353 Clopidogrel Alone and Combined with Everolimus or Tacrolimus Substantially Reduces Obliterative Bronchiolitis (OB) in an Experimental Mouse Model

Abstracts Methods and Materials: From banked BALF specimens, 8 consecutive patients with CLAD (5 BOS and 3 RAS with no evidence of infection, median 24 months after bilateral lung transplantation) and 8 matched stable patients were included in this study. RAS was defined as CLAD (irreversible decline in forced expiratory volume in 1 second (FEV1 ⬍ 80% baseline)) with irreversible decline in total lung capacity (TLC⬍ 90% baseline). BOS was defined as CLAD without RAS. Cytokine expression was measured in BALF by Luminex-based multiplex cytokine array. Results: Median FEV1 and TLC to baseline were 98% and 100% in stable patients, 68% and 102% in BOS and 40% and 63% in RAS. CXCL1, 8, 9, 12 and HGF were upregulated in CLAD compared with stable patients. Strikingly, IL-6/IL-10 ratio and the concentration of RANTES was significantly elevated in RAS compared to BOS (p⫽ 0.025 in both), while lymphotoxin-␣ was upregulated in BOS (p⫽ 0.025).

Conclusions: In CLAD patients, we have noted a distinct difference in cytokine profile between RAS and BOS. IL-6/IL-10 ratio, the concentration of RANTES and lymphotoxin-␣ in BALF might be useful for distinguishing CLAD phenotypes. Hopefully, this type of biologic distinction of these two clinical syndromes will help to develop more specifically directed therapies for patients with underlying CLAD. 351 The JAK1/3-Inhibitor R507 Diminishes Acute and Chronic Rejection While Demonstrating Immune Cell Specificity T. Deuse,1 X. Hua,1 M. Stubbendorff,1 T. Gossler,1 J. Velden,2 V. Taylor,3 G. Park,3 Y. Chen,3 H. Reichenspurner,1 R.C. Robbins,4 S. Schrepfer.1,4 1TSI-Lab, Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany; 2Pathology, University Hospital Hamburg, Hamburg, Germany; 3Rigel Pharmaceuticals, San Francisco; 4 CT Surgery, Stanford University School of Medicine, Stanford. Purpose: Selective inhibition of lymphocyte activation represents an optimal strategy for immunosuppression. This is the first report on the novel JAK3 inhibitor R507 for prevention of acute rejection as well as the development of chronic obliterative airway disease (OAD). Methods and Materials: Heterotopic BN-Lew heart (n⫽24) and luciferase transgeneic Lew-BN orthotopic tracheal transplantations (n⫽24) were performed to study acute cardiac allograft rejection and the development of chronic obliterative airway disease (OAD), respectively. Immunosuppressive efficacy and drug toxicity of R507 (60mg/kg BID) was compared to tacrolimus (4mg/kg QD) and everolimus (4mg/kg QD). Results: Plasma levels of R507 sustained stable in vivo, and in vitro enzyme assays showed selective inhibition of JAK1/3-dependent pathways. In human primary T-cells, R507 inhibits IL2-dependent Stat5 phosphorylation (EC50s of 22nM) and the resulting T-cell proliferation (EC50s of 19nM), while showing only modest activity on general cell proliferation (EC50s⬎2uM). R507 significantly reduced early mononuclear graft infiltration similar to tacrolimus (p⬍0.001 vs. no medication group), decreased histologic rejection scores on day 5, resulting in significant prolonged graft survival (no medication: 7⫾0days, tacrolimus 20⫾3days, R507 22⫾5 days) (p⬍0.001). The Combination Index of 0.584 revealed synergistic effects of R507 with tacrolimus (mean survival of 22⫾6 days). R507 and everolimus similarly inhibited tracheal OAD development (p⫽0.001 vs. no medication) and inhibited donor-specific antibody production (111⫾29 and 138⫾65, vs. no mediaction: 303⫾115). Interestingly, only R507 preserves the respiratory epithelium (86⫾6% preserved respiratory epithelium vs. no preserved epithelium in the everolimus and no medication groups; p⬍0.001). Conclusions: R507 highly effectively diminishes acute cellular rejection

S125 and OAD development while preserving the physiological epithelium and is suitable for combination regimens with tacrolimus. 352 Role of Mitochondrial Activity in the Development of Chronic Allograft Vasculopathy X. Hua,1 T. Deuse,1 M. Stubbendorff,1 T. Gossler,1 J. Velden,2 R.C. Robbins,3 E. Michelakis,4 S. Schrepfer.1,3 1TSI-Lab, Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany; 2 Pathology, University Hospital Hamburg, Hamburg, Germany; 3CT Surgery, Stanford University, Stanford; 4CV Medicine, University of Alberta, Edmonton, Canada. Purpose: This study aimed at investigating the efficacy of dichloracetate (DCA) to prevent the development and progression of chronic allograft vasculopathy (CAV) by regulating the mitochondrial metabolism of aberrant proliferating smooth muscle cells. Methods and Materials: Orthotopic aortic transplantations were performed in the PVG-to-ACI rat model and followed over 120 days. Immunosuppression with DCA (0.75mg/L) or everolimus⫹clopidogrel (4 mg/kg and 1mg/kg per os, respectively) was either started early or delayed when CAV was already present. Through levels were monitored. Aortic luminal obliteration was quantified using computer morphometry and confocal microscopy. Donor-reactive antibodies were quantified by flow cytometry. Results: Untreated animals developed CAV with luminal obliteration of 40.1⫾24.2% after 120 days. Continuous immunosuppression with DCA or everolimus/clopidogrel effectively prevented the development of vasculopathy (7.9⫾4 and 8.1⫾12.9, respectively; p⬍0.001 vs. no treatment). When the start of the immunosuppressive regimen was delayed until postoperative day 80, DCA and everolimus/clopidogrel inhibited a progression of established CAV (6.6⫾8 and 5.3⫾4.6; p⬍0.001 vs. no treatment). Even more importantly, both treatment regimens did also reverse the luminal obliteration (p⬍0.001). Interestingly, delayed treatment of DCA induced apoptosis resulting in shrinking hyperplasia area and inhibition of aberrant SMC proliferation. A stable elevation of donor-reactive IgG-antibody levels was found over 120 days in the absence of treatment. DCA was very well tolerated. Conclusions: DCA prevents the development of CAV and not only inhibits a progression of established disease, but also reverses the established disease by inducing apoptosis. It shows a similar efficacy but a safer drug profile when compared to everolimus/clopidogrel, since only aberrant SMC are affected. 353 Clopidogrel Alone and Combined with Everolimus or Tacrolimus Substantially Reduces Obliterative Bronchiolitis (OB) in an Experimental Mouse Model R. Preidl, S. Eckl, M. Ramsperger-Gleixner, N. Koch, M. Weyand, S.M. Ensminger. Department of Cardiac Surgery, University Hospital, Erlangen-Nürnberg, Germany. Purpose: Obliterative Bronchitis (OB) is the major limiting factor for long-term survival after lung transplantation. The aim of this study was to investigate if platelet inhibition alone and combined with mTOR- or calcineurin-inhibition has a beneficial effect on the development of OB. Methods and Materials: Fully MHC-mismatched C57BL/6 (H2(b)) donor tracheas were orthotopically transplanted into CBA (H2(k)) recipients. Mice received 1mg/kg/d or 20mg/kg/d Clopidogrel alone or combined with 0,05mg/kg/d Everolimus or 12mg/kg/d Tacrolimus. Grafts were analysed by histology, morphometry and immunofluorescence. Alloantibody production was analysed by FACS and cytokines were detected by RT-PCR. Results: Untreated controls showed substantial luminal obliteration on postoperative day 30 in contrast to groups treated with 1mg/kg/d or 20mg/ kg/d Clopidogrel. There was also significantly lower expression of IL12, IL4, TNF␣, IFN␥, PDGF␤, MCP1, P/E-Selectin, ICAM1 and CD40L in the treatment groups. Animals treated with 1mg/kg/d Clopidogrel and Everolimus showed less luminal obliteration compared to mice treated with 1mg/kg/d Clopidogrel [33,97⫹4,01% (1mg/kg/d Clopidogrel ⫹ Everolimus) vs. 42,25⫹5,74 % (1mg/kg/d Clopidogrel) vs. 49,92⫹2,11% (control), n⫽5, p⬍0,05]. Treatment with 1mg/kg/d Clopidogrel and Tacrolimus

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The Journal of Heart and Lung Transplantation, Vol 31, No 4S, April 2012

also resulted in a significant reduction of luminal obliteration [30,950,32% (1mg/kg/d Clopidogrel ⫹ Tacrolimus) vs. 42,25⫹5,74 % (1mg/kg/d Clopidogrel) vs. 49,92⫹2,11% (control), n⫽5, p⬍0,05]. Infiltration with CD4⫹Tcells and macrophages was significantly reduced after treatment with Clopidogrel and Everolimus and a combination of Clopidogrel and Everolimus or Tacrolimus also significantly decreased the amount of alloantibody production. Conclusions: Clopidogrel alone and in combination with Everolimus or Tacrolimus substantially reduces Obliterative Bronchiolitis (OB). 354 Blocking VEGF Receptors 1 and 2 Prevents Inflammatory Response and Experimental Obliterative Airway Disease R. Krebs,1 J. Tikkanen,1 M. Hollmén,1 Y. Wu,2 B. Pytowski,2 K. Lemström.1 1Transplantation Laboratory, University of Helsinki, Helsinki, Finland; 2Department of Experimental Therapeutics, ImClone Systems, New York, NY. Purpose: Obliterative bronchiolitis (OB) is the pulmonary manifestation of chronic rejection. Vascular endothelial growth factor (VEGF) is a proinflammatory angiogenic growth factor. We investigated the roles of VEGF receptors -1 and -2 in OB, using a mouse tracheal allograft model that develops obliterative airway disease (OAD). Methods and Materials: Heterotopic tracheal transplantations were performed from Balb/c to C57 mice. Syngeneic controls were performed from Balb/c to Balb/c mice. Anti-VEGFR antibodies were given every three days (total: 10 ip injections). The control group received IgG, the second group VEGFR-1 Ab (MC-1), the third group VEGFR-2 Ab (DC-101), the fourth group both Abs. Grafts were harvested at 10 and 30 days (n ⫽ 7-8/group; data is analyzed by ANOVA).

Conclusions: Inhibition of the VEGFR -1 and -2 signalling pathways may provide a novel therapeutic strategy to prevent the development of OB. 355 In Vivo Expansion of Regulatory T Cells with ATG and Inhibition of IL-6 Prolongs Non-Human Primate Lung Allografts Survival A. Aoyama,1 M. Tonsho,1 Y. Yamada,1 T.M. Millington,1 S. Boskovic,1 S. Lee,1 R.-N. Smith,2 J.C. Madsen,3 T. Kawai,1 J.S. Allan.4 1Transplant Surgery, Massachusetts General Hospital, Boston, MA; 2Pathology, Massachusetts General Hospital, Boston, MA; 3Transplant Center, Massachusetts General Hospital, Boston, MA; 4Thoracic Surgery, Massachusetts General Hospital, Boston, MA. Purpose: While ex vivo-expanded Tregs have been shown effective to prolong the graft survival or induce tolerance in a number of models, in vivo Treg expansion has not been accomplished in large animals or patients. The aims are to test whether anti-thymocyte globulin (ATG) with inhibition of IL-6 (known to favor Th17 differentiation) can promote Tregs in vivo, and whether those Tregs prolong lung allograft survivals in non-human primates. Methods and Materials: Five cynomolgus monkeys received anti-IL6 receptor mAb (aIL6R) and/or equine ATG. One animal received aIL6R alone, one ATG alone, and 3 ATG⫹aIL6R. Tregs in the peripheral blood were studied by flow cytometry and suppression assay. Eight lung transplants were performed with triple immunosuppression, including three treated with ATG ⫹ aIL6R.

Results: Tregs in peripheral blood was not affected by ATG or aIL6 alone, but co-administration increased Treg transiently, decreasing IFN-␥⫹ cells and increasing IL-4⫹ cells. The increased Tregs were characterized by high CTLA4 / low CD45RA / low CD62L, and were suppressive. Without ATG/aIL6R (n⫽5), lung grafts rejected on day 21, 34, 47, and 61. One animal survived ⬎ 100 days. With ATG and aIL6R (n⫽3), all three animals maintained graft for ⬎ 100 days. One lung biopsy from ATG/ aIL6R-treated animals indicated proliferating Tregs in the lung allograft. Conclusions: In vivo Treg expansion was achieved by co-administration of ATG and aIL6R, which also suppressed IFN- ␥⫹ cells, in cynomolgus monkeys. The increased Tregs were of memory-like phenotype and functional. This novel approach appears beneficial for lung allograft survival and may be feasibly applicable to human transplantation. 356

Results: The combination of VEGFR-1 and VEGFR-2 Abs significantly decreased luminal occlusion of tracheal allografts (86% vs 40%; P⬍0.005) at 30 days. With 1 mg/kg/d tacrolimus, blocking VEGFR-1 and -2 led to a significant decrease of tracheal allograft occlusion (control IgG 40% vs VEGFR-1 Ab 8% vs VEGFR-2 Ab 5% vs VEGFR-1 and -2 Ab 2%) at 30 days (n ⫽ 3-6/group; p⬍0.05 in all cases). Blocking VEGFR-1 and -2, alone and in combination, significantly reduced the number of infiltrating CD4-positive T cells and mRNA expression of CXCL9, CXCL10, MCP-1, CCL21, IL-2, IFN-␥, and CTGF, whereas the mRNA level of IL-10 was increased by VEGFR-1 and-2 blocking Ab combination in tracheal allografts 10 days after transplantation, when compared to the control IgG group (p⬍0.05 in all cases).

Pyruvate Dehydrogenase Kinase 2 Controls Vascular Remodeling T. Deuse,1 X. Hua,1 F. Länger,2 T. Gossler,1 M. Stubbendorff,1 A. Rakovic,3 C. Klein,3 G. Sutendra,4 P. Dromparis,4 L. Maegdefessel,5 P.S. Tsao,5 J. Velden,6 H. Reichenspurner,1 R.C. Robbins,7 F. Haddad,5 E. Michelakis,4 S. Schrepfer.1,7 1TSI-Lab, Cardiovascular Surgery, University Heart Center, Hamburg, Germany; 2Pathology, University School of Medicine Hannover, Hannover, Germany; 3Neurology, University of Luebeck, Luebeck, Germany; 4CV Medicine, University Alberta, Edmonton, Canada; 5CV Medicine, Stanford University, Stanford; 6Pathology, University Hospital Hamburg, Hamburg, Germany; 7CT Surgery, Stanford University, Stanford. Purpose: We hypothesized that SMC may utilize molecular pro-survival mechanisms involving metabolic modulation known from autonomous