Efficient gene delivery to human and rodent islets with double-stranded (ds) AAV-based vectors

Transplantation of allogeneic pancreatic islets is an effective approach to treat type 1 diabetes. To bypass the need for systemic administration of immunosuppression drugs following transplantation, approaches to genetically modify allogeneic islets to express anti-inflammatory, immunosuppressive, or antiapoptotic proteins prior to transplantation are being developed. Adeno-associated viral (AAV) based vectors have been used for gene transfer to islets, but the efficiency of functional transduction is low. Recently, double-stranded (ds) or double-copy (dc) based AAV vectors have been developed that allow for more rapid and efficient AAV-mediated transgene expression following transduction. Here we demonstrate that intact human and murine islets can be transduced with dsAAV2-eGFP efficiently compared to single-stranded AAV2-eGFP. Furthermore, our results demonstrate that murine islets transduced with dsAAV2-eGFP have normal islet glucose responsiveness, viability, and islet insulin content. Transplantation of the dsAAV2-eGFP transduced islet restored normal glycemia in diabetic mice without eliciting an immune response. Significant dsAAV2-mediated eGFP expression was observed in the islet grafts for at least 6 months post-transplant. Finally, we demonstrated that dsAAV serotypes 2, 6, and 8 infect human islets efficiently. Taken together, these results suggest that dsAAV based vectors are highly appropriate for gene transfer to islets to facilitate transplantation.     

Adeno-associated virus vector mediated gene transfer to pancreatic beta cells

Insulin-dependent diabetes mellitus (IDDM) or type 1 diabetes is an autoimmune disease that results in destruction of the insulin-producing pancreatic islet beta cells. Several factors induce the invasion of immune cells into islets and trigger inflammation. Gene therapy approaches targeting the islet cells could be an effective treatment to prevent the onset or reverse type 1 diabetes. Allogeneic islet transplantation provides short-term treatment. However, genetically modified islets, which resist the host immune response, could provide long-term solutions. Adeno-associated virus (AAV) is emerging as a prominent vector system for delivering therapeutic genes for human gene therapy. AAV vector can transduce nondividing cells and provide long-term gene expression by integrating into host chromosome. Therefore, it is an appropriate vector system for islet cell gene therapy. To test the efficacy of AAV vector to transduce pancreatic endocrine cells, we constructed AAV vectors using plasmid pSub201. Wild-type AAV DNA analogue from plasmid psub201 was subcloned into a cloning plasmid pSP72 and AAV vectors were constructed by inserting the transgenes with heterologous promoter in place of AAV open reading frames (rep and cap). In this report we demonstrate the transduction of pancreatic islet cells with AAV vectors encoding bacterial -galactosidase enzyme or enhanced green fluorescent protein (EGFP) as reporter gene. Dispersed porcine and rat islet cells can be transduced by AAV vector, with an efficiency of 47% and 38%, respectively. In particular porcine islet insulin producing beta cells were transduced with an efficiency of 39%. Intact rat islet cells were transduced with an efficiency of 26% as estimated by FACS analysis following transduction with an AAV vector encoding EGFP. Transduction of intact rat islets with an AAV vector did not alter glucose-induced insulin secretion. AAV vector transduction was higher in transformed islet cell lines INS-1 and RIN m5F with an efficiency of 65% and 57%, respectively. These new results suggest that AAV vectors will provide an improved method of gene delivery to pancreatic islets and isolated pancreatic beta cells.     

Prevention of beta cell dysfunction and apoptosis activation in human islets by adenoviral gene transfer of the insulin-like growth factor I

Interleukin-1beta is a potent pro-inflammatory cytokine that has been shown to inhibit islet beta cell function as well as to activate Fas-mediated apoptosis in a nitric oxide-dependent manner. Furthermore, this cytokine is effective in recruiting lymphocytes that mediate beta cell destruction in IDDM onset. The insulin-like growth factor I (IGF-I) has been shown to block IL-1beta actions in vitro. We hypothesized that gene transfer of the insulin-like growth factor I to intact human islets could prevent IL-1beta-induced beta cell dysfunction and sensitization to Fas-triggered apoptosis activation. Intact human islets were infected with adenoviral vectors encoding IGF-I as well as beta-galactosidase and enhanced green fluorescent protein as controls. Adenoviral gene transfer of human IGF-I prevented IL-1beta-mediated nitric oxide production from human islets in vitro as well as the suppression of beta cell function as determined by glucose-stimulated insulin production. Moreover, IGF-I gene transfer prevented IL-1beta-induced, Fas-mediated apoptosis. These results suggest that locally produced IGF-I from cultured islets may be beneficial in maintaining beta cell function and promoting islet survival before and following islet transplantation as a potential therapy for type I diabetes.     

Lipid-mediated gene transfer of viral IL-10 prolongs vascularized cardiac allograft survival by inhibiting donor-specific cellular and humoral immune responses.

The gene encoding the immunosuppressive cytokine viral interleukin-10 (vIL-10) was introduced into BALB/c (H-2d) vascularized cardiac allografts by perfusing the graft vasculature with DNA-liposome complexes, utilizing the experimental cationic lipid gamma AP DLRIE/DOPE and a plasmid encoding vIL-10 under the control of the HCMVie promoter. The DNA to lipid ratio and DNA dose were critical factors in obtaining optimal biologic effects. Gene transfer of vIL-10 with a 3:1 DNA to lipid weight ratio using 375 micrograms DNA significantly prolonged allograft survival in MHC-mismatched C57BL/6 (H-2b) recipients (16.00 days) compared with both unmodified allografts (8.14 days) and vIL-10 antisense controls (8.28 days). Enhanced graft survival was specific to vIL-10 expression since treatment with antisense plasmid or anti-vIL-10 monoclonal antibody (mAb) abrogated the effect. Prolonged survival was associated with a novel histology characterized by a moderate mononuclear infiltrate, edema, and diffuse fibrillar/collagen deposition in the interstitium. Despite these morphologic changes, myocytes remained viable and vessels were patent. Limiting dilution analysis revealed transient infiltration of IL-2 secreting, donor-reactive, helper T lymphocytes (HTL) and cytotoxic T lymphocytes (CTL) in vIL-10 expressing grafts on day 7, that decreased significantly by day 14. Similarly, vIL-10 gene transfer inhibited the accumulation of donor-specific HTL and CTL in the spleen, compared with antisense controls. Prolonged survival was also associated with a marked decrease in IgM and IgG alloantibody production, with little to no IgG isotype switching. These results show that viral IL-10 gene transfer inhibits graft rejection in a clinically relevant model by inhibiting donor-specific cellular and humoral immune responses.     

Viral interleukin-10-engineered autologous hematopoietic stem cell therapy: a novel gene therapy approach to prevent graft rejection

The Epstein-Barr virus-encoded protein BCRF1 (viral interleukin [vIL]-10) is a biologically active homologue of cellular interleukin (IL)-10. In this study, a novel gene therapy approach to prolong allograft survival was designed. Autologous (syngeneic) hematopoietic progenitor/stem cell-enriched (HSC; lineage(-ve)) population derived from CBA/J mouse bone marrow were transduced with retrovirus encoding vIL-10 gene (vIL-10-HSC), ex vivo; vIL-10-HSC were injected (4-6 x 10(6) cells intravenously) into lethally (9.5 Gy) or sublethally (4 Gy) irradiated CBA/J mice. Six weeks after vIL-10-HSC administration, vascular heterotopic heart (C57BL/6) transplantation was performed. Ex vivo, the vIL-10-HSC produced 5.4 +/- 0.5 ng of vIL-10 protein/2 x 10(5) cells per 24 hr. In vivo, serum vIL-10 production was 187 +/- 205 pg/ml during 3-10 weeks after vIL-10-HSC administration. Cardiac allograft survival was prolonged (p < 0.004) in lethally (71 +/- 40 days) and sublethally (114 +/- 15 days) irradiated mice that received vIL-10-HSC compared to controls that received unengineered (UE) HSC or vector DNA-engineered HSC (12-16 days). However, secondary skin graft (C57BL/6) survival was not prolonged in cardiac allograft-tolerant animals. In the vIL-10-HSC-administered group, graft histopathology demonstrated mild arteritis/venulitis (grade 0.7) and rejection (grade 1.0). Intragraft expression of costimulatory molecules (B7.1, B7.2), cytokines (IL-2, IL-4, mIL-10, interferon [IFN]-gamma), and inducible nitric oxide synthase (iNOS) molecules was markedly lower in vIL-10-HSC-treated tolerant grafts that survived more than 100 days compared to vector DNA-HSC- or UE-HSC-treated controls. Furthermore, T lymphocytes derived from vIL-10-HSC-treated tolerant recipients demonstrated hyporeactivity to donor antigens in mixed lymphocyte cultures. Administration of autologous vIL-10-engineered HSC prior to organ transplantation prolonged cardiac allograft survival significantly.     

Islet transplantation in patients with autoimmune diabetes induces homeostatic cytokines that expand autoreactive memory T cells

Successful transplantation requires the prevention of allograft rejection and, in the case of transplantation to treat autoimmune disease, the suppression of autoimmune responses. The standard immunosuppressive treatment regimen given to patients with autoimmune type 1 diabetes who have received an islet transplant results in the loss of T cells. In many other situations, the immune system responds to T cell loss through cytokine-dependant homeostatic proliferation of any remaining T cells. Here we show that T cell loss after islet transplantation in patients with autoimmune type 1 diabetes was associated with both increased serum concentrations of IL-7 and IL-15 and in vivo proliferation of memory CD45RO(+) T cells, highly enriched in autoreactive glutamic acid decarboxylase 65-specific T cell clones. Immunosuppression with FK506 and rapamycin after transplantation resulted in a chronic homeostatic expansion of T cells, which acquired effector function after immunosuppression was removed. In contrast, the cytostatic drug mycophenolate mofetil efficiently blocked homeostatic T cell expansion. We propose that the increased production of cytokines that induce homeostatic expansion could contribute to recurrent autoimmunity in transplanted patients with autoimmune disease and that therapy that prevents the expansion of autoreactive T cells will improve the outcome of islet transplantation.     

Infection of intact human islets by a lentiviral vector.

The transfer of genes encoding immunomodulatory proteins to islets can be used to improve islet function, block apoptosis, and inhibit rejection following transplantation. Adenoviral vectors have been shown to infect intact human islets, but the immunogenicity and transient gene expression of the current adenoviral vectors may hinder their use clinically for islet transplantation. In this report, we compared an HIV-1-based lentiviral vector with the E1-deleted adenoviral vehicle of the Ad5 type for gene transfer to human islets in vitro. We demonstrate that at similar viral particle concentrations per islet that an HIV-based lentiviral vector is able to infect beta-cells within an intact human islet at an efficiency similar to an adenoviral vector. In addition, both the adenoviral and lentiviral vectors were able to express significant levels of soluble interleukin-1 receptor antagonist (IL-1Ra) protein following infection of intact islets. More importantly, there was no impairment of islet beta-cell function following adenoviral and lentiviral infection in responding to glucose stimulation. These results support the utility of replication-defective lentiviral vectors as efficient gene delivery vehicles to islets to faciliate transplantation of islets for therapy of type I diabetes.     

Third-party Mesenchymal Stem Cells Improved Human Islet Transplantation in a Humanized Diabetic Mouse Model

Human islet transplantation can be a permanent treatment of type 1 diabetes if the immune rejection and primary nonfunction (PNF) of transplanted islet grafts were properly addressed. In this study, we determined whether cotransplantation of human bone marrow-derived mesenchymal stem cells (hBMSCs) could prevent immune rejection and improve human islet transplantation in a humanized NOD scid gamma (NSG) mouse model. Human immunity was rebuilt and maintained in NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice up to 13 weeks after intraperitoneal injection of mature human peripheral blood mononuclear cells (PBMCs). The blood glucose control and the levels of serum insulin and c-peptide clearly indicated a better outcome of islet transplantation when islets were cotransplanted with hBMSCs. hBMSCs actively interacted with interleukin-10 (IL-10)-producing CD14⁺ monocytes to suppress the proliferation and activation of T cells in the PBMC/hBMSC coculture and prevent the T cell recruitment into the transplantation site. hBMSCs also increased the percentage of immunosuppressive regulatory T cells (Tregs) and prevented the cytokine-induced loss-of-function of human islets. Taken together, our studies demonstrated that transplantation of islets with hBMSCs is a promising strategy to improve the outcome of human islet transplantation.     

Cytokine gene therapy for myocarditis by in vivo electroporation.

Cytokines are important pathophysiologic and pathogenic factors in cardiovascular disorders, including viral myocarditis. We attempted to treat viral myocarditis with cytokine gene therapy by transferring an inhibitory cytokine, IL-1 receptor antagonist (IL-1ra) or viral IL-10 (vIL-10), by in vivo electroporation, a new method for gene transfer into muscle. Four-week-old male DBA/2 mice were inoculated intraperitoneally with 10 PFU of encephalomyocarditis virus. Immediately after virus inoculation, an expression plasmid carrying IL-1ra or vIL-10 was injected into tibialis anterior muscles followed by electroporation. Serum levels of IL1ra and vIL-10 reached 10.5 and 2.3 ng/ml, respectively, on day 5, when gene expression reached its peak. Histopathological examination showed that myocardial cellular infiltration was improved in mice treated with IL-1ra or vIL-10 compared with the control group. On day 14 after the onset of myocarditis, transfer of IL1ra or vIL-10 expression plasmid had significantly improved the survival rates of the animals. The expression of TNF-alpha was decreased to 0.60-fold (p < 0.005) and inducible nitric oxide synthase (iNOS) 0.43-fold (p < 0.005) by IL-1ra treatment, and the expression of IFN-gamma in the heart was decreased to 0.35-fold (p < 0.05), and iNOS 0.21-fold (p < 0.005), by vIL-10 relative to the controls. These results show that gene therapy with IL-1ra or vIL-10 expression plasmid was effective in the treatment of viral myocarditis, and in vivo electroporation may be a useful method by which to deliver cytokine therapy in cardiovascular diseases.     

Tetracycline-inducible viral interleukin-10 intraocular gene transfer, using adeno-associated virus in experimental autoimmune uveoretinitis

Members of the adeno-associated virus (AAV) family are good candidates for the treatment of ocular diseases because of their relative lack of pathogenicity. We studied the effect of intraocular injection of AAV2-viral IL-10 (vIL-10) on retinal S-antigen-induced experimental autoimmune uveoretinitis (EAU) in Lewis rats. We demonstrated that AAV2/2-GFP injected into the vitreous body transduced the iris and ciliary body, or anterior uvea, and the retina. We showed that intravitreal injection of the AAV2/2-tetON-vIL-10 construct achieved detectable levels of vIL-10 mRNA and protein within the eye and was effective in protecting the rat retina against destruction. This protection was dependent on the level of vIL-10 present in the aqueous humor/ vitreous body. Intravitreal injection of the same construct encased within an AAV5 shell, AAV2/5-tetONvIL- 10, did not confer any degree of protection. It appeared that the AAV2/5 vectors did not transduce the anterior uvea, the site at which inflammatory cells first localize in EAU, nor the ganglion cell layer; induced low expression of vIL-10 mRNA; and did not achieve detectable levels of transgene expression in the aqueous humor/vitreous body. Local treatment with AAV2/2-tetON-vIL-10 did not dampen the systemic immune response, as determined by S-antigen-specific lymphocyte proliferation. Our results show that local intravitreal injection of AAV2/2 is an effective means by which to deliver immunoregulatory molecules into the eye during uveitis, a chronic human ocular disease.     

Altered cutaneous immune parameters in transgenic mice overexpressing viral IL-10 in the epidermis

IL-10 is a pleiotropic cytokine that inhibits several immune parameters, including Th1 cell-mediated immune responses, antigen presentation, and antigen-specific T cell proliferation. Recent data implicate IL-10 as a mediator of suppression of cell-mediated immunity induced by exposure to UVB radiation (280-320 nm). To investigate the effects of IL-10 on the cutaneous immune system, we engineered transgenic mice that overexpress viral IL-10 (vIL-10) in the epidermis. vIL-10 transgenic mice demonstrated a reduced number of I-A(+) epidermal and dermal cells and fewer I-A(+) hapten-bearing cells in regional lymph nodes after hapten painting of the skin. Reduced CD80 and CD86 expression by I-A(+) epidermal cells was also observed. vIL-10 transgenic mice demonstrated a smaller delayed-type hypersensitivity response to allogeneic cells upon challenge but had normal contact hypersensitivity to an epicutaneously applied hapten. Fresh epidermal cells from vIL-10 transgenic mice showed a decreased ability to stimulate allogeneic T cell proliferation, as did splenocytes. Additionally, chronic exposure of mice to UVB radiation led to the development of fewer skin tumors in vIL-10 mice than in WT controls, and vIL-10 transgenic mice had increased splenic NK cell activity against YAC-1targets. These findings support the concept that IL-10 is an important regulator of cutaneous immune function.     

Inhibition of cartilage destruction by double gene transfer of IL-1Ra and IL-10 involves the activin pathway

The objective of the study was to determine the effects and the molecular background of interleukin-1 receptor antagonist (IL-1Ra) and vIL-10 double gene transfer into human synovial fibroblasts from patients with rheumatoid arthritis (RA) using the SCID mouse model for cartilage erosion in RA. RA synovial fibroblasts were transduced with retro- or adenoviruses encoding IL-1Ra and/or viral IL-10 (vIL-10). SCID mice were engrafted subcutaneously with IL-1Ra and vIL-10 transduced human rheumatoid synovial fibroblasts and normal cartilage. In parallel, gene expression analysis before and after gene transfer using RNA arbitrarily primed PCR in combination with cDNA array was performed. vIL-10 and IL-1Ra double gene transfer resulted in inhibition of cartilage invasion and degradation by RA synovial fibroblasts when compared with control transduced and non-transduced implants. Expression of key genes that were altered after double gene transfer were related to the activin pathway. The results demonstrate not only that virus-based gene transfer using a combination of two joint-protective genes is a feasible approach to inhibit cartilage degradation by activated RA synovial fibroblasts, but also that the underlying molecular effects include modulation of the activin pathway.     

Transgenic expression of decoy receptor 3 protects islets from spontaneous and chemical-induced autoimmune destruction in nonobese diabetic mice

Decoy receptor 3 (DCR3) halts both Fas ligand- and LIGHT-induced cell deaths, which are required for pancreatic beta cell damage in autoimmune diabetes. To directly investigate the therapeutic potential of DCR3 in preventing this disease, we generated transgenic nonobese diabetic mice, which overexpressed DCR3 in beta cells. Transgenic DCR3 protected mice from autoimmune and cyclophosphamide-induced diabetes in a dose-dependent manner and significantly reduced the severity of insulitis. Local expression of the transgene did not alter the diabetogenic properties of systemic lymphocytes or the development of T helper 1 or T regulatory cells. The transgenic islets had a higher transplantation success rate and survived for longer than wild-type islets. We have demonstrated for the first time that the immune-evasion function of DCR3 inhibits autoimmunity and that genetic manipulation of grafts may improve the success and survival of islet transplants.     

Adeno-associated virus-mediated human IL-10 gene transfer suppresses the development of experimental autoimmune orchitis

Testicular germ cell-induced autoimmune orchitis is characterized by inflammatory cell infiltration followed by disturbance of spermatogenesis. Experimental autoimmune orchitis (EAO) is an animal model for human immunological male infertility; delayed-type hypersensitivity (DTH) response plays a key role in its induction. Interleukin-10 (IL-10) is a regulatory cytokine that is critical in preventing organ-specific autoimmune inflammation. To determine the effects on EAO of human IL-10 (hIL-10) gene transfer, C3H/He mice immunized by unilateral testicular injury were administered intramuscular (i.m.) injections of adeno-associated viral (AAV) vector-encoding hIL-10 on the day of immunization. Serum hIL-10 was detected beginning at 1 week postinjection, and peaked at 3 weeks. Histological examinations showed a significantly low incidence of orchitis and disturbance of spermatogenesis in AAV hIL-10-treated mice, and the DTH response to autologous testicular cells was significantly suppressed. Immunohistochemical analysis of IFN- and IL-2, T-cell-associated cytokines, in the spleen and testes revealed significantly fewer cytokine-expressing cells after treatment. We conclude that a single i.m. administration of AAV hIL-10 significantly suppresses EAO and hypospermatogenesis by regulating cell-mediated immunity in the testes.     

Leptin- or troglitazone-induced lipopenia protects islets from interleukin 1beta cytotoxicity.

Interleukin 1beta (IL-1beta)-induced beta cell cytotoxicity has been implicated in the autoimmune cytotoxicity of insulin-dependent diabetes mellitus. These cytotoxic effects may be mediated by nitric oxide (NO). Since long-chain fatty acids (FFA), like IL-1beta, upregulate inducible nitric oxide synthase and enhance NO generation in islets, it seemed possible that islets might be protected from IL-1beta-induced damage by lowering their lipid content. We found that IL-1beta-induced NO production varied directly and islet cell viability inversely with islet triglyceride (TG) content. Fat-laden islets of obese rats were most vulnerable to IL-1beta, while moderately fat-depleted islets of food-restricted normal rats were less vulnerable than those of free-feeding normal rats. Severely lipopenic islets of rats made chronically hyperleptinemic by adenoviral leptin gene transfer resisted IL-1beta cytotoxicity even at 300 pg/ml, the maximal concentration. Troglitazone lowered islet TG in cultured islets from both normal rats and obese, leptin-resistant rats and reduced NO production and enhanced cell survival. We conclude that measures that lower islet TG content protect against IL-1beta-induced NO production and cytotoxicity. Leptin or troglitazone could provide in vivo protection against insulin-dependent diabetes mellitus.     

Protective effects of IL-1Ra or vIL-10 gene transfer on a murine model of wear debris-induced osteolysis

The current study evaluated the protective effects of anti-inflammatory cytokine gene transfer on osteolysis provoked by orthopedic biomaterial particles using a murine model of inflammatory bone loss. A section of bone was surgically implanted into an air pouch established on a syngeneic recipient mouse. Inflammation was provoked by introduction of ultra-high-molecular-weight polyethylene (UHMWPE) particles into the pouch, and retroviruses encoding for interleukin-1 receptor antagonist (hIL-1Ra), viral interleukin-10 (vIL-10), or LacZ genes were injected. Pouch fluid and tissue were harvested 7 days later for histological and molecular analyses. The results indicated that IL-1Ra or vIL-10 gene transfer significantly inhibited IL-1beta and tumor necrosis factor (TNF) expression at both mRNA and protein levels. There were significantly lower mRNA expressions of calcitonin receptor and cathepsin K in RNA isolated from hIL-1Ra- or vIL-10-transduced pouches than LacZ-transduced and virus-free controls. Both anti-inflammatory cytokine gene transfers significantly reduced the mRNA expression of M-CSF (70-90%) and RANK (>65%) in comparison with LacZ- and virus-free controls. Histological examination showed that hIL-1Ra or vIL-10 gene transfer dramatically abolished UHMWPE-induced inflammatory cellular infiltration and bone pit erosion compared to LacZ-transduced and virus-free controls. Histochemical staining revealed significantly fewer osteoclast-like cells in samples treated with IL-1Ra or vIL-10 gene transfer. In addition, bone collagen content was markedly preserved in the groups with anti-inflammatory cytokine gene transfers compared with the other two groups. Overall, retrovirus-mediated hIL-1Ra or vIL-10 gene transfer effectively protected against UHMWPE-particle-induced bone resorption, probably due to the inhibition of IL-1/TNF-induced M-CSF production and the consequent osteoclast recruitment and maturation.     

Gene transfer of virally encoded chemokine antagonists vMIP-II and MC148 prolongs cardiac allograft survival and inhibits donor-specific immunity

Introducing immunomodulatory molecules into allografts by gene transfer may avoid the side-effects of systemic immunosuppression. vMIP-II and MC148 are two recently identified chemokine homologues encoded by human herpes virus 8 and Molluscum contagiosum, respectively, that have antagonistic activities against multiple different CC and CXC chemokine receptors. We hypothesized that introduction of these molecules into cardiac allografts may block leukocyte infiltration into the grafts and prolong survival. Vascularized and nonvascularized cardiac allografts in mice were performed and plasmid DNA encoding vMIP-II, MC148 and/or vIL-10 was transferred into the allograft at the time of transplantation. Gene transfer of either vMIP-II or MC148 into cardiac allografts markedly prolonged graft survival. Combining gene transfer of either one of these chemokine antagonists with vIL-10 gene transfer, which has a mechanistically different immunosuppressive action, further enhanced graft survival. vMIP-II and MC148 gene transfer both resulted in a marked decrease of donor-specific cytotoxic T lymphocytes (CTL) infiltrating the grafts and inhibited alloantibody production. These results demonstrate that plasmid-mediated gene transfer of virally encoded chemokine antagonists vMIP-II and MC148 can block donor-specific lymphocyte immunity within cardiac allografts and prolong graft survival. This is a new mechanistic approach to analyze, treat, and prevent graft rejection. Delivery of these or related molecules by gene transfer or conventional pharmacologic means may represent a novel therapeutic modality for alloactivation.