Suppression of Alkali Burn-Induced Corneal Neovascularization by Dendritic Cell Vaccination Targeting VEGF Receptor 2

¹ Laboratory of Retinal Cell Biology, Keio University School of Medicine, Tokyo, Japan; Ophthalmology, Keio University School of Medicine, Tokyo, Japan; Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan
² Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan
³ Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan
⁴ Ophthalmology, Tokyo Medical University, Shinjuku-ku, Japan
⁵ Ophthalmology, Keio University School of Medicine, Tokyo, Japan
⁶ Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Tokyo, 160-8582, Japan

^* To whom correspondence should be addressed. E-mail: ishidasu{at}sc.itc.keio.ac.jp.

	Abstract

Purpose: To investigate whether the induction of cytotoxic T lymphocytes (CTLs) targeting VEGF receptor 2 inhibits corneal neovascularization due to alkali injury. Methods: H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGF receptor 2 (VEGFR2_400-408) was used as an epitope peptide. Dendritic cells (DCs) were harvested from bone marrow progenitors of C57BL/6 mice. Six-week-old C57BL/6 mice received subcutaneous injections of VEGFR2_400-408- or gp70-pulsed mature DCs three times at six-day intervals. After the third immunization, corneal neovascularization was induced by alkali injury. Two weeks after the injury, the corneal vascularized area was evaluated by lectin angiography. To confirm the peptide-specific CTL activities in C57BL/6 mice, CD8⁺ T cells from immunized mice were subjected to ELISA for interferon (IFN)- and tumor necrosis factor (TNF)- production and ⁵¹Cr-release cytotoxicity assay. To determine the in vivo effector T cells, the immunized mice were intraperitoneally injected with an anti-CD4 or -CD8 depletion antibody. Results: Corneal neovascularization was significantly attenuated in mice immunized with VEGFR2_400-408 as compared to those not immunized or immunized with gp70. VEGFR2_400-408 or gp70, but not -gal_96-103, application led to dose-dependent induction of IFN- and TNF- in the CD8⁺ T cells cocultured with stimulator cells. Cytotoxicity assays showed the specific lysis of MHC-matched cells expressing VEGFR2, but not -gal_96-103. In vivo depletion of CD8⁺, but not CD4⁺, T cells significantly reversed the suppressive effect of VEGFR2_400-408 immuinization on corneal neovascularization to the level observed in nonimmunized or gp70-immunized animals. Conclusions: These results indicate the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for corneal chemical injury.

Key Words: cornea, neovascularization, VEGFR2, immunity