Role Of Lentivirus-Mediated Overexpression Of Programmed Death Ligand-1 On Corneal Allograft Survival

Abstract

Purpose: : To investigate the role of lentivirus-mediated overexpression of Programmed Death Ligand-1 (PDL-1) on rat corneal allograft survival.

Methods: : Lentivirus (LV) encoding for PDL-1 (LV.PDL-1) and EGFP (LV.EGFP, as control) were constructed and tested in vitro for mRNA and protein expression by real-time RT-PCR and flow cytometry. A fully allogeneic rat cornea transplant model (DA to LEW) was used for in vivo studies. Ex-vivo cultured corneas from DA rats were transduced with LV.PDL-1 and LV.EGFP and transplanted into LEW recipients. Graft survival and neovascularization was monitored and immunohistochemistry was used to analyze lymphocyte infiltration in the cornea. Moreover, flow cytometric analysis of collagenase digested corneas and draining lymph nodes was applied to study immune cell distribution and function in allo-transplanted animals.

Results: : LV.PDL-1 and LV.EGFP were routinely produced in high titers. An increase of EGFP expression was observed after LV.EGFP transduction for 1, 4 and 20h of incubation of cultured corneas. PDL-1 expression was increased on corneal cells upon LV.PDL-1 transduction compared to vector-treated corneas. In vivo untreated allogeneic controls and allogeneic LV.EGFP transduced corneas were uniformly rejected (MST 13.8±1.7d, n=11 and 12.3±1.9d, n=4, respectively). In contrast, allogeneic LV.PDL-1 transduced corneas showed a high percentage (83%) of graft survival (MST >30d, n=5, 15d, n=1). Interestingly, the dynamics of opacity score close to expected rejection time was similar for LV.PDL-1 and LV.GFP treated groups which assumes that over-expression of PDL-1 does not protect the cornea from lymphocyte infiltration but rather from rejection. Time point analysis revealed reduced expression of pro-inflammatory cytokines (IFN-γ) by infiltrating lymphocytes in PDL-1 transduced corneas compared to allogeneic controls.

Conclusions: : Lentiviral vectors are efficient tools for ex vivo genetic modification of cultured corneas. Local PDL-1 gene transfer is a promising approach for the prevention of corneal allograft rejection.

Keywords: transplantation • immunomodulation/immunoregulation • gene transfer/gene therapy