间充质干细胞线粒体转移赋予天然 T 细胞与工程化 T 细胞的生存优势

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 reg 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 pos and MitoT neg 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. CD3 + MitoT pos cells were resistant to STS-induced apoptosis compared to MitoT neg 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 pos 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.