Immunization of Mice With Dendritic Cells Primed With Epitope Peptide Derived From Vegf Receptor 2 Supresses Choroidal Neovascularization

Abstract

Purpose:: To investigate a potential immunotherapy against VEGF receptor(VEGFR)2 using dendritic cells(DCs) to suppress the development of choroidal neovascularization (CNV).

Methods:: DCs were generated from bone marrow progenitors of C57BL/6 mice. Six-week-old C57BL/6 mice were immunized subcutaneously with 5×10⁵ peptide-pulsed mature DCs and 200µl vaccine mixture,which contained 100µg epitope peptide and 100µl IFA ,three times with seven-day intervals. After the third immunization, laser photocoagulation was performed to induce CNV. One week after photocoagulation, immunized mice were sacrificed and their choroids and splenocytes were prepared for the assays. CNV volume was evaluated by volumetric measurements. Fresh splenocytes, subjected to 1-week in vitro restimulation, served as effectors(cytotxic T lymphocytes;CTLs).The 2×10⁵effectors were co-cultured overnight with 1×10⁶ irradiated (40Gy) fresh splenocytes in 200µl medium at 37°C, 5 %CO₂ with the appropriate peptide concentration. After incubation, supernatant was collected and the concentration of IFN-γ and TNF-α were measured by ELISA.

Results:: The CNV volume in immunized mice with the epitope peptide derived from VEGFR2 (222578±154659µm³) was significantly lower than that in controls (356939±177076µm³).Cytokine assays showed specific production of IFN-γ and TNF-α from the CTLs induced with the epitope peptide.

Conclusions:: Significant suppression of CNV was observed in mice immunized with DCs pulsed with the epitope peptide derived from VEGFR2. The immunogenicity of the epitope peptides in the C57BL/6 mice was confirmed by cytokine assays.These results suggest that immunization targeting VEGFR2 is a novel therapeutic strategy to inhibit CNV.

Keywords: choroid: neovascularization