基于转座子系统构建人类免疫缺陷病毒包膜蛋白嵌合细胞模型及其在嵌合抗原受体T细胞杀伤评估中的应用

Construction of a chimeric cell model expressing human immunodeficiency virus-1 envelope protein based on transposon system and evaluation of chimeric antigen receptor t-cell cytotoxicity in vitro

摘要:

人类免疫缺陷病毒1型(human immunodeficiency virus-1, HIV-1)的包膜蛋白(envelope, env)基因所编码的gp120/gp41复合物，不仅能够介导病毒进入宿主细胞，而且还是广谱中和抗体、基因治疗、膜融合抑制剂以及嵌合抗原受体T细胞(chimeric antigen receptor T-cell, CAR-T)疗法等多种新型抗病毒策略的核心作用靶点。为了建立稳定的HIV包膜蛋白细胞模型，用于靶向识别和杀伤功能的评价，本研究构建了6种定点突变弗林蛋白酶切割位点并敲除gp41胞质尾区序列的gp160(ΔCT)转座子表达质粒。通过与转座酶质粒共转染细胞后，经嘌呤霉素筛选，成功获得gp160(ΔCT)稳定表达细胞株。随后，利用间接流式细胞术评估3种中和抗体与上述6种细胞模型的结合活性，证明gp160(ΔCT)蛋白在细胞膜上表达并能被抗体识别。通过将该细胞模型以不同效靶比分别与3种不同的gp120-CAR-T细胞共孵育，利用钙黄绿素实验检测共培养上清中钙黄绿素释放，ELISA法检测颗粒酶B和穿孔素释放水平。结果表明，NL4-3-gp160(ΔCT)-293T细胞模型，表现出显著的细胞特异性杀伤效应，且呈剂量依赖性。此外，羧基荧光素琥珀酰亚胺酯(carboxyfluorescein succinimidyl ester, CFSE)细胞增殖实验结果显示，3种 CAR-T细胞在该模型细胞的刺激下均呈现显著且剂量依赖的增殖活性。与MLV-gp80(ΔCT)-293T阴性对照相比，本研究构建的膜表面稳定表达HIV-1 gp160(ΔCT)蛋白的HEK293T细胞模型，能够作为CAR-T细胞杀伤的靶细胞并特异性刺激CAR-T细胞增殖。该细胞模型可被用于广谱中和抗体、抗病毒药物及CAR-T细胞等新型疗法的靶向识别和杀伤功能验证，为HIV免疫学研究及细胞治疗药效评价提供了关键的实验平台。

Abstract:

The gp120/gp41 complex on the surface of human immunodeficiency virus type I (HIV-1) mediates viral entry into host cells and it is a primary target for the development of vaccines and antiviral interventions including broadly neutralizing antibodies (bNAbs), gene therapy, membrane fusion inhibitors, and CAR-T cell-based therapies. Therefore, stable cell models displaying the HIV envelope protein are needed to enable CAR-T cells to recognize and trigger their cytotoxic responses. We constructed a transposon-based plasmid system for the stable expression of six envelope proteins. A gp160(ΔCT) variant with a substituted furin cleavage site and a deleted gp41 cytoplasmic tail was constructed. Stable cell lines were generated by co-transfecting HEK293T cells with the transposon plasmid and a transposase plasmid, followed by puromycin selection. The membrane surface expression of the proteins and their recognition by the bNAbs were confirmed via indirect flow cytometry, which measured the binding activities of three neutralizing antibodies to gp160(ΔCT) proteins from diverse HIV-1 strains. The cell model was co-incubated with CAR-T cells at varying effector-to-target ratios. Subsequent calcein-AM release assays and ELISA quantification of granzyme B and perforin confirmed the dose-dependent and antigen-specific cytotoxicity mediated by all three CAR-T cell constructs. Furthermore, flow cytometric analysis using CFSE labeling revealed that all three CAR-T cell products exhibited pronounced, dose-dependent proliferation upon stimulation with the gp160(ΔCT) cell model. Collectively, the HEK293T model stably expressing membrane-anchored HIV-1 gp160(ΔCT) functioned as an effective target for CAR-T cells and robustly induced antigen-specific CAR-T cell proliferation, in comparison with the MLV-gp80(ΔCT)-293T negative control. The gp160(ΔCT)-expressing HEK293T model thus provides a robust platform for benchmarking the targeted recognition and cytotoxic activity of antiviral antibodies, agents, and CAR-T therapies in HIV research.