Dynamics of monocytes/macrophages and T lymphocytes in acutely rejecting rat renal allografts

We examined the infiltration of acutely rejecting renal allografts (DA→LEW) by ED1⁺ and ED2⁺ macrophages and T lymphocytes at intervals of 24 h after transplantation. Donor and recipient macrophages were differentiated by MHC class II antigen expression in double-staining experiments with ED1. Proliferation was assayed after pulse-labelling with BrdU. We subdivided allograft infiltration into three consecutive phases: 1) During phase I on days 1 to 2 after allogeneic kidney transplantation, perivascular infiltrates developed that contained numerous donor and recipient macrophages. Allograft rejection could already be diagnosed 24?h after transplantation by perivascular infiltration of T lymphocytes, whereas T cells were rarely found in isografts. 2) Phase II of allograft rejection from day 3 to 4 was characterized by massive propagation of the infiltrate. About equal numbers of interstitial donor and recipient macrophages were counted. Both macrophages and T lymphocytes proliferated in situ and macrophages outnumbered T cells until complete rejection. 3) During phase III the allograft was destroyed. Large intravascular monocytes surprisingly expressed the ED2 antigen. In the interstitium of viable graft regions, the population of recipient macrophages grew, whereas the population of donor macrophages and of T lymphocytes decreased.     

Renal urokinase-type plasminogen activator (uPA) receptor but not uPA deficiency strongly attenuates ischemia reperfusion injury and acute kidney allograft rejection

Central mechanisms leading to ischemia induced allograft rejection are apoptosis and inflammation, processes highly regulated by the urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR). Recently, up-regulation of uPA and uPAR has been shown to correlate with allograft rejection in human biopsies. However, the causal connection of uPA/uPAR in mediating transplant rejection and underlying molecular mechanisms remain poorly understood. In this study, we evaluated the role of uPA/uPAR in a mice model for kidney ischemia reperfusion (IR) injury and for acute kidney allograft rejection. uPAR but not uPA deficiency protected from IR injury. In the allogenic kidney transplant model, uPAR but not uPA deficiency of the allograft caused superior recipient survival and strongly attenuated loss of renal function. uPAR-deficient allografts showed reduced generation of reactive oxygen species and apoptosis. Moreover, neutrophil and monocyte/macrophage infiltration was strongly attenuated and up-regulation of the adhesion molecule ICAM-1 was completely abrogated in uPAR-deficient allografts. Inadequate ICAM-1 up-regulation in uPAR(-/-) primary aortic endothelial cells after C5a and TNF-alpha stimulation was confirmed by in vitro experiments. Our results demonstrate that the local renal uPAR plays an important role in the apoptotic and inflammatory responses mediating IR-injury and transplant rejection.     

Combined CXCR3/CCR5 blockade attenuates acute and chronic rejection

Chemokine-chemokine receptor interactions orchestrate mononuclear cells recruitment to the allograft, leading to acute and chronic rejection. Despite biologic redundancy, several experimental studies have demonstrated the importance of CXCR3 and CCR5 in acute rejection of allografts. In these studies, deficiency or blockade of CXCR3 or CCR5 led to prolongation of allograft survival, yet allografts were ultimately lost to acute rejection. Given the above findings and the specificity of mononuclear cells bearing CXCR3 and CCR5, we hypothesized that combined blockade of CXCR3 and CCR5 will lead to indefinite (>100 days) graft survival in a full MHC-mismatched murine cardiac allograft model. The donor hearts in the control group were rejected in 6 +/- 1 days after transplantation. Combined blockade of CXCR3 and CCR5 prolonged allograft survival >15-fold vs the control group; all allografts survived for >100 days. More importantly, the donor hearts did not display any intimal lesions characteristic of chronic rejection. Further analysis of the donor hearts in the CXCR3/CCR5 blockade group demonstrated graft infiltration with CD4(+)CD25(+) T cells expressing the Foxp3 gene. Depletion of CD25(+) cells in the combined CXCR3 and CCR5 blockade group resulted in acute rejection of the allografts in 22 +/- 2 days. Combined CXCR3 and CCR5 blockade also reduced alloantigen-specific T lymphocyte proliferation. Combined CXCR3 and CCR5 blockade is effective in preventing acute and chronic rejection in a robust murine model. This effect is mediated, in part, by CD25(+) regulatory T cell recruitment and control of T lymphocyte proliferation.     

A role for fractalkine and its receptor (CX3CR1) in cardiac allograft rejection

The hallmark of acute allograft rejection is infiltration of the inflamed graft by circulating leukocytes. We studied the role of fractalkine (FKN) and its receptor, CX(3)CR1, in allograft rejection. FKN expression was negligible in nonrejecting cardiac isografts but was significantly enhanced in rejecting allografts. At early time points, FKN expression was particularly prominent on vascular tissues and endothelium. As rejection progressed, FKN expression was further increased, with prominent anti-FKN staining seen around vessels and on cardiac myocytes. To determine the capacity of FKN on endothelial cells to promote leukocyte adhesion, we performed adhesion assays with PBMC and monolayers of TNF-alpha-activated murine endothelial cells under low-shear conditions. Treatment with either anti-FKN or anti-CX(3)CR1-blocking Ab significantly inhibited PBMC binding, indicating that a large proportion of leukocyte binding to murine endothelium occurs via the FKN and CX(3)CR1 adhesion receptors. To determine the functional significance of FKN in rejection, we treated cardiac allograft recipients with daily injections of anti-CX(3)CR1 Ab. Treatment with the anti-CX(3)CR1 Ab significantly prolonged allograft survival from 7 +/- 1 to 49 +/- 30 days (p < 0.0008). These studies identify a critical role for FKN in the pathogenesis of acute rejection and suggest that FKN may be a useful therapeutic target in rejection.     

A histological and electron-microscopic study of the cell types involved in rejection of skin allografts in ammocoetes

The rejection of skin allografts by the larval lamprey, Lampetra reissneri, was studied by light- and electron-microscopy, with particular attention to the cell types involved in the reaction. In all allografts, melanophores were destroyed within 20-60 days (the mean survival time, 36 +/- 12 days). Neither the epidermis nor the underlying collagenous lamella was invaded by host cells until the 60th day. A heavy infiltration of host leucocytes was observed in the allografts in melanophore and adipose layers and in the bundles of muscles. Throughout all stages from 10 to 60 days after the grafting, the cells of the polymorphonuclear leucocyte (PMN) series and eosinophilic granulocytes predominated, but macrophages were not observed at any stages examined. Plasma cells occurred occasionally at later stages (40-60 days) of allograft rejection, but lymphocytes were rarely found at any stages of graft rejection. These observations, combined with the recent finding of the antibody-enhanced phagocytic activity of granulocyte-series cells in the lamprey, indicate that PMNs, but not lymphocytes, function as the major effector cells in allograft rejection in this phylogenetically oldest class of contemporary vertebrates.     

Role of nitric oxide and superoxide in acute cardiac allograft rejection in rats

The role of NO and superoxide (O(2)(-)) in tissue injury during cardiac allograft rejection was investigated by using a rat ex vivo organ perfusion system. Excessive NO production and inducible NO synthase (iNOS) expression were observed in cardiac allografts at 5 days after cardiac transplantation, but not in cardiac isografts, as identified by electron spin resonance spectroscopy and Northern blotting. Cardiac isografts or allografts obtained on Day 5 after transplantation were perfused with Krebs bicarbonate buffer with or without various antidotes for NO or O(2)-, including N(omega)-monomethyl-L-arginine (L-NMMA; 1 mM), 2-phenyl-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; 100 microM), 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP; a xanthine oxidase inhibitor; 100 microM), and superoxide dismutase (SOD; 100 units/ml). Treatment of the cardiac allografts with PTIO showed most remarkable improvement of the cardiac injury as revealed by significant reduction in aspartate transaminase, lactate dehydrogenase, and creatine phosphokinase concentrations in the perfusate. Similar but less potent protective effect on the allograft injury was observed by treatment with L-NMMA, AHPP, and SOD. Immunohistochemical analyses for iNOS and nitrotyrosine indicated that iNOS is mainly expressed by macrophages infiltrating the allograft tissues, and nitrotyrosine formation was demonstrated not only in macrophages but also in cardiac myocytes of the allografts, providing indirect evidence for the generation of peroxynitrite during allograft rejection. Our results suggest that tissue injury in rat cardiac allografts during acute rejection is mediated by both NO and O(2)(-), possibly through peroxynitrite formation.     

Monokine induced by IFN-gamma is a dominant factor directing T cells into murine cardiac allografts during acute rejection.

The use of chemokine antagonism as a strategy to inhibit leukocyte trafficking into inflammatory sites requires identification of the dominant chemokines mediating recruitment. The chemokine(s) directing T cells into cardiac allografts during acute rejection remain(s) unidentified. The role of the CXC chemokines IFN-gamma inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig) in acute rejection of A/J (H-2(a)) cardiac grafts by C57BL/6 (H-2(b)) recipients was tested. Intra-allograft expression of Mig was observed at day 2 posttransplant and increased to the time of rejection at day 7 posttransplant. IP-10 mRNA and protein production were 2.5- to 8-fold lower than Mig. Whereas allografts were rejected at day 7-9 in control recipients, treatment with rabbit antiserum to Mig, but not to IP-10, prolonged allograft survival up to day 19 posttransplant. At day 7 posttransplant, allografts from Mig antiserum-treated recipients had marked reduction in T cell infiltration. At the time of rejection in Mig antiserum-treated recipients (i.e., days 17-19), intra-allograft expression of macrophage-inflammatory protein-1alpha, -1beta, and their ligand CCR5 was high, whereas expression of CXCR3, the Mig receptor, was virtually absent. Mig was produced by the allograft endothelium as well as by recipient allograft-infiltrating macrophages and neutrophils, indicating the synergistic interactions between innate and adaptive immune compartments during acute rejection. Collectively, these results indicate that Mig is a dominant recruiting factor for alloantigen-primed T cells into cardiac allografts during acute rejection. Although Mig antagonism delays acute heart allograft rejection, the results also suggest that the alloimmune response circumvents Mig antagonism through alternative mechanisms.     

In vivo microscopic assessment of cremasteric microcirculation during hindlimb allograft rejection in rats.

Experimental and clinical studies of vascular allogenic extremity transplantation have yielded disappointing results and have not been clinically useful. With recent advances in transplantation immunology, considerable interest has focused on the understanding of leukocyte-endothelial interaction at the microcirculatory level. The objective of this study was to characterize the alterations in leukocyte-endothelial interaction in the early stages of rat hindlimb allograft rejection. To study the changes at the microcirculatory level, a new microsurgical model was developed; the cremaster muscle was incorporated into the transplanted hindlimb. The purpose of this study was to report on the microcirculatory changes during rat hindlimb allograft rejection. A total of 24 transplantations were performed among the four experimental groups. In a control group, 12 rat hindlimb-cremaster grafts were transplanted between genetically identical animals, Lewis to Lewis. Microcirculatory measurements of graft survival were taken at 24 hours (group 1A, n = 6) and at 72 hours (group 1B, n = 6). In the rejection control group, 12 transplantations were performed across a major histocompatibility barrier between Lewis-Brown Norway and Lewis rats. Microcirculatory measurements were taken at 24 (group 2A, n = 6) and 72 hours (group 2A, n = 6) as above. The following parameters were evaluated to discover the leukocyte-endothelial interaction: endothelial edema index and the number of rolling, adherent, and transmigrating leukocytes and lymphocytes in the postcapillary venule. Physical signs of limb rejection, such as edema, erythema, scaling, plaque formation on the skin, hair loss, and skin surface temperature, were monitored. Microcirculatory signs of rejection included the following. There was a significant increase in the number of adherent leukocytes in allograft transplants at both 24 hours (205 percent; 2.05 +/- 0.38) and 72 hours (431 percent; 9.11 +/- 3.41) when compared with isograft controls (1.00 +/- 0.89 at 24 hours; 2.11 +/- 0.34 at 72 hours) (p < 0.05). The activation of leukocyte transmigration increased more than 7-fold in muscle allografts at 24 hours (0.55 +/- 0.25 versus 4.16 +/- 1.89) and more than 6-fold at 72 hours (0.72 +/- 0.38 versus 4.38 +/- 1.28) after transplantation (p < 0.05). Endothelial edema index, a measure of endothelial swelling and cellular deposit accumulation, increased more than 119 percent in the allograft group 72 hours after transplantation (1.23 +/- 0.07 versus 1.46 +/- 0.09) (p < 0.05). The first clinical signs of limb rejection were scaling of the skin or hair loss; they were observed between the seventh and ninth postoperative days. The composite rat hindlimb-cremaster model presented in this study introduces a new in vivo approach to monitor acute graft rejection using the intravital microscopy system. This is a valuable model for defining the timing, sequence, and correlation between immunologic events and clinical signs during the acute phase of allograft rejection.     

Renal allograft rejection: immunohistochemistry of inflammatory cellular subsets and vascular lesions

We have examined sixty-seven surgically removed allograft kidneys to identify the different leukocyte subsets of interstitial infiltration and the early vascular lesions which characterize renal allograft rejection. Histochemical and immunohistochemical results (mouse monoclonal antibodies anti: Leu 1, Leu 3a-3b, Leu 7, Leu 2a, OK Ia-Dr, OKB2, Leu M1, Leu M3; rabbit heteroclonal antibodies anti -: IgA, IgG, IgM, C3, fibrinogen, lysozyme; lectins-ABC: RCA, WGA, UEA) and routine histochemical staining have shown an increase of T-helper and T-activated lymphocyte subsets in acute rejection. Neutrophilic leucocytes were present in hyperacute rejection; macrophages were also noted. In chronic rejection, several lymphocyte subsets, in different ratios, were identified. Monocyte/macrophage leukocytes were the most abundant cell population. IgA deposits were noted on tubular epithelia in hyperacute and chronic rejections. IgM deposits were observed in vascular walls in chronic rejection. C3 and fibrinogen deposits were seen in glomerular capillaries and in arterial walls, although in different ratios, in all cases of renal allograft rejections. We have generally seen weak reactions to IgG deposits. Histochemical analysis of lectin receptors has given different results according to the type of rejection considered. In hyperacute rejection, receptors for WGA were found both on glomerular endothelial cells and on the tubular brush border. In the latter, receptors for RCA were also found. In acute rejection, receptors for UEA and WGA were found in a lower number of cases of acute vascular rejection. In acute cellular rejection, receptors for RCA, UEA and WGA were recognized in tubular epithelia. In acute vascular rejection, as well as in chronic rejection, only receptors for WGA were present on tubular epithelia and on capillary loop endothelium. The use of anti-human lysozyme antibodies has yielded the following results: in acute and hyperacute rejection, when renal failure occurred, we saw a high ratio of lysozyme, either coarsely granular or diffuse in the proximal tubular epithelia. Lysozyme was found in myelocyte/macrophage cells within capillary loops and arterial walls, when acute necrotizing vasculitis was present. In acute rejection, proximal tubular cells were lysozyme-negative or lysozyme-positive only segmentally, especially when obliterative vasculitis by fibrointimal proliferation was present and renal function progressively failed. In most of the chronic rejections, tubular cells were lysozyme-negative.     

Differential expression of the IFN-gamma-inducible CXCR3-binding chemokines,IFN-inducible protein 10, monokine induced by IFN,and IFN-inducible T cell alpha chemoattractant in human cardiac allografts: association with cardiac allograft vasculopathy and acute rejection

CXCR3 chemokines exert potent biological effects on both immune and vascular cells. The dual targets suggest their important roles in cardiac allograft vasculopathy (CAV) and rejection. Therefore, we investigated expression of IFN-inducible protein 10 (IP-10), IFN-inducible T cell alpha chemoattractant (I-TAC), monokine induced by IFN (Mig), and their receptor CXCR3 in consecutive endomyocardial biopsies (n = 133) from human cardiac allografts and corresponding normal donor hearts (n = 11) before transplantation. Allografts, but not normal hearts, contained IP-10, Mig, and I-TAC mRNA. Persistent elevation of IP-10 and I-TAC was associated with CAV. Allografts with CAV had an IP-10-GAPDH ratio 3.7 +/- 0.8 compared with 0.8 +/- 0.2 in those without CAV (p = 0.004). Similarly, I-TAC mRNA levels were persistently elevated in allografts with CAV (6.7 +/- 1.9 in allografts with vs 1.5 +/- 0.3 in those without CAV, p = 0.01). In contrast, Mig mRNA was induced only during rejection (2.4 +/- 0.9 with vs 0.6 +/- 0.2 without rejection, p = 0.015). In addition, IP-10 mRNA increased above baseline during rejection (4.1 +/- 2.3 in rejecting vs 1.8 +/- 1.2 in nonrejecting biopsies, p = 0.038). I-TAC did not defer significantly with rejection. CXCR3 mRNA persistently elevated after cardiac transplantation. Double immunohistochemistry revealed differential cellular distribution of CXCR3 chemokines. Intragraft vascular cells expressed high levels of IP-10 and I-TAC, while Mig localized predominantly in infiltrating macrophages. CXCR3 was localized in vascular and infiltrating cells. CXCR3 chemokines are induced in cardiac allografts and differentially associated with CAV and rejection. Differential cellular distribution of these chemokines in allografts indicates their central roles in multiple pathways involving CAV and rejection. This chemokine pathway may serve as a monitor and target for novel therapies to prevent CAV and rejection.     

Immunological disruption of antiangiogenic signals by recruited allospecific T cells leads to corneal allograft rejection

Corneal transplantation is the most common solid organ transplantation. The immunologically privileged nature of the cornea results in high success rates. However, T cell-mediated rejection is the most common cause of corneal graft failure. Using antiangiogenesis treatment to prevent corneal neovascularization, which revokes immune privilege, prevents corneal allograft rejection. Endostatin is an antiangiogenic factor that maintains corneal avascularity. In this study, we directly test the role of antiangiogenic and immunological signals in corneal allograft survival, specifically the potential correlation of endostatin production and T cell recruitment. We report that 75% of the corneal allografts of BALB/c mice rejected after postoperative day (POD) 20, whereas all syngeneic grafts survived through POD60. This correlates with endogenous endostatin, which increased and remained high in syngeneic grafts but decreased after POD10 in allografts. Immunostaining demonstrated that early recruitment of allospecific T cells into allografts around POD10 correlated with decreased endostatin production. In Rag(-/-) mice, both allogeneic and syngeneic corneal grafts survived; endostatin remained high throughout. However, after T cell transfer, the allografts eventually rejected, and endostatin decreased. Furthermore, exogenous endostatin treatment delayed allograft rejection and promoted survival secondary to angiogenesis inhibition. Our results suggest that endostatin plays an important role in corneal allograft survival by inhibiting neovascularization and that early recruitment of allospecific T cells into the grafts promotes destruction of endostatin-producing cells, resulting in corneal neovascularization, massive infiltration of effector T cells, and ultimately graft rejection. Therefore, combined antiangiogenesis and immune suppression will be more effective in maintaining corneal allograft survival.     

Prolonged blockade of CD40-CD40 ligand interactions by gene transfer of CD40Ig results in long-term heart allograft survival and donor-specific hyporesponsiveness, but does not prevent chronic rejection

Previous work on blockade of CD40-CD40 ligand interaction in mice and primates with anti-CD40 ligand mAbs has resulted in a moderate prolongation of allograft survival without the development of true allograft tolerance. In this study, we show in rats that adenovirus-mediated gene transfer of CD40Ig sequences into the graft resulted in prolonged (>200 days) expression of CD40Ig and in long-term (>300 days) survival. Recipients expressing CD40Ig displayed strongly (>90%) inhibited mixed leukocyte reactions and alloantibody production at early (days 5 and 17) and late time points (>100 day) after transplantation, but showed limited inhibition of leukocyte infiltration and cytokine production as evaluated by immunohistology at early time points (day 5). Recipients of long-surviving hearts showed donor-specific hyporesponsiveness since acceptance of second cardiac allografts was donor specific. Nevertheless, long-term allografts (>100 days) displayed signs of chronic rejection vasculopathy. Occluded vessels showed leukocyte infiltration, mainly composed of CD4(+) and CD8(+) cells, macrophages, and mast cells. These recipients also showed antidonor CTL activity. Recipients expressing CD40Ig did not show nonspecific immunosuppression, as they were able to mount anticognate immune responses that were partially inhibited at early time points and were normal thereafter. We conclude that gene transfer-mediated expression of CD40Ig resulted in a highly efficient inhibition of acute heart allograft rejection in rats. This treatment induced donor-specific inhibition of certain alloreactive mechanisms in the short-, but not the long-term, which resulted in long-term survival of allografts concomitant with the development of chronic rejection.     

Characterization of acute renal allograft rejection by proteomic analysis of renal tissue in rat

Rapid and reliable biomarkers of renal allograft rejection have not been available. This study aimed to investigate biomarkers in renal allograft tissue using proteomic analysis. Orthotopic kidney transplantations were performed using Fisher (F344) or Lewis rats as donors and Lewis rats as recipients. Syngenic control group (Group I) constituted F344-to-F344 orthotopic kidney allo-transplantations (n = 8); and allogenic group (Group II) consisted of F344-to-Lewis orthotopic kidney allo-transplantations (n = 8). Renal tissues were harvested 7 days after transplantation. Samples were analyzed using 2-D electrophoresis and matrix assisted laser desorption ionization-time of flight mass spectrometry. 6 differentially expressed proteins were identified between allogenic group and syngenic control group. A rat model of acute renal allograft rejection was successfully set up. Differentially expressed proteins in renal allograft tissue of rat were detected using proteomic analysis and might serve as novel diagnostic and therapeutic targets in human. Quantitative proteomics, using MALDL-TOF-MS methodology has the potential to provide a profiling and a deeper understanding of acute renal rejection.     

Prolonged renal allograft survival by donor interleukin-6 deficiency: association with decreased alloantibodies and increased intragraft T regulatory cells

Both humoral and cellular immune responses are involved in renal allograft rejection. Interleukin (IL)-6 is a regulatory cytokine for both B and Foxp3 (forkhead box P3)-expressing regulatory T (Treg) cells. This study was designed to investigate the impact of donor IL-6 production on renal allograft survival. Donor kidneys from IL-6 knockout (KO) vs. wild-type (WT) C57BL/6 mice (H-2(b)) were orthotopically transplanted to nephrotomized BALB/c mice (H-2(d)). Alloantibodies and Treg cells were examined by fluorescence-activated cell sorting analysis. Graft survival was determined by the time to graft failure. Here, we showed that a deficiency in IL-6 expression in donor kidneys significantly prolonged renal allograft survival compared with WT controls. IL-6 protein was upregulated in renal tubules and endothelium of renal allografts following rejection, which correlated with an increase in serum IL-6 compared with that in those receiving KO grafts or naive controls. The absence of graft-producing IL-6 or lower levels of serum IL-6 in the recipients receiving IL-6 KO allografts was associated with decreased circulating anti-graft alloantibodies and increased the percentage of intragraft CD4(+)CD25(+)Foxp3(+) Treg cells compared with those with WT allografts. In conclusion, the lack of graft-producing IL-6 significantly prolongs renal allograft survival, which is associated with reduced alloantibody production and/or increased intragraft Treg cell population, implying that targeting donor IL-6 may effectively prevent both humoral and cellular rejection of kidney transplants.     

Antigen-induced suppression: The role of Class I major histocompatibility antigens

The role of Class I major histocompatibility (MHC) antigens in the induction of specific suppression of graft rejection has been investigated. Two experimental transplantation models have been used - fully vascularized heterotopic cardiac allografts in the mouse and fully vascularized orthotopic renal allografts in the rat. Preparations of cells expressing Class I MHC antigens, for example highly purified preparations of rat erythrocytes or platelets or mouse L cells (H2k) transfected with the D locus Class I gene of the b haplotype, LDb-1 cells, were used to pretreat recipients prior to transplantation. The function of the allograft was monitored in order to assess any beneficial effects induced by Class I MHC antigens. The results obtained implicate Class I MHC as important in the induction of specific immunosuppression of vascularized allograft rejection.     

Marked prolongation of cardiac allograft survival by dendritic cells genetically engineered with NF-kappa B oligodeoxyribonucleotide decoys and adenoviral vectors encoding CTLA4-Ig

Bone marrow-derived dendritic cells (DCs) can be genetically engineered using adenoviral (Ad) vectors to express immunosuppressive molecules that promote T cell unresponsiveness. The success of these DCs for therapy of allograft rejection has been limited in part by the potential of the adenovirus to promote DC maturation and the inherent ability of the DC to undergo maturation following in vivo administration. DC maturation occurs via NF-kappaB-dependent mechanisms, which can be blocked by double-stranded "decoy" oligodeoxyribonucleotides (ODNs) containing binding sites for NF-kappaB. Herein, we describe the combined use of NF-kappaB ODNs and rAd vectors encoding CTLA4-Ig (Ad CTLA4-Ig) to generate stably immature murine myeloid DCs that secrete the potent costimulation blocking agent. These Ad CTLA4-Ig-transduced ODN DCs exhibit markedly impaired allostimulatory ability and promote apoptosis of activated T cells. Furthermore, administration of Ad CTLA4-Ig ODN-treated donor DCs (C57BL10; B10(H-2b)) before transplant significantly prolongs MHC-mismatched (C3HHeJ; C3H(H-2k)) vascularized heart allograft survival, with long-term (>100 days) donor-specific graft survival in 40% of recipients. The mechanism(s) responsible for DC tolerogenicity, which may involve activation-induced apoptosis of alloreactive T cells, do not lead to skewing of intragraft Th cytokine responses. Use of NF-kappaB antisense decoys in conjunction with rAd encoding a potent costimulation blocking agent offers promise for therapy of allograft rejection or autoimmune disease with minimization of systemic immunosuppression.