67. IL-17 Neutralization Prevents Autoimmune Diabetes in NOD Mice But Does not Delay Allograft Rejection in Islet Transplantation

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 205 Background: Immunotherapeutic approaches to treat melanoma have become increasingly sophisticated with attempts to manipulate the immune response via high dose IL-2, adoptive transfer, and gene therapy. Although, many of these approaches show promise, clinical response appears modest, with adoptive transfer of melanoma antigen specific CD8 ⫹ T-cells showing the most impressive results. CD8 ⫹ cytotoxic lymphocyte therapies are limited by lack of support from the remaining components of the immune system such as dendritic cells, memory cells, B cells, and CD4 ⫹ T cells. Increasing evidence indicates that CD4 ⫹ T helper cells play a crucial function as a facilitator in the anti-tumor response, particularly through the induction and maintenance of CD8 ⫹ T cell immunity. Yet despite, their important role there have been limited efforts to exploit CD4 ⫹ T cells to enhance the immune response to cancer. Results: In this study, we identified an MHC Class II HLA-DR4 restricted CD4 ⫹ T cell receptor (TCR) capable of reacting against a naturally occurring melanocyte differentiation antigen (MDA), Tyrosinase Related Protein-1 (TRP-1). Human lymphocytes specific and reactive to a 21-residue epitope of TRP-1 (positions 277-297) were isolated from the peripheral blood of a metastatic melanoma patient by limiting dilution. We then identified the gene sequence of the individual ␣ and ␤ subunits of the TCR from these lymphocytes via a 5’ race strategy. Next, we constructed a bicistronic lentivirus containing the genes for the ␣ and ␤ subunits, and successfully transduced both transformed human T-cells (Jurkat cells) and peripheral blood mononuclear cells (PBMC). At an MOI of 10:1 we were able to achieve 94% transduction efficiency of our TCR into Jurkat cells, and at a MOI of 100:1 we achieved 56% transduction efficiency into PBMCs. Furthermore, transduced PBMCs were found to recognize TRP-1 peptide, melanoma tumor lysates, and TRP-1 positive tumor lines as measured by specific interferon ␥ (IFN ␥) production on ELISA. Following these in vitro studies, a series of bone marrow transplant experiments were conducted using a TRP-1 TCR transgenic mouse developed by insertion of the TRP-1 TCR gene into fertilized murine oocytes. Initial characterization of the naïve TRP-1 Tg mice by flow cytometry showed no presence of TRP-1 TCR specific lymphocytes in the peripheral blood using a peptide-antibody complex (TRP-1 Pentamer). However, 2.6% of lymphocytes showed pentamer specificity in the thymus. To test the role of the thymus to positively select the transgenic TCR, whole body irradiated DR4 transgenic and C57BL/6 recipient mice received a mix of DR4 Tg and TRP-1 Tg bone marrow. Initial results at the four week time point show a slight increase in TRP-1 pentamer specific CD4 ⫹ cells in the DR4 Tg mice (2.3% vs. 0.2%). By 12 weeks, however, expression had dramatically increased to 37%. Conclusions: Herein we demonstrate the development of a CD4 ⫹ T cell model for studying gene transfer into hematopoietic stem cells. We demonstrate the efficient transduction of our TCR using lentiviral constructs, the functional expression of our TCR in transduced PBMCs, and the successful transplantation of our TCR Tg bone marrow into MHC specific transplant recipients (DR4 Tg mice). These results establish the basis for a lentiviral delivery system which could be used to deliver MDA specific CD4 ⫹ TCRs into hematopoietic stem cells for use in the treatment of patients with metastatic melanoma. 66. NITRITE PROTECTS RAT LUNG GRAFTS AGAINST ISCHEMIA REPERFUSION INJURY. Ryujiro Sugimoto1, Atsunori Nakao1, Junichi Kohmoto1, Yinna Wang1, Mark T. Gladwin2, Timothy R. Billiar6, Kenneth R. McCurry1; 1Department of Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA; 2Vascular Medicine Branch and Critical Care Medicine Department, NIH, Bathesda, MD; 6 Department of Surgery, University of Pittsburgh, Pittsburgh, PA Introduction: The nitrite anion (NO 2 ⫺), which has been considered to be a biologically inert metabolite of NO, has recently been demonstrated to have protective effects in normothermic ischemia/

reperfusion (I/R) injury. Its beneficial effects have been postulated to be related to its reduction to NO under hypoxic and ischemic conditions by deoxyhemoglobin and deoxymyoglobin and potentially other proteins with heme prosthetic groups, such as xanthine oxidoreductase. Little data exist, however, regarding the therapeutic effect of nitrite in cold ischemia and warm perfusion such as occurs at the time of organ transplantation. Since I/R injury following organ transplantation, particularly in lung transplantation, remains a major source of morbidity and mortality, we tested the hypothesis that administration of sodium nitrite would protect transplanted lung grafts from I/R injury following prolonged cold preservation. Methods: Orthotopic left lung transplantation was performed in a syngeneic Lewis to Lewis rat combination. Grafts were preserved in low potassium dextran solution with (nitrite) or without (control) sodium nitrite (10 ␮M) at 4 oC for 6 hrs. Additionally, groups of recipients also received sodium nitrite (480 nmol/body; nitrite IV) or sodium nitrate (480 nmol/body; control IV) intravenously 5 minutes before graft reperfusion. Recipients were sacrificed 1 or 2 hrs after transplantation. The efficacy of sodium nitrite was evaluated by pulmonary vein (PV) blood gas analysis (under 100% O 2) for graft function, real time RT-PCR for inflammatory mediators, myeloperoxidase (MPO) activity for neutrophil accumulation in grafts and histopathology. Cell signaling through MAP kinase activation was also evaluated by Western blot analysis. Results: Graft PV PaO 2 in the nitrite/nitrite IV group was significantly higher compared to those in control/control IV group 2hrs after transplantation. Intragraft mRNA for IL-6, IL-1␤, TNF-␣, iNOS, COX-2 and Egr-1 were upregulated within 2 hrs after transplantation in control/control IV group. In contrast, the increase of these inflammatory mediators was markedly inhibited in nitrite/nitrite IV group. Intragraft MPO activity in nitrite/control IV group and nitrite/nitrite IV group was significantly less than in control/control IV group. Infiltration of graft lungs by neutrophils and ED1⫹ macrophages was significantly reduced in nitrite/nitrite IV group, as assessed by histology. ERK, p38 and JNK were all activated in controls 1hr after transplantation while exposure of grafts and recipients to nitrite (nitrite/nitrite IV group) reduced ERK and p38 activation. Conclusions: These data demonstrate that exposure of lung grafts and lung graft recipients to sodium nitrite potently limits lung ischemia/reperfusion injury following extended cold preservation and transplantation. Furthermore, mechanistically these data demonstrate an association of this cytoprotective effect with downregulation of ERK and p38 activation.

The Efficacy of Nitrite in the Lung I/R Injury (*P < 0.05 vs Control/Control IV) Blood Gas (2 hrs)

PV pO 2 (mmHg)

NM control/control IV control/nitrite IV nitrite/control IV nitrite/nitrite IV

297 ⫾ 17.7 99.2 ⫾ 17.6

mRNA level (2 hrs)

TNF-a

IL-6

MPO (2 hrs)

IL1-␤

(?OD/min/mg protein)

0.8 ⫾ 0.2 14.8 ⫾ 3.1

1 ⫾ 0.3 716 ⫾ 104

0.9 ⫾ 0.1 21.4 ⫾ 3.7

0.15 ⫾ 0.05 3.95 ⫾ 0.81

96.5 ⫾ 25.5

17.8 ⫾ 8.1

570 ⫾ 200

15.9 ⫾ 2.5

2.89 ⫾ 1.34

155.1 ⫾ 36.2

18.3 ⫾ 9.2

577 ⫾ 119

18.3 ⫾ 4.0

2.65 ⫾ 0.47*

234.1 ⫾ 53.7*

6.8 ⫾ 1.2*

367 ⫾ 80.3*

9.4 ⫾ 1.4*

2.06 ⫾ 0.73*

67. IL-17 NEUTRALIZATION PREVENTS AUTOIMMUNE DIABETES IN NOD MICE BUT DOES NOT DELAY ALLOGRAFT REJECTION IN ISLET TRANSPLANTATION. Juliet Emamaullee, Colin Anderson, A.M. James Shapiro; University of Alberta, Edmonton, AB, Canada Objectives: Interleukin-17 is an effector cytokine which is produced by Th17 cells and is associated with inflammatory processes. High levels of IL-17 have been associated with many autoimmune conditions in humans and animal models, including EAE and type 1

206 ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS diabetes. Anti-IL-17 has been shown to reduce inflammation in animal models of autoimmune disease. The effect of anti-IL-17 on the development of diabetes in NOD mice, a model of spontaneous autoimmune diabetes, and during allogeneic islet transplantation was investigated in the present study. Materials and Methods: For studies using NOD mice, anti-IL-17 or isotype control was administered over a 12 day period to either 5 or 10 week old female mice. In addition, anti-IL-17 treatment was investigated in newly diabetic NOD mice and following syngeneic islet transplantation in diabetic NOD mice. For allograft studies, either anti-IL-17 or isotype treatment was given following transplantation of 500 islets from BALB/c donors into streptozotocin-induced diabetic B6 mice. Serum cytokine levels and islet histological analysis for immune markers was performed following diabetes onset. Results: Anti-IL-17 significantly reduced diabetes onset in NOD mice when treatment was started at 10 weeks of age, with 4/10 anti-IL-17 treated animals becoming diabetic versus 9/10 isotype-control treated animals by 6 months of age (p⬍0.001). An increase in FoxP3⫹ regulatory T-cells was detected in the islets of normoglycemic animals that had received anti-IL17 treatment beginning at 10 weeks of age. A significant increase in Th2 cytokines and a decrease in Th1 cytokines was observed in this cohort as well. Anti-IL-17 treatment did not prevent diabetes onset in 5 week old NOD mice, cause remission from diabetes, or prevent recurrent autoimmunity following syngeneic islet transplantation. Anti-IL-17 therapy had no significant impact on islet allograft survival. Conclusion: Anti-IL-17 potently prevents spontaneous autoimmune diabetes in NOD mice at the time of disease onset (10-12 weeks), suggesting that Th17 cells dominate the early phase of islet autoimmunity. The discrepancy between results obtained in islet autoimmunity versus allogeneic islet destruction reported here suggests that IL-17 neutralization may specifically regulate autoreactive immune responses. Further development of this therapy may allow for specific inhibition of autoimmune disease in clinical islet transplantation.

Introduction: Ischemia/reperfusion (I/R) injury remains a major problem in solid organ transplantation, as it contributes to postoperative graft dysfunction and influences both long-term graft and patient survival. We have recently demonstrated that the Toll-like receptor 4 (TLR4) plays a central role in mediating the early inflammatory response after cold I/R. CD14 is a cell surface co-receptor that acts in concert with TLR4. High mobility group box-1 (HMGB1) is a DNA-binding protein that has recently been shown to serve as endogenous mediator of inflammation that signals through TLR4 and possibly CD14. While the importance of TLR4 is evident, the roles of CD14 and HMGB1 in cold I/R are unknown. We now hypothesize that both CD14 and HMGB1 contribute to the early inflammatory response after cold I/R. Methods: To evaluate the role of CD14, we performed syngeneic, heterotopic cardiac transplants in CD14 knock-out (KO) mice (CD14KO¡CD14KO, n⫽6) and C57/Bl6 wildtype (WT) mice (WT¡WT, n⫽6). Donor hearts were subjected to 2 hours of cold ischemia in UW solution. After 3 hours of reperfusion, grafts were retrieved and serum was harvested. Levels of serum cytokines were measured by ELISA. Quantitative RT-PCR was used to measure transcript levels of intra-graft inflammatory mediators. ␤-actin was used as a normalizing control for RT-PCR. To assess the role of HMGB1, syngeneic, heterotopic cardiac transplants were performed in C3H/HeOuJ mice. Immunohistochemical staining for myocardial HMGB1 in control, untransplanted hearts and in transplanted grafts after 3 hours of reperfusion was performed. In a separate experiment, either anti-HMGB1 neutralizing antibody (600 ␮g, n⫽6) or control isotype IgG (600 ␮g, n⫽6) was administered at the time of reperfusion. Serum cytokine levels were measured by ELISA. Results: Compared to WT control mice, CD14 KO mice exhibited significantly lower (p ⬍ 0.05) systemic IL-6 (1554 ⫾ 68 vs. 972 ⫾ 230 pg/ml) and JE/MCP-1 (818 ⫾ 135 vs. 389 ⫾ 86 pg/ml) levels after I/R. Intra-graft TNF␣ (103.6 ⫾ 16.7 vs. 29.8 ⫾ 2.4) and IL-1␤ (114.8 ⫾ 23.9 vs. 56.8 ⫾ 15.4) mRNA levels were also significantly lower (p⬍0.05) in CD14KO grafts compared to WT grafts. Trends toward lower levels of ICAM-1 (113.9 ⫾ 22.9 vs. 75.0 ⫾ 11.3) and iNOS (100.1 ⫾ 29.5 vs. 85.5 ⫾ 9.0) were also observed in CD14KO grafts compared to controls. After I/R, transplanted grafts revealed striking translocation of HMGB1 out of the nucleus in cardiac myocytes by immunohistochemical staining. Further, administration of anti-HMGB1 neutralizing antibody at the time of reperfusion resulted in reduced systemic inflammation (p⬍0.05) compared to animals receiving control IgG, as measured by serum levels of IL-6 (1456 ⫾ 139 vs. 1037 ⫾ 42 pg/ml) and JE/MCP-1 (543 ⫾ 110 vs. 270 ⫾ 66 pg/ml). Conclusions: TLR4 plays a central role in the early inflammatory response after cold I/R. These results demonstrate that both CD14, a co-receptor that acts in concert with TLR4, and HMGB1, an endogenous ligand of TLR4, contribute to the early inflammatory response after cold I/R. A better understanding of the molecular events that mediate the inflammatory response in cold I/R may allow for the development of therapeutics aimed at preventing the adverse consequences of I/R injury. 69. 20-HETE HAS A ROLE IN LIMITING RENAL INJURY FROM ISCHEMIA/REPERFUSUION. Brian D. Shames, Richard J. Roman, Shaoying Chen, Scott K. Van Why; Medical College of Wisconsin, Milwaukee, WI

68. MECHANISMS OF TOLL-LIKE RECEPTOR 4- MEDIATED INFLAMMATION AFTER COLD ISCHEMIA/ REPERFUSION (I/R)ROLE OF CD14 AND HMGB1. David J. Kaczorowski, Atsu Nakao, Raghuveer Vallabhaneni, Brian S. Zuckerbraun, Kenneth R. McCurry, Timothy R. Billiar; University of Pittsburgh, Pittsburgh, PA

Background: Brown-Norway (BN) rats have profound resistance to renal ischemia/reperfusion (IR) injury. We have found this protection to be linked, in part, to chromosome 5 of the BN rat. Cytochrome P450 genes, residing on chromosome 5, metabolize arachidonic acid to 20-HETE. 20-HETE has a role in exacerbating myocardial and brian ischemic injury. In this study, we explored whether 20-HETE plays a role in the kidney after IR injury. Methods: Rat strains studied were the BN, Dahl salt-sensitive (SSMCW), a consomic strain with BN chromosome 5 introgressed into the SSMCW genetic background (SSBN5), and the commonly studied Sprague-Dawley (SD). The production of 20-HETE was measured in SSBN5 rats and