3/CD28 stimulation enhanced IL-2 production, ROI formation and activation of NF-kB and AP-1 in neonatal T cells, levels remained lower. Results suggest that neonatal T cells are at different stags of development and differentiation, and also their activation requirements may differ.
P.l.01.21 CD28 positive avlan y6 T cells K. Koskela, P. Arstila, 0. Lassila. Turku Immunobgy Centre and Department of Medical Microbiollog)! Turku University, Turku, Finland
Introduction:Activation of y8 T cells is poorly understood. Major part of our knowledge in T cell activation is based on UP T cell studies. In the chicken yS T cell activation is shown to be dependent on CD4+ a/l T cells and cytokines produced by them. Avian CD28 is expressed and capable of giving costimulatory signal to (YBT cells. We have studied the function of CD28 molecule in yS T cells. Mefariels and Methods:Phenotype of avian peripheral mononuclear cells was determined by triple immunofluorescence and flowcytometry. Enriched yS T cell subset was obtained by depletion of a,9 T cells with magnetic beads. Further CD28 positive cells were depleted. These cells were stimulated with PMA and anti-CD28 mAb. In antigen specific stimulations PBLs of CFA primed chickens were stimulated with either MT (mycobacterium tuberculosis sonicate) or PPD (mycobacterial purified protein derivative). Reeuftaz Most avian peripheral y6 Tcells do not express CD28 costimulatory molecule. We have found a small CD28+ yS T cell subset that expressed CD8 and CD5 at higher level than CD28 negative cells. yS T cells responded to PMA and anti-CD28 costimulation but when CD28+ cells were removed all responsiveness disappears. This emphasises the importance of the CD28+ yS T cells. We tested whether this CD26+ y6 T cell subpcpulation becomes independent from up T cells during antigen specific activation. Our results revealed that the activation of both CD28+ and CD28- yS T cells was entirely dependent on the presence of UP T cells. Conclusion:Although avian CD26+ yS T cells can receive costimulatory signal through CD28 the results indicate a central role for CD4+ @J T cells in the physiological function of all y6 T cell subsets. P.l.01.22 Co-etlmulatory effect In T cell prollferatlon Induced by oxldleed LDL Ruihua Wu, Ann Kari Lefvert, Johan Frostegard. Medicine of Dept. Karolinska Hospifal, Stockhofm, Sweden LDL may undergo oxidatively modified in vtvo. Oxidised LDL is immunogenic and correlated closely to development of atherosclerosis. Oxidised LDL can stimulate human peripheral T cells to increased DNA synthesis. This process depended on antigen presenting cells (APC) and is restricted by MHC class II molecule. In this study, we have measured signal transduction of oxidised