Immunohistological Investigation of Infiltrating Cells after Viral Interleukin-10 Transduction in Experimental Keratoplasty

Abstract

Abstract: : Purpose: To evaluate immunohistological changes after transplantation of ex-vivo gene-modified donor corneas expressing anti-inflammatory Interleukin-10 in a perforating keratoplasty model of the rat. Methods: Wistar-Furth rat donor corneas were genetically modified ex-vivo by Adenovirus (Ad)-mediated gene transfer (1x10⁸ pfu/cornea, 3h at 37°C) and transplanted in MHC-class I/II incompatible Lewis rats. Three groups were compared: grafts transduced with i) anti-inflammatory EBV-derived Interleukin-10 (AdvIL-10) (n=12), ii) control vector (Ad dl312) (n=13) and iii) untreated grafts (n=12). At postoperative days 10, 17 and 24 four rats of each group were killed and OCT-fixed corneas were analyzed by immunohistochemistry. Frozen sections were immunostained using the Avidin-Biotin Complex method with monoclonal antibodies against CD4, CD8, CD25 and CD45 cells, natural killer cells and monocytes. Cells were counted in one HPF with a magnification of 40. Results: The number of CD4 and CD8 cells was initially diminished in the therapeutic AdvIL-10 group. This effect disappeared at day 17 for the CD8 and at day 24 for the CD4 cells. For the other cell types no significant difference between the AdvIL-10 group and the control groups could be detected. Conclusions: Our results indicate that ex-vivo AdvIL-10 cornea transduction may affect early CD4 and CD8 cell infiltration. However, no long term difference of infiltration cells was observed.

Keywords: gene transfer/gene therapy • immunohistochemistry • transplantation