- Email: [email protected]
Gene Transfer of Antisense B7.1 Attenuates Acute Rejection Against Splenic Allografts in Rats
Gene Transfer of Antisense B7.1 Attenuates Acute Rejection Against Splenic Allografts in Rats C. Liu, S. Pan, H. Jiang, and X. Sun ABSTRACT Blockade of CD80-CD28 costimulatory pathway induces unresponsiveness of T cells to alloantigens and protects allografts against immune rejection in numerous animal models. The aim of this study was to investigate whether blocking expression of B7.1 (CD80) on donor splenocytes by an antisense technique protected splenic allografts against immune rejection. Splenic grafts from Wistar-Furth rats were intra-arterially transfused with an antisense B7.1 expression vector, before they were transplanted into Sprague-Dawley rats. The rats were sacrificed at scheduled times, and the splenic allografts histologically examined. Antisense gene transfer resulted in marked down-regulation of B7.1 in donor spleens, hyporesponsiveness of recipient T cells, and attenuated acute immune rejection against splenic allografts. No obvious damage to skin, liver, or gut due to graft-versus-host disease was detected in the recipients. In conclusion, blocking expression of B7.1 in donor spleens by antisense gene therapy represented a potential alloantigen-specific immunosuppressive strategy to inhibit acute rejection against splenic allografts.
S
PLEEN TRANSPLANTATION has been used to treat hemophilia since the first report by Hathaway in 19691 and subsequently applied in clinical centers.2 However, the main obstacle to its wide application is delayed dysfunction of grafted spleens due to immune rejection and administration of immunosuppressive drugs.3 Therefore, induction of tolerance to splenic allografts and antigen-specific immunosuppression are important goals in spleen transplantation to avoid the long-term adverse effects of nonspecific immunosuppressive agents. Costimulatory pathways between antigen-presenting cells (APCs) and T cells have been shown to play a crucial role in T-cell activation and immune responses.4 –7 Among these receptor-ligand pairs, CD80(B7.1)-CD28 is a major target to achieve antigen-specific immunosuppression in experimental organ transplantation.8 Blockade of the CD80CD28 signaling pathway by administration of soluble CTLA4Ig or anti-CD80 antibody induces a state of anergy in alloantigen-activated T cells,9 reduces vascular damage,10 and effectively prolongs organ allograft survivals in experimental animals.11–16 Therefore, blockade of costimulatory signals seems a feasible strategy to control allograft rejection and induce tolerance; however, these treatments require a substantial amount of agents for a period after organ grafting.16 –18 Gene therapy leads to sustained expression of transgenic proteins in situ by localized delivery, thus
representing a powerful strategy. It has been demonstrated that blockade of costimulatory pathway by a single administration of adenoviral vectors encoding CTLA4Ig allowed long-term liver allograft acceptance for more than 300 days in a high-responding ACI to Lewis rat strain combination.19,20 We have previously reported that gene transfer of an immunoregulatory cytokine, interleukin-4, delayed acute rejection to splenic allografts.21 The present study sought to investigate the benefit of gene transfer of an antisense expression vector to blockade B7.1 expression in the donor spleen. MATERIALS AND METHODS Rats Male Wistar-Furth rats (donors; 180 to 240 g) and male SpragueDawley rats (recipients; 200 to 260 g) were obtained from our From the Department of General Surgery (C.L.), the Fourth Affiliated Hospital, and the Hepatosplenic Surgery Center/ Department of General Surgery (S.P., H.J., X.S.), the First Clinical Medical School, Harbin Medical University, Harbin, China. Supported by grants from the National Natural Scientific Foundation of China (30471681, 30571808) and the Science and Technology Bureau of Heilongjiang Province, China (WH05C02). Address reprint requests to X. Sun, the Hepatosplenic Surgery Center, the First Clinical Medical School, Harbin Medical University, Harbin 15001, China. E-mail: [email protected]
© 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.08.094
Transplantation Proceedings, 39, 3391–3395 (2007)
3391
3392 Animal Research Center. The animals maintained under standard conditions were fed rodent chow and water. All surgical procedures and administered care were in accordance with institutional guidelines.
LIU, PAN, JIANG ET AL they were developed with FAST DAB (3,3=-diaminobenzidine tetrahydrochloride) and CoCl2 enhancer tablets (Sigma). Sections were counterstained, mounted, and examined by microscopy.
Western Blot Analysis Plasmid Construction We produced a 537 bp cDNA fragment encoding the C-terminus of rat B7.1. First, total RNA was extracted from the splenocytes of Wistar rats with TRIZOL (Takara Bio Inc, Japan) according to the manufacturer’s instruction, and reverse transcribed to generate cDNA. The cDNA was used as a template to perform polymerase chain reaction (PCR) with primers 5=-gctcgagctatggcttacagttgccagctg-3= and 5=-ggatccaagagaggcgaggctttgg-3=. The PCR product was subcloned into pGEMT (Promega Corporation), and then into pcDNA3 at XhoI and BamHI sites in an antisense orientation. The integrity of the final expression plasmid was confirmed by DNA sequence analysis.
The tissues were minced and homogenized in protein lysate buffer. Debris was removed by centrifugation at 10,000g for 10 minutes at 4°C. The lysates were resolved on 12% polyacrylamide sodium dodecylsulphate gels and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked with 3% BSA overnight, and then incubated with anti-B7.1 antibody and subsequently with alkaline phosphatase-conjugated secondary antibody. They were developed by 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Tiangen Biotech Co Ltd, Beijing, China). Blots were stained with an anti-tubulin antibody to confirm that each lane contained similar amounts of tumor homogenate.
Mixed Leukocyte Reaction Preparation of DNA/Liposome Complex The preparation of DNA/liposome has been described previously.21,22 Briefly, equal volumes of purified plasmids and Lipofect AMINE Regent (Invitrogen) were incubated and mixed at room temperature for 30 minutes before the mixture was dissolved in cold WMO-1 reserving solution (4°C) at a final concentration of 200 g/mL.
The methodology of mixed leukocyte reaction has been described previously.23
Statistical Methods Results were expressed as mean values ⫾ standard errors of mean (SEM), and a least significance difference test was used to evaluate statistical significance. P ⬍ 0.05 was considered significant.
Operation Procedures The methodology has been described previously.21 Briefly, the vascularized donor spleen with partial portal vein/splenic vein and partial abdominal aorta/splenic artery was excised after in situ perfusion with cold WMO-1 (4°C) solution. The donor spleens were randomly divided into two groups, where 1 mL DNA/ liposome solution containing antisense B7.1/pcDNA3 (n ⫽ 20) or empty pcDNA3 vector (n ⫽ 20) was transfused into the spleens through the splenic artery and left inside the spleen for 2 hour. Then the donor spleen was transplanted to the recipient by end-side anastomoses of the donor abdominal aorta to the recipient abdominal aorta and of the donor portal vein to the recipient inferior vena. The transplanted spleen was fixed in the right iliac fossa.
Histological Examination The splenic allografts were fixed in 10% buffered formalin, embedded in paraffin, stained with hematoxylin/eosin, and examined by light microscopy. Histopathologic scoring was assessed blindly by two independent pathologists to express the sum of the individual grades from 0 (no symptoms); 1 (mild); 2 (moderate); to 3 (severe) for each of the following five signs: mononuclear cell infiltration, dilation/destruction of periarterial lymphatic sheath (PALS), mural thrombosis in splenic arteries, increased splenocytes in white pulp, and fibroplastic proliferation. Biopsies of recipient skin, liver, and gut were also histologically examined as described above.
Immunohistochemical Analysis Spleen sections (5 m) were blocked with 2% bovine serum albumin (BSA), incubated overnight with anti-B7.1 antibody, and subsequently incubated for 30 minutes with appropriate secondary antibodies using the PowerVision Two-step Histostaining Reagent (Zhongshan Golden Bridge Biotechnology Co, Ltd, Beijing, China). Then
RESULTS Down-regulation of B7.1 Expression in situ by Antisense Gene Therapy
Donor spleens were perfused with a DNA/liposome transfection vehicle containing 200 g of antisense B7.1 expression plasmid DNA 2 hours before transplantation. Immunohistochemical analysis of splenic sections prepared 3 days after operation revealed antisense therapy resulted in inhibition of B7.1 expressed endogenously in splenocytes (Fig 1B) compared with controls (Fig 1A), as was confirmed by Western blot analysis (Fig 1C). Antisense Gene Therapy Attenuates Acute Immune Rejection Against Splenic Allografts and Splenic Allograft Does Not Induce Graft-Versus-Host Disease
To investigate whether the antisense B7.1 therapy inhibited immune rejection of allografted spleens, recipient rats were sacrificed on days 1, 3, 7, and 14 after transplantation (each time five rats were randomly killed). The excised spleens were histologically examined. The typical signs of acute rejection include mononuclear cell infiltration, increased splenocytes in white pulp, dilation or destruction of PALS, mural thrombosis in splenic arteries. Gene transfer of antisense B7.1 markedly attenuated the acute immune rejection. Representative photographs taken from splenic allografts treated with pcDNA3 (histological scores: 6), and antisense B7.1/pcDNA3 (histological scores: 3) at 7 days after transplantation are shown in Fig 2A and 2B, respectively. The mean histological scores of splenic allografts treated with antisense B7.1/pcDNA3 were significantly
GENE TRANSFER OF ANTISENSE B7.1
3393
Fig 1. Gene transfer of antisense B7.1/pcDNA3 down-regulates expression of B7.1 in splenocytes. Representative illustrations were taken from the splenic allografts preoperatively perfused with pcDNA3 (A) or antisense B7.1/ pcDNA3 (B), 3 days after transplantation by immunohistochemical analysis with an anti-B7.1 antibody. (C) The expression of B7.1 was confirmed by Western blot analysis with an anti-B7.1 antibody. Splenic homogenates were prepared from donor spleens perfused with pcDNA3 (lane 1) or antisense B7.1/pcDNA3 (lane 2). Blots were stained with an antitubulin antibody to confirm that each lane contained similar amounts of homogenate.
lower than those in controls at all indicated times (3, 7, and 14 days after transplantation; P ⬍ .01; Fig 2C). The skin, liver, and gut biopsies taken from recipients showed no obvious damage, indicating that after gene transfection, the splenic allografts did not induce acute graft-versus-host disease (GVHD).
Fig 2. Antisense gene transfer attenuates immune rejection against splenic allografts. Representative histological illustrations were taken from donor spleens perfused with pcDNA3 (A, histological score: 6) and antisense B7.1/ pcDNA3 (B, histological score: 3) 7 days after transplantation. (C) Histopathologic scoring of splenic allografts was assessed as described in Materials and Methods. (D) Mixed lymphocyte reaction in specific response to stimulator splenocytes. Data are represented by mean ⫾ SEM. Significant decrease from control treated spleens is denoted by asterisk (P ⬍ .01).
Antisense Gene Therapy Reduced Responsiveness of T Cells From Recipients
We next examined the responses of splenocytes from recipient rats to allogeneic splenocytes from donor rats. As shown in Fig 2D, the splenocytes from the rats that had been
3394
transplanted with antisense B7.1-treated donor spleens showed significantly weaker proliferation after stimulation by allogeneic splenocytes compared with controls (P ⬍ .01). DISCUSSION
The present study demonstrated that blockade of B7.1 expression by antisense gene delivery attenuated immune rejection against splenic allografts. The antisense gene therapy resulted in downregulated B7.1 expression in splenic allografts and hyporesponsiveness of recipient T cells. The spleen is the largest peripheral lymphoid organ, and the risk of rejection in splenic grafts is greater than other types of transplants. Immune rejection against splenic grafts and immunosuppressive agents against rejection damage lymphocytes in the spleens, contributing to delayed dysfunction of splenic allografts after long-term application24 or might also retard tolerance to allografts.25 Spontaneous tolerance of splenic allografts in inbred strains of rats have been reported by others.26,27 In those studies, different donor–recipient combinations revealed rejection of spleen “isografts” in Lewis rats.28 In the present study, we used Wistar and Sprague-Dawley rats, but we did not see spontaneous tolerance of splenic allografts, implying that tolerance or rejection of splenic allografts relies on specific combinations. Although outbred Sprague-Dawley rats are not an optimal model to study transplantation, the use of Wistar and Sprague-Dawley rats as donors and recipients seemed to be acceptable as they had been successfully applied in transplantation of various types of organs, including kidney,29 abdominal aorta,30 liver,31 skin,32 pancreas,33 intestine,34 and spleen.21 As the splenic allografts carry a large number of immune cells, we investigated whether spleen transplantation could lead to GVHD, which usually occurred in bone marrow and stem cell transplantations.35 Classically, acute GVHD is characterized by selective damage to liver, skin, and gut.36 However, we did not detect obvious damage to these three organs in the present study. The incidence of GVHD as a complication of solid organ transplantation varies by organ. The rates of GVHD range from 1% to 2% in small bowel and liver transplants,37 with far fewer cases reported in kidney and pancreas transplants.38 The rejection of allotransplanted organs is mediated predominantly by host T cells that are specifically activated by donor alloantigen.39 The first signal for T-cell activation is the interaction of alloantigen on the surface of APCs with the T-cell receptor. Full activation of T cells additionally requires CD80-CD28 interactions.40 The ischemia-reperfusion injury, a frequent process during solid organ transplantation, leads to enhanced expression of costimulatory molecules,41,42 which further enhances the immune response against allografts. CTLA4Ig and antibodies against costimulatory molecules have been used to block the interactions between costimulatory molecules and their ligands, thus inhibiting allograft rejection.8,12,15,16 The method in the present study that directly targeted the costimulatory
LIU, PAN, JIANG ET AL
molecule, B7.1, on the surface of donor splenocytes represented an alternative to achieve immune tolerance. Antisense technology has been increasingly recognized as a reliable tool to manipulate gene expression in the therapeutic area.43 It has been demonstrated that antisense oligonucleotides targeting costimulatory molecules, CD40, CD80, and CD86, produced immune suppression in allotransplantation and autoimmunity.44 Although the most efficient way to introduce transgenes into grafts is via recombinant viral vectors, their immunological response, safety issues, and toxicity preclude use in humans.45,46 Plasmid DNA with low immunogenicity does not in principle pose the health risks entailed by viral infection. It is easy to propagate on a large scale at high quality. The advantages of localized gene delivery are obvious, and the unique anatomic features of the spleen facilitate regional gene transfer. The method used in this study was modified based on the report by Dalesandro et al,22 where a complex of CAT reporter gene/liposome was perfused into a donor heart graft and preserved for 41 ⫾ 6 minutes. The transgenes were found to be expressed at 24 hours after transplantation. The method to deliver the antisense B7.1 expression vector by local perfusion is a more practical and safer approach with several advantages, such as gene modification to the grafts during cold preservation and avoidance of abnormal systemic expression in recipients.
REFERENCES 1. Xiang WZ, Jie ZW, Sheng XS: Clinical observation on hemophilia A treatment by cadaveric spleen transplantation. Transplant Proc 34:1929, 2002 2. Jiang HC, Zhu AL, Xu J, et al: Living related-donor spleen transplantation to treat 5 patients with hemophilia A. [Chinese] Chin J Hepatobil Surg 7:475, 2001 3. Chen Zi, Xia S, Liu C: The effects of different recipes of immunosuppressants on rejection and survival in splenic transplantation. [Chinese] Chinese J Hepatobil Surg 6:358, 2000 4. Clark LB, Foy TM, Noelle RJ: CD40 and its ligand. Adv Immunol 63:43, 1996 5. Lenschow DJ, Walunas TL, Bluestone JA: CD28/B7 system of T cell costimulation. Annu Rev Immunol 14:233, 1996 6. Carreno BM, Collins M: The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annu Rev Immunol 20:29, 2002 7. Frauwirth KA, Thompson CB: Activation and inhibition of lymphocytes by costimulation. J Clin Invest 109:295, 2002 8. Sayegh MH, Turka LA: The role of T-cell costimulatory activation pathways in transplant rejection. N Engl J Med 338:1813, 1998 9. Schwartz RH: A cell culture model for T lymphocyte clonal anergy. Science 248:1349, 1990 10. Rademacher J, Cansolino L, Lillo E, et al: Blockade of B7:CD28 costimulatory pathway reduces the vascular damage in an experimental model of chronic rejection. [Italian] Minerva Chir 60:487, 2005 11. Lin H, Bolling SF, Linsley PS, et al: Long-term acceptance of major histocompatibility complex mismatched cardiac allografts induced by CTLA4Ig plus donor-specific transfusion. J Exp Med 178:1801, 1993 12. Pearson TC, Alexander DZ, Winn KJ, et al: Transplantation tolerance induced by CTLA4-Ig. Transplantation 57:1701, 1994
GENE TRANSFER OF ANTISENSE B7.1 13. Larsen CP, Elwood ET, Alexander DZ, et al: Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381:434, 1996 14. Kirk AD, Tadaki DK, Celniker A, et al: Induction therapy with monoclonal antibodies specific for CD80 and CD86 delays the onset of acute renal allograft rejection in non-human primates. Transplantation 72:377, 2001 15. Li Y, Li XC, Zheng XX, et al: Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nat Med 5:1298, 1999 16. Kirk AD, Harlan DM, Armstrong NN, et al: CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci U S A 94:8789, 1997 17. Kenyon NS, Chatzipetrou M, Masetti M, et al: Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154. Proc Natl Acad Sci U S A 96:8132, 1999 18. Kirk AD, Burkly LC, Batty DS, et al: Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates. Nat Med 5:686, 1999 19. Yanagida N, Nomura M, Yamashita K, et al: Tolerance induction by a single donor pretreatment with the adenovirus vector encoding CTLA4Ig gene in rat orthotopic liver transplantation. Transplant Proc 33:573, 2001 20. Nomura M, Yamashita K, Murakami M, et al: Induction of donor-specific tolerance by adenovirus-mediated CD40Ig gene therapy in rat liver transplantation. Transplantation 73:1403, 2002 21. Jiang H, Liu C, Xu J, et al: Gene transfer of interleukin-4 delays acute rejection of splenic allografts in rats. Transplant Proc 36:1600, 2004 22. Dalesandro J, Akimoto H, Gorman CM, et al: Gene therapy for donor hearts: ex vivo liposome-mediated transfection. J Thorac Cardiovasc Surg 111:416, 1996 23. Jiang H, Hou L, Qiao H, et al: Administration of tolerogenic dendritic cells induced by interleukin- 10 prolongs splenic allograft survival. Transplant Proc 36:3255, 2004 24. Nemlander A, Soots A, Von Willebrand E, et al: Effect of cyclosporin A on the in situ inflammatory response of rat renal allograft rejection. Scand J Immunol 16:91, 1982 25. Jenkins MK, Schwartz RH, Pardoll DM: Effects of cyclosporine A on T cell development and clonal deletion. Science 241:1655, 1988 26. Stepkowski SM, Duncan WR, Smith JP, et al: Suppressor cells in the thoracic duct lymph of tolerant, spleen-grafted rats. Can J Surg 27:22, 1984 27. Duncan WR, Stepkowski SM, Bitter-Suermann H: Induction of transplantation tolerance in rats by spleen allografts. I. Evidence that rats tolerant of spleen allografts contain two phenotypically distinct T suppressor cells. Transplantation 41:626, 1986 28. Bitter-Suermann H, Lewis MG: Rejection of skin and spleen “isografts” in strain of Lewis rats. Transplantation 30:158, 1980 29. Teng D, Lu Y, Gao R, et al: Conversion from cyclosporine to mycophenolate mofetil improves expression of A20 in the rat kidney allografts undergoing chronic rejection. Transplant Proc 38:2164, 2006
3395 30. Gao C, Li Y: SDF-1 plays a key role in the repairing and remodeling process on rat allo-orthotopic abdominal aorta grafts. Transplant Proc 39:268, 2007 31. Wu SL, Yu L, Jiao XY, et al: The suppressive effect of resveratrol on protein kinase C theta in peripheral blood T lymphocytes in a rat liver transplantation model. Transplant Proc 38:3052, 2006 32. Diaz-Peromingo JA, Gonzalez-Quintela A: Influence of gadolinium-induced kupffer cell blockade on portal venous tolerance in rat skin allograft transplantation. Eur Surg Res 37:45, 2005 33. Ozden H, Kabay B, Guven G, et al: Interleukin-10 gene transfection of donor pancreas grafts protects against rejection after heterotopic pancreas transplantation in a rat model. Eur Surg Res 37:220, 2005 34. Zhang X, Li J, Li N: Growth hormone improves graft mucosal structure and recipient protein metabolism in rat small bowel transplantation. Chin Med J (Engl) 115:732, 2002 35. Aharoni R, Teitelbaum D, Arnon R, et al: Copolymer 1 inhibits manifestations of graft rejection. Transplantation 72:598, 2001 36. Bolaños-Meade J, Vogelsang GB: Acute graft-versus-host disease. Clin Adv Hematol Oncol 2:672, 2004 37. Key T, Taylor CJ, Bradley JA, et al: Recipients who receive a human leukocyte antigen-B compatible cadaveric liver allograft are at high risk of developing acute graft-versus-host disease. Transplantation 78:1809, 2004 38. Weinstein A, Dexter D, KuKuruga DL, et al: Acute graftversus-host disease in pancreas transplantation: a comparison of two case presentations and a review of the literature. Transplantation 82:127, 2006 39. Versluis DJ, Ten Kate FJW, Wenting GJ, et al: Mononuclear cells infiltrating kidney allografts in the absence of rejection. Transplant Int 1:205, 1988 40. Yamashita K, Masunaga T, Yanagida N, et al: Long-term acceptance of rat cardiac allografts on the basis of adenovirus mediated CD40Ig plus CTLA4Ig gene therapies. Transplantation 76:1089, 2003 41. Kojima N, Sato M, Suzuki A, et al: Enhanced expression of B7-1, B7-2, and intercellular adhesion molecule 1 in sinusoidal endothelial cells by warm ischemia/reperfusion injury in rat liver. Hepatology 34:751, 2001 42. Bartlett AS, McCall JL, Ameratunga R, et al: Analysis of intragraft gene and protein expression of the costimulatory molecules, CD80, CD86 and CD154, in orthotopic liver transplant recipients. Am J Transplant 3:1363, 2003 43. Giles R: Antisense oligonucleotide technology: from EST to therapeutics. Curr Opin Mol Ther 2:238, 2000 44. Machen J, Harnaha J, Lakomy R, et al: Antisense oligonucleotides down-regulating costimulation confer diabetes-preventive properties to nonobese diabetic mouse dendritic cells. J Immunology 173:4331, 2004 45. Zabner J, Ramsey BW, Meeker DP: Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis. J Clin Invest 97:1504, 1996 46. Fisher KJ, Choi H, Burda J: Recombinant adenovirus deleted of all viral genes for gene therapy of cystic fibrosis. Virology 217:11, 1996
S
PLEEN TRANSPLANTATION has been used to treat hemophilia since the first report by Hathaway in 19691 and subsequently applied in clinical centers.2 However, the main obstacle to its wide application is delayed dysfunction of grafted spleens due to immune rejection and administration of immunosuppressive drugs.3 Therefore, induction of tolerance to splenic allografts and antigen-specific immunosuppression are important goals in spleen transplantation to avoid the long-term adverse effects of nonspecific immunosuppressive agents. Costimulatory pathways between antigen-presenting cells (APCs) and T cells have been shown to play a crucial role in T-cell activation and immune responses.4 –7 Among these receptor-ligand pairs, CD80(B7.1)-CD28 is a major target to achieve antigen-specific immunosuppression in experimental organ transplantation.8 Blockade of the CD80CD28 signaling pathway by administration of soluble CTLA4Ig or anti-CD80 antibody induces a state of anergy in alloantigen-activated T cells,9 reduces vascular damage,10 and effectively prolongs organ allograft survivals in experimental animals.11–16 Therefore, blockade of costimulatory signals seems a feasible strategy to control allograft rejection and induce tolerance; however, these treatments require a substantial amount of agents for a period after organ grafting.16 –18 Gene therapy leads to sustained expression of transgenic proteins in situ by localized delivery, thus
representing a powerful strategy. It has been demonstrated that blockade of costimulatory pathway by a single administration of adenoviral vectors encoding CTLA4Ig allowed long-term liver allograft acceptance for more than 300 days in a high-responding ACI to Lewis rat strain combination.19,20 We have previously reported that gene transfer of an immunoregulatory cytokine, interleukin-4, delayed acute rejection to splenic allografts.21 The present study sought to investigate the benefit of gene transfer of an antisense expression vector to blockade B7.1 expression in the donor spleen. MATERIALS AND METHODS Rats Male Wistar-Furth rats (donors; 180 to 240 g) and male SpragueDawley rats (recipients; 200 to 260 g) were obtained from our From the Department of General Surgery (C.L.), the Fourth Affiliated Hospital, and the Hepatosplenic Surgery Center/ Department of General Surgery (S.P., H.J., X.S.), the First Clinical Medical School, Harbin Medical University, Harbin, China. Supported by grants from the National Natural Scientific Foundation of China (30471681, 30571808) and the Science and Technology Bureau of Heilongjiang Province, China (WH05C02). Address reprint requests to X. Sun, the Hepatosplenic Surgery Center, the First Clinical Medical School, Harbin Medical University, Harbin 15001, China. E-mail: [email protected]
© 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.08.094
Transplantation Proceedings, 39, 3391–3395 (2007)
3391
3392 Animal Research Center. The animals maintained under standard conditions were fed rodent chow and water. All surgical procedures and administered care were in accordance with institutional guidelines.
LIU, PAN, JIANG ET AL they were developed with FAST DAB (3,3=-diaminobenzidine tetrahydrochloride) and CoCl2 enhancer tablets (Sigma). Sections were counterstained, mounted, and examined by microscopy.
Western Blot Analysis Plasmid Construction We produced a 537 bp cDNA fragment encoding the C-terminus of rat B7.1. First, total RNA was extracted from the splenocytes of Wistar rats with TRIZOL (Takara Bio Inc, Japan) according to the manufacturer’s instruction, and reverse transcribed to generate cDNA. The cDNA was used as a template to perform polymerase chain reaction (PCR) with primers 5=-gctcgagctatggcttacagttgccagctg-3= and 5=-ggatccaagagaggcgaggctttgg-3=. The PCR product was subcloned into pGEMT (Promega Corporation), and then into pcDNA3 at XhoI and BamHI sites in an antisense orientation. The integrity of the final expression plasmid was confirmed by DNA sequence analysis.
The tissues were minced and homogenized in protein lysate buffer. Debris was removed by centrifugation at 10,000g for 10 minutes at 4°C. The lysates were resolved on 12% polyacrylamide sodium dodecylsulphate gels and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked with 3% BSA overnight, and then incubated with anti-B7.1 antibody and subsequently with alkaline phosphatase-conjugated secondary antibody. They were developed by 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Tiangen Biotech Co Ltd, Beijing, China). Blots were stained with an anti-tubulin antibody to confirm that each lane contained similar amounts of tumor homogenate.
Mixed Leukocyte Reaction Preparation of DNA/Liposome Complex The preparation of DNA/liposome has been described previously.21,22 Briefly, equal volumes of purified plasmids and Lipofect AMINE Regent (Invitrogen) were incubated and mixed at room temperature for 30 minutes before the mixture was dissolved in cold WMO-1 reserving solution (4°C) at a final concentration of 200 g/mL.
The methodology of mixed leukocyte reaction has been described previously.23
Statistical Methods Results were expressed as mean values ⫾ standard errors of mean (SEM), and a least significance difference test was used to evaluate statistical significance. P ⬍ 0.05 was considered significant.
Operation Procedures The methodology has been described previously.21 Briefly, the vascularized donor spleen with partial portal vein/splenic vein and partial abdominal aorta/splenic artery was excised after in situ perfusion with cold WMO-1 (4°C) solution. The donor spleens were randomly divided into two groups, where 1 mL DNA/ liposome solution containing antisense B7.1/pcDNA3 (n ⫽ 20) or empty pcDNA3 vector (n ⫽ 20) was transfused into the spleens through the splenic artery and left inside the spleen for 2 hour. Then the donor spleen was transplanted to the recipient by end-side anastomoses of the donor abdominal aorta to the recipient abdominal aorta and of the donor portal vein to the recipient inferior vena. The transplanted spleen was fixed in the right iliac fossa.
Histological Examination The splenic allografts were fixed in 10% buffered formalin, embedded in paraffin, stained with hematoxylin/eosin, and examined by light microscopy. Histopathologic scoring was assessed blindly by two independent pathologists to express the sum of the individual grades from 0 (no symptoms); 1 (mild); 2 (moderate); to 3 (severe) for each of the following five signs: mononuclear cell infiltration, dilation/destruction of periarterial lymphatic sheath (PALS), mural thrombosis in splenic arteries, increased splenocytes in white pulp, and fibroplastic proliferation. Biopsies of recipient skin, liver, and gut were also histologically examined as described above.
Immunohistochemical Analysis Spleen sections (5 m) were blocked with 2% bovine serum albumin (BSA), incubated overnight with anti-B7.1 antibody, and subsequently incubated for 30 minutes with appropriate secondary antibodies using the PowerVision Two-step Histostaining Reagent (Zhongshan Golden Bridge Biotechnology Co, Ltd, Beijing, China). Then
RESULTS Down-regulation of B7.1 Expression in situ by Antisense Gene Therapy
Donor spleens were perfused with a DNA/liposome transfection vehicle containing 200 g of antisense B7.1 expression plasmid DNA 2 hours before transplantation. Immunohistochemical analysis of splenic sections prepared 3 days after operation revealed antisense therapy resulted in inhibition of B7.1 expressed endogenously in splenocytes (Fig 1B) compared with controls (Fig 1A), as was confirmed by Western blot analysis (Fig 1C). Antisense Gene Therapy Attenuates Acute Immune Rejection Against Splenic Allografts and Splenic Allograft Does Not Induce Graft-Versus-Host Disease
To investigate whether the antisense B7.1 therapy inhibited immune rejection of allografted spleens, recipient rats were sacrificed on days 1, 3, 7, and 14 after transplantation (each time five rats were randomly killed). The excised spleens were histologically examined. The typical signs of acute rejection include mononuclear cell infiltration, increased splenocytes in white pulp, dilation or destruction of PALS, mural thrombosis in splenic arteries. Gene transfer of antisense B7.1 markedly attenuated the acute immune rejection. Representative photographs taken from splenic allografts treated with pcDNA3 (histological scores: 6), and antisense B7.1/pcDNA3 (histological scores: 3) at 7 days after transplantation are shown in Fig 2A and 2B, respectively. The mean histological scores of splenic allografts treated with antisense B7.1/pcDNA3 were significantly
GENE TRANSFER OF ANTISENSE B7.1
3393
Fig 1. Gene transfer of antisense B7.1/pcDNA3 down-regulates expression of B7.1 in splenocytes. Representative illustrations were taken from the splenic allografts preoperatively perfused with pcDNA3 (A) or antisense B7.1/ pcDNA3 (B), 3 days after transplantation by immunohistochemical analysis with an anti-B7.1 antibody. (C) The expression of B7.1 was confirmed by Western blot analysis with an anti-B7.1 antibody. Splenic homogenates were prepared from donor spleens perfused with pcDNA3 (lane 1) or antisense B7.1/pcDNA3 (lane 2). Blots were stained with an antitubulin antibody to confirm that each lane contained similar amounts of homogenate.
lower than those in controls at all indicated times (3, 7, and 14 days after transplantation; P ⬍ .01; Fig 2C). The skin, liver, and gut biopsies taken from recipients showed no obvious damage, indicating that after gene transfection, the splenic allografts did not induce acute graft-versus-host disease (GVHD).
Fig 2. Antisense gene transfer attenuates immune rejection against splenic allografts. Representative histological illustrations were taken from donor spleens perfused with pcDNA3 (A, histological score: 6) and antisense B7.1/ pcDNA3 (B, histological score: 3) 7 days after transplantation. (C) Histopathologic scoring of splenic allografts was assessed as described in Materials and Methods. (D) Mixed lymphocyte reaction in specific response to stimulator splenocytes. Data are represented by mean ⫾ SEM. Significant decrease from control treated spleens is denoted by asterisk (P ⬍ .01).
Antisense Gene Therapy Reduced Responsiveness of T Cells From Recipients
We next examined the responses of splenocytes from recipient rats to allogeneic splenocytes from donor rats. As shown in Fig 2D, the splenocytes from the rats that had been
3394
transplanted with antisense B7.1-treated donor spleens showed significantly weaker proliferation after stimulation by allogeneic splenocytes compared with controls (P ⬍ .01). DISCUSSION
The present study demonstrated that blockade of B7.1 expression by antisense gene delivery attenuated immune rejection against splenic allografts. The antisense gene therapy resulted in downregulated B7.1 expression in splenic allografts and hyporesponsiveness of recipient T cells. The spleen is the largest peripheral lymphoid organ, and the risk of rejection in splenic grafts is greater than other types of transplants. Immune rejection against splenic grafts and immunosuppressive agents against rejection damage lymphocytes in the spleens, contributing to delayed dysfunction of splenic allografts after long-term application24 or might also retard tolerance to allografts.25 Spontaneous tolerance of splenic allografts in inbred strains of rats have been reported by others.26,27 In those studies, different donor–recipient combinations revealed rejection of spleen “isografts” in Lewis rats.28 In the present study, we used Wistar and Sprague-Dawley rats, but we did not see spontaneous tolerance of splenic allografts, implying that tolerance or rejection of splenic allografts relies on specific combinations. Although outbred Sprague-Dawley rats are not an optimal model to study transplantation, the use of Wistar and Sprague-Dawley rats as donors and recipients seemed to be acceptable as they had been successfully applied in transplantation of various types of organs, including kidney,29 abdominal aorta,30 liver,31 skin,32 pancreas,33 intestine,34 and spleen.21 As the splenic allografts carry a large number of immune cells, we investigated whether spleen transplantation could lead to GVHD, which usually occurred in bone marrow and stem cell transplantations.35 Classically, acute GVHD is characterized by selective damage to liver, skin, and gut.36 However, we did not detect obvious damage to these three organs in the present study. The incidence of GVHD as a complication of solid organ transplantation varies by organ. The rates of GVHD range from 1% to 2% in small bowel and liver transplants,37 with far fewer cases reported in kidney and pancreas transplants.38 The rejection of allotransplanted organs is mediated predominantly by host T cells that are specifically activated by donor alloantigen.39 The first signal for T-cell activation is the interaction of alloantigen on the surface of APCs with the T-cell receptor. Full activation of T cells additionally requires CD80-CD28 interactions.40 The ischemia-reperfusion injury, a frequent process during solid organ transplantation, leads to enhanced expression of costimulatory molecules,41,42 which further enhances the immune response against allografts. CTLA4Ig and antibodies against costimulatory molecules have been used to block the interactions between costimulatory molecules and their ligands, thus inhibiting allograft rejection.8,12,15,16 The method in the present study that directly targeted the costimulatory
LIU, PAN, JIANG ET AL
molecule, B7.1, on the surface of donor splenocytes represented an alternative to achieve immune tolerance. Antisense technology has been increasingly recognized as a reliable tool to manipulate gene expression in the therapeutic area.43 It has been demonstrated that antisense oligonucleotides targeting costimulatory molecules, CD40, CD80, and CD86, produced immune suppression in allotransplantation and autoimmunity.44 Although the most efficient way to introduce transgenes into grafts is via recombinant viral vectors, their immunological response, safety issues, and toxicity preclude use in humans.45,46 Plasmid DNA with low immunogenicity does not in principle pose the health risks entailed by viral infection. It is easy to propagate on a large scale at high quality. The advantages of localized gene delivery are obvious, and the unique anatomic features of the spleen facilitate regional gene transfer. The method used in this study was modified based on the report by Dalesandro et al,22 where a complex of CAT reporter gene/liposome was perfused into a donor heart graft and preserved for 41 ⫾ 6 minutes. The transgenes were found to be expressed at 24 hours after transplantation. The method to deliver the antisense B7.1 expression vector by local perfusion is a more practical and safer approach with several advantages, such as gene modification to the grafts during cold preservation and avoidance of abnormal systemic expression in recipients.
REFERENCES 1. Xiang WZ, Jie ZW, Sheng XS: Clinical observation on hemophilia A treatment by cadaveric spleen transplantation. Transplant Proc 34:1929, 2002 2. Jiang HC, Zhu AL, Xu J, et al: Living related-donor spleen transplantation to treat 5 patients with hemophilia A. [Chinese] Chin J Hepatobil Surg 7:475, 2001 3. Chen Zi, Xia S, Liu C: The effects of different recipes of immunosuppressants on rejection and survival in splenic transplantation. [Chinese] Chinese J Hepatobil Surg 6:358, 2000 4. Clark LB, Foy TM, Noelle RJ: CD40 and its ligand. Adv Immunol 63:43, 1996 5. Lenschow DJ, Walunas TL, Bluestone JA: CD28/B7 system of T cell costimulation. Annu Rev Immunol 14:233, 1996 6. Carreno BM, Collins M: The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annu Rev Immunol 20:29, 2002 7. Frauwirth KA, Thompson CB: Activation and inhibition of lymphocytes by costimulation. J Clin Invest 109:295, 2002 8. Sayegh MH, Turka LA: The role of T-cell costimulatory activation pathways in transplant rejection. N Engl J Med 338:1813, 1998 9. Schwartz RH: A cell culture model for T lymphocyte clonal anergy. Science 248:1349, 1990 10. Rademacher J, Cansolino L, Lillo E, et al: Blockade of B7:CD28 costimulatory pathway reduces the vascular damage in an experimental model of chronic rejection. [Italian] Minerva Chir 60:487, 2005 11. Lin H, Bolling SF, Linsley PS, et al: Long-term acceptance of major histocompatibility complex mismatched cardiac allografts induced by CTLA4Ig plus donor-specific transfusion. J Exp Med 178:1801, 1993 12. Pearson TC, Alexander DZ, Winn KJ, et al: Transplantation tolerance induced by CTLA4-Ig. Transplantation 57:1701, 1994
GENE TRANSFER OF ANTISENSE B7.1 13. Larsen CP, Elwood ET, Alexander DZ, et al: Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381:434, 1996 14. Kirk AD, Tadaki DK, Celniker A, et al: Induction therapy with monoclonal antibodies specific for CD80 and CD86 delays the onset of acute renal allograft rejection in non-human primates. Transplantation 72:377, 2001 15. Li Y, Li XC, Zheng XX, et al: Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nat Med 5:1298, 1999 16. Kirk AD, Harlan DM, Armstrong NN, et al: CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci U S A 94:8789, 1997 17. Kenyon NS, Chatzipetrou M, Masetti M, et al: Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154. Proc Natl Acad Sci U S A 96:8132, 1999 18. Kirk AD, Burkly LC, Batty DS, et al: Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates. Nat Med 5:686, 1999 19. Yanagida N, Nomura M, Yamashita K, et al: Tolerance induction by a single donor pretreatment with the adenovirus vector encoding CTLA4Ig gene in rat orthotopic liver transplantation. Transplant Proc 33:573, 2001 20. Nomura M, Yamashita K, Murakami M, et al: Induction of donor-specific tolerance by adenovirus-mediated CD40Ig gene therapy in rat liver transplantation. Transplantation 73:1403, 2002 21. Jiang H, Liu C, Xu J, et al: Gene transfer of interleukin-4 delays acute rejection of splenic allografts in rats. Transplant Proc 36:1600, 2004 22. Dalesandro J, Akimoto H, Gorman CM, et al: Gene therapy for donor hearts: ex vivo liposome-mediated transfection. J Thorac Cardiovasc Surg 111:416, 1996 23. Jiang H, Hou L, Qiao H, et al: Administration of tolerogenic dendritic cells induced by interleukin- 10 prolongs splenic allograft survival. Transplant Proc 36:3255, 2004 24. Nemlander A, Soots A, Von Willebrand E, et al: Effect of cyclosporin A on the in situ inflammatory response of rat renal allograft rejection. Scand J Immunol 16:91, 1982 25. Jenkins MK, Schwartz RH, Pardoll DM: Effects of cyclosporine A on T cell development and clonal deletion. Science 241:1655, 1988 26. Stepkowski SM, Duncan WR, Smith JP, et al: Suppressor cells in the thoracic duct lymph of tolerant, spleen-grafted rats. Can J Surg 27:22, 1984 27. Duncan WR, Stepkowski SM, Bitter-Suermann H: Induction of transplantation tolerance in rats by spleen allografts. I. Evidence that rats tolerant of spleen allografts contain two phenotypically distinct T suppressor cells. Transplantation 41:626, 1986 28. Bitter-Suermann H, Lewis MG: Rejection of skin and spleen “isografts” in strain of Lewis rats. Transplantation 30:158, 1980 29. Teng D, Lu Y, Gao R, et al: Conversion from cyclosporine to mycophenolate mofetil improves expression of A20 in the rat kidney allografts undergoing chronic rejection. Transplant Proc 38:2164, 2006
3395 30. Gao C, Li Y: SDF-1 plays a key role in the repairing and remodeling process on rat allo-orthotopic abdominal aorta grafts. Transplant Proc 39:268, 2007 31. Wu SL, Yu L, Jiao XY, et al: The suppressive effect of resveratrol on protein kinase C theta in peripheral blood T lymphocytes in a rat liver transplantation model. Transplant Proc 38:3052, 2006 32. Diaz-Peromingo JA, Gonzalez-Quintela A: Influence of gadolinium-induced kupffer cell blockade on portal venous tolerance in rat skin allograft transplantation. Eur Surg Res 37:45, 2005 33. Ozden H, Kabay B, Guven G, et al: Interleukin-10 gene transfection of donor pancreas grafts protects against rejection after heterotopic pancreas transplantation in a rat model. Eur Surg Res 37:220, 2005 34. Zhang X, Li J, Li N: Growth hormone improves graft mucosal structure and recipient protein metabolism in rat small bowel transplantation. Chin Med J (Engl) 115:732, 2002 35. Aharoni R, Teitelbaum D, Arnon R, et al: Copolymer 1 inhibits manifestations of graft rejection. Transplantation 72:598, 2001 36. Bolaños-Meade J, Vogelsang GB: Acute graft-versus-host disease. Clin Adv Hematol Oncol 2:672, 2004 37. Key T, Taylor CJ, Bradley JA, et al: Recipients who receive a human leukocyte antigen-B compatible cadaveric liver allograft are at high risk of developing acute graft-versus-host disease. Transplantation 78:1809, 2004 38. Weinstein A, Dexter D, KuKuruga DL, et al: Acute graftversus-host disease in pancreas transplantation: a comparison of two case presentations and a review of the literature. Transplantation 82:127, 2006 39. Versluis DJ, Ten Kate FJW, Wenting GJ, et al: Mononuclear cells infiltrating kidney allografts in the absence of rejection. Transplant Int 1:205, 1988 40. Yamashita K, Masunaga T, Yanagida N, et al: Long-term acceptance of rat cardiac allografts on the basis of adenovirus mediated CD40Ig plus CTLA4Ig gene therapies. Transplantation 76:1089, 2003 41. Kojima N, Sato M, Suzuki A, et al: Enhanced expression of B7-1, B7-2, and intercellular adhesion molecule 1 in sinusoidal endothelial cells by warm ischemia/reperfusion injury in rat liver. Hepatology 34:751, 2001 42. Bartlett AS, McCall JL, Ameratunga R, et al: Analysis of intragraft gene and protein expression of the costimulatory molecules, CD80, CD86 and CD154, in orthotopic liver transplant recipients. Am J Transplant 3:1363, 2003 43. Giles R: Antisense oligonucleotide technology: from EST to therapeutics. Curr Opin Mol Ther 2:238, 2000 44. Machen J, Harnaha J, Lakomy R, et al: Antisense oligonucleotides down-regulating costimulation confer diabetes-preventive properties to nonobese diabetic mouse dendritic cells. J Immunology 173:4331, 2004 45. Zabner J, Ramsey BW, Meeker DP: Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis. J Clin Invest 97:1504, 1996 46. Fisher KJ, Choi H, Burda J: Recombinant adenovirus deleted of all viral genes for gene therapy of cystic fibrosis. Virology 217:11, 1996