CTLA4Ig Gene Research Articles

Tolerance induction by a single donor pretreatment with the adenovirus vector encoding CTLA4Ig gene in rat orthotopic liver transplantation

Tolerance induction by a single donor pretreatment with the adenovirus vector encoding CTLA4Ig gene in rat orthotopic liver transplantation

Nonviral gene gun–mediated CTLA4-Ig gene transfer for modification of donor organs

Nonviral gene gun–mediated CTLA4-Ig gene transfer for modification of donor organs

Adenovirus-mediated gene transfer of CTLA-4Ig fusion protein in the suppression of experimental autoimmune arthritis.

Blockade of CD28-B7 interactions with soluble CTLA-4Ig fusion protein (which binds and blocks both B7-1 and B7-2 costimulatory molecules on antigen-presenting cells) has been shown to ameliorate experimental autoimmune diseases such as lupus, experimental autoimmune encephalomyelitis, diabetes, and, in our laboratory, collagen-induced arthritis (CIA). Because prolonged inhibition of this costimulatory pathway may be required, and the adenovirus-mediated gene-transfer technology is very efficient in achieving sustained expression of proteins in vivo, we examined the effects of adenovirally delivered CTLA-4Ig in established murine CIA. Replication-deficient recombinant adenoviruses encoding a chimeric CTLA-4Ig fusion protein, or beta-galactosidase as control, were injected intravenously into male DBA/1 mice once at arthritis onset. Disease activity was monitored by the assessment of clinical score, paw thickness, and type II collagen (CII)-specific cellular and humoral responses for 3 weeks. Groups of mice were also serially injected with a CTLA-4Ig fusion protein and an anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) monoclonal antibody (mAb), and disease activity was compared with that in the adenovirally transfused groups. Both the adenovirally delivered and the recombinant CTLA-4Ig fusion protein suppressed established CIA, whereas anti-CTLA-4 mAb and the control beta-galactosidase adenovirus did not significantly affect the disease course. CII-specific lymphocyte proliferation, interferon-gamma production, and anti-CII antibody levels, both IgG1 and IgG2a, were significantly reduced by CTLA-4Ig treatment. Blockade of the B7-CD28 costimulatory pathway by adenovirus-mediated CTLA-4Ig gene transfer is as effective as the recombinant fusion protein in treating established CIA, without the need for repeated administrations. Significant reduction in pathogenic cellular and humoral responses is achieved even after the onset of arthritis, thus suggesting the valuable therapeutic potential of this gene-transfer method in human rheumatoid arthritis.

Tolerance to cardiac allografts via local and systemic mechanisms after adenovirus-mediated CTLA4Ig expression.

Blockade of the CD28/B7 T cell costimulatory pathway prolongs allograft survival and induces tolerance in some animal models. We analyzed the efficacy of a CTLA4Ig-expressing adenovirus in preventing cardiac allorejection in rats, the mechanisms underlying heart transplant acceptance, and whether the effects of CTLA4Ig were restricted to the graft microenvironment or were systemic. CTLA4Ig gene transfer into the myocardium allowed indefinite graft survival (>100 days vs 9 +/- 1 days for controls) in 90% of cases, whereas CTLA4Ig protein injected systemically only prolonged cardiac allograft survival (by up to 22 days). CTLA4Ig could be detected in the graft and in the serum for at least 1 year after gene transfer. CTLA4Ig gene transfer induced local intragraft immunomodulation at day 5 after transplantation, as shown by decreased expression of the IL-2R and MHC II Ags; decreased levels of mRNA encoding for IFN-gamma, inducible NO synthase, and TGF-beta; and inhibited proliferative responses of graft-infiltrating cells. Systemic immune responses were also down-modulated, as shown by the suppression of Ab production against donor alloantigens and cognate Ags, up to at least 120 days after gene transfer. Alloantigenic and mitogenic proliferative responses of graft-infiltrating cells and total splenocytes were inhibited and were not reversed by IL-2. In contrast, lymph node cells and T cells purified from splenocytes showed normal proliferation. Recipients of long-term grafts treated with adenovirus coding for CTLA4Ig showed organ and donor-specific tolerance. These data show that expression of CTLA4Ig was high and long lasting after adenovirus-mediated gene transfer. This expression resulted in down-modulation of responses against cognate Ags, efficient suppression of local and systemic allograft immune responses, and ultimate induction of donor-specific tolerance.

Adenovirus-mediated gene transfer of CTLA4Ig gene results in prolonged survival of heart allograft.

It has been demonstrated that the administration of CTLA4Ig protein can induce the suppression of allograft and xenograft rejection. The purpose of this study is to determine the effect of adenovirus-mediated gene transfer with CTLA4Ig gene on the transgene expression and suppression of alloimmune response in allogeneic cardiac transplantation of rats. Adenoviral vectors with beta galactosidase or human CTLA4Ig cDNA (Adex/LacZ, Adex/hCTLA4Ig) were constructed. These vectors were transduced to the liver of Lewis (LEW) rats by intravenous injection. Then LEW rats received heterotopic cardiac transplantation from Dark Agouti rats. Experiments were performed using both CTLA4Ig gene transduction and/or immunosuppressant FK506. The transgene expression after adenovirus-mediated transfer with CTLA4Ig cDNA lasted for several months, compared with several weeks after that in controls, which was proportional to the blood concentration of CTLA4Ig protein. By the administration of 1 x 10(9) PFU of Adex/hCTLA4Ig, the survival of cardiac grafts was significantly prolonged, compared with controls or the use of 1 x 10(8) PFU of Adex/hCTLA4Ig. In the rats with beating grafts over 100 days, the blood concentrations of CTLA4Ig were undetectable. The combination therapy using a low titer Adex/hCTLA4Ig and low-dose of FK506 was synergistically effective on this cardiac transplantation model. In conclusion, adenovirus-mediated gene transfer with CTLA4Ig gene was efficient for the prolongation of both transgene expression and allograft survival.

Expression of a soluble form of CTLA4 on macrophage and its biological activity.

Interaction between cytotoxic T lymphocyte-associated antigen-4 (CTLA4, CD152) and B7 molecules (B7-1 and B7-2) is of importance in the cellular events of lymphocyte, including antigen-specific T-cell activation and induction of autoreactive T-cell. We describe here the first introduction of a murine soluble CTLA4 gene, CTLA4Ig, to Mm1 cells, a macrophagic cell line. CTLA4Ig was successfully expressed on Mml cells and the expressed CTLA4Ig was found to be functionally active in their binding to B7 molecules by flow cytometry and immunofluorescence studies. The biological activity of CTLA4Ig from the transfected Mm1 cells was studied and showed inhibitory activity on mixed lymphocyte culture. A high CTLA4Ig producing macrophagic cell line was obtained. As Mm1 cells were regarded as difficult for gene transfection and there had so far been no report on expression of CTLA4Ig gene on Mm1 cells, these results suggested that the CTLA4Ig expressing Mm1 cells could be useful for analysis of CTLA4 and B7 molecule interaction in both macrophage and T-cell.

Prolonged cardiac allograft survival in rats systemically injected adenoviral vectors containing CTLA4Ig-gene.

CTLA4Ig, a soluble recombinant fusion protein that contains the extracellular domain of the CTLA4 and Fc portion of IgG1, strongly adheres to the B7 molecule to block CD28-mediated costimulatory signals and inhibits in vitro and in vivo immune responses. In vivo gene transfer using adenovirus vector achieves a high transfection rate into organ cells that usually contain adenoviral receptors. In this study, we investigated expression levels of the transfected gene and the survival times of the allografts in cardiac recipients systemically administered adenoviral vectors containing CTLA4Ig. Hearts from DA rats (RT-1a) were transplanted into a cervical location in LEW recipients (RT1(1)). The adenoviral vectors containing CTLA4Ig was injected via a recipient vein immediately after grafting. The serum level of CTLA4Ig reached to maximum at 51-93 microg/ml 3 to 7 days after gene-transfection and declined after 14 days, although detectable levels were observed up to 49 days. The median survival time of the allografts in the gene-transfected group were significantly prolonged (27 days) in compared to the control group (6 days). In addition, down-regulation of IL-2 and IFN-gamma mRNAs and persistence of IL-4 and IL-10 transcripts were observed in the graft infiltrating cells. The adenovirous-mediated CTLA4Ig gene transfer into a recipient liver by systemic administration resulted in remarkable prolongation of cardiac allograft survival. Its action mechanisms may be mediated by inhibition of CD28-associated signal transduction, reduction of Th1-type cytokine production, and continuous expression of Th2-type cytokines in the activating lymphocytes.

Effect of adenovirus-mediated gene transfer with CTLA4-Ig gene in organ transplantation

Effect of adenovirus-mediated gene transfer with CTLA4-Ig gene in organ transplantation

Cardiac allograft tolerance induced by intra-arterial infusion of recombinant adenoviral CTLA4Ig.

Systemic administration of soluble recombinant fusion protein of cytotoxic T lymphocyte antigen 4 (CTLA4Ig) induces blockade of the CD28/B7 costimulatory pathway and promotes survival of allogeneic and xenogeneic grafts. We tested the efficacy of local expression of CTLA4Ig gene in the myocardium, induced by transduction with a recombinant adenovirus encoding the CTLA4Ig gene, on the survival of rat cardiac allografts. The donor hearts were perfused ex vivo with recombinant adenovirus encoding CTLA4Ig cDNA (AdCTLA4Ig) via intra-aorta coronary artery before transplantation. The distribution and duration of CTLA4Ig transgene expression in the myocardium was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) or in situ RT-PCR after transplantation. In situ RT-PCR demonstrated abundant expression of CTLA4Ig transgene in the endo-myocardium of AdCTLA4Ig-perfused cardiac grafts. Lewis and Brown Norway cardiac allografts transduced with AdCTLA4Ig survived indefinitely in nonimmunosuppressed Wistar Furth recipients. However, donor-strain skin grafts were rejected by long-term recipients of cardiac allografts, which also triggered the rejection of the primary heart grafts. A single ex vivo intra-aortic infusion of recombinant adenovirus encoding the CTLA4Ig gene induced efficient transduction of the endo-myocardium and promoted the permanent survival of cardiac allografts in nonimmunosuppressed hosts. Despite the beneficial effect of local immunosuppression on cardiac allograft survival, the strategy failed to promote a state of donor-specific peripheral tolerance.

Prevention of autoimmune recurrence and rejection by adenovirus-mediated CTLA4Ig gene transfer to the pancreatic graft in BB rat.

Type 1 diabetes is the result of a selective destruction of pancreatic islets by autoreactive T-cells. Therefore, in the context of islet or pancreas transplantation, newly transplanted beta-cells are threatened by both recurrent autoimmune and alloimmune responses in recipients with type 1 diabetes. In the present study, using spontaneously diabetic BB rats, we demonstrate that whereas isolated islets are susceptible to autoimmune recurrence and rejection, pancreaticoduodenal grafts are resistant to these biological processes. This resistance is mediated by lymphohematopoietic cells transplanted with the graft, since inactivation of these passenger cells by irradiation uniformly rendered the pancreaticoduodenal grafts susceptible to recurrent autoimmunity. We further studied the impact of local immunomodulation on autoimmune recurrence and rejection by ex vivo adenovirus-mediated CTLA4Ig gene transfer to pancreaticoduodenal grafts. Syngeneic DR-BB pancreaticoduodenal grafts transduced with AdmCTLA4Ig were rescued from recurrent autoimmunity. In fully histoincompatible LEW-->BB transplants, in which rejection and recurrence should be able to act synergistically, AdmCTLA4Ig transduced LEW-pancreaticoduodenal allografts enjoyed markedly prolonged survival in diabetic BB recipients. In situ reverse transcription-polymerase chain reaction revealed that transferred CTLA4Ig gene was strongly expressed in both endocrine and exocrine tissues on day 3. These results indicate the potential utility of local CD28-B7 costimulatory blockade for prevention of alloimmune and autoimmune destruction of pancreatic grafts in type 1 diabetic hosts.

Adenovirus-mediated gene transfer of CTLA4IG gene results in prolonged survival of heart allograft

Adenovirus-mediated gene transfer of CTLA4IG gene results in prolonged survival of heart allograft

TRANSDUCTION OF DENDRITIC CELLS WITH ADENOVIRAL VECTORS ENCODING CTLA4-Ig MARKEDLY REDUCES THEIR ALLOSTIMULATORY ACTIVITY

109 Background: Dendritic cells (DC) lacking sufficient expression of B7-1(CD80) and B7-2(CD86) can induce alloantigen-specific T cell anergyin vitro and prolong cardiac allograft survival. The failure of these cells to induce allograft tolerance may due to the “late” upregulation of costimulatory molecules on the DC following their interaction with host T cells. Aim: In this study, we examined whether genetic modification of donor DC with an adenoviral vector encoding murine CTLA4-Ig(Ad-CTLA4-Ig) could potentiate their tolerogenity. Methods: DC were propagated from bone marrow (BM) of B10 (H-2b) mice with GM-CSF+IL-4(“IL-4 DC”) or GM-CSF+TGFβ1 (“TGFβ DC”). Compared with IL-4 DC, TGFβ DC were 1) DEC 205+, MHC class II+, CD40low and B71/2low, 2) poor stimulators of allogeneic T cell proliferation and CTL production, 3) capable of phenotypic and functional maturation following addition of IL-4, or withdrawal of TGFβ1. DC were infected with Ad LacZ at different moi, and intracellular LacZ activity was determined by X Gal staining 24 hr after infection. The capacity of the Adtransduced DC to induce T cell proliferation was analyzed by three-day primary MLR using C3H T cells as responders. Cytotoxic T cell (CTL) generation was determined by 4 hr 51Cr release assay, using EL4(H-2b) cells as targels. In vivo migration and survival of Ad-LacZ transduced B10 DC were examined by immunohistochemical staining with α IAb mAb. Results. Both IL-4 DC and TGFβ DC were efficiently transduced by Ad LacZ. More than 85% of the DC were X Gal positive at moi 100-50, 60-70% at moi 20, 40-50% at moi 10. Ad-LacZ transduction enhanced costimulatory molecule expression on TGFβ DC, but not on IL-4 DC, in dose dependent manner. The allostimulatory function of the DC in MLR and induction of CTL responses was significantly enhanced by Ad LacZ transduction at 100 moi. However, both IL-4 DC and TGFβ DC transduced with Ad-CTLA4-Ig at moi 50 exhibited 60-70% reduction in their capacity to induce allogeneic T cell proliferation and CTL responses. The supernatant from Ad-CTLA4-Ig transduced IL-4 DC dramatically inhibited C3H T cell proliferation stimulated by unmodified B10 IL-4 DC. The poor allostimulatory function of TGFβ DC in MLR was further inhibited by Ad-CTLA4-Ig transduction. The migration pattern of B10 Ad-LacZ transduced DC and non-transduced DC, determined by donor MHC class II (IAb) staining, was very similar after their local injection into C3H recipients. Conclusions: BM-derived DC can be efficiently transduced and functionally modified with an adenoviral vector encoding the CTLA4-Ig gene. Ad-CTLA4-Ig transduction shows potential for maximizing the tolerogenic activity of DC.

Effect of Gene Gun-Mediated CTLA4IG and Fas Ligand Gene Transfection on Concordant Xenogeneic Islet Graft Rejection

Effect of Gene Gun-Mediated CTLA4IG and Fas Ligand Gene Transfection on Concordant Xenogeneic Islet Graft Rejection

Effect of CTLA4-Ig gene transfer using adenoviral vector in organ and cell transplantation

Effect of CTLA4-Ig gene transfer using adenoviral vector in organ and cell transplantation

Evidence that the adenoviral vector containing the CTLA4-Ig gene improves transgene expression and graft survival

Rejection of organ allografts is dependent on T cell activation, which requires T-cell recepter engagement by antigen and costimulatory signals delivered by T-cell surface molecules such as CD28. CTLA4-Ig is a recombinant fusion protein that contains the extracellular domain of human CTLA4 (a homologue of CD28) fused to a human IgG 1 heavy chain. It has previously been shown to block the CD28-mediated costimulatory signal and to inhibit immune responses in vitro and in vivo. 1,2 Recently, the replication-defective adenovirus vector has been shown to be efficient in transferring exogenous genes into a variety of cells both in vitro and in vivo. 3 In this study, we examined the ability of adenovirus vector containing CTLA4-Ig (Adex/CTLA4-1g) to modify the rejection process observed in ACI to LEW rats liver transplantation.

Effect of CTLA4-Ig gene transfer using adenoviral vector in organ and cell transplantation

Effect of CTLA4-Ig gene transfer using adenoviral vector in organ and cell transplantation