CTLA4Ig-Gene Transfection Inhibits ObliterativeAirway Disease in RatsYusuke Kita, MD, Kazuya Suzuki, MD, Hiroshi Nogimura, MD,Tsuyoshi Takahashi, MD, and Teruhisa Kazui, MDDepartment of Thoracic Surgery, Haibara General Hospital, Shizuoka, and First Department of Surgery, Hamamatsu UniversitySchool of Medicine, Hamamatsu, Japan

Background. Obliterative airway disease (OAD) is amajor cause of long-term morbidity following lung trans-plantation. Its pathologic characteristics are small-airwayinflammation and occlusion by fibrous tissue. However,the pathogenesis is uncertain and therapy is ineffective.This study presents the effects of CTLA4Ig-gene therapyon OAD in heterotopically transplanted rat trachealallografts.

Methods. Dark Agouti (DA, RT1a) allografts and Lewis(LEW, RT1l) isografts were transplanted into Lewis re-cipients. The tracheal graft was transplanted heterotopi-cally into the subcutaneous pocket into the back. Adeno-viral vectors (1.0�109 pfu) containing the CTLA4Ig-gene(AdCTLA4Ig) or the LacZ-gene (AdLacZ) were injectedinto the tail vein immediately after grafting. Grafts wereharvested and examined after more than 35 days for

mononuclear cell infiltration development and lumenocclusion with fibrosis.

Results. Fully allogenic DA tracheas, treated withAdCTLA4Ig had significantly lower pathologic scoresand infiltrating scores than the control allografts. Thepathologic findings of the grafts, treated withAdCTLA4Ig, were very similar to those of the syngeneicgrafts. The animals experienced no adverse events duringfollow-up. No evidence of vector-mediated tissue dam-age was seen in any graft.

Conclusions. Adenoviral vectors containing theCTLA4Ig-gene markedly inhibited the obliteration of theairway lumen. OAD may be associated with T-cell re-sponses against graft tissue and alloimmune injury.

(Ann Thorac Surg 2003;75:1123–7)© 2003 by The Society of Thoracic Surgeons

Obliterative airway disease (OAD) is the most com-mon complication after lung transplantation [1,2].

Despite contemporary immune suppression, some lungtransplant recipients develop fatal chronic rejection. Thepathologic OAD hallmark is fibrous small cartilaginousairways and variable peribronchiolar inflammatory infil-trate. This lesion differs from that of acute rejection,which is defined by perivascular mononuclear cell infil-tration, possibly reflecting different immune targets ormechanisms [3]. Previous clinical studies support theconcept that the OAD results from alloimmune-mediatedinjury [4 – 6] and graft function stabilization can beachieved with immune suppression [7, 8]. Graft rejectionmay lead to OAD [9, 10] and T cells play an importantrole in disease development [11].

CTLA4Ig is a soluble recombinant fusion protein, con-taining the CTLA4 extracellular domain and IgG1 Fcportion. CTLA4Ig strongly attaches to the B7 molecule toblock CD28-mediated costimulatory signals, and inhibitsthe lymphocyte activation and immune responses [12–14]. CTLA4Ig administration to recipients with organgrafts has achieved prolonged graft survival in severalrodent models [15–19]. In vivo gene transfer using adeno-viral vectors achieves a high transfection rate into organcells, which usually contain adenoviral receptors.

In this study, we investigated the effects of systemicallyadministered adenoviral vectors containing CTLA4Iggene (AdCTLA4Ig) for OAD, using the rat model ofheterotopic tracheal transplantation.

Material and Methods

Adenoviral VectorThe recombinant adenovirus, AxCAhCTLA4Ig(AdCTLA4Ig) and AxCALacZ (AdLacZ), were providedby Dr. S. Hayashi (Nagoya University School of Medi-cine, Nagoya, Japan), Dr. H. Hamada (Japanese Founda-tion for Cancer Research, Tokyo, Japan), and Dr. I. Saito(Laboratory of Molecular Genetics, Institute of MedicalScience, University of Tokyo, Tokyo, Japan). The adeno-virus containing the expression cassette for humanCTLA4Ig cDNA or Escherichia coli �-galactosidase gene(LacZ) was constructed by homologous recombinationbetween the expression cosmid cassette (pAdex/CAhCTLA4Ig) and the parental virus genome [20]. Therecombinant viruses were subsequently propagated with293 cells. The prepared vector solutions were stored at�80°C.

Experimental AnimalsAll laboratory animals received humane care in compli-ance with the Principles of Laboratory Animal Careformulated by the Institute of Laboratory Animal Re-sources and the Guide for the Care and Use of Labora-tory Animals prepared by the National Academy of

Accepted for publication Oct 25, 2002.

Address reprint requests to Dr Kita, Department of Thoracic Surgery,Haibara General Hospital, 2887-1 Hosoe, Haibara-cho, Haibara-gun,Shizuoka-pref, Japan 421-0493; e-mail: kita21@aqua.ocn.ne.jp.

© 2003 by The Society of Thoracic Surgeons 0003-4975/03/$30.00Published by Elsevier Science Inc PII S0003-4975(02)04707-0

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Science and published by the National Institutes ofHealth (National Institutes of Health, publication No.86-23, revised 1985). The experimental protocols wereapproved by the Institutional Animal Care Use Commit-tee, National Children’s Medical Research Center (To-kyo, Japan). The animals were purchased from Japan SLCCo. (Shizuoka, Japan) and maintained under standardconditions.

Heterotopic Tracheal TransplantationAdult male Dark Agouti (DA, RT-la) rats and Lewis(LEW, RT-ll) rats were used as donors and recipients,respectively. Under ether anesthesia, the neck and upperchest of donor rats were incised and bluntly dissected.The trachea was resected from the larynx inferior borderto the carina and placed in ice-cold phosphate-bufferedsaline (PBS) with penicillin (100 U/ml), streptomycinsulfate (100 �g/ml), and amphotericin B (0.25 g/ml) (Sig-ma, St. Louis, MO). The isolated donor tracheas weretransplanted into subcutaneous pockets in the recipientback, essentially as described [9]. In brief, after therecipient’s back skin was prepared, about 1-cm skinincisions were made and subcutaneous pockets wereformed by blunt dissection. The trachea grafts wereplanted heterotopically into the pockets without primaryvascularization, and the wounds were closed with 3-0 silksutures.

Experimental GroupsThe recipients were divided into the following groups:group 1 comprised DA-to-LEW rats injected with 1�109

plaque-forming units (p.f.u.) of control vector, AdLacZ;group 2 comprised DA-to-LEW rats administered 1�109

p.f.u. of AdCTLA4Ig; and group 3 comprised syngeneicgrafts (LEW-to-LEW) without any treatment. The vectorswere administered through the recipient tail vein imme-diately after grafting. The day of grafting was regarded asday 0 and the grafts were harvested weekly and exam-ined over the course of 35 days.

Histologic StudiesFor histologic observation, the grafts were removed on 3,5, 7, 14, 21, 28, and 35 days after transplantation anddivided into four pieces. One of the middle pieces wasfixed in formalin and paraffin-embedded for hematoxy-lin-eosin staining, the other was snap-frozen in tetraflu-oroethane and stored at �80°C for immunohistochemicalstaining. Graft infiltrating cells in the thin cryocut section(6 �m) were stained with the monoclonal antibodies, CD2(a mixture of MRC OX-54 and 55; Serotec, Oxford, UK).Color development was performed with nickel-cobalt-diaminobenzidine product (DAB; 049-22833 [Wako,Osaka, Japan]).

ScoringSlides of grafts were examined two blinded reviewersand given numeric T-cell infiltration scores (on a scale of0 to 4) based on the degree of CD2 positive mononuclearcell infiltration (Table 1). Hematoxylin-eosin stainingsections were examined by the same, blinded reviewers

and given numeric pathologic scores (on a scale of 0 to 4)based on the presence or absence of mononuclear cellinfiltrate, epithelial injury, and fibrosis (Table 1).

Determination of Serum CTLA4Ig LevelsTo determine the time course of the serum CTLA4Iglevels, we injected 1�109 p.f.u. of AdCTLA4Ig into therecipient rats. Blood samples were collected on day 0before injection and 1, 3, 5, 7, 10, 14, 21, and 35 days afterinjection. The serum concentration of CTLA4Ig was as-sayed by enzyme-linked immunosorbent assay, as de-scribed previously [21].

Results

To examine the lymphocyte role in OAD development,tracheal grafts were transplanted into recipients andremoved after grafting for histologic study. The controlgrafts (group 1) revealed that the epithelial squamousmetaplasia or subepithelial thickening were accerelatedfrom day 5 to 7. Severe epithelial abnormalities becameprominent until day 14, and a marked fibrous prolifera-tion and lumenal obstruction were complete before day28, whereas few epithelial abnormalities and no fibrouschange were observed in group 2 and group 3 by hema-toxylin-eosin staining (Fig 1). We compared these differ-ences by pathologic scoring criteria (Table 1) [11]. Wecompared an average of six grafts in each group. DAtracheas treated with AdCTLA4Ig (group 2), harvested onday 5, 7, 14, 21, and 28 had significantly lower pathologicscores than the control, group 1 (day 5; p � 0.05, day 7 to28; p � 0.01, unpaired Student’s t-test; Fig 2). The patho-logic findings of group 2 were very similar to those of thesyngeneic grafts (group 3). CD2 immunohistologic stain-ing was performed to distinguish lymphocytes fromother infiltrating monocytes. CD2 was expressed on bothresting and activated T cells. To examine the lymphocyteinfiltration, we used the infiltrating scoring criteria (Table1) [11]. DA tracheas treated with AdCTLA4Ig (group 2),

Table 1. Scoring Criteria

Pathologic scoring criteria0. Normal.1. Mononuclear cell infiltrate within graft tissue.2. Epithelial squamous metaplasia and/or subepithelial

thickening.3. Severe epitherial abnormalities (severe metaplasia or

denudation).4. Lumenal fibrosis.

Infiltrating scoring criteria0. No CD2� T-cell infiltrate.1. Surrounding host connective tissue contains T cells. No

significant graft T-cell infiltrate.2. Host connective tissue contains T cells. Graft infiltration

� 10 T cells/high power field.3. Graft infiltration of 10–100 T cells/high power field, with

modest subepithelial extension.4. Graft infiltration of �100 T cells/high power field, with

heavy subepithelial extension.

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harvested on day 3, 5, and 7 had significantly lower scoresthan the control, group 1 (p � 0.01, unpaired t-test; Fig 3).However, after day 14, the scores of group 2 were notsignificantly different from those of group 1. The infiltrat-ing scores of group 3 were also significantly lower thanthose of group 1 from day 3 to 7, whereas the scores ofgroup 3 were not significantly different from those ofgroup 2 on any day investigated (Fig 3). The animals

experienced no adverse events during follow-up. Nopathologic changes appeared at autopsy or in postmor-tem analysis of the liver, heart, lung, skin, or spleen. Noevidence of vector-mediated tissue damage was seen inany graft.

The time course of serum CTLA4Ig levels is illustrated

Fig 1. Photomicrographs from tracheas ofgroup 1 (a, d, g, j), group 2 (b, e, h, k), andgroup 3 (c, f, i, l) on day 7 (a–c), day 14(d–f), and day 28 (g–l). These panels weremagnified �100 (a–i) or �50 (j–l). On day7, control grafts (group 1) exhibited submu-cosal edema (a), whereas no subepithelialthickening in AdCTLA4Ig group (b) andsyngeneic grafts (c) (hematoxylin and eo-sin). On day 14, epithelial damage andfibrous proliferation were prominent ingroup 1 (d), whereas there was no fibrouschange in groups 2 (e) and 3 (f). On day28, complete luminal obliteration was seenin the control (g, j). However, the lumenwas completely patent in groups 2 (h, k)and 3 (i, l).

Fig 2. Pathologic scoring criteria (mean � standard error). We com-pared the average of six grafts in each group. Grafts treated withAdCTLA4Ig (group 2 [circles]), harvested on day 5, 7, 14, 21, and28 had significantly lower scores than the control, group 1 (p �0.05 [#], p � 0.01 [*], unpaired Student’s t-test [diamonds]). Thepathologic findings of group 2 were very similar to those of synge-neic grafts (group 3 [triangles]).

Fig 3. The infiltrating scoring criteria (mean � standard error). Wecompared the average of six grafts in each group. Grafts treated withAdCTLA4Ig (group 2 [circles]), harvested on day 3, 5, and 7, hadsignificantly lower scores than the control, group 1 (p � 0.01 [*],unpaired Student’s t-test [diamonds]). However, after day 14, thescores were not significantly different between the control and others.The scores of group 3 (triangles) were not significantly differentfrom those of group 2 on any day investigated.

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in Figure 4. The mean level of 3 rats reached maximum at91 �g/ml on day 5 and declined after 14 days. Detectablelevels were observed more than 35 days. The rats trans-fected with control vector (AdLacZ) did not have anymeasurable CTLA4Ig at any time (data not shown).

Comment

OAD is a significant complication, associated with T-cellresponses against graft tissue. The chronic inflammationmay lead to fibroblast recruitment, extracellular matrixformation, and bronchiole fibrosis. In rat allografts, bron-chial epithelium starts to express major histocompat-ibility complex (MHC) class II antigens during acuterejection and after insufficient cyclosporine treatment[22]. The expression of MHC class II antigen may beinduced by local alloreactive T cells. These antigens onepithelial cells may stimulate the activation of the graftrejection [23].

Monocyte infiltration is a landmark feature of organrejection. The cellular infiltrations are compromised pre-dominantly of macrophages and T lymphocytes, whichare responsible for the immunologic response to foreignbodies. For this to occur, leukocytes must undergo ex-travasation, interact with extracellular matrix proteins ofthe basement membrane, and spread in the graft tissue.AdCTLA4Ig-treatment prevents leukocyte infiltration atthe early stage of graft rejection. This result suggests thatperipheral leukocytes, especially T lymphocytes, wereprevented from activation by the CTLA4Ig-protein andstopped the extravasation. We are studying whether thistherapy is effective for ongoing rejection, such as whenmonocyte infiltration has been established.

In vivo gene expression by adenoviral vector wasefficient. The AdCTLA4Ig enabled the persistent geneexpression in vivo without repeated vector administra-tion. The CTLA4Ig-gene was apparently expressed con-tinuously in the recipients more than 7 weeks. Once theAdCTLA4Ig was injected systemically, intrahepatic ex-

pression of CTLA4Ig would be prominent. In our previ-ous study, the injection of AdLacZ through mouse tailvein demonstrated high gene-expression levels in theliver by X-gal staining (� 95% of hepatocytes), althoughsome expression was seen in the heart, lung, and spleen.No expression in the thymus, small intestine, pancreas,kidney, or brain was detected (data not shown). TheCTLA4Ig protein might be produced in the gene-transfected hepatocytes and released into blood. In thisstudy, we demonstrated the time course of serumCTLA4Ig levels after AdCTLA4Ig administration. Thenimmunosuppressive activity was induced in the allo-grafts. This effect may be similar to that obtained byrepeated administration of exogenous CTLA4Ig protein.However, AdCTLA4Ig induces efficient immunosuppres-sion with its one injection.

As the limitation of adenoviral vector, in vivo geneexpression is generally transient. The immune systemmay play a major role in the limiting gene-transfertechnology. CTLA4Ig would be effective to prolong geneexpression but not permanent. We are now studying theadministration timing and the dose of CTLA4Ig.

In this heterotopic tracheal implant model, all theuntreated allografts develop nearly complete occlusion ofthe airway lumen with fibroblastic tissue and collagenscar by day 28 after transplantation, with the featuresresembling human obliterative bronchiolitis (OB). Mono-nuclear cell infiltration, epithelium denudation, fibro-proliferation, and airway obliteration are subsequentlyfound. These phenomena suggest that graft rejectionmay lead to OAD. Allografts undergo the obliterativechanges and structural destruction, whereas almost allthe syngeneic grafts and the AdCTLA4Ig-treated graftsrelatively retain their normal structures. The surgicalprocedure of transplantation is not so responsible for thechanges observed in this model. Direct epithelial injuryby infection may also trigger the development of bron-chiolitis obliterans. No airway pathology other than thereimplantation response was seen in our syngeneic con-trols. After the grafts were denervated, we did not seeevidence of mucus stasis that might have been predis-posed to infection.

T cells play an important role in disease development[11]. A peak of CD2� cell infiltration in this untreatedgroup was recorded from 7 to 14 days. These results arein concordance with the transbronchial lung biopsy find-ings of human allograft rejection. Once the developmenttoward OAD originated, the process was rapidly pro-ceeded. It seems that OAD is a continuation of acuterejection in this model. Single dose injection ofAdCTLA4Ig on day 0 resulted in significantly lowerscores up to 30 days after implantation, although therewere no significant differences in infiltrating scores after14 postoperative days. For complete inhibition of OAD,we are investigating the combination therapy by using animmunosuppressant, such as FTY720. Simultaneouslyblocking other costimulatory pathways, such as gp39/CD40, ICAM-1/LFA-1 and CD2/CD48, also enhance theT-cell anergy.

In conclusion, the present study demonstrated that

Fig 4. Time course of serum CTLA4Ig levels in recipients after injec-tion of AdCTLA4Ig. Values represent the mean � standard error ofthree rats. The mean level reached to maximum (91 �g/ml) on day 5and declined gradually.

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AdCTLA4Ig-gene transfer into recipient’s liver by sys-temic administration proved to be useful in this hetero-topic tracheal allograft model. AdCTLA4Ig may be effec-tive to inhibit OAD.

The authors gratefully acknowledge Drs Seiichi Suzuki, Xiao-Kang Li, Naoko Funeshima, and Shin Enosawa (Department ofExperimental Surgery & Bioengineering, National Children’sMedical Research Center, Tokyo, Japan) for their critical com-ments and useful suggestions. We also thank Drs Shuji Hayashiand Izumu Saito for providing AxCALacZ; and Dr HirofumiHamada for providing AxCAhCTLA4Ig adenoviral vector.

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