Effects of interleukin-12p40 gene transfer on rat corneal allograft survival.
OBJECTIVE
Despite the immunologically privileged nature of the cornea, graft rejection remains the major cause of human corneal allograft failure. Gene therapy is an interesting approach to introduce immunoregulatory molecules into the graft or the recipient to prevent rejection. In this study we investigated the immmunomodulatory effects of adenovirus-mediated gene transfer of a Th1 antagonist, interleukin-12p40 (IL-12p40), in vitro and on allogeneic graft survival in a rat experimental keratoplasty model.


METHODS
Donor corneas were transduced with an E1/E3 deleted adenoviral (Ad) vector encoding the IL-12p40 gene (AdIL-12p40) and assayed for the expression of the therapeutic gene. Cell culture supernatants containing IL-12p40 protein were generated by transducing human corneal endothelial cells with AdIL-12p40 and analysed for their capacity to inhibit production of IFN-gamma by naive T cells. The effect of both local (ex vivo Ad-mediated gene transfer) and systemic (i.p.-injection) over-expression of IL-12p40 was investigated by analysing the survival of corneal allografts transplanted from Wistar-Furth rats to fully MHC-class I/II incompatible Lewis rats. Moreover, the intra-graft mRNA-expression profile of cytokines and T cell markers was investigated at different time points after gene transfer.


RESULTS
Adenovirus-mediated gene transfer in cultured corneas led to significant IL-12p40 protein expression as determined by specific ELISA. Moreover we could show that IL-12p40 protein containing supernatants significantly inhibited the production of IFN-gamma by alloreactive naive T cells. Interestingly, neither ex vivo genetic modification of cultured corneas before transplantation nor systemic AdIL-12p40 treatment of recipients receiving allogeneic corneas did improve corneal allograft survival. Real-time RT-PCR analysis of ex vivo modified cornea allografts on day 7 after transplantation showed significantly higher IL-4 mRNA-expression levels in the AdIL-12p40 group compared to the control group. Other significant differences in mRNA-expression levels of intra-graft CD3, CD25, IFN-gamma, TNF-alpha, and IL-10 could not be detected, neither on day 7 nor on the day of rejection.


CONCLUSIONS
Despite the capacity of IL-12p40 protein to inhibit the production of IFN-gamma of naive T cells in vitro and some Th1/Th2 shift in vivo, no prolongation of allogeneic graft survival of both AdIL-12p40 modified rat corneas and systemically treated rats could be obtained after transplantation. The possible binding of Ad-mediated IL-12p40 with ubiquitously expressed IL-12p35 in vivo might therefore limit the application of IL-12p40 for the prevention of transplant rejection.
Ritter
Thomas
T
Institute of Medical Immunology, Charité-University Medicine Berlin, Monbijoustrasse 2a, 10117 Berlin, Germany. thomas.ritter@nuigalway.ie
Yang
Jun
J
Dannowski
Haike
H
Vogt
Katrin
K
Volk
Hans-Dieter
HD
Pleyer
Uwe
U
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D016039:Corneal Transplantation / Q000276:immunology* / Q000379:methods*; D016207:Cytokines / Q000096:biosynthesis / Q000276:immunology; D005260:Female; D018014:Gene Transfer Techniques; D006084:Graft Rejection / Q000517:prevention & control; D006085:Graft Survival / Q000235:genetics / Q000276:immunology*; D007371:Interferon-gamma / Q000096:biosynthesis / Q000276:immunology; D053762:Interleukin-12 Subunit p40 / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011917:Rats, Inbred Lew; D011920:Rats, Inbred WF; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D016207:Cytokines; D053762:Interleukin-12 Subunit p40; D012333:RNA, Messenger; D007371:Interferon-gamma