Depletion of recipient CD8+ T cells does not alter the development of cardiac allograft vasculopathy

The Journal of Heart and Lung Transplantation Volume 20, Number 2 sponder CD8⫹ T lymphocytes. Utilizing a recently developed method to culture primary mouse vascular endothelium from thoracic aorta, we co-cultured primed, alloreactive CD8⫹ T lymphocytes with wild type or Fas-deficient mouse vascular endothelium of C57BL/6 origin. Endothelium was activated three days prior to co-culture with IFN-g (500 u/ml) to simulate inflammatory conditions. Apoptosis of vascular endothelium was analyzed by TUNEL staining after 2 and 4 days of co-culture and compared to baseline endothelial apoptosis. Results: Mouse vascular endothelium expresses both Fas and low levels of MHC Class I on its surface and expression was upregulated after activation. Co-culture with primed CBA/J CD8⫹ T lymphocytes induced apoptosis of 11.4% and 19.6% of C57BL/6 wild-type vascular endothelial cells. Similar levels of apoptosis were observed when Fas-deficient vascular endothelial cells were used as targets (9.9% and 18.5%, respectively). Conclusions: We have demonstrated that CD 8⫹ T cells, that have been activated by professional APCs, have the ability to induce apoptosis of allogeneic vascular endothelium. Although mouse vascular endothelium expresses high levels of Fas on its surface, comparable levels of endothelial apoptosis were observed when the Fas/FasLigand pathway was eliminated. We conclude that blockade of the Fas/Fas Ligand death pathway will not likely serve as a strategy to ameliorate immunologic allograft destruction in solid organ transplantation. Ongoing work focuses on defining mechanisms of CTL induced apoptosis of vascular endothelium. 204 DEPLETION OF RECIPIENT CD8ⴙ T CELLS DOES NOT ALTER THE DEVELOPMENT OF CARDIAC ALLOGRAFT VASCULOPATHY W.Y. Szeto, A.M. Krasinskas, D. Kreisel, A.S. Krupnick, S.H. Popma, B.R. Rosengard; University of Pennsylvania, Philadelphia, PA, USA PURPOSE: Cardiac allograft vasculopathy (CAV) is the primary cause of late death after heart transplantation. In a rat heterotopic heart transplant model, the majority of chimeric hearts bearing recipient-type antigen presenting cells (APCs) do not acutely reject, but develop CAV in long-surviving untreated recipients. This finding suggests that CAV is triggered either by the CD8⫹ direct pathway or by the CD4⫹ indirect pathway. To test whether CAV is a CD8⫹-dependent process, chimeric heart recipients underwent CD8⫹ T cell depletion. METHODS: Chimeric hearts were created via bone marrow transplantation in two fully MHC-mismatched rat strain combinations. DA recipients were thymectomized at 5 to 6 weeks of age and treated with a murine monoclonal antibody Ox8 to achieve chronic depletion of CD8⫹ T cells. PVG hearts bearing DA APCs [PVG(DA)] and autologously reconstituted PVG hearts [PVG(PVG)] were transplanted into the abdomen of the recipients and palpated daily. The grafts were harvested at cessation of heartbeat or at 100 days and evaluated histologically for the presence of acute cellular rejection and/or CAV. The severity of CAV was graded from a score of 0 (none) to 5 (severe). RESULTS: CD8⫹ T cell depletion of recipients was confirmed by flow cytometry. PVG(DA) hearts that survived greater than 100 days developed CAV in both untreated and CD8⫹ T cell depleted recipients. In contrast, all grafts survived indefinitely in

Abstracts

219

thymectomized recipients of PVG(DA) hearts, and CAV was significantly reduced. All PVG(PVG) hearts acutely rejected by both untreated and CD8⫹ T cell-depleted recipients. CONCLUSION: In this model, recipient CD8⫹ T cells are not required in the development of CAV. These findings suggest that CD4⫹ indirect allorecognition is the primary pathway responsible for CAV. Group Donor

Recipient Treatment Graft Survival (Days)

Number of Vessels Scored

CAV Score

% Vessels Involved

1 2 3 4 5

DA DA DA DA DA

----133 128 76 * p⬍0.005 vs Group 3

Acute Rej Acute Rej 2.60⫾0.16 1.52⫾0.15* 2.51⫾0.22** **p⫽0.73 vs Group 3

Acute Rej Acute Rej 78⫾4% 58⫾4%* 74⫾5%*** ***p⫽0.49 vs Group 3

PVG(PVG) PVG(PVG) PVG(DA) PVG(DA) PVG(DA)

none thy⫹Ox8 none thy thy⫹Ox8

5,6,7 6,7,7 12,16,⬎100x5 ⬎100x7 15,16x3,⬎100x4 thy⫽ thymectomy

205 UPREGULATION OF PAI-1 IS MEDIATED THROUGH TGF ␤ /SMAD PATHWAY IN TRANSPLANT ARTERIOPATHY C. Dong1, S. Zhu1, W. Yoon1, T. Wang1, R.J. Alvarez2, P.J. Goldschmidt-Clermont1, 1Duke University Medical Center, Durham, NC; 2The Ohio State University, Columbus, OH, USA Plasminogen activator inhibitor 1 (PAI-1) is the primary physiological inhibitor of plasminogen activator in vivo. Increased PAI-1 expression is associated with arteriosclerosis. TGF ␤ induces PAI-1 production via Smads. Hypothesis: TGF ␤ /Smad signaling is the major pathway leading to increased PAI-1 production in transplant arteriopathy (TA). Methods: In vitro: Expression of TGF ␤ receptors (T ␤ Rs), Smad2, 3 & 4 were examined by Western blotting (WB) in human aortic smooth muscle cells (SMCs); phosphorylation of Smad2 & PAI-1 mRNA in SMCs in response to TGF ␤ treatment were detected by WB using anti-phosphorylated Smad2 (p-Smad2) antibody & by TaqMan real-time RT-PCR using specific primers & probe for PAI-1, respectively. In vivo: TGF ␤1, T␤Rs, Smad2, 3, 4, 7, PAI-1 & p-Smad2 were examined by immunohistochemistry in 2 native aortas, 4 rat aortic syngrafts and 9 allografts 45 days post-transplantation. Results: In vitro: T␤RI & II, Smad2, 3 & 4 were present in SMCs. p-Smad2 and a 2 fold increase in PAI-1 mRNA were observed 1h after TGF ␤ treatment. In vivo: Marked intimal thickening & medial SMC loss were observed in aortic allografts, whereas syngrafts exhibited minimal lesions. T␤RI & II, Smad2, 3, 4 & 7 were detected in nearly all cells concerning all vessels. There was no difference between native aortas, syngrafts & allografts. TGF ␤ was detected in endothelial cells (ECs), infiltrating cells and certain mesenchymal cells in all allografts, but not in native & syngeneic vessels. Intensive cytoplasmic immunoreactivity for PAI-1 and nuclear staining for p-Smad2 were observed in ⬎90% ECs, intimal cells, residual medial SMCs & occasional adventitial cells in all allografts. p-Smad2 co-localized with PAI-1. By contrast, p-Sma2 was virtually negative in native & syngeneic aortas. A low basal level of PAI-1 was observed in ECs, medial SMCs & adventitial cells in these vessels. Conclusions: 1) p-Smad2 staining in aortic allografts indicates the activation of TGF ␤ signaling in TA; 2) co-localization of PAI-1 & p-Smad2 suggests that PAI-1 upregulation is mediated mainly by TGF ␤ /Smad Pathway in TA; 3) low basal level of PAI-1 in native aortas & syngrafts is independent of Smad signaling.