[Adenovirus-mediated CTLA4Ig and OX40Ig gene transfer induces long-term survival of cardiac allografts in rats].
OBJECTIVE
To investigate the potential role of CTLA4Ig gene and OX40Ig protein in inducing transplantation tolerance and the mechanisms thereof.


METHODS
Thirty Lewis rats underwent transplantation of the hearts of DA rats and then randomly divided into five equal groups: control group, blank virus AdEGFP treated group (adenovirus containing EGFP at the dose of 1-5 x 10(9) pfu/ml was infused via portal vein immediately after the operation), AdCTLA4Ig treated group, AdOX40Ig treated group, and AdCTLA4Ig-IRES-OX40Ig treated group. The cardiac allograft survival was monitored by daily palpation. The total cessation of beating was defined as rejection and was confirmed by histology. Peripheral venous blood samples were collected 0, 3, 7, 10, 14, 21 and 28 days after the administration of adenovirus. ELISA was used to detect the expression of CTLA4Ig and OX40Ig. Twenty days after the heart transplantation single splenocyte suspension was prepared from surviving Lewis rats to be used as responder. The spleens of the normal donor-DA rats and the third strain DA rats to prepare single cell suspension of the same density to perform mixed lymphocyte reaction (MLR). Then recombinant IL-2 was added into the mixed MLR system to observe t\if the MLR could be reversed. Twenty days after the heart transplantation the splenocytes of the tolerating Lewis rats were injected into the lingual vein of the normal Lewis rats to observe the delayed type hypersensitivity (DTH) of the transferred Lewis rat to normal rat splenocytes. RT-PCR was used to detect the mRNA expression of IL-2, interferon-gamma, IL-4, and IL-10.


RESULTS
The survival time of the AdCTLA4Ig-IRES-OX40Ig treated group was 151.5 d +/- 42.6 d, significantly longer than those of the AdOX40Ig treated group (60.2 d +/- 11.4 d (P = 0.003), AdCTLA4Ig (43.2 d +/- 11.1 d, P = 0.0026), control group (5.7 d +/- 0.5 d, P = 0.000 43), and AdEGFP treated group (5.2 d +/- 0.4 d, P = 0.000 43). CTLA4Ig and/or OX40Ig proteins were expressed at a high level in the adenoviral treated rats. Compared with the control group the splenocytes of the AdCTLA4Ig-IRES-OX40Ig, AdCTLA4Ig, and AdOX40Ig treated groups displayed donor-specific hyporesponsiveness (P = 0.0016, 0.0026 and 0.001), which could be partly reversed by the addition of exogenous IL-2. Moreover, the hyporesponsiveness could be transferred to the same strain rats through adoptive transfer. In comparison with the normal controls, the expression of Th1 type cytokines, such as IL-2 and IFN-gamma, was significantly decreased in the tolerating rats and significantly increased in the rats with rejection; however the expression of the Th2 type cytokines, such as IL03 and IL-10, was significantly increased in the tolerating rats and significantly decreased in the rats with rejection, showing a deviation of Th1/Th2 type cytokines.


CONCLUSIONS
AdCTLA4Ig-IRES-OX40Ig-mediated genes transfer renders prolonged expression of CTLA4Ig and OX40Ig in Lewis recipient rats, leading to a long-term survival of cardiac allografts. The induced tolerance is donor-specific, and the mechanisms may be associated with T cell anergy, deviation of Th1/Th2, and the regulatory T cells.
Wang
Guang-ming
GM
Department of Immunology, Peking University Health Science Center, Beijing 100083, China.
Sun
Tao
T
Zhang
Yuan-yuan
YY
Yuan
Guo-hong
GH
Feng
You-gang
YG
Hao
Jie
J
Gao
Xiang
X
Xie
Shu-sheng
SS
D000069594:Abatacept; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000943:Antigens, Differentiation / Q000097:blood / Q000235:genetics* / Q000502:physiology; D016207:Cytokines / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D006085:Graft Survival / Q000235:genetics / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016027:Heart Transplantation / Q000379:methods*; D018796:Immunoconjugates / Q000097:blood / Q000235:genetics*; D008297:Male; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011897:Random Allocation; D051381:Rats; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018417:Th1 Cells / Q000378:metabolism; D018418:Th2 Cells / Q000378:metabolism
D000943:Antigens, Differentiation; D016207:Cytokines; D018796:Immunoconjugates; C406562:OX40Ig; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D000069594:Abatacept