- Email: [email protected]
CD26 enzymatic activity abrogates accelerated rejection of rat cardiac allografts
25 June 1997- Poster presentations
Novel immunosuppressives and xenotransplantation
also demonstrate that this is not due to general hyporesponsivaness in the high grade RA subset.
09:00-18:30112:00-14:00
Forum lounges
P.5.23 Novel immunosuppressives and xenotransplantation I P.5.23.01 I M l t o g e n l c i t y of anti-TCR/CD3 m A b s attributable to ineffective c r o s s - l i n k i n g of low-affinity Fc~ receptors J. Pischei 1, U. Zengerie 1, T. Takai 2, R. Mailhammer 3 S. Thierfelder 1, U. Kummer 1.1GSF-Institut fOr Immunologie, Marchioninistr. 25, 81377 MOnchen, FRG, 2Department of Biotechnology, Faculty of Engineering, Okayama Universi~ Tsushima-Naka 3-1-1, Okayama 700, Japan, 3GSF-Institut for Experimentelle Hdmatologie, Marchioninistr. 25, 81377 MOnchen, FRG Introduction: OKT3, a mAb reacting with the CD3 complex, has proved effeotive for immunosuppression in clinical practice for about 10 years. However, parallel with this beneficial effect, toxic effects which can result in significant morbidity have been reported upon initial treatment of the patients. In mice, anti-CD3 therapy has been mainly based on the use of the hamster anti-CD3 mAb 145-2Cll which shares many properties with OKT3. In this context little attention has been given to the therapeutic and/or toxic profile of anti-CD3 isotypas different from 145-2Cll. Materials end Methods: In this study we have compared the effects of in vivo administration of 145-2Cll and 17A2 (a rat IgG2b anti-mouse CD3 mAb) on several parameters, including TCR/CD3 modulation, T cell depletion, T cell activation and activation-induced apoptosis. We have extended these in vivo expedmants using mice genetically deficient in FcyRII. The strength of antl-CD3 mAb binding to low-affinity FcyR has been quantitated in an anti-CD3-induced T cell proliferation assay using the Fc~,RII/lll-blocking mAb 2.4G2. Results: We have found that both anti-CD3 mAbs have similar kinetics of TCR/CD3 down-regulation, albeit 145-2Cll was significantly more efficient in this respect. In addition, 17A2 exhibits depleting properties, whereas 145-2Cll tdggers a complete signal of cellular activation which subsequently leads to a massive expansion of the T cell pool followed by a marked T cell depletion due to apoptosis. The depleting and mitoganic properties of 17A2 and 145-2Cll, respectively, are both dependent upon interaction with low-affinity FcyR and could be totally abrogated by administration of mAb 2.4G2. However, FcyRII/lll blockade by 2.4G2 induced only a partial inhibition of 17A2 induced IL-2 production of T cells in vitro. In contrast, in an analogous in vitro expedment 2.4G2 completely neutralized the ability of 145-2Cll to tdgger IL-2 secretion. Furthermore, we have also observed that 17A2 and 145-2Cll retain their mechanisms of action in FcyRII KO-mico. Conclusion: Taken together, these results suggest that the number of productive contacts between antibody-coated target cells and FcyR-bearing effector cells significantly impacts on the nature of the immunosuppression of anti-CD3 mAbs. In effect, nonmitogenic anti-CD3 mAbs with a high affinity for low-affinity FcyR may be effectively trapped on effector cells and therefore directly removed from the circulation. In contrast, the mitoganic properties of anti-CD3 mAbs may be a result of their low FcyR binding affinities. The results and conclusions may open major therapeutic perspectives for the human situation. First, the immunosuppressive effects of nonmitoganic anti-CD3 mAbs may be as potent as those of mitogenic anti-CD3 mAbs though without the risk of severe side effects. Second, mitoganic anti-CD3 mAbs might be exploited clinically to stimulate T cell function in immunocompromised states and to enhance hematopoiesis in myeiodysplastic disorders. 1 P.5.23.02 J Inhibition of DPP IV/CD26 e n z y m a t i c a c t i v i t y a b r o g a t e s a c c e l e r a t e d rejection of rat c a r d i a c allografta I. De Meester 1, S. Korom 2, T.H.W. Stadlbauer 2, F. Goossans 1, A. Haemers 1, J.W. Kupiec-Weglinski 2, S. Scharp61.1 Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium, 2Surgical Research Lab. Harvard Med. School, Dept of Surge~ Brigham and Women's Hospital, Boston, MA, USA Introduction: Dipeptidyl peptidase IV (EC 3.4.14.5) is an ecto-enzyme that occurs as an integral membrane protein on the cell surface. It cleaves off
487
dipeptides from the aminoterminus of pepfides with a proline, hydroxyproline or alanine at the penultimate position. DPP IV is identified as the activation antigen CD26, one of the major costimulatory molecules inveived in T cell activation. Although in vitro inhibitor-studies provide substantial evidence for a role of the catalytic activity in costimulation, this issue remains a matter of debate. Recently, we showed that in vivo inhibition of DPP IV abrogates acute cardiac allogreft rejection at day 7, and prolongs cardiac transplant (l"x) survival to 14.0 +/- 0.9 days (p < 0.0001). In this study we investigated the effect of DPP IV inhibition on the accelerated rejection in sensitized allograft recipients. Matedata and Methods: Following accelerated rejection model was used: LEW rats sensitized with BN skin Tx (day -7) receive LBNF1 cardiac Tx (transplanted at day 0). In untreated animals rejection occurs within 36 hr. Prodipine (Pro-Pro-diphenylphosphonate) was given sc 30 mg/ret at day -7, followed by 20 mg/rat at days -4, -1 and +4. AIIo-antibody responses were measured in the serum by flow cytometry. Results: In vivo targetting DPP IV activity with the low molecular weight specific inhibitor Prodipine reduced plasma DPP IV activities to <15% of pretreatment values. Treatment abrogated accelerated rejection. Cardiac Tx survival was prolonged in 7 out of 13 recipients (mean survival time +/-SD = 5.1 +/- 2.1 days, p < 0.01). Prodipine prevented systemic IgM allo-Ab responses and the effect was so endudng that the usual second IgM peak by day 21 post "Ix was not observed, despite the recovery of DPP IV (>70% of pretreatmant value) at that time. The effects of Prodipine treatment on serum cytokine levels are currently under investigation. Conclusion: Our findings on prolonged graft acceptance in sensitized transplant recipients (accelerated cardiac rejection model in the rat) upon DPP IV inhibition demonstrate the involvement of CD26/DPP IV in the in vivo immune response upon solid organ transplantation.
1:).5.23.03 1 Dualistic capacity of the novel immunosuppresslve drug m y c o p h e n o l a t e mofetll (CellCept ®) R.A. Blaheta 1, K. Leckel 1, B. Wittig 2, M. Scholz 1, D. Zenker 1, S. Weber 1, A. Encke 1, B.H. Markus 1.1j. W. Goethe-Universi~ Dept. Gen. Surge~ Frankfurt am Main, Germany, 1DKFZ, Heidelberg, Germany Introduction: Cellular rejection is charactedzed by 1) proliferation and differentiation of the recipient lymphocytes and 2) infiltration of the lymphocytas via the endothelium into the donor organ. Immunosuppressive drugs as Cyclospodne A or tacrolimus (FK 506) block cellular activation but have no effect on the infiltration process. The novel drug mycophenotate mofetil (MMF) effectively suppresses cell mitosis by blocking inosine-monophosphate-dehydrogenase (IMD), the key enzyme for the pudne biosynthesis. Because IMD additionally regulates the glycoprotein synthesis, some of which are adhesion molecules, an influence of MMF on the cellular infiltration process might also be possible. We therefore investigated in vitro, whether and in what way MMF interferes into the lymphocytic infiltration cascade. Materials and Methods: CD4 and CD8 T-lyrnphocytes were added to endothelial cells (EC), derived from human umbilical veins, together with MMF (0-100 /zM). Lymphocyte binding was analyzed by the monolayer invasion assay, expression of endothelial adhesion receptors by FACS measurement. Activity of the lymphocytic counter receptors in presence of MMF was evaluated by the adhesion spot assay, using isolated chimeric proteins containing the extrecallular domain of E-seleotin, P-selectin, ICAM-1 or VCAM-1. Horizontal locomotion of CD4 and CD8 T-ceils was investigated by videomicroscopy. Results: MMF dose-dependently suppressed loose (moderately) and firm (strongly) attachment of CD4 and CD8 T-lymphocytes to allogeneic EC. The effect was more pronounced when EC were pretreated with ~,-IFN, IL-1 or IL-I+TNF(~. Moreover, MMF-incubation of both lymphocytes and EC lead to a stronger reduction of cell binding than MMF-incubafion of either lymphocytes or EC. Fluorometdc analysis of the endothelial adhesion receptors ICAM-I, VCAM-I, E-selectin and P-salectin showed that MMF moderately influenced ICAM-I (optimal dosage: 1-10 /~M), VCAM-I (optimal dosage: 0.1-I /~M) and P-saleotin (optimal dosage: 0.1 /~M) expression but strongly reduced de novo synthesis of E-seleotin (0.001-10 /~M, p < 0.05). Invastigafion of the lymphocytic counter receptors by the adhesion spot assay demonstrated that MMF did not downregulate binding of CD4 or CD8 T-lymphocytes to E-salectin, but differentially suppressed the cellular interaction via ICAM-1, VCAM-1 or P-selectin proteins. Videomicroscopic analysis of the locomotory activity of CD4 and CD8 T-cells revealed an additional mode of action of MMF on the intmcellular cytoskeleton (optimal dosage: 1/~M). Concluelon: The results presented clearly demonstrate that MMF -in strong contrast to CsA or tacrolimus- blocks organ rejection in a dualistic way. Beside its antiproliterative effect MMF also possesses distinct suppressive properties with regard to the cellular infiltration process. The unique charaotedstice of MMF might allow new immunosuppresslve strategies. Fur~er experiments should evaluate the therapeutic value of MMF on other infiltration-dopendant processes such as tumor metastasis.
Novel immunosuppressives and xenotransplantation
also demonstrate that this is not due to general hyporesponsivaness in the high grade RA subset.
09:00-18:30112:00-14:00
Forum lounges
P.5.23 Novel immunosuppressives and xenotransplantation I P.5.23.01 I M l t o g e n l c i t y of anti-TCR/CD3 m A b s attributable to ineffective c r o s s - l i n k i n g of low-affinity Fc~ receptors J. Pischei 1, U. Zengerie 1, T. Takai 2, R. Mailhammer 3 S. Thierfelder 1, U. Kummer 1.1GSF-Institut fOr Immunologie, Marchioninistr. 25, 81377 MOnchen, FRG, 2Department of Biotechnology, Faculty of Engineering, Okayama Universi~ Tsushima-Naka 3-1-1, Okayama 700, Japan, 3GSF-Institut for Experimentelle Hdmatologie, Marchioninistr. 25, 81377 MOnchen, FRG Introduction: OKT3, a mAb reacting with the CD3 complex, has proved effeotive for immunosuppression in clinical practice for about 10 years. However, parallel with this beneficial effect, toxic effects which can result in significant morbidity have been reported upon initial treatment of the patients. In mice, anti-CD3 therapy has been mainly based on the use of the hamster anti-CD3 mAb 145-2Cll which shares many properties with OKT3. In this context little attention has been given to the therapeutic and/or toxic profile of anti-CD3 isotypas different from 145-2Cll. Materials end Methods: In this study we have compared the effects of in vivo administration of 145-2Cll and 17A2 (a rat IgG2b anti-mouse CD3 mAb) on several parameters, including TCR/CD3 modulation, T cell depletion, T cell activation and activation-induced apoptosis. We have extended these in vivo expedmants using mice genetically deficient in FcyRII. The strength of antl-CD3 mAb binding to low-affinity FcyR has been quantitated in an anti-CD3-induced T cell proliferation assay using the Fc~,RII/lll-blocking mAb 2.4G2. Results: We have found that both anti-CD3 mAbs have similar kinetics of TCR/CD3 down-regulation, albeit 145-2Cll was significantly more efficient in this respect. In addition, 17A2 exhibits depleting properties, whereas 145-2Cll tdggers a complete signal of cellular activation which subsequently leads to a massive expansion of the T cell pool followed by a marked T cell depletion due to apoptosis. The depleting and mitoganic properties of 17A2 and 145-2Cll, respectively, are both dependent upon interaction with low-affinity FcyR and could be totally abrogated by administration of mAb 2.4G2. However, FcyRII/lll blockade by 2.4G2 induced only a partial inhibition of 17A2 induced IL-2 production of T cells in vitro. In contrast, in an analogous in vitro expedment 2.4G2 completely neutralized the ability of 145-2Cll to tdgger IL-2 secretion. Furthermore, we have also observed that 17A2 and 145-2Cll retain their mechanisms of action in FcyRII KO-mico. Conclusion: Taken together, these results suggest that the number of productive contacts between antibody-coated target cells and FcyR-bearing effector cells significantly impacts on the nature of the immunosuppression of anti-CD3 mAbs. In effect, nonmitogenic anti-CD3 mAbs with a high affinity for low-affinity FcyR may be effectively trapped on effector cells and therefore directly removed from the circulation. In contrast, the mitoganic properties of anti-CD3 mAbs may be a result of their low FcyR binding affinities. The results and conclusions may open major therapeutic perspectives for the human situation. First, the immunosuppressive effects of nonmitoganic anti-CD3 mAbs may be as potent as those of mitogenic anti-CD3 mAbs though without the risk of severe side effects. Second, mitoganic anti-CD3 mAbs might be exploited clinically to stimulate T cell function in immunocompromised states and to enhance hematopoiesis in myeiodysplastic disorders. 1 P.5.23.02 J Inhibition of DPP IV/CD26 e n z y m a t i c a c t i v i t y a b r o g a t e s a c c e l e r a t e d rejection of rat c a r d i a c allografta I. De Meester 1, S. Korom 2, T.H.W. Stadlbauer 2, F. Goossans 1, A. Haemers 1, J.W. Kupiec-Weglinski 2, S. Scharp61.1 Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium, 2Surgical Research Lab. Harvard Med. School, Dept of Surge~ Brigham and Women's Hospital, Boston, MA, USA Introduction: Dipeptidyl peptidase IV (EC 3.4.14.5) is an ecto-enzyme that occurs as an integral membrane protein on the cell surface. It cleaves off
487
dipeptides from the aminoterminus of pepfides with a proline, hydroxyproline or alanine at the penultimate position. DPP IV is identified as the activation antigen CD26, one of the major costimulatory molecules inveived in T cell activation. Although in vitro inhibitor-studies provide substantial evidence for a role of the catalytic activity in costimulation, this issue remains a matter of debate. Recently, we showed that in vivo inhibition of DPP IV abrogates acute cardiac allogreft rejection at day 7, and prolongs cardiac transplant (l"x) survival to 14.0 +/- 0.9 days (p < 0.0001). In this study we investigated the effect of DPP IV inhibition on the accelerated rejection in sensitized allograft recipients. Matedata and Methods: Following accelerated rejection model was used: LEW rats sensitized with BN skin Tx (day -7) receive LBNF1 cardiac Tx (transplanted at day 0). In untreated animals rejection occurs within 36 hr. Prodipine (Pro-Pro-diphenylphosphonate) was given sc 30 mg/ret at day -7, followed by 20 mg/rat at days -4, -1 and +4. AIIo-antibody responses were measured in the serum by flow cytometry. Results: In vivo targetting DPP IV activity with the low molecular weight specific inhibitor Prodipine reduced plasma DPP IV activities to <15% of pretreatment values. Treatment abrogated accelerated rejection. Cardiac Tx survival was prolonged in 7 out of 13 recipients (mean survival time +/-SD = 5.1 +/- 2.1 days, p < 0.01). Prodipine prevented systemic IgM allo-Ab responses and the effect was so endudng that the usual second IgM peak by day 21 post "Ix was not observed, despite the recovery of DPP IV (>70% of pretreatmant value) at that time. The effects of Prodipine treatment on serum cytokine levels are currently under investigation. Conclusion: Our findings on prolonged graft acceptance in sensitized transplant recipients (accelerated cardiac rejection model in the rat) upon DPP IV inhibition demonstrate the involvement of CD26/DPP IV in the in vivo immune response upon solid organ transplantation.
1:).5.23.03 1 Dualistic capacity of the novel immunosuppresslve drug m y c o p h e n o l a t e mofetll (CellCept ®) R.A. Blaheta 1, K. Leckel 1, B. Wittig 2, M. Scholz 1, D. Zenker 1, S. Weber 1, A. Encke 1, B.H. Markus 1.1j. W. Goethe-Universi~ Dept. Gen. Surge~ Frankfurt am Main, Germany, 1DKFZ, Heidelberg, Germany Introduction: Cellular rejection is charactedzed by 1) proliferation and differentiation of the recipient lymphocytes and 2) infiltration of the lymphocytas via the endothelium into the donor organ. Immunosuppressive drugs as Cyclospodne A or tacrolimus (FK 506) block cellular activation but have no effect on the infiltration process. The novel drug mycophenotate mofetil (MMF) effectively suppresses cell mitosis by blocking inosine-monophosphate-dehydrogenase (IMD), the key enzyme for the pudne biosynthesis. Because IMD additionally regulates the glycoprotein synthesis, some of which are adhesion molecules, an influence of MMF on the cellular infiltration process might also be possible. We therefore investigated in vitro, whether and in what way MMF interferes into the lymphocytic infiltration cascade. Materials and Methods: CD4 and CD8 T-lyrnphocytes were added to endothelial cells (EC), derived from human umbilical veins, together with MMF (0-100 /zM). Lymphocyte binding was analyzed by the monolayer invasion assay, expression of endothelial adhesion receptors by FACS measurement. Activity of the lymphocytic counter receptors in presence of MMF was evaluated by the adhesion spot assay, using isolated chimeric proteins containing the extrecallular domain of E-seleotin, P-selectin, ICAM-1 or VCAM-1. Horizontal locomotion of CD4 and CD8 T-ceils was investigated by videomicroscopy. Results: MMF dose-dependently suppressed loose (moderately) and firm (strongly) attachment of CD4 and CD8 T-lymphocytes to allogeneic EC. The effect was more pronounced when EC were pretreated with ~,-IFN, IL-1 or IL-I+TNF(~. Moreover, MMF-incubation of both lymphocytes and EC lead to a stronger reduction of cell binding than MMF-incubafion of either lymphocytes or EC. Fluorometdc analysis of the endothelial adhesion receptors ICAM-I, VCAM-I, E-selectin and P-salectin showed that MMF moderately influenced ICAM-I (optimal dosage: 1-10 /~M), VCAM-I (optimal dosage: 0.1-I /~M) and P-saleotin (optimal dosage: 0.1 /~M) expression but strongly reduced de novo synthesis of E-seleotin (0.001-10 /~M, p < 0.05). Invastigafion of the lymphocytic counter receptors by the adhesion spot assay demonstrated that MMF did not downregulate binding of CD4 or CD8 T-lymphocytes to E-salectin, but differentially suppressed the cellular interaction via ICAM-1, VCAM-1 or P-selectin proteins. Videomicroscopic analysis of the locomotory activity of CD4 and CD8 T-cells revealed an additional mode of action of MMF on the intmcellular cytoskeleton (optimal dosage: 1/~M). Concluelon: The results presented clearly demonstrate that MMF -in strong contrast to CsA or tacrolimus- blocks organ rejection in a dualistic way. Beside its antiproliterative effect MMF also possesses distinct suppressive properties with regard to the cellular infiltration process. The unique charaotedstice of MMF might allow new immunosuppresslve strategies. Fur~er experiments should evaluate the therapeutic value of MMF on other infiltration-dopendant processes such as tumor metastasis.