CTLA4Ig Gene Transfer Research Articles

Modified AdCTLA-4 vector blocks alloimmune response in vitro.

Gene transfer of the costimulatory blockade molecule CTLA-4Ig into cold-preserved rat liver allografts results in indefinite allograft survival. Despite local delivery, this mode of immunomodulation is associated with systemic immunosuppression. In an effort to restrict immunosuppression to the graft, we have constructed a novel adenoviral vector, AdCTLA-4ex-TAG, in which the Ig sequence of CTLA-4Ig DNA has been deleted to destabilize the gene product to promote rapid extrahepatic degradation while maintaining its immunosuppressive activity within the graft milieu. (1) Vector construction. CTLA-4 extracellular binding domain (CTLA-4ex) was prepared by PCR-based cloning methods and fused in frame to a genetic element encoding an epitope TAG allowing identification of the transgene product CTLA-4exTAG. CTLA-4exTAG was subcloned into a shuttle vector enabling isolation of AdCTLA-4exTAG. (2) In vitro transfection: AdCTLA-4exTAG was transfected into MH(1)C(1) cells and then supernatant was recovered for Western blot analysis. (3) In vitro alloimmune response was characterized by CFSE proliferation assay. (4) Extrahepatic effect of AdCTLA-4exTAG was characterized by the ability to control tumor growth after implantation of a regressive, immune sensitive cancer cell line (REGb) in the skin of BDIX rats after liver transduction with AdCTLA-4exTAG. Expression and secretion of the recombinant protein were documented by Western blot after infection of the MH(1)C(1) cell line() with AdCTLA-4exTAG. Addition of infected MH(1)C(1) cell supernatant resulted in abrogation of alloimmune response as shown by markedly diminished division of CD4(+) T cells in a CFSE proliferation assay. Extrahepatic tumor regressed normally after liver transduction with AdCTLA-4exTAG. These results show efficient in vitro expression of CTLA-4exTAG after transfection with AdCTLA-4exTAG. The modified protein retains its ability to abrogate in vitro alloimmune response. Efficient control of extrahepatic tumor growth after liver-directed delivery of AdCTLA-4exTAG suggests that the immunosuppressive effect of this vector is restricted to the liver. These results set the ground for the utilization of this novel adenoviral vector in the transplant setting.

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.

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.

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 is highly efficient and prolongs cardiac allograft survival

Adenovirus-mediated gene transfer of CTLA4Ig is highly efficient and prolongs cardiac allograft survival

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

Adenovirus-mediated gene transfer of CTLA4Ig to liver allografts results in prolonged survival and local T-Cell anergy

Adenovirus-mediated gene transfer of CTLA4Ig to liver allografts results in prolonged survival and local T-Cell anergy

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