Mochimaru Hiroshi, Nagai Norihiro, Hasegawa Go, Kudo-Saito Chie, Yaguchi Tomonori, Usui Yoshihiko, Kurihara Toshihide, Koto Takashi, Satofuka Shingo, Shinoda Hajime, Ozawa Yoko, Tsubota Kazuo, Kawakami Yutaka, Ishida Susumu
Laboratory of Retinal Cell Biology, Keio University School of Medicine, Shinanomachi, Tokyo, Japan.
Invest Ophthalmol Vis Sci. 2007 Oct;48(10):4795-801. doi: 10.1167/iovs.07-0425.
To investigate whether the induction of cellular immunity against vascular endothelial growth factor receptor (VEGFR) 2 inhibits the development of choroidal neovascularization (CNV).
H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGFR2 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 the epitope peptide-pulsed mature DCs three times at 6-day intervals. After the third immunization, laser photocoagulation was performed to induce CNV. One week after photocoagulation, mice were killed to harvest the choroid and splenocytes. CNV volume was evaluated by volumetric measurements. To confirm the specific immunogenicity of the epitope peptides in C57BL/6 mice, CD8 T cells isolated from harvested splenocytes were restimulated to measure interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production through enzyme-linked immunospot assay and ELISA. To determine the T-cell subset responsible for the immunotherapy, mice were intraperitoneally injected with an anti-CD4 or anti-CD8 depletion antibody.
CNV volume was significantly lower in mice immunized with the VEGFR2 epitope peptide than in those not immunized or immunized with a control peptide gp70. Cytokine assays showed the peptide-specific production of IFN-gamma and TNF-alpha from the CD8 T cells in a dose-dependent manner. In vivo depletion of CD8, but not CD4, T cells significantly reversed the suppressive effect of the VEGFR2 peptide-pulsed DC vaccination on CNV to the level observed in nonimmunized or gp70-immunized animals.
These results indicate that the VEGFR2 peptide-specific induction of cellular immunity inhibits CNV through the cytotoxicity of CD8 T cells. Results of the present study suggested the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for CNV.
研究诱导针对血管内皮生长因子受体（VEGFR）2的细胞免疫是否能抑制脉络膜新生血管（CNV）的形成。
将对应于VEGFR2第400至408位氨基酸的H-2Db限制性肽用作表位肽。从C57BL/6小鼠的骨髓祖细胞中收获树突状细胞（DC）。6周龄的C57BL/6小鼠每隔6天皮下注射3次表位肽脉冲成熟DC。第三次免疫后，进行激光光凝诱导CNV。光凝后1周，处死小鼠以收获脉络膜和脾细胞。通过体积测量评估CNV体积。为了确认表位肽在C57BL/6小鼠中的特异性免疫原性，对从收获的脾细胞中分离的CD8 T细胞进行再刺激，通过酶联免疫斑点测定和酶联免疫吸附测定来测量干扰素（IFN）-γ和肿瘤坏死因子（TNF）-α的产生。为了确定负责免疫治疗的T细胞亚群，给小鼠腹腔注射抗CD4或抗CD8清除抗体。
用VEGFR2表位肽免疫的小鼠的CNV体积明显低于未免疫或用对照肽gp70免疫的小鼠。细胞因子检测显示，CD8 T细胞以剂量依赖方式产生肽特异性的IFN-γ和TNF-α。体内清除CD8而不是CD4 T细胞显著逆转了VEGFR2肽脉冲DC疫苗接种对CNV的抑制作用，使其达到未免疫或gp70免疫动物中观察到的水平。
这些结果表明，VEGFR2肽特异性诱导的细胞免疫通过CD8 T细胞的细胞毒性抑制CNV。本研究结果提示，以VEGFR2为靶点的DC疫苗接种作为一种新的CNV治疗策略具有可能性。
