Effect of combined OX40Ig and CTLA4Ig gene local transfer on allograft rejection and the underlying mechanisms

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journal homepage: www.JournalofSurgicalResearch.com

Effect of combined OX40Ig and CTLA4Ig gene local transfer on allograft rejection and the underlying mechanisms Jin Zhang, MD,a,1 Qing Miao, MD,b,1 Yang Yang, MD,a Bo Xiao, MD,a Bei Liu, MD,a Jiao Cao, MD,a Xiao-Yan Hao, MD,a Si-Wang Wang, MD,b,** and Shu-Zhong Guo, MDa,* a b

Department of Plastic Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, PR China Institute of Materia Medica, Fourth Military Medical University, Xi’an, PR China

article info

abstract

Article history:

Background: OX40Ig and CTLA4Ig fusion proteins have been suggested to induce immune

Received 12 March 2012

tolerance and prevent rejection in allografts. The present study aims to investigate and

Received in revised form

compare the effects of ex vivo combined OX40Ig and CTLA4Ig lentivirus-mediated

26 April 2012

gene transfer on the long-term survival of the graft, as well as potential underlying

Accepted 9 May 2012

mechanisms.

Available online 31 May 2012

Methods: We ex vivo transferred Brown Norway rats’ superficial groin free flap with lentivirus vectors expressing OX40Ig or CTLA4Ig, or OX40Ig and CTLA4Ig combined, and

Keywords:

transplanted the free flaps to Lewis rats. Short-course rapamycin was administered after

Allotransplantation

transfection and transplantation. RT-PCR and Western blot were employed to evaluate

Gene therapy

expression of OX40Ig and CTLA4Ig. We assessed the survival time of the grafts and the

Co-stimulatory signal

degree of acute graft rejection after indicated treatment. Mixed lymphocyte reaction, flow

OX40Ig

cytometry, and ELISA were also used to evaluate systemic immune reactions.

CTLA4Ig

Results: Ex vivo transfer of OX40Ig or CTLA4Ig lentivirus vectors led to local expression of corresponding mRNA and proteins in the donor flap without affecting other organs of the recipient. The graft survival time was significantly expanded and rejection was markedly attenuated after transfection. Mixed lymphocyte reaction, flow cytometry (CD4þ and CD8þ T lymphocyte proportions), and serum ELISA analysis (IL-2, IFN-g, IL-4, and IL-10) also showed decreased immune response following transfection. Combined OX40Ig and CTLA4Ig transfer exerted superior effect on improving graft survival and preventing graft rejection, inhibiting the immune response and decreasing the production of proinflammatory cytokines, compared with singular transfer of either OX40Ig or CTLA4Ig. Conclusion: Combined ex vivo transfer of OX40Ig and CTLA4Ig lentivirus vectors provided superior benefits on long-term survival and restoration of the graft through inhibiting immune response and decreasing the production of proinflammatory cytokines. Crown Copyright ª 2012 Published by Elsevier Inc. All rights reserved.

* Corresponding author. Department of Plastic Surgery, Xijing Hospital, Fourth Military Medical University, #17 West Changle Road, 710032 Xi’an, PR China. Tel./fax: þ86 29 84775301. ** Corresponding author. Institute of Materia Medica, Fourth Military Medical University, #17 West Changle Road, 710032 Xi’an, PR China. E-mail addresses: [email protected] (S.-W. Wang), [email protected] (S.-Z. Guo). 1 These authors contributed equally to this work. 0022-4804/$ e see front matter Crown Copyright ª 2012 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.jss.2012.05.034

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Introduction

Allofacial tissue transplantation has raised great interest due to the rapid increase in the incidence of facial trauma, neoplasm resection, congenital malformation, and injuries. With progress in composite tissue allotransplantation, there comes new hope for patients with these severe disfigurements. Since the year 2005, we and others have successfully reported several pioneering clinical practices in human facial transplantation. To the best of the authors’ knowledge, there are a total of 18 patients who have received partial composite facial transplantation, with promising results [1e8]. It is of special interest to note a research team from Brigham and Women’s Hospital who has very recently shed light on the breakthrough of full facial transplantation and graft functional restoration in three individuals [9]. Although the facial transplantation technique is complicated and formidable, the real dilemma of allofacial transplantation lies in the big shortage of appropriate healthy donors. Of equal importance, acute and chronic rejections are the major risks of mortality after facial transplantation. Reported allogeneic facial transplantation cases all develop acute rejection after the operation, especially our recipient, with three acute rejections before death [2,4]. Prolonged systemic administration of high doses of corticosteroids, tacrolimus, mycophenolate mofetil, or antilymphocyte serum and extracorporeal photopheresis could reverse the rejections, while it may lead to corresponding deleterious complications, such as opportunistic infections, organ impairments, and malignancies [10]. Therefore, it is urgent to establish alternative adjuvant therapeutic strategies that could effectively attenuate acute and chronic infection, preserve graft functions, and minimize potential adverse effects. Local gene therapy has the potential to prevent graft rejection by manipulating the immune response in the microenvironment. The therapeutic efficacy of local gene transduction is high due to its high bioavailability and minimal toxicity. Extensive studies have well documented that activated T lymphocyte is the predominant immune cell that mediates acute and chronic composite tissue allograft rejection. Blockage of co-stimulatory signals suppresses T-lymphocyte activation and induces antigen-specific tolerance. Several co-stimulatory signal-based agents have become commercially available, such as abatacept and belatacept [11]. OX40 (CD137) and cytotoxic T-lymphocyte antigen 4 (CTLA4) are the most important molecules participating in the process of T-lymphocyte co-stimulation. Blockage of OX40- and CTLA4-mediated costimulatory signals provides promising intervention targets against rejection after allotransplantation [12e14]. Their fusion proteins with immunoglobulin (OX40Ig and CTLA4Ig) have been well described to transmit inhibitory signals to reduce immune reactivity and inhibit allograft rejection in various experimental transplantations [15e17]. Our recent studies on allogeneic skin flap transplantation in rats suggest that local inhibition of either OX40 or CTLA4 by ex vivo transfer of OX40Ig or CTLA4Ig gene recombinant adenovirus vectors combined with short-course rapamycin treatment promotes long-term survival of the graft [18,19]. The primary aim of the present study is to investigate whether combined ex vivo perfusion of lentiviral-mediated

OX40Ig- and CTLA4Ig-expressing vectors induces superior long-term survival of composite tissue allografts. Potential mechanisms of action for OX40Ig and CTLA4Ig were also assessed in the present study.

2.

Materials and methods

2.1.

Animals

Adult male Brown Norway (BN, RT.1n) and Lewis (RT.11) rats weighing 250e300 g were purchased from Vital River Inc, Beijing, China. This study conformed to the Guidelines for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No. 85-23, revised 1985). Animal experiments were also performed with permission from Fourth Military Medical University authorities. All rats were exposed to a 12-h light-dark cycle and provided rat chow and water ad libitum.

2.2.

Lentivirus vector recombination

The gene sequences encoding rat OX40 and CTLA4 were retrieved in GenBank and were synthesized and reconstructed with IgG Fc fragments. These recombinants were subsequently constructed with pCDH1-MCS1-EF1-copGFP (pCDH1) lentivirus vectors using PCDH Cdna cloning and Expression Lentivectors System according to the instructions from the manufacturer (System Bioscience Inc, Tianjin, China). pCDH1OX40Ig and pCDH1-CTLA4Ig lentivirus-expressing vectors purified using cesium chloride gradient ultracentrifugation were titered using an HEK293 plaque assay. Titers of virus are given in plaque-forming units. All these experiments were performed in biosafety level 2 facilities.

2.3.

Surgical techniques

The superficial groin free flaps removed from donor Brown Norway rats were transplanted into recipient Lewis rats. The surgical procedure and viral ex vivo infection methods were performed according to previous studies [20,21]. Briefly, rats were anesthetized, and the groin and leg areas were prepared and draped in a sterile fashion. After the flaps were harvested, a 0.2-mm infusion catheter was placed in the artery and secured with 8-0 nylon sutures. After cannulation of the afferent artery, the cannula was connected to a perfusion pump (Veryark, Nanning, China). The flap bed was first perfused with 5 mL of phosphate-buffered saline (PBS) within 5 min through the artery while leaving the vein unclamped; 1 mL of lentivirus was then perfused through the artery at the speed of 1 mL/h while keeping the vein clamped. The perfusion time was 1 h; the incubation time was also 1 h. The flap was then flushed with 2 mL of PBS to wash away the unincorporated virions, and the native vessels were reanastomosed using interrupted 11-0 nylon sutures. All of these manipulations were done at room temperature (20 C). The whole ischemic time was 3 h. The operative area was securely dressed, and the rat was placed in a biohazard level 2 animal

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care room. The allografts were evaluated daily for evidence of rejection. The endpoint of the study was animal death.

2.4.

Table 1 e Specific primer sequences for OX40Ig, CTLA4Ig, and b-actin. Primer

Experimental protocols

OX40Ig

Lewis recipients were classified into six groups (Fig. 1): group I, no treatment (control group); group II, 1  108 pCDH1-EGFP vector ex vivo transfer (mock group); group III, 2 mg/kg rapamycin (rapamycin group); group IV, 1  108 pCDH1-OX40Ig vector ex vivo transfer and 2 mg/kg rapamycin (OX40Ig group); group V, 1  108 pCDH1-CTLA4Ig vector ex vivo transfer and 2 mg/kg rapamycin (CTLA4Ig group); group VI, 1  108 pCDH1OX40Ig vector in combination with 1  108 pCDH1-CTLA4Ig vector ex vivo transfer and 2 mg/kg rapamycin (combined group). Rapamycin (Sigma, St. Louis, MO) was intraperitoneally injected daily at a dose of 2 mg/kg from day 0 to day 7 after flap allotransplantation.

2.5. Expressions of OX40Ig and CTLA4Ig in graft and internal organs To determine whether ex vivo transfer with OX40Ig and CTLA4Ig lentivirus vectors induces OX40Ig and CTLA4Ig expression, an immunofluorescent assay of the immobilized donor flaps was performed on day 7 after the operation. The allograft flap samples were fixed in 4% paraformaldehyde and incubated with rabbit anti-OX40 and mouse anti-CTLA4 antibodies followed by corresponding secondary antibodies, respectively. To test whether the induced OX40Ig and CTLA4Ig expressions were systemic or local, samples from the recipients’ liver, spleen, and the transplanted flap were subjected to reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. The specific primers for OX40Ig and CTLA4Ig are listed in Table 1. b-actin was used for equal

control mock rapamycin OX40Ig CTLA4Ig combined days 0

7

Fig. 1 e Treatment and experimental groups. Control group: no treatment; Mock group: pCDH1-EGFP vector ex vivo transfer; Rapamycin group: 2 mg/kg rapamycin treatment; OX40Ig group: pCDH1-OX40Ig vector ex vivo transfer and 2 mg/kg rapamycin treatment; CTLA4Ig group: pCDH1-CTLA4Ig vector ex vivo transfer and 2 mg/kg rapamycin treatment; Combined group: pCDH1-OX40Ig and pCDH1-CTLA4Ig vectors ex vivo transfer together with 2 mg/kg rapamycin treatment. Rapamycin was intraperitoneally injected daily at a dose of 2 mg/kg from day 0 to day 7 after flap allotransplantation.

CTLA4Ig

b-actin

Sequence forward: 5ʹ-GGTTGGGTGCCTGGTCTA-3ʹ reverse: 5ʹ-TATGGTGAGCCGCTGTGA-3ʹ forward: 5ʹTTACTCTACTCCCTGAGGAGCTCAGCACATTTGCC-3ʹ reverse: 5ʹACTAGGTCTTGGTACGGGCCTAAGACCATTCATAT-3ʹ forward: 5ʹATCTGGCACCACACCTTCTACAATGAGCTGCG-3ʹ reverse: 5ʹCGTCATACTCCTGCTTGCTGATCCACATCTGC-3ʹ

loading. PCR reactions were carried out with 1 unit of Taq polymerase in a total volume of 50 mL, and amplifications were carried out in a Peltier thermal cycler from MJ Research (MJ Research, Inc., South San Francisco, CA). OX40Ig and CTLA4Ig amplifications were done for 35 cycles (95 C for 10 s, 60 C for 20 s, and 72 C for 15 s, followed by extension at 72 C for 10 min) and b-actin amplification for 20 cycles (94 C for 35 s, 58 C for 35 s, and 72 C for 40 s, followed by extension at 72 C for 10 min). PCR products were analyzed on a 2% agarose gel and visualized by ethidium bromide staining. Western blot analysis for OX40Ig and CTLA4Ig proteins were performed as we described previously [18,19].

2.6.

Graft histology

On day 7 and day 28 after transplantation, full-thickness flap tissue sections were removed and fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin wax. Sections of 5 mm were cut and stained with hematoxylin-eosin for evaluation under light microscopy. The histologic changes in each tissue were assessed based on the Banff 2007 working classification of skin-containing composite tissue allograft pathology [22]. Accordingly, the degree of acute rejection of skin was generally divided into five grades: grade 0, no or rare inflammatory infiltrates; grade I, mild perivascular infiltration, with no involvement of the overlying epidermis; grade II, moderate to severe perivascular inflammation with or without mild epidermal or adnexal involvement (limited to spongiosis and exocytosis), and no epidermal dyskeratosis or apoptosis; grade III, dense inflammation and epidermal involvement with epithelial apoptosis, and dyskeratosis or keratinolysis; and grade IV, necrotizing acute rejection, with frank necrosis of epidermis or other skin structures. An independent, masked observer evaluated the degree of rejection.

2.7.

Mixed lymphocyte reaction

Mixed lymphocyte reaction (MLR) was performed as we described previously [18,19,21]. Lymphocytes were isolated from spleens in each group. A total of 1  106 lymphocytes from the recipients were mixed with 1  106 lymphocytes from allogeneic Brown Norway and Sprague-Dawley rats that had been irradiated (4000 rad) in vitro and cultured in tissue culture plates in 2 mL of culture medium. After 6 d, 200 mL of

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each tube was transferred in triplicate in 96-well, roundbottomed plates and pulsed for 18 h with 3H-thymidine (1 mCi/well; Atomic Energy Research Establishment, Beijing, China). Cells were then harvested, and their 3H-thymidine uptake was measured with a b counter. In each experiment, thymidine uptake was measured with control combinations of Lewis lymphocytes and irradiated Lewis spleen cells. The stimulation index was calculated as follows: stimulation index ¼ counts per minute in allogeneic mixed lymphocyte reaction/counts per minute in control combination.

2.8.

Flow cytometry analysis

On day 7 after transplantation, spleens from each group were harvested. Single cell suspensions were prepared by grinding the tissues with the plunger in a 5-mL disposable syringe and then suspended in RPMI 1640 medium. Splenocytes were treated with a hemolysis buffer to remove red blood cells. Splenocytes were incubated with FITC-labeled anti-CD4 (BD Biosciences, Franklin Lakes, NJ) or PE-labeled anti-CD8 (BD Biosciences) monoclonal antibodies for 30 min at 4 C in the dark. Cells were then washed three times in PBS and resuspended in flow cytometry buffer. A total of 106 cells were assayed using FACSCalibur flow cytometry (BD Biosciences).

2.9.

ELISA assays

On day 7 after transplantation, serum concentrations of IL-2, IFN-g, IL-4, and IL-10 were measured by means of enzymelinked immunosorbent assay (ELISA) with Quantikine Rat immunoassay kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.

2.10.

Statistical analysis

Statistical analysis was performed with SPSS Statistical Program version 13.0 (SPSS Science, Chicago, IL). Data were expressed as mean  standard error of mean. Graft survival was compared between different groups using Kaplan-Meier analysis and the log-rank test. Other data were analyzed using 1-way ANOVA with post hoc comparisons. Differences were considered significant at P < 0.05.

3.

Results

3.1. Combined OX40Ig and CTLA4Ig therapy significantly prolongs graft survival The survival data for all groups are depicted in Fig. 2. The survival time in the mock group was not significantly different from that in the control group (P > 0.05). Thus, the lentivirus perfusion itself had no influence on graft survival. Rapamycin singular therapy prolonged graft survival compared with the control group (P < 0.05) (Fig. 2A). Singular perfusion of OX40Ig or CTLA4Ig lentivirus vector together with short-course rapamycin treatment significantly prolonged graft survival compared with the mock group (P < 0.05). Additionally, OX40Ig and CTLA4Ig in combination therapy expanded the survival

Fig. 2 e Survival data for control (A) (n [ 7), mock (n [ 6), rapamycin (n [ 6), and (B) OX40Ig (n [ 6), CTLA4Ig (n [ 5), and combined (n [ 5) groups. OX40Ig or CTLA4Ig transfer significantly prolonged the graft survival compared with the baseline. Combined OX40Ig and CTLA4Ig transfer led to superior survival time (65 d) compared with the OX40Ig (39 d) and CTLA4Ig (45 d) singular transfer group, respectively. (Color version of figure is available online.)

time to 65 d compared with 39 d in the OX40Ig (P < 0.05) and 45 d in the CTLA4Ig (P < 0.05) singular therapy group, respectively (Fig. 2B).

3.2.

OX40Ig and CTLA4Ig are locally expressed

On day 7 following the allotransplantation, immunofluorescent staining revealed no detectable expression of OX40Ig or CTLA4Ig in the control and mock groups. In the combinedtreated group, OX40Ig and CTLA4Ig fluorescence could be observed in the transplanted flaps, particularly adjacent to the microvessels (Fig. 3A). RT-PCR and Western blot analysis also indicated OX40Ig and CTLA4Ig mRNA and protein are expressed in the flap graft. In contrast, neither OX40Ig nor CTLA4Ig can be detected in the recipients’ livers and spleens in the combined-treated group (Fig. 3B).

3.3.

OX40Ig and CTLA4Ig treatment inhibits rejection

Full-thickness biopsy specimens were harvested from the allografts for histopathologic evaluation on postoperative

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Fig. 3 e Expression of OX40Ig and CTLA4Ig in the donor flap and the recipient liver and spleen after ex vivo transduction. Immunofluorescent staining, (A) RT-PCR, and Western blot analysis (B) indicated OX40Ig and CTLA4Ig were locally expressed in the transplanted flaps. No detectable OX40Ig or CTLA4Ig was observed in the recipients’ livers and spleens. (Color version of figure is available online.)

days 7 and 28. On day 7, the skin demonstrated an extensive, massive perivascular inflammatory infiltration in the dermis and epidermis in the control and mock groups. These samples were all consistent with Banff grade III or IV rejection (Fig. 4A, B, a, and b). Rapamycin singular used as an immunosuppressant partly relieved the rejection (Fig. 4C and c). In other groups, the flap grafts had significantly less damage and exhibited no macroscopic evidence of rejection. Major histologic changes were mild inflammatory infiltration in the dermis (Fig. 4DeF and def). On day 28 after transplantation, grafts in the control, mock, and rapamycin groups died out, while histologic examination of the biopsy specimen in the OX40Ig (Fig. 5A and a), CTLA4Ig (Fig. 5B and b), and combined (Fig. 5C and c) groups still revealed a viable flap with mild inflammatory infiltration. The graft was almost intact, especially in the combined-treated group.

3.4.

Effects of OX40Ig and CTLA4Ig on MLR assay

To determine whether OX40Ig and CTLA4Ig lentivirus vector transfer induces systemic unresponsiveness, the functional

status of T cells was evaluated by MLR in vitro. The stimulation index of Lewis rats receiving rapamycin was 8.78  1.15, which was significantly lower compared with control group animals (16.31  3.62) (Fig. 6). Splenocytes from the OX40Ig, CTLA4Ig, and combined perfused Lewis rats responded poorly to the splenocytes of Brown Norway donors, with stimulation indexes of 7.95  1.32, 4.75  0.92, and 3.12  0.89, respectively (P < 0.05 versus control). The stimulation index of cells taken from each group in response to Sprague-Dawley third-party stimulator cells was not statistically significant (P > 0.05).

3.5. OX40Ig and CTLA4Ig lentivirus vector transfer decreases CD4þ cell proportions and CD4/CD8 ratio Rat spleens were harvested and examined for CD4þ and CD8þ phenotypes by flow cytometry on day 7 posttransplantation (Fig. 7). On gross examination, spleens from the combined group were much smaller than the others. Flow cytometry analysis showed single therapy with rapamycin did not change CD4þ cell proportion while OX40Ig or CTLA4Ig

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Fig. 4 e Gross (AeF) and corresponding histologic (aef) examinations of the graft on day 7 following allotransplantation. The skin demonstrated an extensive, massive perivascular inflammatory infiltration in the dermis and epidermis in the control and mock groups. In the transfected groups, the flap grafts had significantly less damage and exhibited no macroscopic evidence of rejection. (Color version of figure is available online.)

treatments significantly decreased the percentage of CD4þ cells in the spleen. The combined treatment group demonstrated the greatest reduction of CD4þ cells (48% reduction versus control). There was no significant difference in CD8þ

cells between rats receiving OX40Ig or CTLA4Ig perfusion compared with controls. Likewise, OX40Ig or CTLA4Ig treatments decreased the CD4/CD8 ratio and combined treatment demonstrated the greatest reduction.

Fig. 5 e Gross (AeC) and corresponding histologic (aec) examinations of the graft on day 28 following allotransplantation. Grafts in the control, mock, and rapamycin groups died out; histologic examination of the biopsy specimen in the OX40Ig, CTLA4Ig, and combined groups still revealed a viable flap with mild inflammatory infiltration. (Color version of figure is available online.)

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Fig. 6 e Functional status of T cells evaluated by mixed lymphocyte reaction in vitro. Lymphocytes were isolated from spleens of Lewis rats and mixed with irradiated allogeneic spleen cells from Brown Norway and SpragueDawley rats. Stimulation indexes of rats receiving rapamycin, OX40Ig, CTLA4Ig, and combined treatments were significantly reduced compared to those of control and mock groups *P<0.05 verses control.

3.6.

OX40Ig and CTLA4Ig affect Th cell cytokines

Immune-related cytokines reflect the situation of immune rejection and play important roles in regulating immune response. Using ELISA assays, the serum concentrations of Th1 cytokines (IL-2 and IFN-g) and Th2 cytokines (IL-4 and IL-10) from the recipients, either untreated; treated with rapamycin, OX40Ig, or CTLA4Ig; or treated with some combination of those were measured on day 7 after transplantation. Treatment with rapamycin alone diminished the IL-2 and IFN-g by 28.4% and 22.3%, but a greater reduction was achieved by combination with OX40Ig (48.6% and 56.7% reduction) or CTLA4Ig (55.6% and 58.2% reduction). Additionally, triple combination with rapamycin, OX40Ig, and CTLA4Ig treatment produced a higher-magnitude reduction (77.8% for IL-2 and 86.1% for IFN-g). For IL-4 and IL-10, rapamycin singular use caused 40.1% and 18.75% increase, respectively. Either OX40Ig or CTLA4Ig perfusion also increased serum IL-4 and IL-10 contents. The greatest increase was achieved by triple rapamycin, OX40Ig, and CTLA4Ig treatment (102.4% for IL-4 and 128.6% for IL-10) (Fig. 8A and B).

4.

Discussion

Numerous preclinical investigations and clinical trials suggest the OX40- and CTLA4-based target regimen acts as a potent therapeutic strategy against rejection after allotransplantation [23e26]. Fusion proteins of OX40Ig and CTLA4Ig have been described to suppress the co-stimulatory signals and induce systemic immune tolerance in various allograft settings. There are preliminary studies showing that systemic administration of trimeric OX40 fusion protein (a product of OX40Ig) or belatacept (a product of CTLA4Ig) might cause hypophosphatemia, diarrhea, and cough in at least 10% of volunteers. Therefore

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local OX40- and CTLA4-based target therapy exerts great potential to overcome rejection while minimizing undesirable side effects. Local OX40Ig or CTLA4Ig gene therapy in the microenvironment provides potential approaches to preventing graft rejection. Inducible expression of OX40Ig and CTLA4Ig by transduction of relevant vectors has been reported to trigger immunological unresponsiveness to allogeneic antigens. Our recent studies suggested local expression of OX40Ig or CTLA4Ig at the site of the flap by an ex vivo adenoviral-mediated gene transfer technique prevents allogeneic flap rejection and restores graft survival [18,19]. In light of these previous studies, the present study also indicated that ex vivo transduction of either OX40Ig or CTLA4Ig lentivirus-carrying vectors into microvascular free flaps supplemented with short-course rapamycin therapy prolonged graft survival. It is noted that the survival time after combined OX40Ig and CTLA4Ig ex vivo transfer was significantly expanded to 65 d compared with 39 d in the OX40Ig and 45 d in the CTLA4Ig singular transfer group, respectively. Herein comes the hypothesis that combined OX40Ig and CTLA4Ig ex vivo transfer induces superior longterm survival of composite tissue allografts. We therefore performed histologic examination to assess the degree of rejection after allotransplantation. According to the Banff 2007 working classification, the grafts were rapidly rejected and the rats developed grade III to grade IV rejection on day 7 after the surgery. Rapamycin as a single immunosuppressant showed slight effect on the rejection process while local induction of OX40Ig or CTLA4Ig led to grade II rejection. As for the graft in the combined OX40Ig and CTLA4Igetreated group, there was no remarkable evidence of rejection. On biopsy, we also observed splenomegalia in the control, mock, and rapamycin groups. Previous studies suggested that systemic administration of CTLA4Ig inhibited the proliferation of CD4þ T cells in the peripheral lymph nodules in rodent and nonhuman primate models [27e32]. However, the distinct effect of OX40Ig on CD4þ T cells is unclear and it remains to be investigated whether local administration of CTLA4Ig also inhibited CD4þ T-cell proliferation. Our flow cytometry analysis showed that transduction of OX40Ig decreased the percentage of CD4þ T cells but did not affect CD8þ T cells. We found that local ex vivo administration of CTLA4Ig-carrying vectors also significantly reduced the proportion of CD4þ T cells and CD4/CD8 ratio in the spleen. We also reported for the first time that combined OX40Ig and CTLA4Ig local transfer produced synergistic effects on CD4þ T cells and CD4/CD8 ratio in splenocyte populations. On day 28 after transplantation, grafts in the first three groups were completely rejected and animals died out. Biopsy specimens from the remaining groups still revealed a viable flap with mild inflammatory infiltration of grade I or grade II rejection. These results revealed that local induction of OX40Ig or CTLA4Ig suppresses immune response, induces immune tolerance, and prevents rejection in the allotransplantation settings. Combined OX40Ig and CTLA4Ig transfer provoked superior protective effects on the grafts and promoted longer survival. Besides blockage of co-stimulatory signals, the immunosuppressive effects of OX40Ig and CTLA4Ig are also related to induction of differentiation and deviation of Th1/Th2 (T helper) cells [33,34]. The differentiation bias of Th1/Th2

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Fig. 7 e Proportions of CD4D and CD8D T lymphocyte populations after indicated treatments. Flow cytometry analysis showed OX40Ig or CTLA4Ig treatments significantly decreased the percentage of CD4D cells and CD4/CD8 ratio in the spleen. Combined treatment group demonstrated the greatest reduction. There were no significant changes in CD8D cell proportions after treatments. (Color version of figure is available online.)

cells by OX40Ig and CTLA4Ig might induce immune tolerance and would be responsible for the significant lengthening of allograft survival [35]. Several proinflammatory factors and cytokines synthesized by Th subtypes have been described to mediate immune response or tolerance. In murine cardiac transplant models, the immunomodulatory role of CTLA4Ig was related to the upregulation of Th2 cytokines (such as IL-4 and IL-10) and downregulation of Th1 cytokine expression (such as IL-2 and IFN-g) [36]. In our study, we have confirmed effects of CTLA4Ig on the production of Th1 and Th2 cytokines in the rat microvascular free flap allotransplantation models. Similarly, the serum levels of IL-2 and IFN-g were significantly lower, whereas the levels of IL-4 and IL-10 were statistically higher, in the OX40Ig group compared with the control groups. To our interest, combined OX40Ig and CTLA4Ig treatment resulted in more remarkable changes in serum IFN-g and IL-4 secretion. These results showed that the rise in Th2 cytokines and the inhibition of Th1 cytokines as a response to OX40Ig and CTLA4Ig may help to induce immune tolerance in allograft transplantation. Combined

OX40Ig and CTLA4Ig treatment significantly prolonged the graft survival, at least in part, through promoting the differentiation of Th2 subpopulations. Collectively, we demonstrated that ex vivo transduction of OX40Ig and CTLA4Ig lentivirus vectors could not only alleviate rejection to the grafts, but also ensure that expression of OX40Ig and CTLA4Ig mRNA and proteins were indeed locally and specifically in the donor’s organs. Local expression of the lentivirus-mediated OX40Ig and CTLA4Ig gene transfer can inhibit the rejection of allografts and the effects of the combination are superior to those of the use of either alone. Therefore, OX40Ig- and CTLA4Ig-based gene therapy is a promising biologic dressing for preventing allotransplantation rejection. This protocol can effectively suppress acute rejection, prolong the survival of recipients, and induce immune tolerance. The combined effectiveness of OX40Ig and CTLA4Ig therapy is marked in this study. Interruption of the co-stimulatory signals, downregulation of Th1 cellerelated cytokines, and upregulation of Th2 cellerelated cytokine expression might contribute to these beneficial effects.

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Fig. 8 e Effects of OX40Ig and CTLA4Ig perfusion on the production of proinflammatory cytokines. OX40Ig or CTLA4Ig treatments decreased Th1 cytokines (A) while increasing Th2 cytokines (B). Combined OX40Ig and CTLA4Ig treatment provoked a greater decrease in Th1 cytokines and increase in Th2 cytokines.

Acknowledgment The present study was supported by grants from National Natural Science Foundation of China (30830102).

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