Combined therapy of CTLA4Ig-gene transfection with FTY720 administration in rat lung allografts

Combined Therapy of CTLA4Ig-Gene Transfection With FTY720 Administration in Rat Lung Allografts Y. Kita, H. Nogimura, M. Ida, Y. Kageyama, S. Ohi, Y. Ito, K. Matsushita, T. Takahashi, K. Suzuki, T. Kazui, S. Hayashi, X.-K. Li, and S. Suzuki

F

TY720 is a synthetic drug that is made by the chemical modification of ISP-1. ISP-1 is purified from culture filtrates of Isaria sinclairii, an ascomycete, which is a member of the “vegetable wasp and plant worm” family. Isaria sinclairii acts as a parasite on the larva of the cicada, Meimuna opalifera Walker.1,2 The chemical structure of FTY720 is completely different from conventional immunosuppressants and it has the ability to induce reduction of peripheral lymphocytes and long-term acceptance of rat and dog allografts.3 CTLA4Ig is a soluble recombinant fusion protein that was constructed with an extracellular domain of human CTLA4 and the Fc portion of human IgG1.4 To activate T cells, two kinds of signals are required: (1) by antigenpresenting cells (APCs), with MHC molecules to the T-cell receptor (TCR); and (2) by the costimulatory signal. One of the best known costimulatory signals is from B7-1 (CD80) on activated APCs. B7-1 binds to CD28 on helper T cells.4 –9 The engagement of antigen/MHC with TCR in the absence of costimulatory signals induces T-cell anergy. CTLA4 is a membrane-type adhesion molecule, highly homologous to CD28, and is expressed on activated cytotoxic T cells (CTL). CTLA4 acts as a negative regulator of CTL.10,11 CTLA4Ig adheres strongly to the B7 molecule and blocks CD28-mediated costimulatory signals.12,13 The administration of CTLA4Ig to recipients with organ grafts has resulted in prolonged graft survival in several models.14 –18 Immunoregulatory gene transfer by adenoviral vector is one of the alternative approaches to modulate immune responses. Adenoviral vectors lead to efficient levels of gene expression in vivo, especially in the liver.19 –24 Adenoviral vectors have been reported to be highly effective in several animal models and in human tissues.25–28 Although transfected virus is confined by the host immune responses,29,30 adenoviral vector is better used to achieve a high transfectional rate into organ cells, which usually contain adenoviral receptors and their intracellular uptake pathway. In contrast to retroviral vectors or DNA–protein complex injection, the recombinant adenoviral vectors are capable of transfecting almost 100% of the hepatocytes when injected intravenously. Ex vivo transfection of adenoviral vectors containing the CTLA4Ig gene (AdCTLA4Ig) into rat liver

allografts can induce long-term graft acceptance in recipients.31 There are some negative aspects in the use of adenoviral vectors. Transgene expression is diminished in a timedependent manner.26,27 This phenomenon must be caused by the activation of host immune responses due to viral infection. When the vector containing ␤-galactosidase (␤gal) was injected into the rat myocardium directly, transgene expression was reported to decrease within 4 weeks.32,33 CTLA4Ig blocks the B7/CD28-mediated costimulated signal to inhibit immune responses. AdCTLA4Ig was effective as one of the immunosuppressants. However, it diminishes in a time-dependent manner, and therefore we considered the combined therapy of AdCTLA4Ig with FTY720. The present study was conducted to evaluate the combined effect of CTLA4Ig-gene transfection and perioperative administration of FTY720 in rat lung allografting. We also investigated whether preoperative administration of FTY720 may prolong the period of transfected gene expression in the rat lung allograft. MATERIALS AND METHODS Adenoviral Vector The recombinant adenoviruses, AxCAhCTLA4Ig (AdCTLA4Ig) and AxCALacZ (AdLacZ), were provided by Dr S. Hayashi (Department of Surgery II, Nagoya University School of Medicine, Nagoya, Japan), Dr H. Hamada (Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan), and Dr I. Saito (Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan). The adenovirus containing the expression cassette for human CTLA4Ig cDNA or Escherichia coli From the Departments of Thoracic Surgery (Y.K., H.N.) and Respiratory Disease (M.I.), Haibara General Hospital, Shizuoka, Japan; First Department of Surgery (Y.K., S.O., Y.I., K.M., T.T., K.S., T.K.); Hamamatsu University School of Medicine, Hamamatsu, Japan; Department of Surgery II, Nagoya University, School of Medicine, Nagoya, Japan (S.H.); and Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center, Tokyo, Japan (X.-K.L., S.S.). Address reprint requests to Dr Yusuke Kita, Department of Thoracic Surgery, Haibara General Hospital, 2887-1 Hosoe, Haibara-cho, Haibara-gun, 421-0493 Shizuoka, Japan.

© 2002 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0041-1345/02/$–see front matter PII S0041-1345(02)02918-4

Transplantation Proceedings, 34, 1437–1440 (2002)

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␤-galactosidase gene (LacZ) was constructed by homologous recombination between the expression cosmid cassette (pAdex/ CAhCTLA4Ig) and the parental virus genome. The recombinant viruses were subsequently propagated with 293 cells. The prepared vector solutions were stored at ⫺80°C.

Drugs FTY720, synthesized by Taito Co, Ltd (Tokyo, Japan) in cooperation with Yoshitomi Pharmaceutical Industries, Ltd (Osaka, Japan), was in powder form. For in vivo use, the drug was dissolved in physiologic saline and administered orally to recipient rats.

Lung Transplantation Donor left lungs were transplanted into the recipient orthotopically. Adult male DA (RT-1a) and LEW (RT-1l) rats, weighing 250 to 300 g, were used as donors and recipients, respectively. The animals were maintained under standard conditions and fed rodent chow and water. The rats were anesthetized with intraperitoneal administration of pentobarbital (20 mg/kg) and ketamine (25 mg/kg). After intratracheal intubation, the lungs were ventilated with room air at a tidal volume 3 mL and respiratory rate of 80 to 90 breaths/min. First, left thoracotomy was performed at the fifth intercostal space of the donor. The inferior pulmonary ligament was divided and the hilum was dissected. Heparin (1000 U/kg) was injected intravenously. The recipient was then placed in the left-side-up position. Left thoracotomy was performed at the fourth intercostal space. After the hilum was dissected, the bronchus was ligated and transected. Pulmonary artery (PA) and vein (PV) were cross-clamped, incised, and rinsed with heparinized saline. The lung graft was harvested and soaked in cold heparinized saline. A cuff technique was used for PA and PV, as previously described.34 Briefly, a cuff was first attached to the graft PV, inserted into the incised PV of the recipient, and fixed with a ligature of 6-0 silk. PA was connected in the same way and the circulation was restored. The main bronchus was sutured with an 8-0 polypropylene continuous suture for the cartilaginous ring and 9-0 interrupted sutures for the membranous wall. The thorax was closed and, after spontaneous respiration was resumed, the chest tube and the intratracheal tube were removed.

Experimental Groups The recipients were divided into the following groups: group 1 (n ⫽ 6), DA-to-LEW rats injected with 1 ⫻ 109 plaque-forming units (pfu) of control vector (AdLacZ); group 2 (n ⫽ 6), DA-to-LEW rats administered FTY720 that was dissolved in saline and administered orally at a dose of 5 mg/kg 1 day before and the day of transplantation (days ⫺1 and 0); group 3 (n ⫽ 10), DA-to-LEW rats administered 1 ⫻ 109 pfu of AdCTLA4Ig; and group 4 (n ⫽ 10), DA-to-LEW rats administered 1 ⫻ 109 pfu of AdCTLA4Ig with perioperative FTY720 administration. The vectors were administered via the recipient tail vein immediately after grafting. The day of grafting was regarded as day 0. The grafts were harvested and examined over the course of 100 days.

Histologic Studies For histologic observation, the grafts were removed for light microscopic examination and divided into two pieces. One piece was fixed in formalin and paraffin-embedded for hematoxylin– eosin staining, and the other was snap frozen in tetrafluoroethane and stored at ⫺80°C for immunohistochemical staining. A thin

KITA, NOGIMURA, IDA ET AL cryocut section (6 ␮m) of the frozen piece was stained with the following monoclonal antibodies: mouse IgG1 isotypes and CD2 (a mixture of MRC OX-54 and -55; Serotec), CD4 (W3/25; Cosmo Bio, Tokyo, Japan), CD8 (MRC OX-8, Serotec, Oxford, UK), or CD25 (MRC OX-39; Serotec). Color development was performed with nickel– cobalt– diaminobenzidine product (DAB; 049-22833, Wako, Osaka, Japan).

Measurement of Numbers of Peripheral Lymphocytes For study of the combined effect of FTY720 and AdCTLA4Ig, peripheral blood was collected periodically from the tail veins of allografted recipients. We counted the number of peripheral leukocytes by Turk staining, and measured the proportion of lymphocytes by Giemsa staining of the smear of recipient’s peripheral blood.

Determination of Serum CTLA4Ig Levels To determine the time course of serum CTLA4Ig levels, we collected the blood samples on day 0 before injection and 1, 3, 5, 7, 11, 14, 21, 35, and 49 days after injection. The serum concentration of CTLA4Ig was assayed by ELISA; a 96-well microtiter plate (Pro-Bind Assay Plate, Falcon 3915, Becton Dickinson, Lincoln Park, NJ) was coated with anti-human CTLA4 antibody (purified mouse monoclonal antibody [34581A], Pharmingen, San Diego, Calif) by incubating at 4°C for 3 days with 50 ␮L of the antibody solution (10 ␮g/mL in PBS) to each well. After washing with PBS, 50 ␮L of the serum sample was added to each well. The plate was incubated for 2 hours at room temperature in a humid atmosphere and washed again. The second antibody, anti-human IgG1 Fc conjugated with horseradish peroxidase (MH1715, Dai Nihon Seiyaku, Osaka, Japan) diluted to 1:200 with 3% BSA in PBS with 0.02% sodium azide, was added at 50 ␮L/well and incubated further for 2 hours at room temperature. After washing, 100 ␮L of o-phenylenediamine dihydrochloride (P-8412; Sigma, St Louis, Mo) solution (3 mg/mL in PBS) with 0.15 ␮L/mL H2O2 was added to each well and incubated for 10 to 30 minutes at room temperature. One hundred microliters of 1 mol/L H2SO4 was then added to each well and the absorbance values were obtained with a microplate reader (Model 450, Big-Rad, Tokyo, Japan) at 450 nm. CTLA4Ig concentrations in each serum sample were quantified by comparing the absorbance values with controls.

RESULTS

Recipient rats treated with control vector (group 1) rejected their grafts at 5 to 7 days after grafting. Graft survival in the FTY720-treated rats (group 2) ranged from 6 to 10 days. Survival in the AdCTLA4Ig-transfected group (group 3) was prolonged significantly to 27 days and that in the AdCTLA4Ig ⫹ FTY720 group (group 4) was prolonged markedly to 54 days of median survival time (P ⬍ .01 versus group 1 by unpaired t test). There were four permanent graft survival cases in the group 4, and one permanent survival in group 3 (Table 1). No deaths were associated with viral infection. The grafts were removed for histologic study on days 3, 5, 7, 14, 21, and 28 after transplantation. Control grafts (group 1) showed a marked infiltration of mononuclear cells around the vessels at days 3 to 7, whereas little cell infiltration was observed in groups 3 and 4 by hematoxylin–

COMBINED THERAPY

1439 Table 1. Graft Survival Time

Group

1. 2. 3. 4.

AdLacZ (control) FTY720 AdCTLA4Ig AdCTLA4Ig ⫹ FTY720

n

Survival Days

Median (d)

P Value* Versus Group 1

6 6 10 10

5 ⫻ 2, 6 ⫻ 3, 7 6 ⫻ 4, 7, 10 12, 18, 20, 24, 26, 28, 38, 42, 48, ⬎100 24, 38, 42, 49, 52, 56, ⬎100 ⫻ 4

6 6 27 54

— NS ⬍.01 ⬍.01†

*Unpaired t test; †P ⬍ .05 vs group 3. NS, not significant.

eosin staining. Immunohistologic staining demonstrated that both CD2⫹ and CD25⫹ cells decreased in number in groups 3 and 4. CD2 was expressed in both resting and activated T cells, and CD25 in activated T cells. CD2⫹ and CD25⫹ cells were prominently observed in group 1, whereas there were few monocytes in groups 3 and 4 at day 5 posttransplant. In addition, no evidence of vector-mediated tissue damage was seen in any graft. In the recipient rats treated with control vector (group 1) and in the adCTLA4Ig-transfected group (group 3), the number of lymphocytes in the peripheral blood was increased for 5 to 7 days after the transplantation and decreased thereafter. Conversely, in the FTY720-treated group (group 2), lymphocytes were decreased for ⬎4 weeks after administration. In the AdCTLA4Ig ⫹ FTY720 group (group 4), peripheral lymphocytes also decreased in number, but no significant difference was found in the FTY720 group (group 2). The CTLA4Ig protein levels in the peripheral blood from groups 3 and 4 were observed in the recipient serum by ELISA up to 49 days after gene administration. In group 3, the levels reached a maximum at 70 to 80 ␮g/mL at 5 to 7 days after administration and averaged 3 cases daily, the maximum level was 77 ␮g/mL at day 7. In group 4, the maximum levels of three cases ranged from 88 to 116 ␮g/mL on days 5 to 10, and the highest level of averaged three cases was at 94 ␮g/mL on day 5. Levels in the AdCTLA4Ig ⫹ FTY720 group (group 4) were higher than those of the AdCTLA4Ig only group (group 3) at each day with significantly higher levels on days 5, 10, 14, 28, and 49. The rats transfected with control vector (group 1) had no measurable CTLA4Ig at any timepoint (data not shown). DISCUSSION

In vivo gene expression using a recombinant adenovirus was highly efficient but transient in many models. In view of this limitation, the immunosuppressive product, CTLA4Ig, would be highly effective for prolonging gene expression by adenovirus-mediated gene transfer. If other immunosuppressants are combined, the immunosuppressive effects become higher. The chemical structure of FTY720 is different from that of conventional immunosuppressants. FTY720 did not affect interleukin-2 (IL-2) production and its mechanisms of action appeared to be distinguishable from CTLA4Ig, CsA, and other immunosuppressants. The present study demonstrated that FTY720 results in long-term expression of the transgene and prolonged survival of lung allografts.

One advantage of using AdCTLA4Ig is the persistence of gene expression in vivo without need for repeated administration of the vector. Olthoff et al31 injected AdCTLA4Ig or recombinant CTLA4Ig protein intraperitoneally into rats and confirmed that the serum level of CTLA4Ig was tenfold higher in the AdCTLA4Ig-transfected group than in the recombinant CTLA4Ig-administered group. The also noted that CTLA4Ig level in the AdCTLA4Ig group remained high at day 20 after transfection and declined by ⬍10 ␮g/mL from day 40. Systemic administration of AdCTLA4Ig into the recipients resulted in intrahepatic expression of CTLA4Ig, which was released into blood circulation, and eventually induced immunosuppressive activity in cardiac allografting. The lung allograft in the present study did not achieve indefinite survival, as did orthotopic liver allografts.31 Transduction of AdCTLA4Ig in combination with short-course administration of an immunosuppressant may be beneficial for further prolonging graft survival. Hale et al35 demonstrated that CTLA4Ig was highly effective when combined with cyclosporine (CsA) or sirolimus (SRL) in MHC-mismatched rat allografting. It has also been suggested that simultaneous blockage of the costimulatory pathways, including gp39/ CD40, ICAM-1/LFA-1, and CD2/CD48, enhance the induction of T-cell anergy. Wang et al36 found that FTY720 potentiates the immunosuppressive effects of CsA and/or SRL both in vitro and in vivo. Previous in vitro studies indicated that ⬎4 ⫻ 10⫺6 mol/L FTY720 was needed to induce apoptosis, as documented by the fragmentation of chromosomal DNA.33 In this study, we examined the peripheral number of lymphocytes as a marker of FTY720 effects. FTY720 reduce peripheral lymphocytes, but CTLA4Ig does not. When CTLA4Ig-gene transfection was combined with FTY720 administration, the number of lymphocytes showed little difference between FTY720-only and combination therapy. This suggests that CTLA4Ig may not enhance FTY720 effects, although FTY720 and CTLA4Ig have different mechanisms as immunosuppressants. Expression of the CTLA4Ig gene in animals could inhibit the production of neutralizing antibody or the activation of cell-mediated immunity against the gene-expressing cells. We believed that FTY720 would emphasize the effects of CTLA4Ig-gene transfection. Preoperative administration of FTY720, may enhance and prolong the effect of gene therapy. FTY720 reduces lymphocyte function, which may induce prominent prolongation of CTLA4Ig expression. In addition, FTY720 may have the potential to enhance other types of gene

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expression due to its unique function. We are presently investigating these alternate mechanisms of combined therapy. CONCLUSIONS

The present study demonstrated that AdCTLA4Ig-gene transfection in combination with perioperative administration of FTY720 results in a mutual immunosuppressive effect in rat lung allografts. FTY720 appears to be highly effective for prolonging gene expression via adenovirusmediated gene transfer. ACKNOWLEDGMENT The authors gratefully acknowledge Dr S. Enosawa, Dr H. Kimura, Dr T. Okuyama, and Dr N. Funeshima, (Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center, Tokyo, Japan) for their critical comments and useful suggestions. We also thank Dr I. Saito for providing the AxCALacZ, and Dr H. Hamada for providing the AxCAhCTLA4Ig adenoviral vector.

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