- Email: [email protected]
Expression of CD80 and CD86 on CD28+ and CD28− T cells: A role in clonal expansion?
52
Co-stimulation
1P.l .01.05 1 Analysis of the role of ICAM- as a costlmulatory molecule through a cell model: CAMY-1 and CAMY-2 (two C%50-negative Jurkat-T cell clones) C. Vilardell, M. Juan l, A. Miralles, E. Palou, J. Esparza. R. Vilella, F. Lozano, J. Vives, A. Gaya, J. Yagiie. Servei d’lmmunologia, Hospital Clinic, Barcelona, Spain, ’ Unitat d’lmmunologia, Hospital Germans Tdas i Pujol. Badalona, Spain Introduction: CD50 (or ICAM-3) is a surface glycoprotein of 120 KD with an expression almost restricted to leucocytes. Its role in initial interactions of immune recognition is supported by the fact that is the only ICAM molecule ready to bind LFA-1 before any lymphocyte activation happens. Moreover ICAM- has been described as a signal transduction molecule in T cells and its cytoplasmic association with p56rCkand ~59~ suggests an important role in T cell activation. To characterize the main elements responsibles for signal transduction and their molecular association is our main goal. To better analyse the role of CD50 as a costimulatory molecule, we have obtained two CDSO-negative Jurkat T cell clones, which can be used as a host to transfect different CD50 constructs and study their specific function through the transduction pathway. Materiala and Methods:These clones, named CAMY-1 and CAMY-2, have been obtained by cell sorting and limiting dilution. They have been characterized for their expression of several surface markers such as CD3, CD54, CD102, CD45, LFA-1, CD69 and CD50 among others. To confirm this CD60 lack of surface expression, we studied the content of m-RNA by Northern Blot and FIT-PCR.Calcium assays allowed us to analyse their ability for activation under several stimuli. Likewise, CD69 induction after stimulation with co-immobilized mAbs (antiCD3 + antiCD50) was another parameter tested. Results: CAMY-1 and CAMY-2 show no significant differences by phenotypical analysis, but their lack of CD50 expression, respect to wild type line Jurkat. They have an undetectable level of m-RNA by Northern Blot and we only detect it after 30 amplification cycles of PCR. Neither they present calcium mobilization after CD50 cross-linking or CD69 induction after CD3 more CD50 cross-linking. By contrast, CD50 crosslinking is able to induce calcium mobilization in CAMY cells transfected with wild type cDNA. An effect previously described in Jurkat T cell line. Also, incubatkm of CAMY cells transfected with ICAMB wild type with co-immobilized OKT-3 + antiCD50 mAb is able to induce a remarkable increase in CD69 expression. Conclusion: 1) Both clones, CAMY-I and CAMY-2, seems to be convtnient experimental models to analyse CD50 role. 2) Preliminary analysis have shown that CAMY transfection with wildtype CD50 restores ICAM- signaling.
P.l .01.06
Expression of CD80 and CD88 on CD28+ and CD28- T cells: A role In clonal expansion?
P. Romero ‘, C. Ortega ‘, F. Garcia-C6zar2, M.D. Gallego 3, J. Peria ‘, I. Molina 3, M. Santamaria ‘. ’ Dpt. of tmmunobg~ Universiiy of Co&b, Spain, 2Center for Blood Research, Harvard University, USA, 3Dpt. of Biochemistry and Molecular Biolog) University of Granada, Spain Complete activation of CD26+ T cells via CD~ITCR molecular complex requires engagement of CD26 molecules by its ligand(s), CD6OICD66 expressed on the surface of antigen presenting cells, which induces a major costimulatory pathway. In the absence of such costimulatory signals, occupancy of the TCR atone can induce anergy or apoptosis in CD26+ cells. After productive activation both, CD80 and CD66 appears on the T cell membrane, and together to HLA Class II expression, provides activated T cells with APC main phenotypical features. However, their role on Tcells remains unknown. We have investigated the expression and it possible significance of CD60 and CD66 on whole T cell population, CD26+ cells and CD28- cells upon activation with anti-CD3 mAbs. Expression time-course, subpopulation distribution and the influence of distinct cytoquines, including, gamma-IFN, 11-4, IL-7, IL-10 and 11-12, CD28+ was analyzed. Results show a differential kinetics of expression between CD80 and CD86 and when comparing CD4+ cells and CD8+ cells. After 1Odin culture, a number of T cells are oositive for both CD80 and CD86 but prooression of culture up to 30 days reveals that CD60 is mainly express on CDh+ c&s, either, CD28+ or CD28-. Antigen-driven expertments canted out with enriched naive T cells from umbilical cord, indicates that functional interactions of CD80/CD86 expressed on T cells are an important requirement for T cell to respond to antigen, since inhibition of natural interactions of CD8O/CD66 expressed on activated T cells limits their expansion, even in the presence of nominal antigen. These and further results will be discussed.
23 June 1997 - Poster presentations and thereby inducing the dysfunction of the transplanted organ. Our project examines, in which way the biliary epithelial cells (BEC) induce a immune response of allogenic lymphocytes. In our established in-vitremodel of liver transplant rejection we use epithelial cells from gallbladders (GBEZ) instead of intrahepatic BEC. Materials and Methods:GBEZ are isolated from gallbladders after routine cholezystektomies. For immunogenic staining we use FITC-labeled CD4, CD8 and HtA monoclonal antibodies. Proliferation of lymphocytes were examined by thymidin-incorporation. To analyse, if specific costimulatory factors like CD80 or CD66 play a role in immunologic activation, we stain for this markers with monoclonal antibodies. Subsequently we try to detect mRNA of costimulatory factors by reverse transcription-polymerase chain reaction analysis (FtT-PCR). To Investigate, if exogenic proteins like tetanus toxoid or CMV-peptides are presented on cell surfaces we use monoclonal antibodies to stain peptide segments. Resutts: GBEZ induces a significant proliferation of allogenic T cells. Blocking studies show, that the reaction of PBL which are preincubated with antibodies against CD8 will reduce proliferation. We find out, that GBEZ express costimulatory factors. Furthermore CD80 mRNA expression was detected by RT-PCR analysis. Freshly isolated GBEZ show no mRNA expression, although CD60 mRNA was detected in cultured cells. First results of protein incorporation show, that GBEZ are able to take up and proceed exogenlc proteins and present them on the epithelial cell surfaces. Conclusion: Epithelial cells show the clinically relevant ability to induce allogenic lymphocyte proliferation. The improvement of CD80 and CD86 indicates, that beside immunologic activation through HLA, other factors will be of importance. The ability to present exogenic peptides, like tetanus toxoid or CMVpeptides, may be also from interest for clinical examinations in liver transplant rejection.
P.l .01.08
C. Pioli, S. Pucci, S. Barile, P. Barattini, C. Goso, D. Frasca, G. Doria. Laboratory of Immunobg~ AMB-PRO-TOSS, ENEA, Rome, Italy
Introduction:Optimal activation of antigen-induced T cell proliferation and cytokine production requires the engagement of co-receptors. As Tcell functions change with age, cytokine production was studied in purified CD4 cells from aging mice upon CD3ICD28 stimulation. Materialsand Methods:CD4 cells from young (3 mos) and old (19 mos) mice were purified by magnetic microbead cell sorting and stimulated by platebound anti-CD3 mAb (145-2Cll) alone or also by soluble anti-CD28 mAb (37.51). At different times of culture, cytokines were measured in supematants by ELISA. Total RNA was extracted from cells, each sample was reverse-transcribed and the cDNA was amplified by PCR. To evaluate the mRNA turn-over, mRNA synthesis was blocked by actinomycin D. Resub It was found that anti-CD3 alone is not sufficient to induce IL2 production in CD4 cells from both young and old mice. However, anti-CD28 mAb, together with anti-CD3 mAb, induces a much higher production of I12 in CD4 cells from young as compared to old mice. Conversely. IFN-y production is also induced by anti-CD3 mAb alone and is higher in CD4 cells from dd as compared to young mice. Upon addition of anti-CD28 mAb, IFN-y production increases in both groups, but it remains much higher in old than in young mice. Also the production of IL4 and IL10 is induced by anti-CD3 mAb alone but it is increased by the addition of anti-CD28 mAb. CD4 cells from old mice produce more IL4 and IL10 as compared to cells from young mice, the kinetics of both cytokines being slower than that of I12 and IFN-y at both ages. The analysis of the mRNA expression in CD4 cells from young and old mice, stimulated by anti-CD3 mAb alone or costimulated by anti-CD28 mAb, conftrms the findings observed at the protein level. Moreover, when CD4 cells are stimulated by anti-CD3 mAb alone or costimulated also by anti-CD28 mAb, the IFN-y, IL4 and IL10 specific mRNAs from old mice are more stable than those from vouna mice. Conduslon:CD4cells from young and old-micgexhibit a different pattern of cytokine production, at both the protein and mRNA levels, when CD3stimulated and CDPI-costimulated. As to 114, IL10 and IFN-y, the patterns are consistent with the differences found in mRNA turn-over between CD4 cells from young and dd mice.
P.l .01.09 P.l .01.07
Functional lnvestlgatlons on the Immunecompetence of blllary eplthellal cells
S. Weber, A. GooB, D. Schleicher, M. Scholz, R.A. Blaheta, A.E. Encke, B.H. Markus. JW Goethe-Universi& Dept. of Geneml Surgery; FrankfwVMain, Germany
Introduction: The biliary epithelium is one of the main targets of immunologically mediated rejection after liver transplantation. The epithelium is destroied
Cytoklne production In CD4 cells from aglng mice as Induced by CD28 costlmulatlon
Expression and costlmulatory capacity of CTLA4 llgands on several dlfferent murlne cells
Gl6rta Ldszld, L&z16 Cervenak. Department of Immunology; L. EdtvvdsUniv G&t, Hungary Successful T cell activation requires at least two independent signals provided bv APC. The 1st sianal is mediated via the TCR after the soecific bindina oi MHC/peptide co;plex. The 2nd signal is delivered through CD28. Twi CD28/CTlA-4 ligands [87-l and 87-21 have already been identified in both humans and mice and a 3rd 87 molecule has also been suggested in humans.
Co-stimulation
1P.l .01.05 1 Analysis of the role of ICAM- as a costlmulatory molecule through a cell model: CAMY-1 and CAMY-2 (two C%50-negative Jurkat-T cell clones) C. Vilardell, M. Juan l, A. Miralles, E. Palou, J. Esparza. R. Vilella, F. Lozano, J. Vives, A. Gaya, J. Yagiie. Servei d’lmmunologia, Hospital Clinic, Barcelona, Spain, ’ Unitat d’lmmunologia, Hospital Germans Tdas i Pujol. Badalona, Spain Introduction: CD50 (or ICAM-3) is a surface glycoprotein of 120 KD with an expression almost restricted to leucocytes. Its role in initial interactions of immune recognition is supported by the fact that is the only ICAM molecule ready to bind LFA-1 before any lymphocyte activation happens. Moreover ICAM- has been described as a signal transduction molecule in T cells and its cytoplasmic association with p56rCkand ~59~ suggests an important role in T cell activation. To characterize the main elements responsibles for signal transduction and their molecular association is our main goal. To better analyse the role of CD50 as a costimulatory molecule, we have obtained two CDSO-negative Jurkat T cell clones, which can be used as a host to transfect different CD50 constructs and study their specific function through the transduction pathway. Materiala and Methods:These clones, named CAMY-1 and CAMY-2, have been obtained by cell sorting and limiting dilution. They have been characterized for their expression of several surface markers such as CD3, CD54, CD102, CD45, LFA-1, CD69 and CD50 among others. To confirm this CD60 lack of surface expression, we studied the content of m-RNA by Northern Blot and FIT-PCR.Calcium assays allowed us to analyse their ability for activation under several stimuli. Likewise, CD69 induction after stimulation with co-immobilized mAbs (antiCD3 + antiCD50) was another parameter tested. Results: CAMY-1 and CAMY-2 show no significant differences by phenotypical analysis, but their lack of CD50 expression, respect to wild type line Jurkat. They have an undetectable level of m-RNA by Northern Blot and we only detect it after 30 amplification cycles of PCR. Neither they present calcium mobilization after CD50 cross-linking or CD69 induction after CD3 more CD50 cross-linking. By contrast, CD50 crosslinking is able to induce calcium mobilization in CAMY cells transfected with wild type cDNA. An effect previously described in Jurkat T cell line. Also, incubatkm of CAMY cells transfected with ICAMB wild type with co-immobilized OKT-3 + antiCD50 mAb is able to induce a remarkable increase in CD69 expression. Conclusion: 1) Both clones, CAMY-I and CAMY-2, seems to be convtnient experimental models to analyse CD50 role. 2) Preliminary analysis have shown that CAMY transfection with wildtype CD50 restores ICAM- signaling.
P.l .01.06
Expression of CD80 and CD88 on CD28+ and CD28- T cells: A role In clonal expansion?
P. Romero ‘, C. Ortega ‘, F. Garcia-C6zar2, M.D. Gallego 3, J. Peria ‘, I. Molina 3, M. Santamaria ‘. ’ Dpt. of tmmunobg~ Universiiy of Co&b, Spain, 2Center for Blood Research, Harvard University, USA, 3Dpt. of Biochemistry and Molecular Biolog) University of Granada, Spain Complete activation of CD26+ T cells via CD~ITCR molecular complex requires engagement of CD26 molecules by its ligand(s), CD6OICD66 expressed on the surface of antigen presenting cells, which induces a major costimulatory pathway. In the absence of such costimulatory signals, occupancy of the TCR atone can induce anergy or apoptosis in CD26+ cells. After productive activation both, CD80 and CD66 appears on the T cell membrane, and together to HLA Class II expression, provides activated T cells with APC main phenotypical features. However, their role on Tcells remains unknown. We have investigated the expression and it possible significance of CD60 and CD66 on whole T cell population, CD26+ cells and CD28- cells upon activation with anti-CD3 mAbs. Expression time-course, subpopulation distribution and the influence of distinct cytoquines, including, gamma-IFN, 11-4, IL-7, IL-10 and 11-12, CD28+ was analyzed. Results show a differential kinetics of expression between CD80 and CD86 and when comparing CD4+ cells and CD8+ cells. After 1Odin culture, a number of T cells are oositive for both CD80 and CD86 but prooression of culture up to 30 days reveals that CD60 is mainly express on CDh+ c&s, either, CD28+ or CD28-. Antigen-driven expertments canted out with enriched naive T cells from umbilical cord, indicates that functional interactions of CD80/CD86 expressed on T cells are an important requirement for T cell to respond to antigen, since inhibition of natural interactions of CD8O/CD66 expressed on activated T cells limits their expansion, even in the presence of nominal antigen. These and further results will be discussed.
23 June 1997 - Poster presentations and thereby inducing the dysfunction of the transplanted organ. Our project examines, in which way the biliary epithelial cells (BEC) induce a immune response of allogenic lymphocytes. In our established in-vitremodel of liver transplant rejection we use epithelial cells from gallbladders (GBEZ) instead of intrahepatic BEC. Materials and Methods:GBEZ are isolated from gallbladders after routine cholezystektomies. For immunogenic staining we use FITC-labeled CD4, CD8 and HtA monoclonal antibodies. Proliferation of lymphocytes were examined by thymidin-incorporation. To analyse, if specific costimulatory factors like CD80 or CD66 play a role in immunologic activation, we stain for this markers with monoclonal antibodies. Subsequently we try to detect mRNA of costimulatory factors by reverse transcription-polymerase chain reaction analysis (FtT-PCR). To Investigate, if exogenic proteins like tetanus toxoid or CMV-peptides are presented on cell surfaces we use monoclonal antibodies to stain peptide segments. Resutts: GBEZ induces a significant proliferation of allogenic T cells. Blocking studies show, that the reaction of PBL which are preincubated with antibodies against CD8 will reduce proliferation. We find out, that GBEZ express costimulatory factors. Furthermore CD80 mRNA expression was detected by RT-PCR analysis. Freshly isolated GBEZ show no mRNA expression, although CD60 mRNA was detected in cultured cells. First results of protein incorporation show, that GBEZ are able to take up and proceed exogenlc proteins and present them on the epithelial cell surfaces. Conclusion: Epithelial cells show the clinically relevant ability to induce allogenic lymphocyte proliferation. The improvement of CD80 and CD86 indicates, that beside immunologic activation through HLA, other factors will be of importance. The ability to present exogenic peptides, like tetanus toxoid or CMVpeptides, may be also from interest for clinical examinations in liver transplant rejection.
P.l .01.08
C. Pioli, S. Pucci, S. Barile, P. Barattini, C. Goso, D. Frasca, G. Doria. Laboratory of Immunobg~ AMB-PRO-TOSS, ENEA, Rome, Italy
Introduction:Optimal activation of antigen-induced T cell proliferation and cytokine production requires the engagement of co-receptors. As Tcell functions change with age, cytokine production was studied in purified CD4 cells from aging mice upon CD3ICD28 stimulation. Materialsand Methods:CD4 cells from young (3 mos) and old (19 mos) mice were purified by magnetic microbead cell sorting and stimulated by platebound anti-CD3 mAb (145-2Cll) alone or also by soluble anti-CD28 mAb (37.51). At different times of culture, cytokines were measured in supematants by ELISA. Total RNA was extracted from cells, each sample was reverse-transcribed and the cDNA was amplified by PCR. To evaluate the mRNA turn-over, mRNA synthesis was blocked by actinomycin D. Resub It was found that anti-CD3 alone is not sufficient to induce IL2 production in CD4 cells from both young and old mice. However, anti-CD28 mAb, together with anti-CD3 mAb, induces a much higher production of I12 in CD4 cells from young as compared to old mice. Conversely. IFN-y production is also induced by anti-CD3 mAb alone and is higher in CD4 cells from dd as compared to young mice. Upon addition of anti-CD28 mAb, IFN-y production increases in both groups, but it remains much higher in old than in young mice. Also the production of IL4 and IL10 is induced by anti-CD3 mAb alone but it is increased by the addition of anti-CD28 mAb. CD4 cells from old mice produce more IL4 and IL10 as compared to cells from young mice, the kinetics of both cytokines being slower than that of I12 and IFN-y at both ages. The analysis of the mRNA expression in CD4 cells from young and old mice, stimulated by anti-CD3 mAb alone or costimulated by anti-CD28 mAb, conftrms the findings observed at the protein level. Moreover, when CD4 cells are stimulated by anti-CD3 mAb alone or costimulated also by anti-CD28 mAb, the IFN-y, IL4 and IL10 specific mRNAs from old mice are more stable than those from vouna mice. Conduslon:CD4cells from young and old-micgexhibit a different pattern of cytokine production, at both the protein and mRNA levels, when CD3stimulated and CDPI-costimulated. As to 114, IL10 and IFN-y, the patterns are consistent with the differences found in mRNA turn-over between CD4 cells from young and dd mice.
P.l .01.09 P.l .01.07
Functional lnvestlgatlons on the Immunecompetence of blllary eplthellal cells
S. Weber, A. GooB, D. Schleicher, M. Scholz, R.A. Blaheta, A.E. Encke, B.H. Markus. JW Goethe-Universi& Dept. of Geneml Surgery; FrankfwVMain, Germany
Introduction: The biliary epithelium is one of the main targets of immunologically mediated rejection after liver transplantation. The epithelium is destroied
Cytoklne production In CD4 cells from aglng mice as Induced by CD28 costlmulatlon
Expression and costlmulatory capacity of CTLA4 llgands on several dlfferent murlne cells
Gl6rta Ldszld, L&z16 Cervenak. Department of Immunology; L. EdtvvdsUniv G&t, Hungary Successful T cell activation requires at least two independent signals provided bv APC. The 1st sianal is mediated via the TCR after the soecific bindina oi MHC/peptide co;plex. The 2nd signal is delivered through CD28. Twi CD28/CTlA-4 ligands [87-l and 87-21 have already been identified in both humans and mice and a 3rd 87 molecule has also been suggested in humans.