- Email: [email protected]
FasL pathway
S80
Abstracts
Methods: b2m- cells were purified from bone marrow of lewis rats using a magnetic activated cell sorting technique. b2m- cells (2.5 x 106 cells in 100l) were transplanted seven days after infarction into a transmural myocardial scar induced by cryoinjury in lewis rats (n⫽9). control group #1(n⫽10) received a 100 l injection of normal saline, and control group #2 (n⫽15) received no injection. the b2m- cells were labeled prior to transplantation using the membrane fluorescent intercalated dye pkh26-gl. repopulation was examined at 6 and 8 weeks after transplantation. differentiation of b2m- cells into cardiac myocytes was determined by the co-localization of troponin and pkh26-gl to the same cell, utilizing immunohistochemistry, uv photo-microscopy and fluorescence confocal laser microscopy on 6m serial sections. area of engraftment within the scar was calculated by planimetry. Results: the treatment group had multiple islands of de-novo formation of myocardium within the fibrous matrix of the transmural scar (mean area, 35% ⫾ 4.2% of scar area at 6 and 8 weeks). these cells co-localized cardiac specific troponin and pkh26-gl. this staining pattern was not observed in the control groups. prior to transplantation, the b2m- cells were troponin negative. Conclusion: This study demonstrates that B2M- cells represent a novel sub-population of bone marrow derived stem cells capable of successful and substantial engraftment in areas of transmural myocardial scar, with de-novo formation of cardiac myocytes.
19 TRANSPLANTATION OF A MIXED POPULATION OF BONE MARROW-DERIVED PROGENITOR CELLS IMPROVES REGIONAL SYSTOLIC FUNCTION IN A RABBIT MODEL OF CRYOINJURED MYOCARDIUM R.B. Thompson,1 S. Emani,1 S. Colgrove,2 B.H. Davis,2 D. Craig,1 Y. Morimoto,2 D. Glower,1 D.A. Taylor,2 1Department of Surgery, Duke University Medical Center, Durham, NC; 2Department of Medicine, Duke University Medical Center, Durham, NC Introduction: Cell therapy with bone marrow derived progenitor cells has been proposed with a number of single cell populations. We hypothesize that a mixed population of CD45- and CD45⫹ cells will provide not only myocardial, but also vascular precursors and thus be a better source of cells to improve both engraftment and function. Methods: Nineteen New Zealand Black rabbits underwent left thoracotomy for cryoinjury and hindlimb bone marrow aspiration. Progenitor cells were isolated and expanded in vitro. FACS analysis of the resultant cell population for injection indicated 80% CD45-(Mesenchymal) and 20% CD45⫹ (Hematopoetic). After two weeks, 108 cells fluorescently labeled with DAPI were injected into the cryoinjured region through a redo thoracotomy in 11 animals (Treated). Media alone was injected into the remaining 8 animals (Untreated). Regional systolic function was measured using micromanometry and sonomicrometry. At four weeks post injection, final functional measurements were obtained. Data were analyzed using ANOVA. Results: All 11 treated animals showed systolic functional improvement. Regional stroke work increased in treated animals (⌬SW ⫽ 9.57⫾2.4 mm*mmHg) but decreased in the untreated animals (⌬SW -1.15⫾1.2 mm*mmHg, p⬍0.007). Systolic shortening increased in treated animals as compared to untreated animals (⌬SS 0.97⫾0.87mm vs ⌬SS-0.05⫾0.05mm, p⬍0.05). Histological analysis revealed DAPI labeled cells in the previously cryoinjured area of myocardium signifying engraftment. Preliminary evaluation of cell phenotype shows cells which resemble fibroblasts and vascular progenitors, without evidence of mature cardiomyocytes.
The Journal of Heart and Lung Transplantation January 2003 Conclusion: A mixed population of bone marrow progenitor cells engraft and significantly improve regional systolic function four weeks after injection into cryoinjured rabbit myocardium. 20 CELLULAR CARDIOMYOPLASTY USING SKELETAL MYOBLASTS OVEREXPRESSING IL-1 RECEPTOR ANTAGONIST ENHANCES EFFECTS ON ADVERSE POSTINFARCTION REMODELING B. Murtuza, K. Suzuki, M.H. Yacoub, Gene Therapy, Harefield Hospital, United Kingdom Cellular cardiomyoplasty (CCM) using skeletal myoblasts (SkM) improves cardiac function in infarcted rodent hearts, though has not been shown to limit cardiac myocyte hypertrophy or interstitial collagen accumulation- adverse remodeling events which lead to ventricular dilatation and failure. Interleukin-1 induces cardiac myocyte hypertrophy and elevated levels correlate with collagen expression and LVEDD following MI. The purpose of this study was to engraft SkM overexpressing interleukin-1 receptor antagonist (IL-1ra) to infarcted myocardium to enhance CCM effects by inhibiting IL-1-mediated adverse remodeling. Methods: A male skeletal myoblast cell line overexpressing secretory IL-1ra was generated. Female nude mice underwent left coronary artery ligation followed by implantation of 0.5 million cells (IL-1ra or control-transfected: CONT) at each of two sites at the infarct border zone (n⫽7). Sham mice underwent LAD ligation only. Results: At 4 weeks, RT-PCR confirmed IL-1ra overexpression in this group. Whilst the CONT group showed some attenuated remodeling, the IL-1ra group had significantly less cardiac myocyte hypertrophy and collagen expression at infarct border zones - both on picrosirius red staining (Table: p⬍0.05 vs. sham; p⬍0.05 vs. Cont) and on RT-PCR for pro ␣1 col(I) mRNA. In addition, echocardiography demonstrated significantly reduced LVEDD and LVESD with improved ejection fraction in the IL-1ra group compared with CONT group. %MI size was similar in all hearts (47.5⫾5.6). Conclusion: Targeted overexpression of IL-1ra in infarcted hearts by transplanted SkM enhances CCM effects by further attenuating cardiac myocyte hypertrophy and collagen accumulation, thereby better preserving ventricular morphology and function.
IL-1ra CONT SHAM
LVEDD (mm)
LVES D (mm)
EF (%)
Myocyte size (area units)
Col Vol # (%)
4.0 ⫾ 0.1 4.5 ⫾ 0.1 4.8 ⫾ 0.2
2.8 ⫾ 0.2 3.4 ⫾ 0.1 3.9 ⫾ 0.2
62.1 ⫾ 2.6 53.8 ⫾ 4.7 40.5 ⫾ 3.1
772.6 ⫾ 50.3 931.3 ⫾ 54.8 1150.7 ⫾ 56.1
25.5 ⫾ 0.9 34.3 ⫾ 2.2 37.2 ⫾ 2.3
21 INTERLEUKIN-4 AND -10 COMBINED GENE THERAPY INDUCES CARDIAC ALLOGRAFT TOLERANCE BY PROMOTING THE ALLOREACTIVE T CELLS APOPTOSIS AND PREVENTING MYOCYTES APOPTOSIS VIA FAS/FASL PATHWAY H. Furukawa, K. Oshima, H. Russell, T. Tung, J. Xu, G. Cui, H. Laks, L. Sen, Surgery, UCLA Medical Center/UCLA School of Medicine, Los Angeles, CA To test the hypothesis that liposome-mediated ex vivo intracoronary interleukin-4 (IL-4) and interleukin-10 (IL-10) combined gene therapy may have synergistic immunoregulatory effect to induce the transplant tolerance, a functional cervical heterotopic heart transplant model of rabbits was used. The human recombinant IL-4 and IL-10 or IL-10 cDNA were complexed with the cationic liposome and intracoronary delivered to the allograft ex vivo. The mean survival of cardiac allograft
The Journal of Heart and Lung Transplantation Volume 22, Number 1S was significantly (p⬍0.01) prolonged from 7⫾1 days in control group (CG) to 28⫾7 days in IL-10 gene therapy group (IL-10G) and 129⫾10 days in IL-4 and IL-10 combined gene therapy group (IL-4&10G). The transgene and protein expression were increased in postoperative day (POD) 5-8, and slowly reduced thereafter. The rejection score in POD3-6 was significantly lower (3.6⫾0.2 in CG, 2.7⫾0.3 in IL-10G and 2.2⫾0.2 in IL-4&10G, p⬍0.05), and even 2.0⫾0.0 in POD⬎31. The percentage of graft infiltrating CD3⫹, CD4⫹ and CD8⫹ T cells in IL-4&10G was significantly lower in entire course (p⬍0.01). In IL4&10G, apoptotic CD3⫹, CD4⫹ and CD8⫹ T cells were increased, and apoptotic myocytes were significantly decreased. The Fas⫹ and Fas ligand (FasL)⫹ apoptotic CD4⫹ and CD8⫹ T cells increased significantly (p⬍0.05) in IL-4&10G, the increase of apoptotic CD4⫹ T cells was more remarkable. The amount of FasL⫹ myocytes in IL-4&10G was significantly reduced (9.1⫾1.1% in CG, 4.7⫾0.4% in IL-10G and 2.6⫾0.3%, p⬍0.05)), and correlated with rejection score of allografts (p⬍0.05). These results suggest that combinational cytokine gene targeting may induce localized overexpression of IL-4 and IL-10 which acts synergistically to promote alloreactive T cells apoptosis and prevent myocytes apoptosis via Fas/FasL pathway, therefore induces allograft tolerance. 22 IL-10 INDUCES SUPPRESSOR OF CYTOKINE SECRETION-3 (SOCS-3) AND ILT-3 IN HUMAN ENDOTHELIAL CELLS AND INHIBITS T CELL COSTIMULATION C. Gleissner, A. Zastrow, R. Klingenberg, N. Xia, T.J. Dengler, Cardiology, Medizinische Universitaetsklinik III, Heidelberg, Germany Interleukin-10 (IL-10), a predominantly immunosuppressive cytokine, has been shown to attenuate lymphocyte responses and to inhibit antigen uptake and presentation, but its effects upon T cell activation mediated by human vascular endothelial cells (EC) are less well defined.We have previously demonstrated IL-10 receptor expression and effective IL-10 signaling in human umbilical vein EC (HUVEC) via STAT-3. IL-10 effects on EC-dependent T cell activation were investigated with a novel T cell proliferation assay utilizing HUVEC transgenic for Fcg receptor type II (CD32). After binding of mitogenic anti-CD3 antibody CD32 on EC, this system allows the study of endothelial costimulation independent of alloantigen. 24 h pretreatment of HUVEC with IL-10 resulted in a dose-dependent inhibition of CD4⫹ or CD8⫹ T cell proliferation, reaching ⬃ 70% inhibition with 10 ng/ml of IL-10. Complete removal of IL-10 after the pre-treatment effected similar levels of inhibition, while addition of IL-10 only at the beginning of T cell stimulation showed significantly less inhibition. Altered binding of comitogenic CD3 antibody or changes in the expression of classical costimulatory molecules on EC could be excluded as causes of costimulation inhibition. RT-PCR and western blot analysis demonstrated that stimulation of human EC with IL-10 induced significant upregulation of SOCS-3 (⬃ 4-fold), a potent negative regulator of cytokine secretion, and de novo expression of ILT-3, a novel surface receptor implicated to mediate the suppressive effect of regulatory CD4⫹CD25⫹ T cells. In summary, IL-10 suppresses the T cell-stimulatory potential of human EC independent of antigen presentation. Kinetics of inhibition and upregulation of SOCS-3 and ILT-3 by IL-10 suggest latered cytokine secretion and cell contact-dependent modulation of costimulatio as underlying mechanisms. 23 INFLUENCE OF AN ANTI-CD25 MAB ON TOLERANCE INDUCTION THROUGH BMT WITH COSTIMULATION BLOCKADE
Abstracts
S81
S. Bigenzahn,1 P. Blaha,1 Z. Koporc,1 M. Schmid,1 E. Selzer,1 H. Bergmeister,1 C. Heusser,2 K. Wagner,2 F. Muehlbacher,1 T. Wekerle,1 1Vienna General Hospital, Vienna, Austria; 2Novartis Pharma, Basel, Switzerland Background: We investigated whether an anti-CD25 (IL2R␣) mAb interferes with tolerance induction through BMT with costimulation blockade. Methods: C57BL/6 mice received 3 Gy total body irradiation, ⬇17x10*6 Balb/c bone marrow cells, anti-CD154 mAb and CTLA4Ig. Groups were additionally treated either with 1) anti-CD25, 2) anti-CD25 plus RAD (Everolimus), 3) RAD alone or 4) IL2 for 4 wks post-BMT. Deletion, macrochimerism and the percentage and origin of CD4⫹CD25⫹ cells were followed by flow cytometry. Tolerance was tested by transplanting donor and 3rd party (C3H) skin. Results: Early multi-lineage chimerism was induced with similar frequency with the standard protocol involving anti-CD154 and CTLA4Ig (13/16), if anti-CD25 (12/14) was given in addition, or with additional IL2 (6/8). Anti-CD25 plus RAD as well as RAD alone established chimerism in 8/8 mice. Deletion of donor-reactive T cells was not blocked by anti-CD25, and was not increased by IL2, suggesting that deletion is not dependent on the IL2-sensitive apoptosis pathway of AICD. CD4⫹CD25⫹ (regulatory T) cells were virtually completely depleted during the first two months in the groups receiving anti-CD25 (p⬍0.0002). Notably, 7 weeks post-BMT, a substantial fraction of the CD4⫹CD25⫹ cells was of donor origin. 9/11 mice of the standard protocol group, 4/7 of the IL2-treated group but only 3/11 mice of the anti-CD25 treated group (p⬍0.05 vs standard protocol) and 5/8 in the group treated with anti-CD25 plus RAD (p⬍0.05 vs anti-CD25 alone) accepted donor skin grafts (observation ongoing). Conclusion: Anti-CD25 or IL2-treatment did not influence deletion and did not abrogate the induction of early macrochimerism, but might influence tolerance development. RAD when combined with anti-CD25 seems to positively influence tolerance induction.
24 DONOR-DERIVED SUPPRESSOR MACROPHAGES FOR RECIPIENT-SPECIFIC IMMUNOSUPPRESSION IN LUNG TRANSPLANTATION IN MINIPIGS G. Warnecke,1 F. Faendrich,2 S.P. Sommer,1 T. Steinkamp,1 G. Zehle,2 M. Ruhnke,2 A.R. Simon,1 J.M. Hohlfeld,1 J. Niedermeyer,1 A. Haverich,1 M. Strueber,1 1Div. of T&CV Surgery, Hannover Medical School, Hannover, Germany; 2Dept. of Surgery, University Kiel, Kiel, Germany Purpose: The purpose of our study was to evaluate donor-derived in vitro stimulated suppressor macrophages, that induce apoptosis at donor-specific recipient T-cell clones in porcine lung transplantation to develop a less invasive tolerance induction protocol. Methods: Single lung transplantation from MHC mismatched donors was performed in 7 minipips. Donor macrophages were isolated, cultured, expanded, stimulated by cytokines for 6 days and infused on postoperative days (POD) 7, 14 and 21 at 5x106 total cells (MphTX). Immunosuppression included methylprednisolone and high dose FK 506 (5 animals) or Cyclosporine A (CsA, 2 animals) and was discontinued on POD 28. Donor cell chimerism and donor-specific tolerance were monitored by flow cytometry and mixed lymphocyte reaction (MLR). Data were compared to our standard CsA controls and to preoperatively irradiated animals that developed donor specific tolerance and chronic graft survival, which has been reported on a previous ISHLT meeting.
Abstracts
Methods: b2m- cells were purified from bone marrow of lewis rats using a magnetic activated cell sorting technique. b2m- cells (2.5 x 106 cells in 100l) were transplanted seven days after infarction into a transmural myocardial scar induced by cryoinjury in lewis rats (n⫽9). control group #1(n⫽10) received a 100 l injection of normal saline, and control group #2 (n⫽15) received no injection. the b2m- cells were labeled prior to transplantation using the membrane fluorescent intercalated dye pkh26-gl. repopulation was examined at 6 and 8 weeks after transplantation. differentiation of b2m- cells into cardiac myocytes was determined by the co-localization of troponin and pkh26-gl to the same cell, utilizing immunohistochemistry, uv photo-microscopy and fluorescence confocal laser microscopy on 6m serial sections. area of engraftment within the scar was calculated by planimetry. Results: the treatment group had multiple islands of de-novo formation of myocardium within the fibrous matrix of the transmural scar (mean area, 35% ⫾ 4.2% of scar area at 6 and 8 weeks). these cells co-localized cardiac specific troponin and pkh26-gl. this staining pattern was not observed in the control groups. prior to transplantation, the b2m- cells were troponin negative. Conclusion: This study demonstrates that B2M- cells represent a novel sub-population of bone marrow derived stem cells capable of successful and substantial engraftment in areas of transmural myocardial scar, with de-novo formation of cardiac myocytes.
19 TRANSPLANTATION OF A MIXED POPULATION OF BONE MARROW-DERIVED PROGENITOR CELLS IMPROVES REGIONAL SYSTOLIC FUNCTION IN A RABBIT MODEL OF CRYOINJURED MYOCARDIUM R.B. Thompson,1 S. Emani,1 S. Colgrove,2 B.H. Davis,2 D. Craig,1 Y. Morimoto,2 D. Glower,1 D.A. Taylor,2 1Department of Surgery, Duke University Medical Center, Durham, NC; 2Department of Medicine, Duke University Medical Center, Durham, NC Introduction: Cell therapy with bone marrow derived progenitor cells has been proposed with a number of single cell populations. We hypothesize that a mixed population of CD45- and CD45⫹ cells will provide not only myocardial, but also vascular precursors and thus be a better source of cells to improve both engraftment and function. Methods: Nineteen New Zealand Black rabbits underwent left thoracotomy for cryoinjury and hindlimb bone marrow aspiration. Progenitor cells were isolated and expanded in vitro. FACS analysis of the resultant cell population for injection indicated 80% CD45-(Mesenchymal) and 20% CD45⫹ (Hematopoetic). After two weeks, 108 cells fluorescently labeled with DAPI were injected into the cryoinjured region through a redo thoracotomy in 11 animals (Treated). Media alone was injected into the remaining 8 animals (Untreated). Regional systolic function was measured using micromanometry and sonomicrometry. At four weeks post injection, final functional measurements were obtained. Data were analyzed using ANOVA. Results: All 11 treated animals showed systolic functional improvement. Regional stroke work increased in treated animals (⌬SW ⫽ 9.57⫾2.4 mm*mmHg) but decreased in the untreated animals (⌬SW -1.15⫾1.2 mm*mmHg, p⬍0.007). Systolic shortening increased in treated animals as compared to untreated animals (⌬SS 0.97⫾0.87mm vs ⌬SS-0.05⫾0.05mm, p⬍0.05). Histological analysis revealed DAPI labeled cells in the previously cryoinjured area of myocardium signifying engraftment. Preliminary evaluation of cell phenotype shows cells which resemble fibroblasts and vascular progenitors, without evidence of mature cardiomyocytes.
The Journal of Heart and Lung Transplantation January 2003 Conclusion: A mixed population of bone marrow progenitor cells engraft and significantly improve regional systolic function four weeks after injection into cryoinjured rabbit myocardium. 20 CELLULAR CARDIOMYOPLASTY USING SKELETAL MYOBLASTS OVEREXPRESSING IL-1 RECEPTOR ANTAGONIST ENHANCES EFFECTS ON ADVERSE POSTINFARCTION REMODELING B. Murtuza, K. Suzuki, M.H. Yacoub, Gene Therapy, Harefield Hospital, United Kingdom Cellular cardiomyoplasty (CCM) using skeletal myoblasts (SkM) improves cardiac function in infarcted rodent hearts, though has not been shown to limit cardiac myocyte hypertrophy or interstitial collagen accumulation- adverse remodeling events which lead to ventricular dilatation and failure. Interleukin-1 induces cardiac myocyte hypertrophy and elevated levels correlate with collagen expression and LVEDD following MI. The purpose of this study was to engraft SkM overexpressing interleukin-1 receptor antagonist (IL-1ra) to infarcted myocardium to enhance CCM effects by inhibiting IL-1-mediated adverse remodeling. Methods: A male skeletal myoblast cell line overexpressing secretory IL-1ra was generated. Female nude mice underwent left coronary artery ligation followed by implantation of 0.5 million cells (IL-1ra or control-transfected: CONT) at each of two sites at the infarct border zone (n⫽7). Sham mice underwent LAD ligation only. Results: At 4 weeks, RT-PCR confirmed IL-1ra overexpression in this group. Whilst the CONT group showed some attenuated remodeling, the IL-1ra group had significantly less cardiac myocyte hypertrophy and collagen expression at infarct border zones - both on picrosirius red staining (Table: p⬍0.05 vs. sham; p⬍0.05 vs. Cont) and on RT-PCR for pro ␣1 col(I) mRNA. In addition, echocardiography demonstrated significantly reduced LVEDD and LVESD with improved ejection fraction in the IL-1ra group compared with CONT group. %MI size was similar in all hearts (47.5⫾5.6). Conclusion: Targeted overexpression of IL-1ra in infarcted hearts by transplanted SkM enhances CCM effects by further attenuating cardiac myocyte hypertrophy and collagen accumulation, thereby better preserving ventricular morphology and function.
IL-1ra CONT SHAM
LVEDD (mm)
LVES D (mm)
EF (%)
Myocyte size (area units)
Col Vol # (%)
4.0 ⫾ 0.1 4.5 ⫾ 0.1 4.8 ⫾ 0.2
2.8 ⫾ 0.2 3.4 ⫾ 0.1 3.9 ⫾ 0.2
62.1 ⫾ 2.6 53.8 ⫾ 4.7 40.5 ⫾ 3.1
772.6 ⫾ 50.3 931.3 ⫾ 54.8 1150.7 ⫾ 56.1
25.5 ⫾ 0.9 34.3 ⫾ 2.2 37.2 ⫾ 2.3
21 INTERLEUKIN-4 AND -10 COMBINED GENE THERAPY INDUCES CARDIAC ALLOGRAFT TOLERANCE BY PROMOTING THE ALLOREACTIVE T CELLS APOPTOSIS AND PREVENTING MYOCYTES APOPTOSIS VIA FAS/FASL PATHWAY H. Furukawa, K. Oshima, H. Russell, T. Tung, J. Xu, G. Cui, H. Laks, L. Sen, Surgery, UCLA Medical Center/UCLA School of Medicine, Los Angeles, CA To test the hypothesis that liposome-mediated ex vivo intracoronary interleukin-4 (IL-4) and interleukin-10 (IL-10) combined gene therapy may have synergistic immunoregulatory effect to induce the transplant tolerance, a functional cervical heterotopic heart transplant model of rabbits was used. The human recombinant IL-4 and IL-10 or IL-10 cDNA were complexed with the cationic liposome and intracoronary delivered to the allograft ex vivo. The mean survival of cardiac allograft
The Journal of Heart and Lung Transplantation Volume 22, Number 1S was significantly (p⬍0.01) prolonged from 7⫾1 days in control group (CG) to 28⫾7 days in IL-10 gene therapy group (IL-10G) and 129⫾10 days in IL-4 and IL-10 combined gene therapy group (IL-4&10G). The transgene and protein expression were increased in postoperative day (POD) 5-8, and slowly reduced thereafter. The rejection score in POD3-6 was significantly lower (3.6⫾0.2 in CG, 2.7⫾0.3 in IL-10G and 2.2⫾0.2 in IL-4&10G, p⬍0.05), and even 2.0⫾0.0 in POD⬎31. The percentage of graft infiltrating CD3⫹, CD4⫹ and CD8⫹ T cells in IL-4&10G was significantly lower in entire course (p⬍0.01). In IL4&10G, apoptotic CD3⫹, CD4⫹ and CD8⫹ T cells were increased, and apoptotic myocytes were significantly decreased. The Fas⫹ and Fas ligand (FasL)⫹ apoptotic CD4⫹ and CD8⫹ T cells increased significantly (p⬍0.05) in IL-4&10G, the increase of apoptotic CD4⫹ T cells was more remarkable. The amount of FasL⫹ myocytes in IL-4&10G was significantly reduced (9.1⫾1.1% in CG, 4.7⫾0.4% in IL-10G and 2.6⫾0.3%, p⬍0.05)), and correlated with rejection score of allografts (p⬍0.05). These results suggest that combinational cytokine gene targeting may induce localized overexpression of IL-4 and IL-10 which acts synergistically to promote alloreactive T cells apoptosis and prevent myocytes apoptosis via Fas/FasL pathway, therefore induces allograft tolerance. 22 IL-10 INDUCES SUPPRESSOR OF CYTOKINE SECRETION-3 (SOCS-3) AND ILT-3 IN HUMAN ENDOTHELIAL CELLS AND INHIBITS T CELL COSTIMULATION C. Gleissner, A. Zastrow, R. Klingenberg, N. Xia, T.J. Dengler, Cardiology, Medizinische Universitaetsklinik III, Heidelberg, Germany Interleukin-10 (IL-10), a predominantly immunosuppressive cytokine, has been shown to attenuate lymphocyte responses and to inhibit antigen uptake and presentation, but its effects upon T cell activation mediated by human vascular endothelial cells (EC) are less well defined.We have previously demonstrated IL-10 receptor expression and effective IL-10 signaling in human umbilical vein EC (HUVEC) via STAT-3. IL-10 effects on EC-dependent T cell activation were investigated with a novel T cell proliferation assay utilizing HUVEC transgenic for Fcg receptor type II (CD32). After binding of mitogenic anti-CD3 antibody CD32 on EC, this system allows the study of endothelial costimulation independent of alloantigen. 24 h pretreatment of HUVEC with IL-10 resulted in a dose-dependent inhibition of CD4⫹ or CD8⫹ T cell proliferation, reaching ⬃ 70% inhibition with 10 ng/ml of IL-10. Complete removal of IL-10 after the pre-treatment effected similar levels of inhibition, while addition of IL-10 only at the beginning of T cell stimulation showed significantly less inhibition. Altered binding of comitogenic CD3 antibody or changes in the expression of classical costimulatory molecules on EC could be excluded as causes of costimulation inhibition. RT-PCR and western blot analysis demonstrated that stimulation of human EC with IL-10 induced significant upregulation of SOCS-3 (⬃ 4-fold), a potent negative regulator of cytokine secretion, and de novo expression of ILT-3, a novel surface receptor implicated to mediate the suppressive effect of regulatory CD4⫹CD25⫹ T cells. In summary, IL-10 suppresses the T cell-stimulatory potential of human EC independent of antigen presentation. Kinetics of inhibition and upregulation of SOCS-3 and ILT-3 by IL-10 suggest latered cytokine secretion and cell contact-dependent modulation of costimulatio as underlying mechanisms. 23 INFLUENCE OF AN ANTI-CD25 MAB ON TOLERANCE INDUCTION THROUGH BMT WITH COSTIMULATION BLOCKADE
Abstracts
S81
S. Bigenzahn,1 P. Blaha,1 Z. Koporc,1 M. Schmid,1 E. Selzer,1 H. Bergmeister,1 C. Heusser,2 K. Wagner,2 F. Muehlbacher,1 T. Wekerle,1 1Vienna General Hospital, Vienna, Austria; 2Novartis Pharma, Basel, Switzerland Background: We investigated whether an anti-CD25 (IL2R␣) mAb interferes with tolerance induction through BMT with costimulation blockade. Methods: C57BL/6 mice received 3 Gy total body irradiation, ⬇17x10*6 Balb/c bone marrow cells, anti-CD154 mAb and CTLA4Ig. Groups were additionally treated either with 1) anti-CD25, 2) anti-CD25 plus RAD (Everolimus), 3) RAD alone or 4) IL2 for 4 wks post-BMT. Deletion, macrochimerism and the percentage and origin of CD4⫹CD25⫹ cells were followed by flow cytometry. Tolerance was tested by transplanting donor and 3rd party (C3H) skin. Results: Early multi-lineage chimerism was induced with similar frequency with the standard protocol involving anti-CD154 and CTLA4Ig (13/16), if anti-CD25 (12/14) was given in addition, or with additional IL2 (6/8). Anti-CD25 plus RAD as well as RAD alone established chimerism in 8/8 mice. Deletion of donor-reactive T cells was not blocked by anti-CD25, and was not increased by IL2, suggesting that deletion is not dependent on the IL2-sensitive apoptosis pathway of AICD. CD4⫹CD25⫹ (regulatory T) cells were virtually completely depleted during the first two months in the groups receiving anti-CD25 (p⬍0.0002). Notably, 7 weeks post-BMT, a substantial fraction of the CD4⫹CD25⫹ cells was of donor origin. 9/11 mice of the standard protocol group, 4/7 of the IL2-treated group but only 3/11 mice of the anti-CD25 treated group (p⬍0.05 vs standard protocol) and 5/8 in the group treated with anti-CD25 plus RAD (p⬍0.05 vs anti-CD25 alone) accepted donor skin grafts (observation ongoing). Conclusion: Anti-CD25 or IL2-treatment did not influence deletion and did not abrogate the induction of early macrochimerism, but might influence tolerance development. RAD when combined with anti-CD25 seems to positively influence tolerance induction.
24 DONOR-DERIVED SUPPRESSOR MACROPHAGES FOR RECIPIENT-SPECIFIC IMMUNOSUPPRESSION IN LUNG TRANSPLANTATION IN MINIPIGS G. Warnecke,1 F. Faendrich,2 S.P. Sommer,1 T. Steinkamp,1 G. Zehle,2 M. Ruhnke,2 A.R. Simon,1 J.M. Hohlfeld,1 J. Niedermeyer,1 A. Haverich,1 M. Strueber,1 1Div. of T&CV Surgery, Hannover Medical School, Hannover, Germany; 2Dept. of Surgery, University Kiel, Kiel, Germany Purpose: The purpose of our study was to evaluate donor-derived in vitro stimulated suppressor macrophages, that induce apoptosis at donor-specific recipient T-cell clones in porcine lung transplantation to develop a less invasive tolerance induction protocol. Methods: Single lung transplantation from MHC mismatched donors was performed in 7 minipips. Donor macrophages were isolated, cultured, expanded, stimulated by cytokines for 6 days and infused on postoperative days (POD) 7, 14 and 21 at 5x106 total cells (MphTX). Immunosuppression included methylprednisolone and high dose FK 506 (5 animals) or Cyclosporine A (CsA, 2 animals) and was discontinued on POD 28. Donor cell chimerism and donor-specific tolerance were monitored by flow cytometry and mixed lymphocyte reaction (MLR). Data were compared to our standard CsA controls and to preoperatively irradiated animals that developed donor specific tolerance and chronic graft survival, which has been reported on a previous ISHLT meeting.