The Fas/FasL system is involved in, but not required for CD4+ T cell–mediated corneal allograft rejection.

Abstract

Abstract: : Purpose: We have previously shown that CD4⁺ T cells are involved as effector cells in corneal allograft rejection, but their exact effector mechanisms remain unclear. The role of Fas/FasL–induced apoptosis in CD4⁺ T cell–mediated graft rejection is examined in this study. Methods: A well–characterized mouse model of penetrating keratoplasty was used to examine effector mechanisms of corneal graft rejection. Wild type or Fas–deficient (lpr) corneal allografts were transplanted onto wild type or FasL–deficient (gld) hosts. CD4⁺ splenocytes from BALB/c–immunized C57BL/6 (B6) mice or from B6 mice that rejected BALB/c allografts were co–cultured in vitro with BALB/c corneal epithelial cells, endothelial cells, or keratocytes. Corneal cells were also incubated with agonistic anti–Fas MAb (Jo2). Apoptosis of corneal cells was detected by Annexin V binding and flow cytometry. Results: When BALB/c corneas were grafted onto FasL–deficient (B6.gld) hosts, 37.5% rejected compared to 100% of wild type B6 hosts (p < 0.05), implicating FasL in the host as a factor in graft rejection. However, when Fas–deficient (B6.lpr) corneas were grafted onto wild type BALB/c hosts, corneas rejected with similar incidence and tempo compated to wild type B6 donors (45–50%; p > 0.05). Agonistic anti–Fas MAb induced apoptosis of corneal endothelial cells and keratocytes in vitro, but not corneal epithelial cells. This apoptosis could be blocked by Fas–Fc chimeric protein. BALB/c corneal endothelial cells, epithelial cells, and keratocytes were all susceptible to allospecific CD4⁺ T cell–mediated apoptosis, none of which however could be blocked by anti–FasL blocking antibody or Fas–Fc. Allospecific CD4⁺ T cells from B6.gld mice did not induce apoptosis in BALB/c corneal endothelial cells or keratocytes, but did kill corneal epithelial cells. Conclusions: Our previous work has shown that CD4⁺ T cells help mediate corneal allograft rejection independently of macrophages, CD8⁺ T cells, or perforin. This study shows that allospecific CD4⁺ T cells contribute to corneal allograft rejection in a process involving FasL–mediated apoptosis. However, this manner of rejection can only take place in the endothelial or stromal layers of the cornea, not the epithelial layer. FasL–mediated apoptosis does not fully explain CD4⁺ T cell–mediated graft rejection, as some grafts still reject in the absence of Fas or FasL. Furthermore, our inability to block Fas–mediated rejection in vitro leads us to believe that other factors are involved in the CD4⁺ T cell–mediated apoptosis of allogeneic corneal cells.

Keywords: transplantation • apoptosis/cell death • cornea: basic science