Table1_Regeneration of T cells from human-induced pluripotent stem cells for CAR-T cell medicated immunotherapy.DOCX

Background: Chimeric antigen receptor (CAR) T cell treatment involves in vitro 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.
Methods: The Alkaline phosphatase assay and MycoEasy rapid mycoplasma detection kit was implemented for detection of hiPSCs and mycoplasma, respectively. The CD34+ HSPCs were harvested in AggreWellTM 400 using a 37-micron reversible strainer. Likewise, the lymphoid progenitor and CD4+CD8+ 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 in vivo antitumor effects of the CAR-T cells were evaluated using experimental animals (mice).
Results: 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 in vivo and in vitro 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.
Conclusion: 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.

研究背景：嵌合抗原受体（Chimeric antigen receptor, CAR）T细胞疗法是指从患者血液中体外制备携带针对肿瘤抗原的合成受体的T细胞。该疗法可绕过主要组织相容性复合体（major histocompatibility complex, MHC）识别肿瘤抗原，使血液系统恶性肿瘤的缓解率降低80%。鉴于CAR-T疗法的疗效，本研究旨在从人诱导多能干细胞（human induced pluripotent stem cells, hiPSC）中制备功能性分化簇（clusters of differentiation, CD）8+ T细胞，并制备具备高抗原特异性细胞毒性的hiPS-CAR-T细胞。
研究方法：分别采用碱性磷酸酶检测法与MycoEasy快速支原体检测试剂盒，对hiPSC与支原体进行检测。使用37微米可逆滤器在AggreWell™ 400培养体系中收集CD34+造血干细胞/祖细胞（hematopoietic stem and progenitor cells, HSPCs）。同样方法收集淋巴系祖细胞与CD4+CD8+双阳性（double positive, DP）T细胞。采用细胞计数试剂盒-8（Cell Counting Kit-8, CCK-8）评估细胞毒性，采用酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）检测干扰素-γ（interferon-γ, IFN-γ）的分泌水平。此外，通过流式细胞术与Transwell小室分别评估细胞周期、迁移与侵袭能力。最后，借助实验动物（小鼠）评估CAR-T细胞的体内抗肿瘤效果。
研究结果：结果显示，无血清、无饲养层的分化体系在分化阶段添加白细胞介素-2（interleukin-2, IL-2）、白细胞介素-15（interleukin-15, IL-15）及激活剂，可高效诱导基于hiPSC的T细胞免疫疗法，促进这些细胞分化为人诱导多能干细胞来源T细胞（hiPS-T细胞）。通过CD19 CAR慢病毒感染hiPSC，可制备hiPSC-CAR-T细胞。本研究通过体内外实验验证了hiPS-CAR-T细胞的功能，结果表明，hiPSC诱导的hiPS-CAR-T细胞与外周血来源的CAR-T细胞在细胞形态与功能上无显著差异。
研究结论：本研究开发了一种高效且具备临床应用价值的培养方法，可从hiPSC中制备功能性CD8+ T细胞，并获得抗原特异性细胞毒性优异的hiPS-CAR-T细胞，其与外周血来源的CAR-T细胞差异极小。本研究结果可为利用hiPSC制备功能性CD8+ T细胞及hiPS-CAR-T细胞以应用于癌症细胞疗法的临床转化开辟新路径。