Artificial mitochondria transfer prevents staurosporine-induced apoptosis of human T lymphocytes.

Background & Aim The immunoregulatory and tissue restoring effects of Mesenchymal stem cells (MSCs) are mediated by the production of bioactive paracrine factors. While soluble proteins and extracellular vesicles are the main contributory, evidence suggests that mitochondrial transfer from MSCs (MitoT) may also play a significant role. In actual fact, we have demonstrated that MitoT to lymphoid cells represent a novel immune reprogramming mechanism that elicits a marked clinical improvement of GVHD. In the current work, we adopt a cell-free approach by evaluating the effect of artificial transfer of mitochondria isolated from umbilical cord-MSC (UC-MSCs) on T lymphocyte survival. Methods, Results & Conclusion Methods: Artificial transfer of mitochondria to peripheral blood mononuclear cells (PBMCs) was performed following the mitoception protocol. Briefly, UC-MSC-derived mitochondria were labelled with a mitoTracker then incubated with cells at a defined ratio. Apoptosis was then induced using Staurosporine (STS) at different concentrations. Cell viability of mitocepted (CD3+Mito+) and mitoception-resistant (CD3+Mito-) lymphocytes was evaluated by flow cytometry following a 7- AAD/annexinV staining. Mitochondria membrane potential (MMP)) was measured using a JC-1 dye. The activation level of caspase-3, a pro-apoptotic protein, was evaluated following its cleavage by western blot, comparing both T cell populations. Results CD3+Mito+ cells showed a significant resistance to STD-induced apoptosis in comparison with mitoception resistant (CD3+Mito-) lymphocytes (22.98% versus 34.48% at 10 μM of STS; and 22.39% versus 43.18% at 20 μM of STS, respectively). Interestingly, mitochondria transfer prevented the collapse of the MMP levels (0,67 versus 0,19 with 10 μM of STS; and 0,60 versus 0,10 with 20 μM of STS when comparing Mito+ and Mito- populations, respectively). When caspase-3 activation was measured following protein cleavage, CD3+Mito- population showed a 1,7 fold increase of caspase-3 cleavage in comparison with Mito+ population at 10 μM of STS. In conclusion, artificial mitochondrial transfer prevents staurosporine-induced apoptosis of human T lymphocytes by interfering with the caspase pathway. These results widens the perspective use of cell free, organelle-based therapies in immune conditions where viability of T lymphocytes is compromised.