CTLA4Ig Gene Transfer Research Articles

Immunological tolerance of human hepatocyte xenograft induced by adenovirus vector-mediated CTLA4Ig gene transfer

Systemic administration of CTLA4Ig has been applied in inducing immunological tolerance of hepatocyte implants, but has potential for systemic immune inhibition. This study was designed to induce hepatocyte immunological tolerance by locally expressing CTLA4Ig in an attempt to improve the effectiveness of cell transplantation. A normal human liver cell line (L02) was transfected with adenovirus vector containing the CTLA4Ig gene (Ad-CTLA4Ig-EGFP) in vitro, and the expression of CTLA4Ig by transfected cells was assessed by fluorescent imaging and immunocytochemical staining. Transfected cells then were injected into the spleen of Sprague-Dawley rats, the survival of cells was determined by immunohistochemistry, and the immune status was examined through CD4+ and CD69+ T cell-counts and ELISA detection of IL-2 in peripheral blood. L02 cells expressed CTLA4Ig in the cytoplasm for >4 weeks. Surviving L02 cells were observed in the experimental group at 3 and 4 weeks post-transplantation, while none was detected in the control group. Furthermore, the percentages of CD4+ and CD4+CD69+ T cells in the CTLA4-transfected group were 24.5% and 45.1%, markedly lower than those in the control group at 4 weeks post-transplantation (P<0.01). Furthermore, the IL-2 level was also lower in the CTLA4-transfected group than in the control group. Adenovirus-mediated CTLA4Ig gene transfer into human hepatocytes has the potential to become an effective method of inducing immunological tolerance in hepatocyte transplantation.

Recombinant adenovirus-mediated gene transfer suppresses experimental arthritis

Collagen Induced Arthritis (CIA) is a widely studied animal model to develop and test novel therapeutic approaches for treating Rheumatoid Arthritis (RA) in humans. Soluble Cytotoxic T-Lymphocyte Antigen 4 (CTLA4-Ig), which binds B7 molecule on antigen presenting cells and blocks CD28 mediated T-lymphocyte activation, has been shown to ameliorate experimental autoimmune diseases such as lupus, diabetes and CIA. Objective of our research was to investigate in vivo the effectiveness of blocking the B7/CD28 T-lymphocyte co-stimulatory pathway, utilizing a gene transfer technology, as a therapeutic strategy against CIA. Replication-deficient adenoviruses encoding a chimeric CTLA4-Ig fusion protein, or beta-galactosidase as control, have been injected intravenously once at arthritis onset. Disease activity has been monitored by the assessment of clinical score, paw thickness and type II collagen (CII) specific cellular and humoral immune responses for 21 days. The adenovirally delivered CTLA4-Ig fusion protein at a dose of 2x10^8 pfu suppressed established CIA, whereas the control beta-galactosidase did not significantly affect the disease course. CII-specific lymphocyte proliferation, IFNgamma production and anti-CII antibodies were significantly reduced by CTLA4-Ig treatment. Our results demonstrate that blockade of the B7/CD28 co-stimulatory pathway by adenovirus-mediated CTLA4-Ig gene transfer is effective in treating established CIA suggesting its potential in treating RA.

Effects of Gene Transfer CTLA4Ig and Anti-CD40L Monoclonal Antibody on Islet Xenograft Rejection in Mice

Blockade of a costimulatory pathway by adenovirus-mediated cytotoxic T lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) gene transfer and anti-CD40L mAb(MR1) have been reported to enhance graft survival in several experimental transplantation models. In this study, we investigated the effects of gene transfer of CTLA4Ig and MR1 on islet xenograft rejection in mice. Recombinant adenovirus AdCTLA4Ig was constructed to express CTLA4Ig. Islet grafts from adult male DA rats transferred with AdCTLA4Ig were transplanted to streptozocin-induced diabetic Balb/c mice. The diabetic mice were treated with MR1 after transplantation. We evaluated the islet xenograft mean survival time as well as changes in interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) levels in transplanted mice. The mean survival of islet xenografts in the MR1 treatment group was 34.9 ± 5.62 days, in the AdCTLA4Ig treatment group it was 56.5 ± 10.64 days, and in the AdCTLA4Ig plus MR1 treatment group it was 112.9 ± 19.26 days, all significantly prolonged compared with an untreated group (8.1 ± 0.83 days). Within 1 week after transplantation the levels of IL-2 and TNF-α showed sharp increases in the untreated group, being significantly higher than those observed prior to transplantation. In conclusion, using both AdCTLA4Ig and MR1 can improve the islet xenograft survival. The beneficial effects of the combined use of the 2 reagents were superior to either 1 alone, possibly related to down-regulated expression of Th1 cell–related cytokines.

Adenovirus-Mediated CTLA4Ig Gene Transfer Improves the Survival of Grafted Human Hepatic Progenitors in Mouse Liver

The invasive nature of surgery and limited numbers of donor livers for end-stage patients has prompted the search for alternative cell therapies for intractable hepatic disease. Hepatocyte ransplantations have been performed for a variety of indications, but sustained benefits have not been observed in most cases. Rat fetal liver epithelial cells (liver stem cells) have demonstrated self-renewal in vivo and functional repopulation of the liver. We have previously isolated and expanded epithelial progenitor cells (EPC) from the human fetal liver to investigate their differentiation potential. In this study, we applied suppression of immunorejection by adenoviral CTLA4Ig gene delivery mediated to examine the survival and differentiation of human fetal EPC transplanted into normal mouse liver. The grafted EPC showed extensive proliferation at both 1 and 2 months after transplantation compared with controls. Moreover, most EPC differentiated into hepatocytes, while a small fraction became bile ductular cells. This finding suggested that human fetal EPC may be a ideal source of cell-based therapy for various liver diseases.

CTLA4Ig gene transfer alleviates abortion in mice by expanding CD4 +CD25 + regulatory T cells and inducing indoleamine 2,3-dioxygenase

Successful pregnancy requires a state of immunological tolerance since normally the maternal immune system does not reject the semi-allogeneic conceptus. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), a ligand for B7, delivers negative signals to antigen presenting cells (APCs) to compete with CD28 for binding to B7 molecules and down-regulate proinflammatory responses, thus inhibiting T cell activation. Using CBA/J × DBA/2 matings as an abortion-prone model, we observed that adenovirus-mediated CTLA4Ig (Ad-CTLA4Ig) gene transfer improves pregnancy outcome. Ad-CTLA4Ig therapy skewed the ability of serum cytokine production toward a Th2 bias. Flow cytometry revealed that Ad-CTLA4Ig administration expanded peripheral CD4 +CD25 + regulatory T cell populations in CBA/J × DBA/2 matings. Furthermore, Ad-CTLA4Ig administration induced indoleamine 2,3-dioxygenase (IDO) and Foxp3 mRNA expression at the materno-fetal interface. Our results demonstrate that adenovirus-mediated CTLA4Ig gene transfer improves pregnancy outcome in a murine model of abortion by expanding the CD4 +CD25 + regulatory T cell population and inducing IDO mRNA expression.

Therapeutic effect of exosomes from indoleamine 2,3‐dioxygenase–positive dendritic cells in collagen‐induced arthritis and delayed‐type hypersensitivity disease models

We have demonstrated previously that dendritic cells (DCs) modified with immunosuppressive cytokines, and exosomes derived from DCs can suppress the onset of murine collagen-induced arthritis (CIA) and reduce the severity of established arthritis. Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme that is important for immune regulation and tolerance maintenance. DCs expressing functional IDO can inhibit T cells by depleting them of essential tryptophan and/or by producing toxic metabolites, as well as by generating Treg cells. This study was undertaken to examine the immunosuppressive effects of bone marrow (BM)-derived DCs genetically modified to express IDO, and of exosomes derived from IDO-positive DCs. BM-derived DCs were adenovirally transduced with IDO or CTLA-4Ig (an inducer of IDO), and the resulting DCs and exosomes were tested for their immunosuppressive ability in the CIA and delayed-type hypersensitivity (DTH) murine models. Both DCs and exosomes derived from DCs overexpressing IDO had an antiinflammatory effect in CIA and DTH murine models. The suppressive effects were partially dependent on B7 costimulatory molecules. In addition, gene transfer of CTLA-4Ig to DCs resulted in induction of IDO in the DCs and in exosomes able to reduce inflammation in an IDO-dependent manner. These results demonstrate that both IDO-expressing DCs and DC-derived exosomes are immunosuppressive and antiinflammatory, and are able to reverse established arthritis. Therefore, exosomes from IDO-positive DCs may represent a novel therapy for rheumatoid arthritis.

Adenoviral-mediated localized CTLA-4Ig gene expression induces long-term allograft pancreas survival and donor-specific immune tolerance in rats

T cell activation following alloantigen recognition plays a critical role in the development of the rejection in all solid organ, tissue and cell transplantation. A recombinant molecule, cytotoxic T lymphocyte antigen 4 antibody (CTLA-4Ig), is known to induce to T-cell into “anergy” by blocking the costimulatory B7-CD28 interaction. Either systemic or localized administration of CTLA-Ig has been shown to prolong allograft survival and induce donor-specific tolerance in some transplant models. In this study, we characterized the expression and immunosuppressive effectiveness of adenoviral-mediated CTLA-4Ig gene transfer. We demonstrated transduction of the allografts with AdCTLA-4Ig resulted in localized expression, permanent graft survival and stable donor-specific tolerance. In addition, by performing simultaneous dual-organ transplantation, we targeted on immunosuppression through a local expression of CTLA-4Ig via adenoviral-mediated gene transfer into pancreatic allografts.

Hydrodynamics‐based delivery of plasmid DNA encoding CTLA4‐Ig prolonged cardiac allograft survival in rats

Although gene therapy using plasmid vectors is thought to be safer compared with viral vectors, poor efficacy of gene transfer is the obstacle preventing wide application of plasmid vectors. However, high levels of foreign gene expression have been achieved by rapid tail vein injection of a large volume of a plasmid DNA solution into rats. Using this technique, we examined the effect of rat CTLA4-Ig gene transfer on prevention of cardiac allograft rejection in this animal model. Recipient Lewis rats were injected with either plasmid pCAGGS-CTLA4-Ig-Glu-tag as a treatment vector or plasmid pCAGGS-signal peptide (SP)-Ig as a control vector by hydrodynamics-based delivery technique on the day before heart transplantation. Hearts from Brown Norway donors were transplanted into the neck of Lewis recipients and graft survival was assessed. The plasma level of CTLA4-Ig reached a peak of nearly 5 microg/mL 1 day after injection, and then slowly decreased but still remained above 0.9 microg/mL until 100 days after injection. The recipient rats treated with the control vector and untreated rats rejected cardiac allografts within 7 days. On the other hand, the median survival time of the grafts treated with pCAGGS-CTLA4Ig-Glu-tag was more than 100 days. Histological examination revealed that long-term survival allografts contained fewer infiltrating lymphocytes. The serum from recipients with long-term survival allograft suppressed allogenic mixed lymphocyte reaction. CTLA4-Ig gene transfer by means of tail vein injection of plasmid DNA into a recipient rat resulted in remarkable prolongation of cardiac allograft survival with persistent plasma level of CTLA4-Ig protein.

Adeno-Associated Virus–Mediated CTLA4Ig Gene Transfer Protects MHC-Mismatched Renal Allografts from Chronic Rejection

Short-term results of renal transplantation have improved considerably in the past 20 yr; however, similar improvements in long-term outcome have not been achieved. The primary cause of late graft loss is chronic rejection that might be treated by gene therapeutic approaches. Ideally, one would like to impair locally the contact between transplant antigen and the host immune system without compromising the generalized immune competence of the recipient. This can be achieved by local expression of the therapeutic protein in the site of interest using gene therapy. Here it is shown that chronic allograft rejection can be prevented effectively by local delivery of recombinant adeno-associated virus (AAV) vectors that encode the CTLA4Ig immunosuppressant protein to the donor kidney in a fully MHC-mismatched rat strain combination. AAV CTLA4Ig prevented progressive proteinuria and protected transplant kidneys from renal structural injury. A population of anergic T cells with regulatory activity, which eventually were responsible for the induction of tolerance, were found in recipient lymph nodes and in the graft as long as 120 d after transplantation. These data indicate that AAV-mediated CTLA4Ig gene transfer to donor graft represents a promising tool to prevent the onset of chronic rejection and circumvent the unwanted systemic adverse effects of the administration of immunomodulatory protein.

Upregulation of CD4+CD25+ T lymphocyte by adenovirus-mediated gene transfer of CTLA4Ig fusion protein in experimental autoimmune myocarditis

Objective: To explore the effects of adenovirus vector-mediated gene transfer of CTLA4Ig fusion protein on CD4+CD25+ T cells in experimental autoimmune myocarditis (EAM).Methods: EAM was induced by porcine cardiac myosin as previously described. Adenovirus vector-mediated CTLA4Ig gene was administrated intravenously in EAM rats on days 1, 4 and 7, with EGFP as control. On day 21, myocardium histopathology was examined and CD4+CD25+ T cells were isolated. Proliferation and suppression assays were used to evaluate the suppressive capacity of CD4+CD25+ T cells in vitro. Relative mRNA level of Foxp3 and TGF-β was determined by quantitative real-time RT-PCR; expression of CTLA-4, B7-1 and B7-2 protein was compared with Western blot in CD4+CD25+ Tregs.Results: Severe inflammatory lesions were observed in the hearts of EGFP-treated EAM rats and the untreated ones, while Ad–CMV–CTLA4Ig alleviated the myocarditis histologically. Adenovirus vector-mediated CTLA4Ig gene transfer up-regulated the proportion of CD4+CD25+ Tregs significantly. T cell proliferation was greatly inhibited in the CTLA4Ig group compared with the untreated and EGFP-treated groups in vitro. CTLA-4 and B7-2 proteins were down-regulated in the CTLA4Ig group, Foxp3 and TGF-β mRNA was up-regulated significantly by CTLA4Ig treatment.Conclusions: Adenovirus vector-mediated CTLA4Ig gene transfer alleviated inflammation in EAM, one of the potential mechanisms is up-regulation of CD4+CD25+ Tregs.

Adenovirus-mediated CTLA4Ig and OX40Ig gene transfer induces long-term survival of cardiac allografts in rats

To investigate the potential role of CTLA4Ig gene and OX40Ig protein in inducing transplantation tolerance and the mechanisms thereof. Thirty Lewis rats underwent transplantation of the hearts of DA rats and then randomly divided into five equal groups: control group, blank virus AdEGFP treated group (adenovirus containing EGFP at the dose of 1-5 x 10(9) pfu/ml was infused via portal vein immediately after the operation), AdCTLA4Ig treated group, AdOX40Ig treated group, and AdCTLA4Ig-IRES-OX40Ig treated group. The cardiac allograft survival was monitored by daily palpation. The total cessation of beating was defined as rejection and was confirmed by histology. Peripheral venous blood samples were collected 0, 3, 7, 10, 14, 21 and 28 days after the administration of adenovirus. ELISA was used to detect the expression of CTLA4Ig and OX40Ig. Twenty days after the heart transplantation single splenocyte suspension was prepared from surviving Lewis rats to be used as responder. The spleens of the normal donor-DA rats and the third strain DA rats to prepare single cell suspension of the same density to perform mixed lymphocyte reaction (MLR). Then recombinant IL-2 was added into the mixed MLR system to observe t\if the MLR could be reversed. Twenty days after the heart transplantation the splenocytes of the tolerating Lewis rats were injected into the lingual vein of the normal Lewis rats to observe the delayed type hypersensitivity (DTH) of the transferred Lewis rat to normal rat splenocytes. RT-PCR was used to detect the mRNA expression of IL-2, interferon-gamma, IL-4, and IL-10. The survival time of the AdCTLA4Ig-IRES-OX40Ig treated group was 151.5 d +/- 42.6 d, significantly longer than those of the AdOX40Ig treated group (60.2 d +/- 11.4 d (P = 0.003), AdCTLA4Ig (43.2 d +/- 11.1 d, P = 0.0026), control group (5.7 d +/- 0.5 d, P = 0.000 43), and AdEGFP treated group (5.2 d +/- 0.4 d, P = 0.000 43). CTLA4Ig and/or OX40Ig proteins were expressed at a high level in the adenoviral treated rats. Compared with the control group the splenocytes of the AdCTLA4Ig-IRES-OX40Ig, AdCTLA4Ig, and AdOX40Ig treated groups displayed donor-specific hyporesponsiveness (P = 0.0016, 0.0026 and 0.001), which could be partly reversed by the addition of exogenous IL-2. Moreover, the hyporesponsiveness could be transferred to the same strain rats through adoptive transfer. In comparison with the normal controls, the expression of Th1 type cytokines, such as IL-2 and IFN-gamma, was significantly decreased in the tolerating rats and significantly increased in the rats with rejection; however the expression of the Th2 type cytokines, such as IL03 and IL-10, was significantly increased in the tolerating rats and significantly decreased in the rats with rejection, showing a deviation of Th1/Th2 type cytokines. AdCTLA4Ig-IRES-OX40Ig-mediated genes transfer renders prolonged expression of CTLA4Ig and OX40Ig in Lewis recipient rats, leading to a long-term survival of cardiac allografts. The induced tolerance is donor-specific, and the mechanisms may be associated with T cell anergy, deviation of Th1/Th2, and the regulatory T cells.

Effect of Adenovirus-Mediated Transfer of the CTLA4IG Gene in Hamster-to-Rat Xenotransplantation

Costimulatory molecules such as CD28 and B7 are essential for T cell activation, as well as playing a role in the T cell receptor and major histocompatibility complex pathway. It is well known that rejection in allotransplantation is diminished by treatment with CTLA4Ig, but whether a similar effect occurs in xenotransplantation remains to be determined. In this study, we investigated whether adenovirus-mediated gene transfer with CTLA4Ig cDNA by intravenous injection to the recipient is effective in suppression in hamster-to-rat cardiac xenotransplantation. With CTLA4Ig gene transfer, the duration of gene expression was clearly prolonged, based on reduced production of antiadenovirus antibody and shrinkage of the spleen. The survival of cardiac xenografts was significantly prolonged with CTLA4Ig gene transfer compared to the control graft, and survival with combination use of FK506 and CTLA4Ig gene transfer in xenotransplantation was also significantly prolonged compared to that with CTLA4Ig gene transfer alone. Cessation of the cardiac graft in the combination treatment occurred in parallel with the elevation of antihamster IgM antibodies in rat sera. These results suggest that adenovirus-mediated CTLA4Ig gene transfer is effective for immunosuppression in hamster-to-rat xenotransplantation.

Adenovirus-Mediated CTLA4Ig or CD40Ig Gene Transfer Delays Pancreatic Islet Rejection in a Rat-to-Mouse Xenotransplantation Model after Systemic but Not Local Expression

Transient costimulation signal blockade of either CD28/CD80-86 interactions and/or CD40/CD154 interactions can prevent islet rejection in some models of both allo- and xenotransplantation. We have used adenoviruses coding for CTLA4Ig or CD40Ig and compared the efficacy of genetic modification of islets to systemic production through either intramuscular (i.m.) or intravenous (i.v.) injection of these vectors in a rat-to-mouse islet transplantation model. When gene transfer was performed into islets, a high level of primary nonfunction was induced. Furthermore, transduced functional grafts were rejected with the same kinetics as nontransduced islets. In contrast, i.m. AdCTLA4Ig and i.v. AdCD40Ig significantly delayed rejection (mean survival time of 54 +/- 26.9 and 67.6 +/- 44.9 days, respectively, vs. 24.3 +/- 9.7 days for unmodified islets, p < 0.05). Combination of ex vivo AdCTLA4Ig islet transduction and i.v. AdCD40Ig did not improve graft survival further. In conclusion, islet graft transduction with adenoviruses coding for costimulation inhibitors resulted in local expression with low serum concentrations of CTLA4Ig or CD40Ig and was unable to protect islet xenografts from rejection. In contrast, i.m. or i.v. gene transfer resulted in high serum concentrations of these molecules and was highly efficient in prolonging xenograft survival. These results contrast with the efficacy of AdCTLA4Ig we observed in a rat islet allotransplantation model and suggest that islet xenograft rejection might be more difficult to control.

Development of donor-specific immunoregulatory T-cells after local CTLA4Ig gene transfer to pancreatic allograft.

CTLA4Ig gene transfer directly to graft tissue might have the potential to avoid the need for systemic immunosuppression. In our previous studies of bio-breeding (BB) rats, local adenovirus-mediated CTLA4Ig gene transfer protected the pancreas from autoimmune and alloimmune responses. This study investigated the potency of local CD28/B7 costimulatory blockade for induction of donor-specific tolerance and further examined the existing mechanisms. Brown Norway (BN; RT1)-pancreaticoduodenal grafts transfected with Ad.CTLA4Ig via intraarterial ex vivo perfusion were transplanted into streptozotocin-induced diabetic Lewis (LEW; RT1) rats. Ad.CTLA4Ig transduced grafts combined with a short course of FK506 resulted in indefinitely prolonged survival (>156 days vs. 19.5 days with FK506 alone). CTLA4Ig was predominantly expressed in grafts on day 4. The expression was gradually diminished and was only slightly detectable at day >100. The proliferative responses against BN antigen were remarkably enhanced among recipients with rejected grafts, but the T-cells from tolerant recipients (>100 days) showed poor cytotoxic responses. On adoptive transfer assay, the splenic T-cells of tolerant recipients were able to suppress the rejection of BN, but not third-party Wistar Furth (WF; RT1) hearts in irradiated (480 cGy) LEW recipients. The percentage of CD4CD25 splenic T-cells was significantly increased in tolerant recipients (13.53 +/- 4.06% vs. 6.06 +/- 0.56% in naive rats). CTLA4Ig gene transfer to the pancreaticoduodenal allograft combined with a short course of FK506 induces donor-specific tolerance. The mechanism of maintaining tolerance could be explained by development of splenic T suppressor cells.

Long-term survival of xenografted neonatal cardiomyocytes by adenovirus-mediated CTLA4-Ig expression and CD40 blockade.

Prolonged survival of xenografted neonatal cardiomyocytes was achieved by blocking the CD28/B7 costimulatory pathway via CTLA4-Ig gene transfer. We examined the long-term survival of xenografted neonatal cardiomyocytes by adenovirus-mediated CTLA4-Ig expression and transient CD40 blockade with anti-CD40L monoclonal antibody (MR1). Neonatal cardiomyocytes derived from Dark Agouti rats were infected with CTLA4-Ig-expressing adenovirus vectors and injected directly into the normal myocardium of C3H/He mice. Mice were also given an intraperitoneal injection of 500 microg MR1 (CTLA+MR group, n=30) or control immunoglobulin (CTLA group, n=30) 1 hour before and 1, 3, and 7 days after cardiomyocyte implantation. As a control, cells infected with beta-Gal-expressing adenovirus vector (RL group, n=15) and cells without infection (control group, n=15) were injected into additional mice. Mice from all groups were killed 2, 4, and 8 weeks after xenotransplantation, and mice from the CTLA+MR and CTLA groups were killed 4 and 6 months after xenotransplantation. Neonatal cardiomyocytes were successfully infected by adenovirus vectors. Immunohistochemical analysis showed that the xenografted cardiomyocytes survived and expressed CTLA4-Ig for 6 months in all mice from the CTLA+MR and CTLA groups. A gap junction between the xenografted and host cardiomyocytes was also confirmed. Conversely, neonatal cardiomyocytes did not survive for even 2 weeks after xenotransplantation in the mice from the RL and control groups. Long-term survival of xenografted neonatal cardiomyocytes was achieved by adenovirus-mediated CTLA4-Ig expression and transient CD40 blockade.

Simultaneous administration of a low-dose mixture of donor bone marrow cells and splenocytes plus adenovirus containing the CTLA4Ig gene result in stable mixed chimerism and long-term survival of cardiac allograft in rats.

T-cell costimulatory blockade combined with donor bone marrow transfusion may induce mixed chimerism, rendering robust tolerance in transplanted organs and cells. However, most protocols entail high doses of donor bone marrow cells (BMCs) or repeated administration of costly agents that block costimulatory pathways, thus delaying clinical development. To circumvent these shortcomings, we developed a strategy in which the dosage of donor BMCs was reduced but compensated by donor splenocytes (SPLCs). Furthermore, repeated administration of costly agents was replaced with a single injection of adenovirus expressing a gene of interest. In rat cardiac transplantation models, cardiac allografts from DA (RT-1a) rats were transplanted heterotopically into the abdomen of LEW (RT-11) recipient rats. Immediately after cardiac transplantation, an adenovirus vector (AdCTLA4Ig; 5 x 10(9) plaque-forming units) containing the gene for CTLA4Ig was administered to recipients (n = 6) simultaneously with a low dose of donor BMCs (1 x 10(8)/rat) and SPLCs (5 x 10(7)/rat) via the portal vein. The treated LEW recipient rats developed long-lasting mixed chimerism (>10% at >100 days) and exhibited long-term cardiac allografts (mean survival time of > 200 days) compared with control recipients. Moreover, recipients displaying long-lasting mixed chimerism accepted subsequent donor skin allografts while promptly rejecting third-party skin allografts. These results suggest that blockade of the CD28-B7 pathway, using adenovirus-mediated CTLA4Ig gene transfer, in concert with a low dosage of donor BMCs and SPLCs, may represent a feasible strategy to induce stable mixed chimerism and permit long-term survival of cardiac allografts.

CTLA4Ig adenoviral gene transfer induces long-term islet rat allograft survival, without tolerance, after systemic but not local intragraft expression.

Genetic engineering using recombinant adenoviruses offers an opportunity to modify islet grafts in order to prevent allograft rejection. We have used an adenovirus coding for CTLA4Ig to compare its efficacy in preventing islet rejection depending on local or systemic production after gene transfer either into the islets or intramuscularly, respectively. Islet allograft survival was also evaluated using recombinant CTLA4Ig administered intraperitoneally or incubated ex vivo with islets prior to transplantation. Transduction of islets with 10(3) or 10(4) plaque-forming units (pfu) per islets of AdCTLA4Ig prolonged islet survival (mean +/- standard deviation [SD] days = 19.5 +/- 5.8 and 19.5 +/- 5.6, respectively, vs. 10.6 +/- 2.4 in control islets, p < 0.001), with low levels of circulating CTLA4Ig. In contrast, long-term survival (>60 days) was obtained after intramuscular injection of AdCTLA4Ig that resulted in sustained high levels of circulating CTLA4Ig. Islets incubated in vitro with CTLA4Ig did not show prolonged survival (10.3 +/- 2.5 days). Graft rejection was delayed after one injection of CTLA4Ig (23 +/- 7.6 days, p < 0.003 vs. control). Recipients of long-term surviving grafts after intramuscular AdCTLA4Ig gene transfer were not tolerant because second islet grafts of donor origin were rejected. These recipients also had a strong inhibition of humoral responses against nominal antigens, whereas animals receiving transduced islets showed normal responses. These data demonstrate that local production of CTLA4Ig after gene transfer was as efficient as a single injection of CTLA4Ig in preventing graft rejection. Furthermore, intramuscular gene transfer of CTLA4Ig was the most efficient way to induce long-term islet graft survival but no donor-specific tolerance was induced.

Suppression of graft coronary arteriosclerosis by gene gun-mediated CTLA4-Ig gene transfer

Suppression of graft coronary arteriosclerosis by gene gun-mediated CTLA4-Ig gene transfer

Prolongation of liver xenograft survival by adenovirus vector-mediated CTLA-4Ig gene transfer

Cytotoxic T lymphocyte-associated antigen-4/immunoglobulin fusion products (CTLA-4Ig), a structural homologue of CD28, has been shown to inhibit cellular and humoral immune responses. In this study, we investigated the efficacy of an adenovirus vector containing the CTLA-4Ig gene (AdCTLA-4Ig) on recipient survival after hamster-to-rat liver xenografting. AdCTLA-4Ig was administrated intravenously immediately after grafting. Gene expression was achieved a maximum of 7 days after vector injection and continued for more than 4 weeks. The proportion of CD25 + T-cells in recipient lymph nodes was significantly reduced 7 days after administration of AdCTLA-4Ig, compared to a group given an adenoviral vector containing LacZ gene (AdLacZ) or to an untreated control group. AdCTLA-4Ig markedly reduced CD2 + T-cell infiltration into the graft and significantly prolonged recipient survival time (9.2±4.12 days), compared to the untreated group (5.4±0.78 days) ( P<0.001) and the AdLacZ-treated group (5.2±0.28 days) ( P<0.001). These results indicate that a blockade of T-cell co-stimulation by AdCTLA-4Ig inhibited T-cell activation and attenuated CD2 + T-cell infiltration into the xenograft, resulting in significant prolongation of recipient survival time. Thus, AdCTLA-4Ig therapy may provide a novel approach to immune regulation.

Ex vivo and systemic transfer of adenovirus-mediated CTLA4Ig gene combined with a short course of FK506 therapy prolongs islet graft survival

Adenovirus-mediated CTLA4Ig gene transfer has been reported to enhance graft survival in several rodent transplantation models. In this study, we investigated the efficacy of ex vivo and systemic transfer of the CTLA4Ig gene by adenoviral vectors in pancreatic islet allo-transplantation. Islet grafts from BN rats were transplanted to chemically induced diabetic LEW rats. First, ex vivo CTLA4Ig gene transfer into isolated islets was performed prior to transplantation. Survival of transduced grafts under the kidney capsule was slightly prolonged (8.6±1.3 days) compared with survival of untransduced grafts (6.7±1.2 days); when combined with a short course of FK506, graft survival was further extended (32.6±10.7 days vs. 13.7±1.0 days with FK506 alone). Secondly, systemic gene transfer was accomplished by intravenous administration immediately after the transplantation procedure. In these animals, islet grafts under the kidney capsule survived longer (15.2±3.3 days) than in controls (6.7±1.2 days), and when FK506 was administered perioperatively, all the islet grafts survived for more than 100 days. In systemically transduced recipients, the survival of islet grafts transplanted into the liver was not significantly different from that of the grafts placed under the kidney capsule. In order to examine organ-specific immunogenicity, heterotopic BN cardiac grafts were transplanted to LEW rats intra-abdominally, with the virus transferred systemically as in the islet model. In contrast to the islet grafts, all the cardiac grafts were accepted for longer than 100 days, even without FK506 therapy. Finally, the LEW recipients with long-surviving islet or cardiac grafts were re-transplanted with islet grafts from the same donor strain (BN) on day 100. The second islet grafts survived longer than 100 days in half of the cardiac recipients, but consistently failed in the islet recipients. We conclude that in this transplant model, CTLA4Ig gene transfer and FK506 treatment synergistically improved islet graft survival, systemic transfer of the gene was more effective than ex vivo transfer to the islets, and donor-specific tolerance could not be achieved for islet transplantation but was achieved for cardiac transplantation.