Suppression of alkali burn-induced corneal neovascularization by dendritic cell vaccination targeting VEGF receptor 2.
OBJECTIVE
To investigate whether the induction of cytotoxic T lymphocytes (CTLs) targeting VEGF receptor 2 inhibits corneal neovascularization caused by 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 6-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)-gamma and tumor necrosis factor (TNF)-alpha production and (51)Cr-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) compared with those not immunized or immunized with gp70. VEGFR2(400-408) or gp70, but not beta-gal(96-103), application led to dose-dependent induction of IFN-gamma and TNF-alpha in the CD8(+) T cells cocultured with stimulator cells. Cytotoxicity assays showed the specific lysis of major histocompatibility complex-matched cells expressing VEGFR2, but not beta-gal(96-103). In vivo depletion of CD8(+), but not CD4(+), T cells significantly reversed the suppressive effect of VEGFR2(400-408) immunization 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.
Mochimaru
Hiroshi
H
Laboratory of Retinal Cell Biology, Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan.
Usui
Tomohiko
T
Yaguchi
Tomonori
T
Nagahama
Yasuharu
Y
Hasegawa
Go
G
Usui
Yoshihiko
Y
Shimmura
Shigeto
S
Tsubota
Kazuo
K
Amano
Shiro
S
Kawakami
Yutaka
Y
Ishida
Susumu
S
D000818:Animals; D002057:Burns, Chemical / Q000276:immunology / Q000517:prevention & control*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D016510:Corneal Neovascularization / Q000276:immunology / Q000517:prevention & control*; D016207:Cytokines / Q000378:metabolism; D003601:Cytotoxicity Tests, Immunologic; D003713:Dendritic Cells / Q000276:immunology*; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005126:Eye Burns / Q000139:chemically induced*; D005434:Flow Cytometry; D008212:Lymphocyte Depletion; D016131:Lymphocyte Subsets; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000276:immunology; D012972:Sodium Hydroxide; D014611:Vaccination; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000276:immunology*
D016207:Cytokines; D000939:Epitopes; D010446:Peptide Fragments; D012972:Sodium Hydroxide; D040301:Vascular Endothelial Growth Factor Receptor-2