Ge Ping-ling, Ma Li-ping, Wang Wei, Li Yun, Zhao Wen-ming
Department of Transfusion, Aerospace Center Hospital, Beijing, China.
Chin Med J (Engl). 2009 May 5;122(9):1039-48.
Arthritogenic T lymphocytes with common T cell receptor (TCR) Vbeta clonotypes, infiltrating in the articulars of rheumatoid arthritis (RA) patients, play a central role in the pathogenesis of RA. TCR Vbeta5.2 and TCR Vbeta8.2 are the main pathogenic T cell clonotypes in the course of collagen-induced arthritis (CIA) progression in Lewis rats. To investigate a TCR-based immunotherapy for RA, we constructed recombinant DNA vaccines encoding TCR Vbeta5.2 and TCR Vbeta8.2, and evaluated the inhibitive effects of the two vaccines on CIA rats.
Genes encoding TCR Vbeta5.2 and TCR Vbeta8.2 were amplified by RT-PCR from spleen lymphocytes of Lewis rats and cloned into the eukaryotic expression vector pTargeT. The expression of vaccines was confirmed by RT-PCR and immunohistochemistry. The inhibitive effects of the vaccines on articulars of CIA rats were assessed with arthritis index evaluation and histology. Interferon gamma (IFN-gamma) and interleukin (IL)-4 production by spleen lymphocytes were tested with enzyme-linked immunospot assay (ELISPOT) technique, the changes in peripheral CD4(+) and CD8(+) lymphocyte populations were tested by flow cytometry, and the level of anti-CII antibody in serum was assayed by enzyme-linked immunosorbent assay (ELISA).
Recombinant DNA vaccines pTargeT-TCR Vbeta5.2 and pTargeT-pTCR Vbeta8.2 were successfully constructed. Both vaccines inhibited CIA, which alleviated the arthritis index score (P < 0.05), decreased the level of IFN-gamma (P < 0.05), and reduced the ratio of CD4(+)/CD8(+) lymphocytes (P < 0.05) and the anti-CII antibody in serum (P < 0.05). In addition, the histological change in DNA-vaccinated rats was less serious than CIA rats. Compared to pTCR Vbeta 8.2 and pTCR Vbeta 5.2 groups, the group that was injected with a combination of the two vaccines showed stronger inhibitive effects on CIA than either individual vaccine.
The recombinant plasmids pTargeT-TCR Vbeta5.2 and pTargeT-TCR Vbeta8.2 have obvious inhibatory effects on CIA rats and better effects could be achieved when the vaccines were used in combination.
具有共同T细胞受体（TCR）Vβ克隆型的致关节炎T淋巴细胞浸润类风湿关节炎（RA）患者的关节，在RA发病机制中起核心作用。TCR Vβ5.2和TCR Vβ8.2是Lewis大鼠胶原诱导性关节炎（CIA）进展过程中的主要致病性T细胞克隆型。为研究基于TCR的RA免疫疗法，我们构建了编码TCR Vβ5.2和TCR Vβ8.2的重组DNA疫苗，并评估了这两种疫苗对CIA大鼠的抑制作用。
通过RT-PCR从Lewis大鼠脾淋巴细胞中扩增编码TCR Vβ5.2和TCR Vβ8.2的基因，并克隆到真核表达载体pTargeT中。通过RT-PCR和免疫组织化学确认疫苗的表达。用关节炎指数评估和组织学评估疫苗对CIA大鼠关节的抑制作用。用酶联免疫斑点分析（ELISPOT）技术检测脾淋巴细胞产生的干扰素γ（IFN-γ）和白细胞介素（IL）-4，用流式细胞术检测外周血CD4(+)和CD8(+)淋巴细胞群体的变化，并用酶联免疫吸附测定（ELISA）检测血清中抗CII抗体的水平。
成功构建了重组DNA疫苗pTargeT-TCR Vβ5.2和pTargeT-pTCR Vβ8.2。两种疫苗均抑制CIA，减轻关节炎指数评分（P < 0.05），降低IFN-γ水平（P < 0.05），降低CD4(+)/CD8(+)淋巴细胞比例（P < 0.05）和血清中抗CII抗体水平（P < 0.05）。此外，接种DNA疫苗的大鼠的组织学变化比CIA大鼠轻。与pTCR Vβ 8.2和pTCR Vβ 5.2组相比，注射两种疫苗组合的组对CIA的抑制作用比单独使用任何一种疫苗都更强。
重组质粒pTargeT-TCR Vβ5.2和pTargeT-TCR Vβ8.2对CIA大鼠有明显的抑制作用，联合使用疫苗可取得更好的效果。
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