体外 Akt 抑制促进了高效 CD19CAR T 细胞的生成，用于过继免疫治疗。

BACKGROUND: Insufficient persistence and effector function of chimeric antigen receptor (CAR)-redirected T cells have been challenging issues for adoptive T cell therapy. Generating potent CAR T cells is of increasing importance in the field. Studies have demonstrated the importance of the Akt pathway in the regulation of T cell differentiation and memory formation. We now investigate whether inhibition of Akt signaling during ex vivo expansion of CAR T cells can promote the generation of CAR T cells with enhanced antitumor activity following adoptive therapy in a murine leukemia xenograft model. METHODS: Various T cell subsets including CD8+ T cells, bulk T cells, central memory T cells and naïve/memory T cells were isolated from PBMC of healthy donors, activated with CD3/CD28 beads, and transduced with a lentiviral vector encoding a second-generation CD19CAR containing a CD28 co-stimulatory domain. The transduced CD19CAR T cells were expanded in the presence of IL-2 (50U/mL) and Akt inhibitor (Akti) (1 μM) that were supplemented every other day. Proliferative/expansion potential, phenotypical characteristics and functionality of the propagated CD19CAR T cells were analyzed in vitro and in vivo after 17-21 day ex vivo expansion. Anti-tumor activity was evaluated after adoptive transfer of the CD19CAR T cells into CD19+ tumor-bearing immunodeficient mice. Tumor signals were monitored with biophotonic imaging, and survival rates were analyzed by the end of the experiments. RESULTS: We found that Akt inhibition did not compromise CD19CAR T cell proliferation and expansion in vitro, independent of the T cell subsets, as comparable CD19CAR T cell expansion was observed after culturing in the presence or absence of Akt inhibitor. Functionally, Akt inhibition did not dampen cell-mediated effector function, while Th1 cytokine production increased. With respect to phenotype, Akti-treated CD19CAR T cells expressed higher levels of CD62L and CD28 as compared to untreated CD19CAR T cells. Once adoptively transferred into CD19+ tumor-bearing mice, Akti treated CD19CAR T cells exhibited more antitumor activity than did untreated CD19CAR T cells. CONCLUSIONS: Inhibition of Akt signaling during ex vivo priming and expansion gives rise to CD19CAR T cell populations that display comparatively higher antitumor activity.

背景：嵌合抗原受体 (CAR) 靶向 T 细胞的持久性和效应功能不足一直是过继性 T 细胞治疗的挑战。在该领域，产生有效的 CAR T 细胞变得越来越重要。研究表明 Akt 途径在 T 细胞分化和记忆形成的调节中起着重要作用。我们现在研究在体外扩增 CAR T 细胞过程中抑制 Akt 信号是否可以促进在小鼠白血病异种移植模型中过继治疗后具有增强抗肿瘤活性的 CAR T 细胞的产生。

方法：从健康供者的 PBMC 中分离出各种 T 细胞亚群，包括 CD8+ T 细胞、 bulk T 细胞、中央记忆 T 细胞和幼稚/记忆 T 细胞，用 CD3/CD28 珠激活，并转导带有 CD28 共刺激结构域的第二代 CD19CAR 的慢病毒载体。在补充有 IL-2（50U/mL）和 Akt 抑制剂（Akti）（1 μM）的情况下，每天补充一次，对转导的 CD19CAR T 细胞进行扩增。在 17-21 天的体外扩增后，分析体外和体内扩增的 CD19CAR T 细胞的增殖/扩增潜力、表型特征和功能。将 CD19CAR T 细胞过继转移到携带 CD19+肿瘤的免疫缺陷小鼠后，评估其抗肿瘤活性。通过生物光子学成像监测肿瘤信号，并在实验结束时分析存活率。

结果：我们发现 Akt 抑制在体外不影响 CD19CAR T 细胞的增殖和扩增，与 T 细胞亚群无关，因为在存在或不存在 Akt 抑制剂的情况下培养后观察到相当的 CD19CAR T 细胞扩增。在功能上，Akt 抑制并没有抑制细胞介导的效应功能，而 Th1 细胞因子的产生增加。在表型方面，与未经处理的 CD19CAR T 细胞相比，Akti 处理的 CD19CAR T 细胞表达更高水平的 CD62L 和 CD28。一旦过继转移到携带 CD19+肿瘤的小鼠体内，与未经处理的 CD19CAR T 细胞相比，Akti 处理的 CD19CAR T 细胞表现出更强的抗肿瘤活性。

结论：在体外启动和扩增过程中抑制 Akt 信号会产生具有相对较高抗肿瘤活性的 CD19CAR T 细胞群体。