Endothelial induction of fgl2 contributes to thrombosis during acute vascular xenograft rejection

Thrombosis is a prominent feature of acute vascular rejection (AVR), the current barrier to survival of pig-to-primate xenografts. Fibrinogen-like protein 2 (fgl2/fibroleukin) is an inducible prothrombinase that plays an important role in the pathogenesis of fibrin deposition during viral hepatitis and cytokine-induced fetal loss. We hypothesized that induction of fgl2 on the vascular endothelium of xenografts contributes to thrombosis associated with AVR. We first examined fgl2 as a source of procoagulant activity in the pig-to-primate combination. The porcine fgl2 (pfgl2) was cloned and its chromosomal locus was identified. Recombinant pfgl2 protein expressed in vitro was detected on the cell surface and generated thrombin from human prothrombin. Studies of pig-to-baboon kidney xenografts undergoing AVR in vivo revealed induction of pfgl2 expression on graft vascular endothelial cells (ECs). Cultured porcine ECs activated by human TNF-alpha in vitro demonstrated induction of pfgl2 expression and enhanced activation of human prothrombin. The availability of gene-targeted fgl2-deficient mice allowed the contribution of fgl2 to the pathogenesis of AVR to be directly examined in vivo. Hearts heterotopically transplanted from fgl2(+/+) and fgl2(+/-) mice into Lewis rats developed AVR with intravascular thrombosis associated with induction of fgl2 in graft vascular ECs. In contrast, xenografts from fgl2(-/-) mice were devoid of thrombosis. These observations collectively suggest that induction of fgl2 on the vascular endothelium plays a role in the pathogenesis of AVR-associated thrombosis. Manipulation of fgl2, in combination with other interventions, may yield novel strategies by which to overcome AVR and extend xenograft survival.     

Absence of recipient CCR5 promotes early and increased allospecific antibody responses to cardiac allografts

Acute rejection is mediated by T cell infiltration of allografts, but mechanisms mediating the delayed rejection of allografts in chemokine receptor-deficient recipients remain unclear. The rejection of vascularized, MHC-mismatched cardiac allografts by CCR5(-/-) recipients was investigated. Heart grafts from A/J (H-2(a)) donors were rejected by wild-type C57BL/6 (H-2(b)) recipients on day 8-10 posttransplant vs day 8-11 by CCR5(-/-) recipients. When compared with grafts from wild-type recipients, however, significant decreases in CD4(+) and CD8(+) T cells and macrophages were observed in rejecting allografts from CCR5-deficient recipients. These decreases were accompanied by significantly lower numbers of alloreactive T cells developing to IFN-gamma-, but not IL-4-producing cells in the CCR5(-/-) recipients, suggesting suboptimal priming of T cells in the knockout recipients. CCR5 was more prominently expressed on activated CD4(+) than CD8(+) T cells in the spleens of allograft wild-type recipients and on CD4(+) T cells infiltrating the cardiac allografts. Rejecting cardiac allografts from wild-type recipients had low level deposition of C3d that was restricted to the graft vessels. Rejecting allografts from CCR5(-/-) recipients had intense C3d deposition in the vessels as well as on capillaries throughout the graft parenchyma similar to that observed during rejection in donor-sensitized recipients. Titers of donor-reactive Abs in the serum of CCR5(-/-) recipients were almost 20-fold higher than those induced in wild-type recipients, and the high titers appeared as early as day 6 posttransplant. These results suggest dysregulation of alloreactive Ab responses and Ab-mediated cardiac allograft rejection in the absence of recipient CCR5.     

Up-regulation of fibrinogen-like protein 2 in porcine endothelial cells by xenogeneic CD40 signal

Acute humoral xenograft rejection (AHXR), characterized by thrombin generation and endothelial cell activation, should be overcome for the success of xenotransplantation. Fibrinogen-like protein 2 (fgl2) expressed on endothelial cells can convert prothrombin to thrombin directly, which indicates that the induced fgl2 expression in activated endothelial cells can contribute to thrombosis. In xenotransplant condition, the interaction between human CD40L and porcine endothelial CD40 can activate endothelial cells. In this study, we investigated the effect of endothelial cell activation through the interaction between human CD40L and porcine CD40 on fgl2 expression and its function as a direct prothrombinase. We found that CD40 stimulation up-regulated fgl2 expression as well as its enzymatic activity in porcine endothelial cells. Moreover, functional studies using knock-down system showed that the major factor converting human prothrombin to thrombin is fgl2 protein expressed on porcine endothelial cells. Overall, this study demonstrates that fgl2 expression can be induced by xenogeneic CD40 signal on endothelial cells and contribute to thrombin generation.     

Donor IFN-gamma receptors are critical for acute CD4(+) T cell-mediated cardiac allograft rejection.

Recent studies using mouse models demonstrate that CD4(+) T cells are sufficient to mediate acute cardiac allograft rejection in the absence of CD8(+) T cells and B cells. However, the mechanistic basis of CD4-mediated rejection is unclear. One potential mechanism of CD4-mediated rejection is via elaboration of proinflammatory cytokines such as IFN-gamma. To determine whether IFN-gamma is a critical cytokine in CD4-mediated acute cardiac allograft rejection, we studied whether the expression of IFN-gamma receptors on the donor heart was required for CD4-mediated rejection. To investigate this possibility, purified CD4(+) T cells were transferred into immune-deficient mice bearing heterotopic cardiac allografts from IFN-gamma receptor-deficient (GRKO) donors. While CD4(+) T cells triggered acute rejection of wild-type heart allografts, they failed to trigger rejection of GRKO heart allografts. The impairment in CD4-mediated rejection of GRKO hearts appeared to primarily involve the efferent phase of the immune response. This conclusion was based on the findings that GRKO stimulator cells provoked normal CD4 proliferation in vitro and that intentional in vivo challenge of CD4 cells with wild-type donor APC or the adoptive transfer of in vitro primed CD4 T cells failed to provoke acute rejection of GRKO allografts. In contrast, unseparated lymph node cells acutely rejected both GRKO and wild-type hearts with similar time courses, illustrating the existence of both IFN-gamma-dependent and IFN-gamma-independent mechanisms of acute allograft rejection.     

Antibody-mediated rejection of cardiac allografts in CCR5-deficient recipients

Rejected MHC-mismatched cardiac allografts in CCR5(-/-) recipients have low T cell infiltration, but intense deposition of C3d in the large vessels and capillaries of the graft, characteristics of Ab-mediated rejection. The roles of donor-specific Ab and CD4 and CD8 T cell responses in the rejection of complete MHC-mismatched heart grafts by CCR5(-/-) recipients were directly investigated. Wild-type C57BL/6 and B6.CCR5(-/-) (H-2(b)) recipients of A/J (H-2(a)) cardiac allografts had equivalent numbers of donor-reactive CD4 T cells producing IFN-gamma, whereas CD4 T cells producing IL-4 were increased in CCR5(-/-) recipients. Numbers of donor-reactive CD8 T cells producing IFN-gamma were reduced 60% in CCR5(-/-) recipients. Day 8 posttransplant serum titers of donor-specific Ab were 15- to 25-fold higher in CCR5(-/-) allograft recipients, and transfer of this serum provoked cardiac allograft rejection in RAG-1(-/-) recipients within 14 days, whereas transfer of either serum from wild-type recipients or immune serum from CCR5-deficient recipients diluted to titers observed in wild-type recipients did not mediate this rejection. Wild-type C57BL/6 and B6.CCR5(-/-) recipients rejected A/J cardiac grafts by day 11, whereas rejection was delayed (day 12-60, mean 21 days) in muMT(-/-)/CCR5(-/-) recipients. These results indicate that the donor-specific Ab produced in CCR5(-/-) heart allograft recipients is sufficient to directly mediate graft rejection, and the absence of recipient CCR5 expression has differential effects on the priming of alloreactive CD4 and CD8 T cells.     

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.     

Long-term cardiac allograft survival across an MHC mismatch after "pruning" of alloreactive CD4 T cells

Specific tolerance to allografts has been achieved by a variety of means. We have previously shown that ex vivo removal of dividing CD4(+) T cells from an MLR or "pruning" delays skin allograft rejection. We tested pruning of alloreactive T cells as a strategy for retaining a broad T cell repertoire while removing alloreactive T cells in a model of cardiac allograft transplant. Using CFSE staining of responder BALB/c cells with stimulator C57BL/6 cells in an MLR, SCID mice were reconstituted with either dividing (D) or nondividing (ND) CD4(+) T cells derived from an MLR and then challenged with heterotopic cardiac allografts. Mice reconstituted with D CD4(+) T cells rejected cardiac allografts from the stimulator strain with a median survival time (MST) of 29 days, while mice reconstituted with ND CD4(+) T cells maintained allografts from the stimulator strain (MST of >100 days) while rejecting third-party allografts (B10.BR) (MST = 11 days). ELISPOT assays demonstrate donor-specific hyporesponsiveness of the ND CD4(+) T cells. TCR beta-chain V region (TRBV) repertoire analysis demonstrates clonal expansion within both rejecting D cardiac allografts and ND cardiac allografts surviving for the long-term. Histology showed greater allograft infiltration by the D CD4(+) T cells. The surviving ND cardiac allografts demonstrated reduced cellular infiltration and reduced incidence of allograft vasculopathy, but with the development of chronic fibrosis. Thus, pruning of alloreactive T cells allows long-term-specific cardiac allograft survival while retaining the ability to reject third-party allografts.     

Immunobiology of allograft rejection in the absence of IFN-gamma: CD8+ effector cells develop independently of CD4+ cells and CD40-CD40 ligand interactions

Both wild-type (WT) and IFN-gamma-deficient (IFN-gamma(-/-)) C57BL/6 mice can rapidly reject BALB/c cardiac allografts. When depleted of CD8(+) cells, both WT and IFN-gamma(-/-) mice rejected their allografts, indicating that these mice share a common CD4-mediated, CD8-independent mechanism of rejection. However, when depleted of CD4(+) cells, WT mice accepted their allografts, while IFN-gamma(-/-) recipients rapidly rejected them. Hence, IFN-gamma(-/-), but not WT mice developed an unusual CD8-mediated, CD4-independent, mechanism of allograft rejection. Allograft rejection in IFN-gamma(-/-) mice was associated with intragraft accumulation of IL-4-producing cells, polymorphonuclear leukocytes, and eosinophils. Furthermore, this form of rejection was resistant to treatment with anti-CD40 ligand (CD40L) mAb, which markedly prolonged graft survival in WT mice. T cell depletion studies verified that anti-CD40L treatment failed to prevent CD8-mediated allograft rejection in IFN-gamma(-/-) mice. However, anti-CD40L treatment did prevent CD4-mediated rejection in IFN-gamma(-/-) mice, although grafts were eventually rejected when CD8(+) T cells repopulated the periphery. The IL-4 production and eosinophil influx into the graft that occurred during CD8-mediated rejection were apparently epiphenomenal, since treatment with anti-IL-4 mAb blocked intragraft accumulation of eosinophils, but did not interfere with allograft rejection. These studies demonstrate that a novel, CD8-mediated mechanism of allograft rejection, which is resistant to experimental immunosuppression, can develop when IFN-gamma is limiting. An understanding of this mechanism is confounded by its association with Th2-like immune events, which contribute unique histopathologic features to the graft but are apparently unnecessary for the process of allograft rejection.     

Combined Adenovirus-Mediated Artificial microRNAs Targeting mfgl2, mFas,and mTNFR1 Protect against Fulminant Hepatic Failure in Mice

Hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) has a poor prognosis with high in-hospital mortality. Hepatic and circulating inflammatory cytokines, such as fibrinogen like protein 2 (fgl2), FasL/Fas, and TNFα/TNFR1, play a significant role in the pathophysiology of ACLF. This study aimed to investigate the therapeutic effect of recombinant adenoviral vectors carrying constructed DNA code for non-native microRNA (miRNA) targeting mouse fgl2 (mfgl2) or both mFas and mTNFR1 on murine hepatitis virus (MHV)-3-induced fulminant hepatitis in BALB/cJ mice. Artificial miRNA eukaryotic expression plasmids against mfgl2, mFas, and mTNFR1 were constructed, and their inhibitory effects on the target genes were confirmed in vitro. pcDNA6.2-mFas-mTNFR1- miRNA，which expresses miRNA against both mFas and mTNFR1 simultaneously，was constructed. To construct a miRNA adenovirus expression vector against mfgl2, pcDNA6.2-mfgl2-miRNA was cloned using Gateway technology. Ad-mFas-mTNFR1- miRNA was also constructed by the same procedure. Adenovirus vectors were delivered by tail-vein injection into MHV-3-infected BALB/cJ mice to evaluate the therapeutic effect. 8 of 18 (44.4%) mice recovered from fulminant viral hepatitis in the combined interference group treated with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA. But only 4 of 18 (22.2%) mice receiving Ad-mfgl2-miRNA and 3 of 18 (16.7%) mice receiving Ad-mFas-mTNFR1- miRNA survived. These adenovirus vectors significantly ameliorated inflammatory infiltration, fibrin deposition, hepatocyte necrosis and apoptosis, and prolonged survival time. Our data illustrated that combined interference using adenovirus-mediated artificial miRNAs targeting mfgl2, mFas, and mTNFR1 might have significant therapeutic potential for the treatment of fulminant hepatitis.     

CD4 T cell-mediated rejection of cardiac allografts in B cell-deficient mice

CD4 T cell-dependent mechanisms promoting allograft rejection include expression of inflammatory functions within the graft and the provision of help for donor-reactive CD8 T cell and Ab responses. These studies tested CD4 T cell-mediated rejection of MHC-mismatched cardiac allografts in the absence of both CD8 T and B lymphocytes. Whereas wild-type C57BL/6 recipients depleted of CD8 T cells rejected A/J cardiac grafts within 10 days, allografts were not rejected in B cell-deficient B6.muMT(-/-) recipients depleted of CD8 T cells. Isolated wild-type C57BL/6 and B6.muMT(-/-) CD4 T cells had nearly equivalent in vivo alloreactive proliferative responses. CD4 T cell numbers in B6.muMT(-/-) spleens were 10% of that in wild-type mice but were only slightly decreased in peripheral lymph nodes. CD8 T cell depletion did not abrogate B6.muMT(-/-) mice rejection of A/J skin allografts and this rejection rendered these recipients able to reject A/J cardiac allografts. Redirection of the alloimmune response to the lymph nodes by splenectomy conferred the ability of B6.muMT(-/-) CD4 T cells to reject cardiac allografts. These results indicate that the low number of splenic CD4 T cells in B6.muMT(-/-) mice underlies the inability to reject cardiac allografts and this inability is overcome by diverting the CD4 T cell response to the peripheral lymph nodes.     

Interleukin-9 stimulates the production of interleukin-5 in CD4+ T cells

We recently showed that interleukin-9 (IL-9), a Th2 cytokine, promotes IL-5-mediated rejection of allografts in mice. This observation led us to investigate the functional link between IL-9 and IL-5 production during alloreactive T cell responses in vitro and in vivo. Firstly, we found that IL-9 was produced by alloreactive Th2 cells, and IL-9 mRNA was detected in skin allograft during Th2-type rejection. We then established that IL-5 production was impaired in alloreactive Th2 cells isolated from IL-9-deficient mice and that optimal IL-5 production after allogeneic stimulation requires a functional IL-9 receptor (IL-9R) on the responding cells. Finally, the production of IL-5 by anti-CD3-stimulated CD4+ T cells was abolished by neutralization of IL-9. Despite the fact that IL-9 promotes IL-5 production by alloreactive T cells, IL-9-deficient recipients of skin allografts still developed eosinophilic graft infiltrates and neither IL-9 nor IL-9R deficiency modified Th2-type allograft rejection.     

Alloimmune injury and rejection of human skin grafts on human peripheral blood lymphocyte-reconstituted non-obese diabetic severe combined immunodeficient beta2-microglobulin-null mice

Small animal models with the capacity to support engraftment of a functional human immune system are needed to facilitate studies of human alloimmunity. In the present investigation, non-obese diabetic (NOD) severe combined immunodeficient (scid) beta2-microglobulin-null (B2mnull) mice engrafted with human peripheral blood lymphocytes (hu-PBL-NOD-scid B2mnull mice) were used as in vivo models for studying human skin allograft rejection. Hu-PBL-NOD-scid B2mnull mice were established by injection of human spleen cells or PBLs and transplanted with full-thickness allogeneic human skin. Human cell engraftment was enhanced by injection of anti-mouse CD122 antibody. The respective contributions of human CD4+ and CD8+ cells in allograft rejection were determined using depleting antibodies. Human skin grafts on unmanipulated NOD-scid B2mnull mice uniformly survived but on chimeric hu-PBL-NOD-scid B2mnull mice exhibited severe immune-mediated injury that often progressed to complete rejection. The alloaggressive hu-PBLs did not require prior in vitro sensitization to elicit targeted effector cell activity. Extensive mononuclear cell infiltration directed towards human-origin endothelium was associated with thrombosis and fibrin necrosis. No evidence of graft-versus-host disease was detected. Either CD4+ or CD8+ T cells may mediate injury and alloimmune rejection of human skin grafts on hu-PBL-NOD-scid B2mnull mice. It is proposed that Hu-PBL-NOD-scid B2mnull mice engrafted with human skin will provide a useful model for analysis of interventions designed to modulate human allograft rejection.     

Cutting edge: blockade of the CD28/B7 costimulatory pathway inhibits intestinal allograft rejection mediated by CD4+ but not CD8+ T cells.

The effect of blocking the CD28/B7 costimulatory pathway on intestinal allograft rejection was examined in mice. Murine CTLA4Ig failed to prevent the rejection of allografts transplanted into wild-type or CD4 knockout (KO) mice but did inhibit allograft rejection by CD8 KO recipients. This effect was associated with decreased intragraft mRNA for IFN-gamma and TNF-alpha and increased mRNA for IL-4 and IL-5. This altered pattern of cytokine production was not observed in allografts from murine CTLA4Ig-treated CD4 KO mice. These data demonstrate that blockade of the CD28/B7 pathway has different effects on intestinal allograft rejection mediated by CD4+ and CD8+ T cells and suggest that these T cell subsets have different costimulatory requirements in vivo. The results also suggest that the inhibition of CD4+ T cell-mediated allograft rejection by CTLA4Ig may be related to down-regulation of Th1 cytokines and/or up-regulation of Th2 cytokines.     

A critical role for protein kinase C-theta-mediated T cell survival in cardiac allograft rejection

Protein kinase C (PKC)-theta mediates the critical TCR signals required for T cell activation. Previously, we have shown that in response to TCR stimulation, PKC-theta-/- T cells undergo apoptosis due to greatly reduced levels of the anti-apoptotic molecule, Bcl-xL. In this study, we demonstrate that PKC-theta-regulated expression of Bcl-xL is essential for T cell-mediated cardiac allograft rejection. Rag1-/- mice reconstituted with wild-type T cells readily rejected fully mismatched cardiac allografts, whereas Rag1-/- mice reconstituted with PKC-theta-/- T cells failed to promote rejection. Transgenic expression of Bcl-xL in PKC-theta-/- T cells was sufficient to restore cardiac allograft rejection, suggesting that PKC-theta-regulated survival is required for T cell-mediated cardiac allograft rejection in this adoptive transfer model. In contrast to adoptive transfer experiments, intact PKC-theta-/- mice displayed delayed, but successful cardiac allograft rejection, suggesting the potential compensation for PKC-theta function. Finally, a subtherapeutic dose of anti-CD154 Ab or CTLA4-Ig, which was not sufficient to prevent cardiac allograft rejection in the wild-type mice, prevented heart rejection in the PKC-theta-/- mice. Thus, in combination with other treatments, inhibition of PKC-theta may facilitate achieving long-term survival of allografts.     

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.     

Prolonged allograft survival in TNF receptor 1-deficient recipients is due to immunoregulatory effects, not to inhibition of direct antigraft cytotoxicity.

TNF-alpha and lymphotoxin (LT)alpha have been shown to be important mediators of allograft rejection. TNF-R1 is the principal receptor for both molecules. Mice with targeted genetic deletions of TNF-R1 demonstrate normal development of T and B lymphocytes but exhibit functional defects in immune responses. However, the role of TNF-R1-mediated signaling in solid organ transplant rejection has not been defined. To investigate this question, we performed vascularized heterotopic allogeneic cardiac transplants in TNF-R1-deficient (TNF-R1(-/-)) and wild-type mice. Because all allografts in our protocol expressed TNF-R1, direct antigraft effects of TNF-alpha and LTalpha were not prevented. However, immunoregulatory effects on recipient inflammatory cells by TNF-R1 engagement was eliminated in TNF-R1(-/-) recipients. In our study, cardiac allograft survival was significantly prolonged in TNF-R1(-/-) recipients. Despite this prolonged allograft survival, we detected increased levels of CD8 T cell markers in allografts from TNF-R1(-/-) recipients, suggesting that effector functions, but not T cell recruitment, were blocked. We also demonstrated the inhibition of multiple chemokines and cytokines in allografts from TNF-R1(-/-) recipients including RANTES, IFN-inducible protein-10, lymphotactin, and IL-1R antagonist, as well as altered levels of chemokine receptors. We correlated gene expression with the physiologic process of allograft rejection using self-organizing maps and identified distinct patterns of gene expression in allografts from TNF-R1(-/-) recipients. These findings indicate that in our experimental system TNF-alpha and LTalpha exert profound immunoregulatory effects through TNF-R1.     

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.