Survival advantage of native and engineered T cells is acquired by mitochondrial transfer from mesenchymal stem cells

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Chimeric antigen receptor T (CAR-T) cells; Induced-apoptosis; Mesenchymal stromal/stem cells; Mitochondria transfer.

Fig. 1

Experimental workflow of MitoT to native and CAR engineered T cells. This schematic diagram illustrates the experimental methodology used to isolate mitochondria from umbilical cord MSCs after their transfer into T cells and subsequently challenge them by staurorsporine or through DNA electroporation for CAR-T generation. Apoptosis was then assessed using various markers and membrane potential measurements

Fig. 2

Transfer of UC-MSC-derived MT modifies T lymphocyte response to stimulus and apoptosis gene expression. A Volcano plot analysis derived from FPKM values showing DE genes (p < 0.05, Student’s t test). Red dots: underexpressed genes (FC MitoT^pos/MitoT^neg < − 1.5). Blue dots: overexpressed genes (FC MitoT^pos/MitoT^neg > 1.5). B Pathway enrichment analysis (PEA) for DE genes between CD3+ MitoT^pos (n = 4) and CD3+ MitoT^neg (n = 4) cells. C Significantly enriched GO terms related to apoptosis, cell death, and/or responses to different stimuli of DE genes (p < 0.05, n = 4) between MitoT^pos and MitoT^neg cells. D Unsupervised hierarchical clustering heatmap of differentially expressed genes associated with GO terms from MitoT^pos (n = 4) and MitoT^neg (n = 4) samples, from whole transcriptome RNA sequencing analysis. Red transcripts represent genes directly involved in the apoptotic process regulation

Fig. 3

MitoT from MSC-derived MT grants apoptosis resistance to T cells. A Experimental design of artificial MitoT (mitoception) used for apoptosis assays. B Representative FACS plots of Annexin V-apoptosis assay on CD3+ MitoT^pos and MitoT^neg populations after 4 h of staurosporine (STS) treatment. C Percentage of apoptotic CD3+ MitoT^pos cells compared to MitoT^neg cells after STS treatment. The left bars represented no STS-treatment control (n = 3). D Representative TUNEL assay (left panel) and percentage of TUNEL+ cells (right panel), by FACS, of CD3+ MitoT^pos cells compared to MitoT^neg cells after STS treatment (n = 3). E Representative western blot of caspase-3 cleavage on MitoT^pos and MitoT^neg CD3+ T lymphocytes after 4 h of STS treatment. β-Actin was used as loading control. F Quantification of proteins showed in E, represented as caspase 3/pro-caspase-3 ratio in CD3+ MitoT^pos cells compared to MitoT^neg cells (n = 3). G Representative FACS plots of the MMP by flow cytometry using JC-1 labeling of T lymphocytes after STS treatment. H Mitochondrial depolarization, as the ratio of red to green fluorescence, in CD3+ MitoT^pos cells compared to MitoT^neg cells after STS treatment by FACS analysis (n = 3). For figures C, D, F and H: graphs show mean ± SEM and statistical analysis by unpaired t-test. All replicates are biological

Fig. 4

MitoT increases the expression of anti-apoptotic Bcl-2/BARD1 pathways. A Fold change of differentially expressed (DE) genes contained within the GO terms related to apoptosis, cell death, and/or responses to different stimuli. B qRT-PCR analysis of BARD1 mRNA expression levels in FACS-sorted CD3+ T cells after 24-h post-mitoception with MSC derived-MT (n = 4). C Representative western blots of BARD1 in FACS-sorted CD3+ MitoT^pos cells and MitoT^neg cells after 24 or 48 h post-mitoception. D Fold change quantification of proteins showed in C (n = 3). E Ingenuity Pathway analysis for Bcl-2 gene network for the DE genes denoted in A. F qRT-PCR of Bcl-2 family gene expression in FACS-sorted CD3+ MitoT^pos cells and MitoT^neg cells after 24 h post-mitoception (n = 4). For figures B and D: graphs show mean ± SEM and statistical analysis by unpaired t-test. For figure F: graph shows mean ± SEM and statistical analysis by unpaired Mann–Whitney test (*p < 0.05, relative to MitoT^neg control). All replicates are biological

Fig. 5

Anti-apoptotic effect on cMyc-tagged CAR-T cells. A Experimental design of artificial MitoT used for apoptosis assay on CAR-T cells. B Representative plots (left panel) and percentage of CAR_pos cells (right panel), by FACS, of CD3+ MitoT^pos cells compared to MitoT^neg cells after cell-electroporation (n = 4). C Representative FACS plots of Annexin V-7AAD apoptosis assay on CD3+ MitoT^pos and MitoT^neg cells in CAR_neg and CAR_pos populations after cell-electroporation. D Percentage of live, apoptotic, and necrotic CD3+ MitoT^pos and MitoT^neg cells in CAR_neg and CAR_pos populations after cell-electroporation (n = 4). E, F Mean of cytotoxicity activity of lymphocytes mitocepted (MitoT^pos) or not (MitoT^neg) and electroporated with 19BBz or Mock control, against Nalm-6-GFP B-cell line, after 8 days of expansion. The effector cells were incubated in a 1:2 (E) or 1:1 (F) ratio with the target cells (E:T) for 48 or 96 h. Right bar graph shows the average of remaining target cells (counts) (n = 4). G Representative plots (left panel) and percentage of CAR_pos IFNγ⁺ cells (right panel), by FACS, of CD3⁺ lymphocytes mitocepted (MitoT^pos) or not (MitoT^neg) after 8 days of expansion (n = 4). H Cytokines quantification, by ELISA, on supernatant from lymphocytes expanded and co-cultured for 24 h with target cells (1:1 ratio) (n = 4). Graphs show mean ± SEM and statistical analysis by unpaired t-test for figures B and D, paired t-test for figures E, F and G, and One-way ANOVA for figure H. All replicates are biological