Chen Yanyan, Huang Pufeng, Niu Mengda, Tian Chuanhuizi, Zhang Tingting, Peng Zhiping
Department of Radiation Medicine, School of Basic Medicine, Chongqing Medical University, Chongqing, China.
Front Bioeng Biotechnol. 2023 May 18;11:1159507. doi: 10.3389/fbioe.2023.1159507. eCollection 2023.
Chimeric antigen receptor (CAR) T cell treatment involves production of T cells from patient blood with synthetic receptors specific to a cancer antigen. They circumvent the major histocompatibility complex to recognize the tumor antigen, reducing hematologic malignancy remission rates by 80%. Considering the efficacy of CAR-T treatment, the present work aimed at generating functional clusters of differentiation (CD)8 + T cells from human induced pluripotent stem cells (hiPSC) and to generate hiPS-CAR-T cells with high antigen-specific cytotoxicity. The Alkaline phosphatase assay and MycoEasy rapid detection kit was implemented for detection of hiPSCs and , respectively. The CD34 HSPCs were harvested in AggreWellTM 400 using a 37-micron reversible strainer. Likewise, the lymphoid progenitor and CD4CD8 DP T cells were also harvested. The Cell Counting Kit-8 (CCK-8) assay was used to mark cytotoxicity and ELISA was used to detect IFN-γ secretion. Further, flow cytometry and transwell chambers were used to assess cell cycle, and migration and invasion. Finally, the antitumor effects of the CAR-T cells were evaluated using experimental animals (mice). Results revealed that a serum-free, feeder layer-free differentiation system significantly yielded hiPSC-based T cell immunotherapy with interleukin-2, interleukin-15, and activators at the differentiation stage to promote the maturation of these cells into human induced pluripotent stem (hiPS)-T cells. The infection of hiPSCs with the CD19 CAR lentivirus resulted in the production of the hiPSC-CAR-T cells. We validated the function of hiPS-CAR-T cells and experimentation which revealed no significant differences in cell morphology and function between hiPSC-derived hiPS-CAR-T cells and peripheral blood-derived CAR-T cells. This study developed a culture method that is efficient and clinically useful to make functional CD8 T cells from hiPSC and to get hiPS-CAR-T cells with high antigen-specific cytotoxicity that are not very different from CAR T cells found in peripheral blood. As a result, our findings may open the way for the clinical use of hiPSC to create functional CD8 T and hiPS-CAR-T cells cells for use in cell-based cancer therapy.
嵌合抗原受体（CAR）T细胞治疗涉及从患者血液中产生T细胞，并使其带有针对癌症抗原的合成受体。它们绕过主要组织相容性复合体来识别肿瘤抗原，使血液系统恶性肿瘤缓解率降低80%。考虑到CAR-T治疗的疗效，本研究旨在从人诱导多能干细胞（hiPSC）中生成功能性分化簇（CD）8 + T细胞，并生成具有高抗原特异性细胞毒性的hiPS-CAR-T细胞。分别使用碱性磷酸酶检测法和MycoEasy快速检测试剂盒检测hiPSC。使用37微米可逆滤网在AggreWellTM 400中收获CD34造血干细胞。同样，也收获了淋巴样祖细胞和CD4CD8双阳性T细胞。使用细胞计数试剂盒-8（CCK-8）检测法标记细胞毒性，使用酶联免疫吸附测定（ELISA）检测γ干扰素分泌。此外，使用流式细胞术和Transwell小室评估细胞周期以及迁移和侵袭能力。最后，使用实验动物（小鼠）评估CAR-T细胞的抗肿瘤作用。结果显示，一种无血清、无饲养层的分化系统在分化阶段显著产生了基于hiPSC的T细胞免疫疗法，该疗法使用白细胞介素-2、白细胞介素-15和激活剂来促进这些细胞成熟为人诱导多能干细胞（hiPS）-T细胞。用CD19 CAR慢病毒感染hiPSC导致产生hiPSC-CAR-T细胞。我们验证了hiPS-CAR-T细胞的功能，实验表明hiPSC来源的hiPS-CAR-T细胞与外周血来源的CAR-T细胞在细胞形态和功能上没有显著差异。本研究开发了一种高效且对临床有用的培养方法，可从hiPSC中制备功能性CD8 T细胞，并获得具有高抗原特异性细胞毒性的hiPS-CAR-T细胞，这些细胞与外周血中的CAR-T细胞没有太大差异。因此，我们的研究结果可能为hiPSC在临床中的应用开辟道路，以创建用于基于细胞的癌症治疗的功能性CD8 T细胞和hiPS-CAR-T细胞。
