130

The Journal of Heart and Lung Transplantation Volume 25, Number 2S

Procedure: Wild type (WT) C57Bl6 (B6) recipients (H2b) were immunized with 400␮g recombinant murine vimentin in 100␮l complete Freund’s adjuvant (CFA), followed by a booster 400␮g vimentin 7days later, to generate high titers of IgG AVA. B-cell knockout mice on B6 background (IgH6) immunized with vimentin had high frequencies of Interleukin-2 and Interferon-␥ producing splenocytes, but no antibodies. Allogeneic or syngeneic control B6 WT recipients received 200␮g hen egg lysozyme (HEL) or vimentin, respectively, in CFA. Two weeks following primary immunization, recipients received a vascularized 129/sv cardiac allograft [MHC identical (H2b), differ only in non-MHC loci] or a B6 isograft. Result: Vimentin immunized (VIM) B6 WT recipients showed accelerated rejection of allografts [8.4(⫾1.5)days;n⫽18], compared to HEL-immunised B6 WT recipients [13.3(⫾2.2)days;n⫽10;P ⬍0.0001,Log-rank test]. In contrast, allografts in VIM IgH6 recipients were rejected with similar kinetics to unimmunised IgH6 controls [11.2(⫾2.4)days vs 11.2(⫾4.1)days;n⫽5;p⫽0.99]. Isografts in VIM B6 WT recipients continued to beat at 90days (n⫽7). At rejection, WT recipients did not produce anti donor-MHC antibodies [MHC-identical;H2b]. However, VIM WT recipients had high titers of AVA; AVA could be eluted from rejected cardiac allografts, as shown by binding of eluate to vimentin in western blots. There was significant C3d deposition in allografts in WT VIM recipients, compared to WT controls (31.3(⫾7.7)vs14.1(⫾4.5)pixel units;P ⬍0.05). Conclusion: The anti-vimentin response significantly accelerates rejection of cardiac allografts by antibody-mediated mechanisms in an MHC-identical murine model, suggesting a pathogenic role for autoantibodies. 130 GRAFT ENDOTHELIAL REPOPULATION BY RECIPIENT CELLS IN THE ABSENCE OF AN ADAPTIVE IMMUNE RESPONSE IS IFN-GAMMA-DEPENDENT AND MAY PROTECT FROM ALLOINJURY G. Warnecke,1 K.J. Wood,1 1Nuffield Dept. of Surgery, University of Oxford, Oxford, United Kingdom Purpose: Endothelial repopulation by recipient-derived cells and transplant arteriosclerosis are generally interdependent, but if the replacement of the donor endothelial cell layer by recipient cells could be achieved without additional damage to the graft, this in itself might be beneficial. Procedures: Thoracic aortae from C57BL/6 (H-2b) CD31 knockout (ko) donors were transplanted after 1 or 24 hrs of cold ischemia into the infrarenal abdominal aorta of C57BL/6 IFN-gamma ko, C57BL/6 wildtype or CBA-rag1 ko (H-2k) recipients. Some grafts were procured from their primary CBA-rag1 recipients after 30 days and re-transplanted into secondary recipients. Grafts were harvested after 30 days and H&E, Elastin van Giesson and immunohistochemistry stainings were performed. Results: Syngeneic transplants (CD31 kowildtype) with 1 hr cold ischemia were harvested after 30 days and showed completely donor-derived CD31-negative endothelium. Prolongation of the cold ischemic time to 24 hrs resulted in endothelial repopulation by recipient-derived, CD31-positive cells, but no additional damage of the aorta graft was evident. This repopulation was completely abrogated in recipients deficient for IFN-gamma. CD31 ko grafts placed into allogeneic immunodeficient CBA-rag1 recipients did not develop transplant vasculopathy after 30 days but underwent endothelial repopulation in experiments with extended ischemia (24 hrs). Upon re-transplantation into immunocompetent secondary CBA recipients, grafts that had retained their C57BL/6 endothelium in CBA-rag1 recipients developed heavy transplant arteriosclerosis. Grafts that were repopulated by CBA endothelium at the time of re-transplantation developed less severe arteriosclerosis.

Abstracts

S89

Conclusion: IFN-gamma-dependent endothelial repopulation may protect vascularized grafts from alloinjury. This phenomenon is independent from the loss of antigen presenting cells from the graft during parking in primary immunodeficient hosts. 131 GENE TRANSFER OF PROGRAMMED DEATH LIGAND-1 (PDL1) PROLONGS ALLOGRAFT SURVIVAL IN A RAT MODEL OF HEART TRANSPLANTATION J. Li, G. Vassalli, Transplantation Research, NIBR, Basel, Switzerland; 1Services de Cardiologie, Chirurgie Expe´rimentale, Chirurgie Cardio-vasculaire, et Centre de Transplantation d’Organes, CHUV, Lausanne, Switzerland Background: Programmed death (PD) and its ligand PDL-1 regulate T cell co-stimulation in the context of antigen presenting cell APC)-T cell interactions, which are essential for efficient T cell activation. We therefore hypothesized that forced PDL-1 overexpression within cardiac grafts might attenuate T cell-mediated acute rejection. Methods: We analyzed the efficacy of an adenoviral vector expressing PDL-1 in delaying cardiac allograft rejection, and the mechanisms underlying the protective effects. PDL-1 gene transfer was performed in F344 rat donor hearts that were transplanted into Lewis recipients. Control donor hearts received either noncoding vector or buffer solution alone. In additional experiments, PDL-1 gene transfer combined with a sub-therapeutic dose of cyclosporine A (CyA) for 5 days was compared with CyA alone. Inflammatory cell infiltrates in the grafts were assessed by immunohistochemistry (for CD4⫹, CD8⫹, and TCR⫹ T cells and ED-1⫹ monocytes/macrophages). Results: Allografts expressing PDL-1 survived for longer periods of time compared to those receiving noncoding adenovirus or buffer solution alone; median survival time (MST), 17 (range: 16 –20) days (n ⫽ 6) vs. 11 (8 –14) days and 9 (8 –13) days (n ⫽ 9 each), respectively; p ⬍0.001). Combined PDL-1 gene transfer with a low-dose CyA was superior to CyA alone; MST, 25 (15– 42) days (n ⫽ 5) vs. 15 (13–17) days (n ⫽ 7; p ⬍0.05). PDL-1 gene transfer was associated with decreased CD4⫹ cell infiltrates, but not CD8 or macrophage infiltrates. Conclusions: PDL-1 gene transfer attenuates acute allograft rejection in a rat model of heart transplantation. 132 INTERPLAY BETWEEN INFLAMMATION AND ANGIOGENESIS IN CARDIAC ALLOGRAFTS – THERAPEUTIC APPLICATIONS FOR VEGFR-1 AND -2 INHIBITION A.I. Nyka ¨ nen,1 O. Raisky,2 M. Hollme´n,1 R. Krebs,1 J.M. Tikkanen,1 Y. Wu,3 D.J. Hicklin,3 P.K. Koskinen,1 K.B. Lemstro ¨ m,1 1Transplanation Laboratory, University of Helsinki, Helsinki, Finland; 2Ho ˆ pital Cardiologique Louis Pradel, Lyon, France; 3ImClone Systems Incorporated, New York, NY Background: Interplay between angiogenesis and inflammation is important in many physiological and pathological situations. We examined the role of angiogenic and proinflammatory vascular endothelial growth factor (VEGF), and its two receptors VEGFR-1 and -2, in angiogenesis, inflammation and development of arteriosclerotic lesions in cardiac allografts. Methods and Results: Our results with mouse chronic rejection heart transplantation model, imply that alloimmune response activates neocapillarization in cardiac allografts. These small blood vessels were found to co-express endothelial marker CD31, stem cell marker c-kit, VEGFR-2. In contrast, VEGFR-1 expression was detected mainly in arterial smooth muscle cells, cardiomyocytes and occasional CD11b⫹ myelomonocytic cells. Specific receptor blocking antibodies