IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Santiago, Chile.
Cell for Cells, Santiago, Chile.
J Transl Med. 2024 Sep 27;22(1):868. doi: 10.1186/s12967-024-05627-4.
Apoptosis, a form of programmed cell death, is critical for the development and homeostasis of the immune system. Chimeric antigen receptor T (CAR-T) cell therapy, approved for hematologic cancers, retains several limitations and challenges associated with ex vivo manipulation, including CAR T-cell susceptibility to apoptosis. Therefore, strategies to improve T-cell survival and persistence are required. Mesenchymal stem/stromal cells (MSCs) exhibit immunoregulatory and tissue-restoring potential. We have previously shown that the transfer of umbilical cord MSC (UC-MSC)-derived mitochondrial (MitoT) prompts the genetic reprogramming of CD3 T cells towards a T cell lineage. The potency of T cells plays an important role in effective immunotherapy, underscoring the need for improving their metabolic fitness. In the present work, we evaluate the effect of MitoT on apoptotis of native T lymphocytes and engineered CAR-T cells.
We used a cell-free approach using artificial MitoT (Mitoception) of UC-MSC derived MT to peripheral blood mononuclear cells (PBMCs) followed by RNA-seq analysis of CD3 MitoT and MitoT sorted cells. Target cell apoptosis was induced with Staurosporine (STS), and cell viability was evaluated with Annexin V/7AAD and TUNEL assays. Changes in apoptotic regulators were assessed by flow cytometry, western blot, and qRT-PCR. The effect of MitoT on 19BBz CAR T-cell apoptosis in response to electroporation with a non-viral transposon-based vector was assessed with Annexin V/7AAD.
Gene expression related to apoptosis, cell death and/or responses to different stimuli was modified in CD3 T cells after Mitoception. CD3MitoT cells were resistant to STS-induced apoptosis compared to MitoT cells, showing a decreased percentage in apoptotic T cells as well as in TUNEL cells. Additionally, MitoT prevented the STS-induced collapse of the mitochondrial membrane potential (MMP) levels, decreased caspase-3 cleavage, increased BCL2 transcript levels and BCL-2-related BARD1 expression in FACS-sorted CD3 T cells. Furthermore, UC-MSC-derived MitoT reduced both early and late apoptosis in CAR-T cells following electroporation, and exhibited an increasing trend in cytotoxic activity levels.
Artificial MitoT prevents STS-induced apoptosis of human CD3 T cells by interfering with the caspase pathway. Furthermore, we observed that MitoT confers protection to apoptosis induced by electroporation in MitoT CAR T-engineered cells, potentially improving their metabolic fitness and resistance to environmental stress. These results widen the physiological perspective of organelle-based therapies in immune conditions while offering potential avenues to enhance CAR-T treatment outcomes where their viability is compromised.
细胞凋亡是一种程序性细胞死亡形式，对于免疫系统的发育和稳态至关重要。嵌合抗原受体 T（CAR-T）细胞疗法已被批准用于血液系统癌症，但仍存在与体外操作相关的一些限制和挑战，包括 CAR-T 细胞对细胞凋亡的敏感性。因此，需要提高 T 细胞的存活率和持久性的策略。间充质干细胞（MSC）具有免疫调节和组织修复潜力。我们之前已经表明，脐带 MSC（UC-MSC）衍生的线粒体（MitoT）的转移促使 CD3 T 细胞向 T 细胞谱系进行基因重编程。T 细胞的效力在有效的免疫治疗中起着重要作用，这凸显了改善其代谢适应性的必要性。在本工作中，我们评估了 MitoT 对天然 T 淋巴细胞和工程 CAR-T 细胞凋亡的影响。
我们使用了一种无细胞方法，即用 UC-MSC 衍生的 MT 的人工 MitoT（Mitoception）处理外周血单核细胞（PBMCs），然后对 CD3 MitoT 和 MitoT 分选细胞进行 RNA-seq 分析。用 Staurosporine（STS）诱导靶细胞凋亡，用 Annexin V/7AAD 和 TUNEL 测定法评估细胞活力。通过流式细胞术、Western blot 和 qRT-PCR 评估凋亡调节剂的变化。用电穿孔用非病毒转座子基载体评估 MitoT 对 19BBz CAR-T 细胞对 STS 诱导的凋亡的影响。
在接受 Mitoception 后，CD3 T 细胞中的与细胞凋亡、细胞死亡和/或对不同刺激的反应相关的基因表达发生改变。与 MitoT 细胞相比，CD3MitoT 细胞对 STS 诱导的凋亡具有抗性，表现为凋亡 T 细胞以及 TUNEL 细胞的百分比降低。此外，MitoT 可防止 STS 诱导的线粒体膜电位（MMP）水平崩溃，减少 caspase-3 切割，增加 FACS 分选 CD3 T 细胞中的 BCL2 转录物水平和 BCL-2 相关 BARD1 表达。此外，UC-MSC 衍生的 MitoT 降低了电穿孔后 CAR-T 细胞中早期和晚期的凋亡，并表现出细胞毒性活性水平增加的趋势。
人工 MitoT 通过干扰半胱氨酸蛋白酶途径来防止 STS 诱导的人 CD3 T 细胞凋亡。此外，我们观察到 MitoT 可赋予经电穿孔的 MitoT CAR-T 工程细胞凋亡诱导的保护作用，这可能会改善其代谢适应性并增强其对环境压力的抵抗力。这些结果拓宽了细胞器疗法在免疫条件下的生理视角，同时为增强 CAR-T 治疗效果提供了潜在途径，因为其活力受到了损害。
