Effects of interleukin-12p40 gene transfer on rat corneal allograft survival
Introduction
Despite the immunologically privileged nature of the cornea, immune mediated graft rejection remains the major cause of human corneal allograft failure. The activation of alloreactive T lymphocytes is considered to play a central role in this process [1], [2], [3]. Interleukin-12 (IL-12) is a key regulatory cytokine involved in the activation of T lymphocytes. IL-12 induces cellular immunity by promoting the proliferation of NK and T cells, the differentiation to Th1 cells and CTL- and NK-mediated cytolytic activity [4]. A significant IL-12 mRNA up-regulation was observed in rejected corneal allografts in rats [5]. In addition, the induction of immunity to intra-graft antigens in the draining lymph nodes (LN) after cornea transplantation occurs via an IL-12 and IFN-γ-dependent mechanism [6]. Therefore, IL-12 seems to be a potential target for intervention of corneal allograft rejection. The biologically active form of IL-12 is a 70 kDa heterodimer (IL-12p70), which consists of disulfide-bonded 40 kDa (p40) and 35 kDa (p35) subunits. It has been demonstrated that over-expression of IL-12p40 blocks the activities of IL-12p70 by competitively binding to the IL-12 receptor thereby reducing IL-12p70 mediated Th1 responses [7]. Moreover, it was shown that transfection of the IL-12p40 gene in myoblasts could prevent their rejection in allogeneic transplant recipients [8]. In addition, local expression of the immunoregulatory IL-12p40 gene also showed prolonged syngeneic islet graft survival in diabetic NOD mice [9].
Gene therapy is an interesting approach to introduce therapeutic genes into transplanted cells or tissues. Intra-graft over-expression of an immunomodulatory gene product may influence the allograft response. In contrast to other tissues routinely transplanted, the cornea has an additional advantage as a target for gene therapy [10], [11], [12], [13]. The cornea can be maintained under standard culture conditions for prolonged periods of up to 1 month, which allows time for genetic modification prior to transplantation. In addition, corneal endothelial cells are of particular interest for gene therapy because they are the major target of allograft rejection [1], [2], [3] and have a critical function in the maintenance of corneal transparency. Thus, modification of the endothelial cell genome with immunoregulatory molecules may influence the survival of corneal allografts. Previously, the recombinant adenovirus (Ad) has been shown to be an effective vehicle for transferring genes into corneal endothelial cells [14], [15]. Recently it has been shown that local over-expression of IL-12p40 in cultured corneas lead to significant prolongation of graft survival in a sheep transplant model [16] however only few mechanistic insights have been provided. Therefore our strategy for local expression of IL-12p40 was designed to inhibit local IL-12 production and subsequent secretion of IFN-γ and to study the underlying mechanisms.
Here we demonstrate that although IL-12p40 protein significantly inhibits the activation of naive T cells in vitro, over-expression of IL-12p40 mediated by recombinant adenovirus either locally or systemically could not protect corneal grafts from rejection in a MHC-class I/II full mismatched rat keratoplasty model.
Section snippets
Objectives
The objectives of this work were the following:
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To generate an adenoviral vector expressing the inhibitory subunit of interleukin-12, IL-12p40
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To investigate if IL-12p40 containing cell culture supernatants can inhibit production of IFN-gamma of naïve T cells
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To analyse if local and systemic over-expression of IL-12p40 can prevent allogeneic graft rejection in a rat corneal transplant model
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To investigate if over-expression of IL-12p40 modulates intra-graft cytokine gene expression
Generation of recombinant Adenovirus encoding for rat IL-12p40
The generation of replication-defective recombinant Ad expressing therapeutic genes has been previously described [15]. Briefly, E1-, E3-deleted Ad Type 5 vector encoding full-length rat IL-12p40 (AdIL-12p40) under the transcriptional control of the hCMV-IE was used. To generate this construct, the cDNA encoding for rat IL-12p40 was cloned into the pACCMV-vector followed by co-transfection together with an adenovirus Type 5 plasmid (pJM17) into 911 cells [17]. The formation of recombinant
Expression of IL-12p40 protein by cultured rat corneas
In order to evaluate the ex vivo duration of therapeutic gene expression, cultured rat corneas (n = 6) were transduced with AdrIL-12p40 (1 × 108 pfu/cornea). Supernatants containing IL-12p40 were harvested every 48 h and analysed for IL-12p40 protein expression. As shown in Fig. 1, AdIL-12p40 gene transfer led to significant cytokine production by transduced corneas. The expression of the therapeutic gene in cultured corneas reached a peak value at day 8 after transduction and continued at least
Discussion
In the present study, we investigated the immunomodulatory effects of adenovirus-mediated gene transfer of IL-12p40 both in vitro and in vivo. Although we demonstrate that IL-12p40 protein significantly inhibits the production of IFN-γ by alloreactive T cells in vitro, neither local gene transfer to cultured corneas before transplantation nor systemic delivery of AdrIL-12p40 resulted in prolongation of allograft survival in a rat keratoplasty model.
Beside the report by Klebe et al. [16] showing
Acknowledgements
The authors thank Dr. Bengt Widegren, Department of Cell and Molecular Biology, Section of Tumor Immunology, University of Lund, Lund, Sweden, for providing the rat IL-12p40 cDNA and Dr. Jürgen Bednarz, Eye Clinic, University Hospital Hamburg-Eppendorf, Germany, for providing human corneal endothelial cells (HCEC). In addition, the authors are grateful to Ms Michaela Schmack for technical assistance. This work was funded in part by DFG Pl 150/10-1 and Ri 764/6-1. Jun Yang was supported in part
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Cited by (0)
- 1
Present address: Brigham and Women's Hospital, Renal Division, Laboratory of Immunogenetics and Transplantation, 75 Francis Street, Boston, MA 02115, USA.
- 2
Present address: Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin-Buch, Robert-Rössle-Str. 10, 13092 Berlin (letters), 13125 Berlin (parcels), Germany.