Allogeneic Sensitization and Tolerance Induction After Corneal Endothelial Cell Transplantation in Mice.

Jun Yamada,Chie Sotozono,Shigeru Kinoshita,Katsuhiko Shinomiya,Morio Ueno,Munetoyo Toda,Junji Hamuro

doi:10.1167/iovs.15-19020

Abstract

We evaluated the allogeneic response after corneal endothelial cell transplantation in the anterior chamber (AC) in a new mouse model by examining the acquisition of a delayed-type hypersensitivity (DTH) response, induction of allogeneic AC-associated immune deviation (ACAID), and acquisition of delayed transplantation tolerance. The corneal eyecups from C57BL/6 mice were prepared. The epithelial layer was detached with EDTA solution and treated with trypsin to release mouse-derived primary corneal endothelial cells (mpCECs). The mpCECs (1 × 104 cells) were transplanted into the AC of the eye or subcutaneously (SC) into the neck of BALB/c mice. In the mouse model of endothelial cell transplantation, the endothelial cells in a 2-mm central area of the cornea were eliminated by cryoinjury. The mpCEC transplant model was evaluated by measuring allogeneic cell survival and corneal thickness. The allospecific DTH response and ACAID induction were evaluated 1 week after transplantation. The long-term transplantation tolerance was evaluated by observing a secondary penetrating keratoplasty (PKP) performed on the same donor C57BL/6 mice. The SC injection of mpCECs induced a DTH response, whereas the AC injection induced ACAID. However, eyes inflamed by cryoinjury showed neither the DTH response nor ACAID following AC injection. The mpCECs survived for at least 1 week after injection. Penetrating keratoplasty allografts at 8 weeks after mpCEC transplantation survived indefinitely (100%). The mpCECs display low allogenicity in the AC and are capable of inducing allogeneic tolerance. Corneal endothelial cell transplantation into the AC may represent a safe technique for allogeneic transplantation.

Highlights

We evaluated the allogeneic response after corneal endothelial cell transplantation in the anterior chamber (AC) in a new mouse model by examining the acquisition of a delayedtype hypersensitivity (DTH) response, induction of allogeneic AC-associated immune deviation (ACAID), and acquisition of delayed transplantation tolerance
Corneal endothelial cell transplantation into the AC may represent a safe technique for allogeneic transplantation
Because corneal endothelial cells (CECs) in humans are severely limited in their proliferative ability,[2,3,4] restoration to clear vision has required corneal transplantation in the form of full-thickness corneal transplantation, Descemet’s stripping automated endothelial keratoplasty (DSAEK), or Descemet’s membrane endothelial keratoplasty (DMEK).[5,6,7]

Summary

Methods

Male BALB/c (H-2d), C57BL/6 (H-2b), and C3H/HeN (H-2k) mice (SLC, Osaka, Japan), aged between 8 and 12 weeks, were used in the experiments. All experiments were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine. The corneal eyecups, excluding the corneal limbus, were dissected from donor C57BL/6 mice and incubated in 20 mM EDTA at 378C for 20 minutes, followed by a wash with sterile Hanks’s balanced salt solution (HBSS). The resulting stroma-endothelium eyecups were incubated in Tryple Express (Invitrogen Co., Carlsbad, CA, USA), containing EDTA-Trypsin, at 378C for 30 minutes. Almost 1.5 3 105 mpCECs could be collected from 10 corneas. The mpCECs were labeled using a PKH26 Red Fluorescent cell linker kit (Sigma-Aldrich Corp., St. Louis, MO, USA) before transplantation. P values less than 0.05 were deemed statistically significant

Results

Discussion

Conclusion