- Email: [email protected]
65. CD4+ T Cell Immunity Following Gene Transfer Into Hematopoietic Stem Cells
204 ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS canonical Wnt signaling in NB, and to determine whether a subset of NB tumor cells maintain canonical Wnt signaling thus rendering the neuroblast phenotype. Methods: Pediatric NB specimens were collected during operative biopsy from consenting patients (IRBapproved protocol). Specimens were fixed and prepared for histological analysis and immunohistochemistry. An established neuroblastic NB cell line, BE(2)-C, that is multipotent for both glia and neurons was utilized to facilitate in vitro analysis. Immunoblotting for the de-phosphorylated active form of -catenin was performed utilizing whole cell lysates. Co-localization for Hnk-1, a neural crest progenitor cell marker, and -catenin was performed by immunofluorescence. A lentiviral reporter construct (SuperTOP.GFP) containing (7) TCF/LEF cassettes driving GFP expression was utilized to identify a subpopulation of NB cells with canonical signaling. Exogenous Wnt3a protein stimulation was used to stimulate Super.TOP transduction followed by FACS for GFP in order to isolate cells with enhanced canonical Wnt (-catenin/TCF/LEF) pathway activation. Results: Nuclear (Canonical) -catenin expression was observed in a subpopulation of neuroblastoma cells from a series of operative specimens (n⫽3) by immunohistochemistry. Immunoblotting detected the expression of active -catenin in the BE(2)-C cells prior to differentiation, however this level did not change following BE(2)-C cell differentiation to either a glial or neural- predominant population as determined by blotting for the marker proteins vimentin and peripherin respectively. Immunofluorescence studies did demonstrate that -catenin was co-expressed in Hnk-1⫹ cells. SuperTOP reporter assays demonstrated that an initial sub-population (14%) of NB cells with functional Wnt/catenin signal transduction could be enriched to 35% when undifferentiated cells were stimulated by exogenous Wnt3a protein prior to FACS analysis. Conclusions: The pediatric embryonal tumor NB is comprised of a subset of cells that demonstrate canonical Wnt signal transduction as evidenced by the nuclear localization of -catenin within a minor population of blast like cells. The colocalization of -catenin and the neural crest progenitor marker HNK-1 suggests that canonical Wnt signaling may identify a subpopulation of NB cells that maintain the neuroblast phenotype. These cells can be enriched from a heterogeneous tumor cell population using Wnt protein stimulation and FACS analysis for -catenin/TCF/LEF signal transduction. The SuperTOP functional reporter of canonical Wnt signaling will facilitate our testing the neuroblast as tumor stem cell hypothesis as well as the functional role of Wnt signaling in NB tumor cell biology.
increased levels of iNOS protein and mRNA (Figure 1,2). Because increased iNOS is known to induce apoptosis via formation of NO and its reactive oxidation intermediate, peroxynitrite, we reasoned that NO may mediate ES-induced apoptosis. Indeed, blocking iNOS activity with L-NIL significantly reduced levels of apoptosis in EStreated cells (Figure 3). Conclusion: Our data show that ESinduced enterocyte apoptosis involves up-regulation of iNOS. Inhibitors of iNOS, therefore, may be effective in protecting the intestinal barrier in NEC from pathogenic bacteria.
64. INDUCIBLE NITRIC OXIDE SYNTHASE (INOS) MEDIATES ENTEROBACTER SAKAZAKII (ES)-INDUCED APOPTOSIS OF ENTEROCYTES. Diana H. Yu, Catherine J. Hunter, Yigit S. Guner, Nikunj K. Chokshi, Anatoly V. Grishin, Henri R. Ford; Childrens Hospital Los Angeles, Los Angeles, CA Introduction: Necrotizing Enterocolitis (NEC) is characterized by intestinal epithelial injury in susceptible neonates, leading to gut barrier failure. Bacterial translocation at the site of injury is implicated in NEC pathogenesis. The use of infant formula contaminated with Enterobacter sakazakii (ES) has been associated with severe outbreaks of NEC. ES has been shown to cause apoptosis in intestinal epithelial cells in vitro. We have previously shown that sustained up-regulation of inducible nitric oxide synthase (iNOS) in NEC leads to enterocyte apoptosis. Thus, we hypothesized that enterocyte apoptosis induced by ES may be mediated by iNOS up-regulation. Methods: Rat intestinal epithelial cells (IEC-6) were treated with ES (10 5 cfu/ml) for 6 h. The cells were harvested and the levels of iNOS mRNA and protein were determined by semi-quantitative PCR and Western blot analysis respectively. Apoptosis was determined using TUNEL assay. iNOS activity was inhibited with L-iminoethyl lysine (L-NIL) (3uM). Results: Previously we have established that exposure of IEC-6 cells to live ES induced apoptosis. Here we show that ES induces expression of iNOS, as judged by
TRANSPLANT/IMMUNOLOGY I 65. CD4 ⴙ T CELL IMMUNITY FOLLOWING GENE TRANSFER INTO HEMATOPOIETIC STEM CELLS. Sung P. Ha1, Kenneth Cornetta1, Hal E. Broxmeyer1, Nicholas P. Restifo2, Christopher E. Touloukian1; 1Indiana University School of Medicine, Indianapolis, IN; 2NIH, Bethesda, MD
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
increased levels of iNOS protein and mRNA (Figure 1,2). Because increased iNOS is known to induce apoptosis via formation of NO and its reactive oxidation intermediate, peroxynitrite, we reasoned that NO may mediate ES-induced apoptosis. Indeed, blocking iNOS activity with L-NIL significantly reduced levels of apoptosis in EStreated cells (Figure 3). Conclusion: Our data show that ESinduced enterocyte apoptosis involves up-regulation of iNOS. Inhibitors of iNOS, therefore, may be effective in protecting the intestinal barrier in NEC from pathogenic bacteria.
64. INDUCIBLE NITRIC OXIDE SYNTHASE (INOS) MEDIATES ENTEROBACTER SAKAZAKII (ES)-INDUCED APOPTOSIS OF ENTEROCYTES. Diana H. Yu, Catherine J. Hunter, Yigit S. Guner, Nikunj K. Chokshi, Anatoly V. Grishin, Henri R. Ford; Childrens Hospital Los Angeles, Los Angeles, CA Introduction: Necrotizing Enterocolitis (NEC) is characterized by intestinal epithelial injury in susceptible neonates, leading to gut barrier failure. Bacterial translocation at the site of injury is implicated in NEC pathogenesis. The use of infant formula contaminated with Enterobacter sakazakii (ES) has been associated with severe outbreaks of NEC. ES has been shown to cause apoptosis in intestinal epithelial cells in vitro. We have previously shown that sustained up-regulation of inducible nitric oxide synthase (iNOS) in NEC leads to enterocyte apoptosis. Thus, we hypothesized that enterocyte apoptosis induced by ES may be mediated by iNOS up-regulation. Methods: Rat intestinal epithelial cells (IEC-6) were treated with ES (10 5 cfu/ml) for 6 h. The cells were harvested and the levels of iNOS mRNA and protein were determined by semi-quantitative PCR and Western blot analysis respectively. Apoptosis was determined using TUNEL assay. iNOS activity was inhibited with L-iminoethyl lysine (L-NIL) (3uM). Results: Previously we have established that exposure of IEC-6 cells to live ES induced apoptosis. Here we show that ES induces expression of iNOS, as judged by
TRANSPLANT/IMMUNOLOGY I 65. CD4 ⴙ T CELL IMMUNITY FOLLOWING GENE TRANSFER INTO HEMATOPOIETIC STEM CELLS. Sung P. Ha1, Kenneth Cornetta1, Hal E. Broxmeyer1, Nicholas P. Restifo2, Christopher E. Touloukian1; 1Indiana University School of Medicine, Indianapolis, IN; 2NIH, Bethesda, MD
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