Prolonged Cardiac Allograft Survival Research Articles

Extracellular Hmgb1 Functions as an Innate Immune-Mediator Implicated in Murine Cardiac Allograft Acute Rejection

Hmgb1, an evolutionarily conserved chromosomal protein, was recently re-discovered to be an innate immune-mediator contributing to both innate and adaptive immune responses. Here, we show a pivotal role for Hmgb1 in acute allograft rejection in a murine cardiac transplantation model. Extracellular Hmgb1 was found to be a potent stimulator for adaptive immune responses. Hmgb1 can be either passively released from damaged cells after organ harvest and ischemia/reperfusion insults, or actively secreted by allograft infiltrated immune cells. After transplantation, allografts show a significant temporal up-regulation of Hmgb1 expression accompanied by inflammatory infiltration, a consequence of graft destruction. These data suggest the involvement of Hmgb1 in acute allograft rejection. In line with these observations, treatment of recipients with rA-box, a specific blockade for endogenous Hmgb1, significantly prolonged cardiac allograft survival as compared to those recipients treated with either rGST or control vehicle. The enhanced graft survival is associated with reduced allograft expression of TNFα, IFNγ and Hmgb1 and impaired Th1 immune response.

Prolongation of Cardiac Allograft Survival by Inhibition of ERK1/2 Signaling in a Mouse Model

It has been demonstrated that in vitro the presence of extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling inhibitor suppresses T cell activation and Th1 development. However, pharmacological interference of ERK1/2 signaling by administration of its small molecule inhibitor has not been tested as a therapeutic target in the prevention of allograft rejection. The immunosuppressive effect of targeting ERK1/2 signaling was tested on cardiac allograft survival in C57BL/6 (H-2b) to Balb/c (H-2d) murine model using PD98059 inhibitor. Phosphorylation/activation of ERK 1/2 and STAT6 proteins were assessed by Western blot. Blockade of ERK1/2 using PD98059 had significant immunosuppressive effect and prolonged survival of mouse cardiac allografts from 8.3+/-0.5 days (vehicle) to 12.6+/-1.3 days (100 mg/kg PD98059; P<0.0001). Combination therapy of PD98059 (100 mg/kg) with cyclosporine (CsA, 15 mg/kg for 20 days) additionally enhanced graft survival (34.4+/-1.2 days) compared to CsA (14.9+/-1.1 days; P<0.0001) or PD98059 monotherapy (P<0.0001). Attenuation of graft rejection by PD98059 correlated to reduction of intragraft ERK phosphorylation and leukocyte infiltration, and to increase in interleukin (IL)-4 or decrease in interferon-gamma production within the grafts. In vitro inhibition of ERK1/2 by PD98059 promoted Th2 differentiation by upregulation IL-4 production but not altering IL-4 stimulating STAT6 pathway. Targeting ERK1/2 signaling results in suppression of alloimmune responses by an unique mechanism that involves Th1/Th2 skewing, suggesting a therapeutic potential of inhibition of ERK1/2 signaling for transplant rejection, particularly in combination with CsA.

Control of Allograft Rejection by Applying a Novel Nuclear Factor-κB Inhibitor, Dehydroxymethylepoxyquinomicin

Nuclear factor (NF)-kappaB plays a crucial role in lymphocyte activation, proliferation, and survival. We examined the immunosuppressive effect of a newly developed NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) in allotransplantation. Purified C57BL/6 (H-2b) T cells were used for in vitro studies examining activation, proliferation, cytokine production and nuclear NF-kappaB and nuclear factor of activated T cells (NFAT) protein levels. A fully major histocompatibility complex incompatible BALB/c (H-2d)-to-C57BL/6 mice cardiac transplantation model was utilized for in vivo studies. DHMEQ was given intraperitoneally to transplant recipients at a various dose starting from day 0. In some, DHMEQ was administered concomitantly with tacrolimus. DHMEQ significantly suppressed alphaCD3 + alphaCD28 monoclonal antibody-triggered T-cell proliferation, CD25/CD69 expressions, and both interleukin-2 and interferon (IFN)-gamma production in a dose-dependent fashion. DHMEQ blocked nuclear translocation of NF-kappaB but not NFAT in activated T cells. Combined treatment with DHMEQ and tacrolimus significantly suppressed T cell activation as compared to that of mono-therapy with either agent alone. Single DHMEQ treatment moderately prolonged cardiac allograft survival. Further, combination of DHMEQ plus tacrolimus markedly prolonged graft mean survival time (MST) to 59.5 days when compared to either DHMEQ (MST: 10 days) or tacrolimus (MST: 13 days) treatment alone. Such effect was associated with inhibition of mixed lymphocyte reaction against donor antigen, IFN-gamma producing splenocytes and graft cellular infiltration as examined at 5 and 12 days posttransplantation. DHMEQ inhibits nuclear translocation of NF-kappaB but not NFAT in activated T cells, and prolongs allograft survival. Blocking both NF-kappaB and NFAT by DHMEQ and tacrolimus induces potent immunosuppression, which may become a new modality in controlling allograft rejection.

Essay: History of transplantation

Over the past 50 years organ transplantation has become established worldwide with ever-improving results, conferring immense benefi t to hundreds of thousands of patients. The general principles of the surgical procedures and organ preservation have been accepted for all organ transplants, but the biology of graft rejection is still only partly understood. The discovery of natural acquired immunological tolerance and experimental methods of producing tolerance by Billingham, Brent, and Medawar raised the hopes of clinicians that there might be a way of inducing in the recipient at the time of organ grafting a state of temporary immunological plasticity similar to the stage that occurs in fetal development. In 1959 mercaptopurine, which is used in the treatment of leukaemia, was shown by Schwarz and Damechek to prevent antibody formation in rabbits challenged with foreign protein. They called this observation drug-induced immunological tolerance. Studies in the UK on kidney-grafted animals treated with mercaptopurine produced a moderate extension of graft survival and led to a practical clinical regimen of treatment with the mercaptopurine analogue azathioprine plus corticosteroids. At 1 year, graft function was around 50%. Kidney transplantation remained confi ned to about ten centres worldwide, and the procedure was viewed with suspicion because of the poor overall results. Then, in the late 1970s, Borel and colleagues discovered the immunosuppressive properties of the fungal cyclic peptide ciclosporin, which prolonged survival of skin grafts in mice. Further studies of this drug in the UK showed prolonged survival of cardiac allografts in rats and pigs and renal transplants in dogs. When ciclosporin was fi rst used in people, based on the dose given to animals, it was severely nephrotoxic. After a worrying learning curve of dose adjustments, ciclosporin was shown to improve the 1-year survival of kidney grafts to around 80%, and for the fi rst time surgeons became confi dent in transplanting the liver and heart with ciclosporin immunosuppression. Ciclosporin was a watershed in the development of transplantation. Instead of a treatment limited to a handful of centres worldwide, transplantation became a muchvalued form of therapy spreading to more than a thousand centres. The kidney was the organ most commonly transplanted, but increasingly good results were obtained with heart, liver, and, eventually, lungs and pancreas. One method of minimising the nephrotoxicity of ciclosporin was to combine this drug with azathioprine and corticosteroids so that the total immunosuppressive activity would be additive, but the individual side-eff ects of the diff erent agents would be less. Gradually, the “halflife” of organ transplants improved. Many new immunosuppressive agents have been investigated experimentally, and several have been introduced into the clinic, including polyclonal and monoclonal antilymphocyte antibodies. There has been an unfortunate tendency to add more and more potent immunosuppressive agents to therapy with concomitant over-immunosuppression. Bone-marrow transplantation has also advanced through new regimens of non-ablative transplantation with mixed chimaerism, initially planned to retain the graft-againstleukaemia eff ect but also used in an important series of clinical experiments in patients subsequently given kidney transplants, in whom tolerance occurred despite the disappearance of the donor chimaeric state in the blood. Besides their expense, conventional drug regimens can cause great hardship to patients and non-compliance is common. Some patients with liver transplants stopped taking their drugs and performed a clinical experiment demonstrating immunological tolerance, surviving many years with good liver function despite the absence of any maintenance immunosuppression. Other patients were not so lucky, and this weaning of maintenance immunosuppression is far more likely to be successful with liver than with kidney transplants, which is consistent with experimental demonstration of liver tolerance without any drugs after orthotopic liver transplantation in pigs and rodents. Lancet 2006; 368: S51–S52

Exogenous IL-10 overexpression reduces perforin production by activated allogenic CD8+cells and prolongs cardiac allograft survival

Perforin is a cytolytic mediator produced by cytotoxic T cells (CD8(+) cells) and natural killer cells. We previously reported that ex vivo IL-10 gene therapy induced apoptosis of allogenic infiltrative CD8(+) cells and significantly prolonged cardiac allograft survival. To further test the hypothesis that localized IL-10 overexpression in cardiac allografts may also effect the alloreactive CD8(+) T cell function by downregulating its perforin production, we used a rabbit functional heterotopic allograft heart transplant model. Human recombinant IL-10 gene complexed with liposome was intracoronary delivered into the cardiac allografts ex vivo. The percentage of apoptotic infiltrative CD8(+) cells in cardiac allografts was increased 6-fold in the gene therapy group vs. the control group, whereas the percentage of perforin-positive CD8(+) cells was decreased 2.9-fold (P < 0.01). Perforin expression level in the allograft myocardium of the gene therapy group was deceased 3.2-fold (P < 0.01). The amount of infiltrative perforin-positive CD8(+) cells and perforin expression level were inversely correlated with IL-10 transgene and protein expression level in the myocardium of cardiac allografts (P < 0.01), the percentage of apoptotic cardiac myocytes (P < 0.01), and the peak left ventricular systolic pressure of cardiac allografts (P < 0.01) but significantly correlated with the infiltrative T cell cytotoxicity (P < 0.01) and allograft rejection score (P < 0.01). These results suggest that localized IL-10 gene therapy prolongs cardiac allograft survival, at least in part, through downregulation of perforin production by activated allogenic CD8(+) T cells. Reduction of cytolytic function of cytotoxic effector cells prevents the apoptosis of cardiac myocytes.

Use of the Inhibitory Effect of Apoptotic Cells on Dendritic Cells for Graft Survival Via T-Cell Deletion and Regulatory T Cells

Tolerance induction against donor allo-antigens (allo-Ag) remains one of the most challenging aspects of transplant immunology. The ability of dendritic cells (DC) to participate in immunity and tolerance makes them an excellent tool for tolerance induction. Here, we employed the immunosuppressive properties of apoptotic cells to deliver simultaneously an inhibitory signal and donor allo-Ag to recipient DC for treatment of allograft rejection. DC that captured apoptotic cells remained immature and activated deficiently anti-donor CD4(+) T cells that were unable to upregulate T-cell activation markers, to secrete IL-2 and IFN-gamma and to survive under homeostatic conditions due to low expression of Bcl-X(L), IL-7R and IL-15R. Administration of donor apoptotic cells decreased the systemic anti-donor T- and B-cell response and prolonged cardiac allograft survival in mice. The effect was donor specific and required the interaction of donor apoptotic cells with recipient quiescent CD8alpha(+) DC. When combined with CD40-CD154-blockade, administration of donor apoptotic cells resulted in indefinite graft survival mediated by generation of regulatory T cells. The use of the inhibitory effects of apoptotic cells on the anti-donor response provides a new approach to treat transplant rejection.

STAT-1 and AP-1 decoy oligonucleotide therapy delays acute rejection and prolongs cardiac allograft survival

Acute myocardial rejection is a cell-mediated process characterized by increased leukocyte recruitment into the graft myocardial tissue. Transcription factors like STAT-1 and AP-1 are critically involved in this process by regulating vascular adhesion molecule expression. The aim of our study was to investigate the effect of decoy oligodeoxynucleotide (dODN) treatment targeting transcription factors AP-1 and STAT-1 on acute cardiac allograft rejection in a rat transplant model. Wistar-Furth (WF) cardiac allografts were transplanted into Lewis (LEW) rats after perfusion with STAT-1 or AP-1 dODN solution (5 micromol/l), buffer or the corresponding mutant control ODNs. Grafts were harvested and processed for histologic and immunohistochemical evaluation. As demonstrated by fluorescence dye-labelled dODN, exposure of the grafts to the dODNs during 45 min of warm ischemia resulted in a dominant uptake of naked DNA by the graft endothelium. Treatment with AP-1 and STAT-1 dODNs, but not with vehicle or the control dODNs, significantly prolonged cardiac allograft survival by approximately 40% from 5.6+/-0.5 days to 7.8+/-1.3 days and 7.4+/-0.5 days, respectively (mean+/-S.D., p<0.01, n=5 in each group). Immunohistochemical examination on days 1, 3 and 6 revealed a marked reduction of infiltrating leukocytes (AP-1 dODN: 85%, STAT-1 dODN: 50%), namely T-cells, in the dODN-perfused grafts at day 3 post transplantation. In addition, as demonstrated by immunohistochemical analysis, endothelial expression of ICAM-1 and VCAM-1 was found to be markedly reduced in dODN-treated grafts. Both AP-1 and STAT-1 dODN treatments suppress graft endothelial adhesion molecule expression, reduce graft infiltration and in turn significantly delay acute rejection. The utilization of dODNs in the cardioplegic solution might be a novel strategy to protect transplanted organs from early damage during transplantation, to preserve organ function and bridge the critical phase after transplantation when standard immunosuppression is not yet completely effective.

Gene transfer of RANTES and MCP-1 chemokine antagonists prolongs cardiac allograft survival

Vascularized organ allografts are rapidly destroyed by host immune cells that are recruited along chemokine gradients. Among chemokines, Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) CC chemokine ligand (CCL5) and monocyte chemoattractant protein (MCP)-1 (CCL2) are upregulated in rejecting cardiac allografts. To antagonize these chemokines, we constructed adenoviral vectors expressing NH(2)-terminal deletion (8ND) mutants of the respective genes. Using the F344-to-LEW rat model, intragraft gene transfer of chemokine analogs prolonged cardiac allograft survival from 10.1+/-0.7 and 10.4+/-0.7 days using non-coding adenovirus and vehicle alone, respectively, to 17.0+/-0.7 days for 8ND-RANTES (P<0.001) and 14.2+/-0.8 days for 8ND-MCP-1 (P<0.01). 8ND-RANTES reduced graft infiltration by monocytes/macrophages, cluster of differentiation (CD) 8alpha(+) and T-cell receptor alphabeta(+) cells, while 8ND-MCP-1 reduced monocytes/macrophages. In mixed leukocyte reactions in vitro, proliferation of host lymphocytes from regional lymph nodes in response to donor splenocytes was unaffected by 8ND-RANTES gene transfer. Using a two-gene approach, the contribution of 8ND-MCP-1 was negligible, consistent with available evidence that 8ND-RANTES inhibits both RANTES and MCP-1 activities. 8ND-RANTES gene transfer and a short course of low-dose cyclosporine A synergistically prolonged graft survival to 37.8+/-5.5 vs 15.4+/-0.5 days with cyclosporine alone (P<0.001). These results suggest a role for anti-chemokine gene therapy as an adjuvant therapy in heart transplantation.

An Antagonist Mutant IL-15/Fc Promotes Transplant Tolerance

IL-15 is a proinflammatory and antiapoptotic T-cell growth factor that plays an important role in a variety of autoimmune disorders and transplant rejection. To inhibit IL-15 function and to target IL-15 receptor (IL-15R) bearing cells, we have generated a unique lytic antagonistic mutant IL-15/Fc fusion protein (mIL-15/Fc). In this study, we further examined the efficacy of mIL-15/Fc in preventing allograft rejection cross minor and major histocompatibility barriers. A short-course treatment with mIL-15/Fc fusion protein is sufficient to prevent cardiac allograft rejection and induce antigen-specific tolerance in minor histocompatibility complex-mismatched recipients, and permit prolonged cardiac allograft survival in fully MHC mismatched recipients. In addition, mIL-15/Fc treatment, in combination with a suboptimal dose of anti-CD154 antibody, confers permanent cardiac allograft engraftment in a fully MHC-mismatched mouse strain combination. In a murine islet allograft model, mIL-15/Fc monotherapy is capable to permit permanent allograft survival in 50% fully MHC-mismatched recipients. Immunochemistry studies demonstrated that prolonged graft survival was accompanied by reduced intragraft mononuclear cell infiltration and pro-inflammatory cytokine gene expression in the mIL-15/Fc treated recipients. Moreover, parallel experiments employing a mutated nonlytic IgG2a Fc demonstrate that the Fc portion of mIL-15/Fc contributes to the overall efficacy of the molecule in vivo.

Lymphoid Sequestration of Alloreactive Memory CD4 T Cells Promotes Cardiac Allograft Survival

Memory T cells specific for donor Ags present a unique challenge in transplantation. In addition to expressing robust immune responses to a transplanted organ, memory T cells may be resistant to the effects of immunosuppressive therapies used to prolong graft survival. In this study, we explore the possibility of controlling deleterious donor-reactive memory CD4 T cells through lymphoid sequestration. We showed that sphingosine 1-phosphate receptor-1 agonist FTY720 induces relocation of circulating memory CD4 T cells into secondary lymphoid organs. Lymphoid sequestration of these donor-reactive memory CD4 T cells prolonged survival of murine heterotopic cardiac allografts and synergizes with conventional costimulatory blockade to further increase graft survival. Despite limited trafficking, memory CD4 T cells remain capable of providing help for the induction of anti-donor CD8 T cell and alloantibody responses. Elimination of antidonor humoral immunity resulted in indefinite allograft survival proving the pathogenicity of alloantibody under these conditions. Overall, this is the first demonstration that FTY720 influences memory CD4 T cell trafficking and attenuates their contribution to allograft rejection. The data have important implications for guiding FTY720 therapy and for designing combinatorial strategies aimed at prolonging allograft survival in sensitized transplant patients with donor-specific memory T cells.

Immune regulatory activity of liver‐derived dendritic cells generated in vivo

Hepatic tolerance is demonstrated by spontaneous acceptance of liver allografts in mice. Hepatic dendritic cells (DC) play a crucial role in determining immunity or tolerance. In this study, we adopted an approach to transfect gene(s) into the mouse liver by tail-vein injection of plasmid-carrying genes. Transfection with GM-CSF expanded liver CD11c+ myeloid DC (LMDC), while liver B220+CD11c- lymphoid DC (LLDC) were expanded after transfection of IL-3 and CD40L. Flow analysis revealed that these liver DC subsets were phenotypically mature following overnight culture. However, in contrast to LMDC, LLDC induced hyporesponsiveness in allogeneic T-cells, with suppressed secretion of both IL-2 and IFN-gamma, and prolonged cardiac allograft survival. This immune regulatory DC population in the liver may play a role in modulating T-cell immunity in the liver.

Ex Vivo Expanded Dendritic Cells Home to T-Cell Zones of Lymphoid Organs and Survive in Vivo after Allogeneic Bone Marrow Transplantation

Little is known about adoptive transfer of allogeneic ex vivo expanded dendritic cells (eDCs). We investigated the trafficking pattern of eDCs in mice after allogeneic bone marrow transplantation by using bioluminescence imaging. eDCs were expanded from bone marrow precursors in the presence of GM-CSF, interleukin-4, and Flt3L and retrovirally transduced to express luciferase (luc) and green fluorescence protein (gfp). Flow cytometry showed polyclonal DC populations after expansion that consisted of CD11c+CD11b+ and CD11c-CD11b+ cells that co-expressed CD40, CD80, CD86, and MHCII. eDCs were functional in mixed lymphocyte reactions and produced tumor necrosis factor-alpha on phytohemagglutinin stimulation. The eDCs were then injected intravenously into BALB/c recipient mice that had received allogeneic bone marrow transplantation 6 weeks previously. On day 1 after transfer, eDCs were detected by bioluminescence imaging throughout the lungs and spleen. In the later course, signals were observed throughout thymus, lower abdomen, and spleen throughout a period of more than 42 days. Immunofluorescence microscopy confirmed CD11c positivity on the gfp+ donor cells, which localized in T-cell zones of mesenteric lymph nodes, Peyer's patches, spleen, and thymus. These findings are important for adoptive immunotherapies because they indicate that eDCs migrate efficiently in vivo and are capable of surviving long term.

Inhibition of Polymorphonuclear Leukocyte–Mediated Graft Damage Synergizes With Short-Term Costimulatory Blockade to Prevent Cardiac Allograft Rejection

The early inflammatory response during reperfusion of cardiac allografts is initiated by the infiltration of polymorphonuclear leukocytes (PMNs) into the graft. The impact of early PMN infiltration on allograft rejection compared with long-term graft survival remains poorly understood. We tested the role of CXCR2, the receptor for 2 PMN attractant chemokines, KC/CXCL1 and MIP-2/CXCL2, on intragraft inflammation and vascularized cardiac allograft rejection in a murine model. Compared with allografts retrieved from control recipients, both PMN infiltration and intragraft proinflammatory cytokine expression were significantly attenuated in allografts from CXCR2-antisera-treated wild-type or from CXCR2(-/-) recipients. Adoptive transfer of alloantigen-primed T cells rapidly infiltrated and rejected allografts in control recipients, but T-cell infiltration was significantly decreased in recipients depleted of PMNs at transplantation. The influence of early PMN-mediated inflammation on the therapeutic efficacy of costimulatory blockade to prevent allograft rejection was tested. Short-term treatment of recipients with anti-CD154 mAb or CTLA-4 Ig induced modest prolongation of cardiac allograft survival. However, CD154 mAb or CTLA-4 Ig treatment, combined with either peritransplantation PMN depletion or antibodies specific for KC/CXCL1 plus MIP-2/CXCL2, prolonged cardiac allograft survival beyond 100 days. Results suggest that strategies attenuating PMN-mediated tissue damage during reperfusion significantly improve the efficacy of short-term costimulatory blockade to prevent T-cell-mediated rejection of cardiac allografts.

Tolerance to Rat Heart Grafts Induced by Intrathymic Immunomodulation Is Mediated by Indirect Recognition Primed CD4+CD25+ Treg Cells

In a rat model (PVG.R8-to-PVG.1U) disparate for one class I antigen, RT.1Aa, we previously demonstrated that intrathymic immunomodulation with donor antigens resulted in prolonged survival of cardiac allografts that underwent chronic rejection. However, long-term survivors developed a regulatory cell population that prevented both acute and chronic rejection when adoptively transferred into secondary graft recipients. The purpose of this study was to characterize these regulatory cells with particular emphasis on CD4+CD25+ Treg cells. Spleens, lymph nodes, and peripheral blood lymphocytes of secondary tolerant recipients were characterized using antibodies to various T cell markers in flow cytometry. In vitro MLR and in vivo adoptive transfer experiments were conducted to investigate the involvement of CD4+CD25+ T cells in the observed tolerance. The presence of various cytokines in the sera of graft recipients and MLR culture supernatants was tested using ELISA. Tolerant recipients compared with naive rats had substantially higher percentages of CD4+CD25+ T cells in the spleen (28+/-3% vs. 11+/-5%) and blood (23+/-6% vs. 9+/-4%). Tolerant animals also had higher levels of serum IL-10 than naive and rejecting animals. CD4+CD25+ T cells from secondary long-term graft survivors inhibited donor-specific proliferative responses in vitro that was associated with high IL-10 production. Importantly, depletion of CD4+CD25+ T cells from splenocytes of tolerant rats abrogated their ability to transfer tolerance to tertiary graft recipients. Our data demonstrate that cardiac allograft tolerance in this model is mediated by CD4+CD25+ Treg cells primed by indirect recognition and is associated with high levels of IL-10.

Expression Profiling of Murine Double-Negative Regulatory T Cells Suggest Mechanisms for Prolonged Cardiac Allograft Survival

Recent studies have demonstrated that both mouse and human alpha beta TCR(+)CD3(+)NK1.1(-)CD4(-)CD8- double-negative regulatory T (DN Treg) cells can suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells. To identify molecules involved in DN Treg cell function, we generated a panel of murine DN Treg clones, which specifically kill activated syngeneic CD8+ T cells. Through serial cultivation of DN Treg clones, mutant clones arose that lost regulatory capacity in vitro and in vivo. Although all allogeneic cardiac grafts in animals preinfused with tolerant CD4/CD8 negative 12 DN Treg clones survived over 100 days, allograft survival is unchanged following infusion of mutant clones (19.5 +/- 11.1 days) compared with untreated controls (22.8 +/- 10.5 days; p < 0.001). Global gene expression differences between functional DN Treg cells and nonfunctional mutants were compared. We found 1099 differentially expressed genes (q < 0.025%), suggesting increased cell proliferation and survival, immune regulation, and chemotaxis, together with decreased expression of genes for Ag presentation, apoptosis, and protein phosphatases involved in signal transduction. Expression of 33 overexpressed and 24 underexpressed genes were confirmed using quantitative real-time PCR. Protein expression of several genes, including Fc epsilon RI gamma subunit and CXCR5, which are >50-fold higher, was also confirmed using FACS. These findings shed light on the mechanisms by which DN Treg cells down-regulate immune responses and prolong cardiac allograft survival.

Prolongation of Cardiac Allograft Survival by Systemic Administration of Immature Recipient Dendritic Cells Deficient in NF-κB Activity

To develop a more applicable approach that uses recipient-derived dendritic cells (DC) for organ transplantation. Systemic administration of immature donor DC that are deficient in costimulatory molecules delays the onset of allograft rejection. However, this approach requires in vitro DC propagation and would not be applicable to deceased donor organ transplantation. DC were propagated from C3H (H2) mouse bone marrow with GM-CSF; their maturation was arrested by treatment with oligodeoxyribonucleotides (ODN) specifically against nuclear factor (NF)-kappaB. The DC were pulsed with B10 (H2) splenocyte lysate. DC phenotype was examined by flow cytometry. Their allostimulatory activity was assessed in vitro by MLR and CTL assays and in vivo by the influence on B10 cardiac allograft survival. Cytokine profiles were analyzed by ELISA and RNase protection assay. NF-kappaB activity in DC nuclear protein was detected by gel shifting assay. Compared with mature DC, NF-kappaB ODN-treated immature DC pulsed with B10 (H2) spleen cell lysate elicited markedly lower proliferative responses and correlated with reduced IFN-gamma and increased IL-10 production. In contrast to administration of mature C3H DC pulsed with B10 antigen that accelerated rejection of B10 cardiac allografts, a single injection of NF-kappaB ODN DC pulsed with donor antigens significantly prolonged allograft survival in an antigen-specific manner. This was associated with induction of T-cell hyporesponsiveness and enhanced T-cell apoptosis. An approach to use recipient DC as a "vaccine" strategy provides a more feasible approach for deceased-donor organ transplantation.

FK778, a novel immunosuppressive agent, reduces early adhesion molecule up-regulation and prolongs cardiac allograft survival*

The adhesion molecules, P-selectin, ICAM-1, and VCAM-1 are important mediators of T-cell adhesion and T-cell co-stimulation. We investigated the effect of the malononitrilamide FK778 on cardiac allograft survival, acute allograft rejection, and adhesion molecule up-regulation in a heterotopic, cardiac transplantation model. Rats received low- or high-dose FK778 or no treatment. Grafts were harvested on the fifth postoperative day for histologic examinations. To assess allograft survival, recipients were treated for a maximum of 10 days and grafts were harvested after cessation of the contractile activity. FK778 low dose showed a mild but significant decrease in mononuclear infiltration but failed to markedly reduce histologic rejection, adhesion molecule up-regulation, or to prolong allograft survival. However, high-dose FK778 treatment significantly reduced early up-regulation of P-selectin, ICAM-1, and VCAM-1, abolished infiltration, reduced histologic rejection and resulted in prolonged cardiac allograft survival. Therefore, FK778 is a novel, highly desirable immunosuppressive drug for transplantation medicine.

Effect of streptavidin on cardiac allograft prolongation is due to host T-Cell suppression

Aim The aim of this study was to evaluate the effectiveness of streptavidin immunomodulation in the high-responder WF-to-Lewis combination. Methods/results We examined the effects of streptavidin on the proliferative response of T cells in coculture studies. Two to 200 μg/mL streptavidin significantly ( P < .001) suppressed the proliferation of Lewis T cells to WF by 76%–83% compared with untreated responders. Next, we studied the survival of WF cardiac allografts in Lewis recipients pretreated with streptavidin. A 5-day course of peritransplantation recipient treatment with streptavidin doses of 8, 12, 20, 40, and 60 mg/kg combined with single dose of 0.5 mL antilymphocyte serum (ALS) significantly ( P < .001) prolonged cardiac allograft survival from MST of 7 ± 0.5 and 8 ± 0.5 days in naive and ALS-treated controls to 15 ± 1, 20 ± 3, 16 ± 3, 17 ± 3, and 23 ± 2 days, respectively. In contrast, posttransplantation administration of 80 mg/kg streptavidin resulted in animal death, suggesting toxicity of this dose. Additionally, 10 mg/kg or 20 mg/kg streptavidin administration for 10 consecutive days resulted in significant graft prolongation (MST of 18 ± 1 and 21 ± 1 days, respectively; P < .001). Conclusion Although peritransplantation streptavidin treatment is effective in prolonging rat cardiac allografts in the high-responder WF-to-Lewis combination, it does not induce permanent graft survival as observed in the low-responder combination of Lewis-to-ACI. Our finding of in vitro immunomodulatory effect of streptavidin on T-cell proliferation suggests that its in vivo effect is partly due to prevention of T-cell activation following antigen exposure.

BAFF binding to T cell-expressed BAFF-R costimulates T cell proliferation and alloresponses.

Binding of the TNF family member, B cell activating factor (BAFF), to its receptor (BAFF-R, TNFRSF13C) is required for generation and maintenance of mature B cells, but there are no data as to any role for the BAFF/BAFF-R pathway in T cell functions. We report that the binding of BAFF to BAFF-R expressed by a subset of primarily CD4(+) T cells costimulates T cell activation and allo-proliferation in vitro and in vivo, and that mice with a mutation in the BAFF-R, or with a targeted deletion of BAFF, show prolonged cardiac allograft survival as compared to wild-type or transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI)(-/-) controls. Taken together, these data indicate the BAFF/BAFF-R pathway contributes to both T and B cell responses and may be an attractive target for control of acute and chronic allograft rejection.

EN-BLOC HEART AND THYMUS TRANSPLANTATION IN CYNOMOLGUS MONKEYS

P1204 Aims: To determine whether cotransplantation of a whole vascularized donor thymic lobe can prolong cardiac allograft survival in cynomolgus monkeys. Background: We have previously shown that transplantation of vascularized donor thymic tissue can prolong cardiac allograft survival in miniature swine. To test this modality in primates and, at the same time, test a procurement technique applicable to human cadaveric heart transplantation, we have developed an en-bloc heart-thymus transplant procedure for cynomolgus monkeys. Methods: The right mediastinal thymus was harvested en-bloc with the heart, preserving the entire arterial supply and venous drainage to the thymus. The en-bloc heart-thymus grafts were transplanted heterotopically into MHC mismatched (by MLR) cynomolgus monkeys. All recipients were treated with rabbit anti-thymocyte globulin at the time of transplant, and daily FK506 for 28 days (target levels 30-50 ng/ml). Some recipients were thymectomized on the day of transplant. Results: Three control animals were transplanted with unmodified heart alone (one was thymectomized at the time of transplant). Cardiac allograft survival was 40, 61 (euthymic), and 62 (thymectomized) days. Three animals underwent en-bloc heart-thymus transplantation (one was thymectomized at the time of transplant). En-bloc heart-thymus allograft survival was 158, 48 (euthymic), and 127 (thymectomized) days. Acute rejection was eventually seen in the donor thymus and heart and all animals maintained donor specific responsiveness by MLR. One of the three en-bloc heart-thymus grafts (survival 158 days) was from an animal that had previously received thymic irradiation, as was evident on an early thymic biopsy which revealed fibrosis. Early biopsy was available on another en-bloc graft (survival 127 days) which showed normal medullary and cortical regions, however without any hassals corpuscles. Conclusions: En-bloc heart-thymus transplantation is technically feasible in non-human primates. Thymic lobe cotransplantation modestly prolonged cardiac allograft survival. The failure to induce stable tolerance by heart-thymus transplantation in this model may be due to compromise of the donor thymus by the effects of radiation or age. We also hypothesize that more effective T cell depletion may be required to prevent acute rejection of the donor thymus and allow it to facilitate specific unresponsiveness.