Abstract
Purpose:
Every corneal layer contributes to immunogenicity of a corneal graft. We previously demonstrated that a composite graft with an allogeneic epithelial layer overexpressing anti-apoptotic p35 resulted in significantly increased graft acceptance and enhanced epithelial barrier integrity (compared to untreated allogeneic epithelia, mouse corneal transplantation model). The purpose of this study was to further study mechanisms regulating the immune-privileged status of the cornea following epithelial gene therapy.
Methods:
4 different groups of composite corneal grafts were compared in C57BL/6 (B6) and BALB/c (B/c) mice (follow-up 3 wks): (#1) syngeneic B/c epithelium (EPI) + B/c stroma-endothelium (S-E) (n=5); (#2) allogeneic B6 EPI, untreated + B6 S-E (n=5); (#3) B6 EPI lenti-GFP [empty vector] + B6 S-E (n=4); (#4) B6 EPI lenti-p35 + B6 S-E (n=4). T cells from ipsilateral cervical lymph nodes were studied in cell suspensions. Isolated lymphocytes were stained with anti-mouse CD4 FITC, CD25 PE, CD69 PE and Foxp3 PECy5. Mixed lymphocyte reaction (MLR) and colorimetric BrdU proliferation assay were included, using magnetically separated T cells and splenocytes, to demonstrate priming of CD4+ T cells. In addition, to determine the functionality of MLR cultures, we employed sandwich ELISA for INF-γ and IL-10 measurements.<br />
Results:
Post-transplantation B6 EPI lenti-p35 + B6 S-E (# 4) and B/c EPI + B/c S-E (# 1) showed improved graft survival and reduced total number of cells in cervical lymph nodes compared to other allogeneic groups, untreated or empty vector. In MLR we detected decreased frequency of CD4+ cells in p35 group being more comparable with naïve and syngeneic transplantation. Moreover, the diminished phenotype of CD4 in p35 group was confirmed by significantly lower proliferation in Brdu assay and lower INF-γ concentration in ELISA.
Conclusions:
Donor (B6) epithelial gene therapy decreases later allosensitization of the recipient by decreased priming of CD4+ cells in cervical lymph nodes. We therefore believe that gene-mediated anti-inflammatory and anti-apoptotic effects could positively affect allosensitization of the recipient’s CD4+ T cells in cervical lymph nodes and thus increase graft survival.