一种用于测量定制T细胞针对HIV Env(+)细胞功能的新型实时CTL检测法。
A novel real-time CTL assay to measure designer T-cell function against HIV Env(+) cells.
作者信息
MacLean Andrew G, Walker Edith, Sahu Gautam K, Skowron Gail, Marx Preston, von Laer Dorothee, Junghans Richard P, Braun Stephen E
机构信息
Tulane National Primate Research Center, Covington, LA, USA.
出版信息
J Med Primatol. 2014 Oct;43(5):341-8. doi: 10.1111/jmp.12137. Epub 2014 Aug 20.
BACKGROUND
To increase the immunosurveillance in HIV infection, we used retroviral vectors expressing CD4-chimeric antigen receptors (CARs) to genetically modify autologous T cells and redirect CTL toward HIV. The CD4 extracellular domain targets envelope and the intracellular signaling domains activate T cells. The maC46 fusion inhibitor binds HIV and blocks viral replication.
METHODS
We stimulated rhesus PBMCs with antibodies to CD3/CD28 and cotransduced T cells with CD4-CAR and maC46 vectors. CD4-CAR-transduced T cells were added to Env(+) 293T cells at E:T of 1:1. Killing of target cells was measured as reduced impedance.
RESULTS
We observed gene expression in 60-70% of rhesus CD3(+) CD8(+) T cells with the individual vectors and in 35% of the cells with both vectors. CD4-CAR-transduced populations specifically killed Env(+) cells.
CONCLUSIONS
In these studies, we showed that designer T cells were redirected to kill Env(+) cells. Control of viremia without HAART would revolutionize treatment for HIV patients.
背景
为增强对HIV感染的免疫监视，我们使用表达CD4嵌合抗原受体（CAR）的逆转录病毒载体对自体T细胞进行基因改造，使细胞毒性T淋巴细胞（CTL）重新靶向HIV。CD4细胞外结构域靶向包膜，细胞内信号结构域激活T细胞。maC46融合抑制剂可结合HIV并阻断病毒复制。
方法
我们用抗CD3/CD28抗体刺激恒河猴外周血单个核细胞（PBMC），并用CD4-CAR和maC46载体共转导T细胞。将转导了CD4-CAR的T细胞以1:1的效应细胞与靶细胞比例（E:T）加入Env(+) 293T细胞中。通过测量阻抗降低来检测靶细胞的杀伤情况。
结果
我们观察到，单独使用载体时，60%-70%的恒河猴CD3(+) CD8(+) T细胞有基因表达，同时使用两种载体时，35%的细胞有基因表达。转导了CD4-CAR的细胞群体可特异性杀伤Env(+)细胞。
结论
在这些研究中，我们表明经过设计的T细胞可重新定向以杀伤Env(+)细胞。无需高效抗逆转录病毒治疗（HAART）即可控制病毒血症将彻底改变HIV患者的治疗方式。
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