Mitochondrial transfer from MSCs to T cells induces Treg differentiation and restricts inflammatory response

Abstract

Mesenchymal stem cells (MSCs) have fueled ample translation for the treatment of immune-mediated diseases. They exert immunoregulatory and tissue-restoring effects. MSC-mediated transfer of mitochondria (MitoT) has been demonstrated to rescue target organs from tissue damage, yet the mechanism remains to be fully resolved. Therefore, we explored the effect of MitoT on lymphoid cells. Here, we describe dose-dependent MitoT from mitochondria-labeled MSCs mainly to CD4⁺ T cells, rather than CD8⁺ T cells or CD19⁺ B cells. Artificial transfer of isolated MSC-derived mitochondria increases the expression of mRNA transcripts involved in T-cell activation and T regulatory cell differentiation including FOXP3, IL2RA, CTLA4, and TGFβ1, leading to an increase in a highly suppressive CD25⁺ FoxP3⁺ population. In a GVHD mouse model, transplantation of MitoT-induced human T cells leads to significant improvement in survival and reduction in tissue damage and organ T CD4⁺ , CD8⁺ , and IFN-γ⁺ expressing cell infiltration. These findings point to a unique CD4⁺ T-cell reprogramming mechanism with pre-clinical proof-of-concept data that pave the way for the exploration of organelle-based therapies in immune diseases.

Keywords: T regulatory cells; graft-versus-host disease; immunosuppression; mesenchymal stem cells; mitochondrial transfer.

Figure 1. MSCs efficiently transfer mitochondria in a dose‐dependent manner to T and B lymphocytes

1. Schematic representation of the experimental design used for co‐culture experiments.

2. Representative confocal microscopy of human PBMCs stained with DAPI (white arrows) co‐cultured with previously labeled UC‐MSC with MitoTracker‐Green (top image) and specific antibody for human CD90 (bottom image).

3. Percentage of mitochondrial transfer (MitoT) on total human CD45⁺ cells after 24 h co‐culture with MitoTracker‐Green labeled bone marrow (BM), menstrual (Mens) or umbilical cord (UC)‐MSCs (MSC:PBMC ratio 1:25) (n = 3 biological replicates, with three different donors of BM, Mens and UC‐MSCs).

4. FACS analysis of MitoT on T CD3⁺ (n = 4), B CD19⁺ (n = 4) and natural killer (NK) CD56⁺ (n = 3) cells after 24 h co‐culture with MitoGreen labeled UC‐MSCs (ratio 1:25) (Biological replicates, with different donors of UC‐MSCs).

5. FACS analysis of MitoT on CD3⁺ CD4⁺ or CD3⁺ CD8⁺ T cell populations after 24 h co‐culture with labeled UC‐MSCs (ratio 1:25) (n = 3 biological replicates, with three different donors of UC‐MSCs).

6. Representative FACS plots of MitoT on CD45⁺ CD3⁺ T cells at increasing amounts of UC‐MSC.

7. Percentage of MitoT on CD45⁺ CD3⁺ human cells after 24 h co‐culture with increasing ratios of UC‐MSC:PBMCs (n = 5 biological replicates, with five different UC‐MSCs and 5 different PBMCs).

8. FACS analysis of MitoT on CD45⁺ CD3⁺ T cells after short times of co‐culture with UC‐MSCs at two different temperatures, at a ratio 1:25 (n = 3 biological replicates, with three different UC‐MSCs and three different PBMCs).

9. Mean fluorescence intensity (MFI) of MitoTracker‐Green+ UC‐MSCs at baseline or after 72 h PBMC co‐culture. Representative FACS plot (left panel) and average of MFI (n = 4 biological replicates, with four different UC‐MSCs) (right panel).

10. Confocal microscopy of human PBMCs after co‐culture with MitoTracker‐Green stained UC‐MSCs. Images showed MSC‐mitochondria (green) inside the CD3⁺ T cells (red).

11. qPCR analysis of human β2‐microglobulin (B2M), human specific mitochondrial (Mito) and mouse specific (β‐actin) gene expression levels in UC‐MSCs, mouse derived spleen cells or FACS sorted CD45⁺ mouse cells after 24 h co‐culture with UC‐MSCs (n = 3 biological replicates). n/d = not detected.

12. In vivo experimental design for MitoT in Balb/c mice transplanted with MitoTracker Green labeled MSCs.

13. FACS analysis of in vivo MitoT from UC‐MSCs to Balb/c mouse mesenteric lymph node and spleen cells, 24 h after intraperitoneal transplantation of 5 × 10⁶ MitoTracker‐labeled MSCs or without cells (control) (n = 1).

Data information: In (C–E, G–I and K), graphs show mean ± SEM and statistical analysis by Student's t‐test.

Figure EV1. Extended characterization of MitoT from MSC to lymphocytes

1. Representative gating strategy showing MitoT to different cell type populations analyzed.

2. FACS analysis of MitoT on T CD3⁺ (n = 3), B CD19⁺ (n = 3) and NK CD56⁺ (n = 2) cells after 24 h co‐culture with MitoGreen labeled bone marrow (BM) or menstrual (Mens) MSCs (ratio 1:25).

3. Percentage of MitoT on CD45⁺ CD3⁺ human cells after 1–3 days of co‐culture with UC‐MSCs (ratio 1:25) (n = 3 biological replicates).

4. Mean fluorescence intensity (MFI) of T CD3⁺ MitoT^pos cells after short times of co‐culture with UC‐MSCs. Average bar graph (n = 3, left panel) and representative FACS plot (right panel).

5. Representative FACS plots of MitoT on CD45⁺ CD3⁺ cells after short times of co‐culture with MitoTracker Green labeled UC‐MSCs.

6. Representative FACS plot, out of five independent experiments, on human CD3⁺ cells cultured for 24 h with MitoGreen labeled UC‐MSC supernatant.

7. Representative FACS analysis of MitoT from MitoTracker stained PBMC to CD90⁺ UC‐MSC, 24 h following the co‐culture (ratio 1:25).

8. Representative FACS analysis of MitoT from MitoGreen stained MSC to CellTrace Violet (CTV) labeled MSC, after 24 h co‐culture (ratio 1:1).

9. Percentage of MitoT on CD3⁺ T cells from UC‐MSCs or PBMCs pre‐treated with increasing concentrations of the anti‐oxidant Pterostilbene (Ptsb), after 4 h co‐culture (n = 2 biological replicates).

10. Percentage of MitoT on CD45⁺ CD3⁺ human cells after 1–3 days of co‐culture with UC‐MSCs (ratio 1:25) with or without pro‐inflammatory licensing cytokines (n = 3 biological replicates).

Data information: All graphs show mean ± SEM and statistical analysis by Student's t‐test.

Figure 2. CD3⁺ T cells are permissive to artificial MitoT (Mitoception) resulting in an increase of their mitochondrial content

1. Schematic representation of the experimental design of the Mitoception protocol.

2. Confocal microscopy images of human CD3⁺ T cells with endogenous MT‐labeled with MitoTracker Red CMXRos (white arrows, left panel) or after Mitoception with MitoTracker Green labeled‐MT from UC‐MSCs (white asterisk, right panel).

3. FACS analysis of MitoT on CD3⁺ human T cells after Mitoception with increasing amounts of UC‐MSC‐MT corresponding to the same cell number ratios used in co‐culture experiments (n = 4 biological replicates, with 4 different donors of UC‐MSCs). Graph shows mean ± SEM and statistical analysis by Student's t‐test.

4. Representative transmission electron microscopy images of FACS‐sorted human CD3⁺ MitoT^neg and CD3⁺ MitoT^pos cells following Mitoception with UC‐MSC‐MT. Red arrows show human mitochondria.

5. Quantification of human MT in sorted CD3⁺ MitoT^neg and MitoT^pos cells after Mitoception with UC‐MSCs, by transmission electron microscopy (n = 25 independent cells analyzed per group). Graph shows mean ± SEM and statistical analysis by Student's t‐test.

Figure EV2. Characterization of MSC‐derived mitochondria activity and fate. Functional assays on isolated MT

1. Representative confocal microscopy of isolated MSC‐MT stained with MitoTracker Red CMXRos at different magnifications (40× left and middle panel, and 63× right panels).

2. Representative gating strategy showing isolated mitochondria from 1 × 10⁶ MSCs per condition by FACS.

3. Mean fluorescence intensity (MFI) of TMRM dye on unstained MSC‐MT, MSC‐MT stained with TMRM or MSC‐MT stained with TMRM previously treated with 0.2 μM of CCCP (carbonyl cyanide 3‐chlorophenylhydrazone). Representative FACS plot (left panel) and average of MFI (n = 3 biological replicates, with three different UC‐MSCs) (right panel).

4. ATP production of isolated MSC‐MT from increasing amounts of MSC cells (n = 3 biological replicates run in triplicates). Graph shows mean ± SEM and statistical analysis by one‐way ANOVA with Tukey's post‐test.

5. Analysis of mitochondrial morphology parameters of human MT in sorted CD3⁺ MitoT^neg and MitoT^pos cells after Mitoception with UC‐MSCs, by transmission electron microscopy (n = 20 independent cells analyzed per group).

6. qRT‐PCR analysis of genes related to mitochondrial fusion/fission in FACS‐sorted CD3⁺ MitoT^neg and CD3⁺ MitoT^pos cells from healthy human PBMCs (n = 3 biological replicates, with three different donors of PBMCs), after 24, 48 and 72 h post Mitoception. The graph depicts fold expression in CD3⁺ MitoT^pos cells relative to CD3⁺ MitoT^neg cells, which are set at 1 (red dotted line).

7. Western Blot analysis of protein levels related to mitochondrial fusion/fission in FACS‐sorted CD3⁺ MitoT^neg and CD3⁺ MitoT^pos cells from healthy human PBMCs. Representative blots at 24 h post Mitoception (left panel) and bar graph showing 24, 48 and 72 h post Mitoception (right panel) (n = 3 biological replicates, with three different donors of PBMCs). The graph depicts fold expression in CD3⁺ MitoT^pos cells relative to CD3⁺ MitoT^neg cells, which are set at 1.

Data information: Graphs show mean ± SEM and statistical analysis by Student's t‐test.

Figure EV3. Functional analysis of MitoT on CD3⁺ T cells. Metabolic changes, transmigration capacity and proliferation analysis on MitoT^pos cells

1. A–C

   Extracellular Acidification Rate (ECAR) analysis measured in a Seahorse XFp extracellular flux analyzer in FACS‐sorted MitoT^pos and MitoT^neg CD3⁺ T cells mitocepted with UC‐MSC mitochondria (n = 3 biological replicates, ran in quadruplicates).

2. D–F

   Oxygen Consumption Rate (OCR) analysis measured in a Seahorse XFp extracellular flux analyzer in FACS‐sorted MitoT^pos and MitoT^neg CD3⁺ T cells mitocepted with UC‐MSC mitochondria (n = 3 biological replicates, ran in quadruplicates).

3. G

   Glycolysis/OXPHOS ratio on FACS‐sorted MitoT^pos and MitoT^neg CD3⁺ T cells mitocepted with UC‐MSC‐MT (n = 3 biological replicates, ran in quadruplicates).

4. H

   L(+)‐Lactate concentrations from supernatants of sorted CD3⁺ MitoT^neg and MitoT^pos T cells, previously mitocepted with UC‐MSC‐MT (n = 2 biological replicates, each one in triplicate).

5. I

   Number of migrated CD4⁺ MitoT^neg and MitoT^pos sorted T cells from 100.000 cells seeded on media non‐activated or activated with anti‐CD3 and IL‐2 for 3 days (n = 3 biological replicates, in duplicates).

6. J

   Percentage of CD3⁺ T cell proliferation after increasing Mitoception ratios, quantified by measuring the corresponding decrease in CellTrace Violet (CTV) proliferation dye intensity by flow cytometry (n = 3 biological replicates with three different PBMC donors).

7. K

   Percentage of proliferation of FACS‐sorted MitoT^neg and MitoT^pos CD3⁺ cells, after 5 days in culture with anti‐CD3/CD28 beads (1/20) and IL‐2 (50 U/ml) in the presence or absence of PHA (15 μg/ml) (round or triangle dots respectively) (n = 5 biological replicates with five different PBMC donors).

Data information: All graphs show mean ± SEM and statistical analysis by Student's t‐test.

Figure 3. Bioinformatic analysis of MitoT cells identifies changes in mRNA expression associated with T cell activation and differentiation pathways

1. Differentially expressed (DE) genes from whole transcriptome RNA sequencing analysis in FACS‐sorted CD3⁺ MitoT^neg and CD3⁺ MitoT^pos cells from healthy human peripheral blood samples (n = 4 biological replicates, with 4 different donors of PBMCs).

2. Volcano plot analysis derived from FPKM values showing DE genes (P < 0.05, Student's t test) in sorted MitoT^neg and MitoT^pos CD3⁺ cells (from four different donors of PBMCs).

3. Heatmap depiction of RNA‐Seq analysis of significantly (P < 0.01, Student's t test) DE coding genes from sorted MitoT^pos and MitoT^neg CD3⁺ cells (from four different donors of PBMCs).

4. Significantly enriched pathways related to T cell regulation and activation for DE genes (P < 0.05, Student's t test) between MitoT^pos and MitoT^neg CD3⁺ cells, from the GO Biological Process 2015 database (from 4 different donors of PBMCs).

5. Differentially expressed genes from the significantly enriched pathways listed in Fig 2D. Highlighted in red are key markers of Treg phenotype.

6. qRT‐PCR analysis of genes related to T cell activation after Mitoception with UC‐MSC‐MT (n = 4 biological replicates, with four different donors of PBMCs). The graph depicts fold expression in CD3⁺ MitoT^pos cells relative to CD3⁺ MitoT^neg cells, which are set at 1. Graph shows mean ± SEM and statistical analysis by Student's t‐test (*P < 0.05).

7. Correlation analysis of 14 sampled genes between qRT‐PCR and RNA‐Seq, from MitoT^pos and MitoT^neg CD3⁺ cells (from 4 different donors of PBMCs). Graph depicts best‐fit line and 95% confidence intervals by Pearson correlation analysis.

Figure EV4. MitoT‐driven Treg differentiation. Analysis of gene set enrichment of RNA‐Seq data

1. Interaction network from FOXP3‐associated DE coding genes in sorted MitoTposCD3⁺ compared to MitoTnegCD3⁺ T cells (n = 4 different donors of PBMCs), using Genemania.

2. Gene Set Enrichment Analysis (GSEA) of co‐expressed modules identified by webCEMiTool showing, the enriched module activity between sorted MitoTpos and MitoTnegCD3⁺ T cells (from 4 different donors of PBMCs).

3. Top over‐represented functional categories according to webCEMiTool and MSigDB, identified co‐expressed gene modules.

4. Gene co‐expression/interaction networks of module 34 (M34) displaying FOXP3 as hub gene.

5. FACS analysis of MitoT on CD4⁺ and naïve (CD4⁺ CD45RA⁺ CD45RO⁻) T cells after Mitoception with UC‐MSC mitochondria, represented as bar graph (left panel, n = 3 biological replicates) and representative FACS plot (right panel). Graph shows mean ± SEM and statistical analysis by Student's t‐test.

6. FACS analysis of Treg induction after 5–7 days differentiation in plain media of FACS‐sorted CD4⁺ naïve MitoTneg and MitoTpos cells previously mitocepted with Fibroblasts or same donor PBMCs mitochondria (n = 3 biological replicates for each condition).

7. Representative proliferation FACS plots of CTV‐stained PBMC co‐cultured with MitoTneg or MitoTpos cells (ratio 1:1) after 5–7 days of CD4⁺ naïve cell activation with differentiation media.

Figure 4. Efficient MitoT to T cells underlies functional changes including enhanced immunosuppression through regulatory T cell (Treg) induction

1. Representative bright‐field microscopy images of FACS‐sorted CD4⁺ naïve MitoT^neg (upper panel) and MitoT^pos (lower panel) cells after 5 days in culture with plain media (no cytokines added). Red arrows are indicative of activation/proliferation clusters.

2. Representative FACS plots of Treg (CD127^lowCD25⁺ FoxP3⁺) populations after T cell differentiation on media with or without cytokines and antibody supplements (anti‐CD3, IL‐2 and TGF‐b1).

3. FACS analysis of Treg induction after 5–7 days differentiation of FACS‐sorted CD4⁺ naïve MitoT^neg and MitoT^pos cells in plain (n = 5) or differentiation media (n = 6) (Biological replicates, with different donors of PBMCs).

4. ICOS expression on FACS‐sorted CD4⁺ naïve MitoT^neg and MitoT^pos cells after 5–7 days in culture with differentiation media. Representative FACS plot (left panel) and bar graph (right panel) of ICOS high population (n = 4 biological replicates).

5. CCR7 expression on sorted CD4⁺ naïve MitoT^neg and MitoT^pos cells after 5–7 days in culture with differentiation media. Representative FACS plot (left panel) and bar graph (right panel) of CCR7⁺ population (n = 4 biological replicates).

6. FACS analysis of Th1 (CD4⁺ IFN‐γ⁺) induction after 5–7 days differentiation of FACS‐sorted CD4⁺ naïve MitoT^neg and MitoT^pos cells in plain media (n = 3 biological replicates). n/s= not significant.

Data information: In (C–F) graphs show mean ± SEM and statistical analysis by Student's t‐test.

Figure 5. MitoT‐induced Treg cells inhibit the proliferation of target PBMCs

1. Schematic representation of the experimental design used for the immunosuppression assay.

2. Representative proliferation FACS histogram of CTV‐stained PBMC co‐cultured with MitoT^neg or MitoT^pos cells (ratio 1:1) after 5–7 days of CD4⁺ naïve cell activation with Treg induction/differentiation media (anti‐CD3, IL‐2 and TGF‐b1). PBMC alone represents positive control of proliferation and PBMC co‐culture with MitoT^neg correspond to the inhibition control with induced Treg cells.

3. Percentage of immunosuppression of CTV‐stained PBMC co‐cultured with MitoT^neg or MitoT^pos cells after 5–7 days of CD4⁺ naïve cell activation with differentiation media (ratio MitoT cells:PBMC) (n = 3 biological replicates, with different donors of PBMCs). Graph shows mean ± SEM and statistical analysis by Student's t‐test.

Figure 6. Xeno‐GVHD induction with MitoT^pos‐PBMCs is associated with improved survival and reduced tissue inflammation in a mouse model of disease

1. A

   In vivo experimental design of human xenogenic model of GVHD in NSG immunodeficient mice, with MSC‐mitocepted human PBMC transplant.

2. B

   Representative FACS plots of Mitoception (% of MitoTracker+) in CD4⁺ or CD8⁺ T cells from mitocepted PBMC compared to control PBMC, before intravenous injection.

3. C

   Percent of mouse weight after GVHD induction with IV injection of mitocepted PBMC or control PBMC cells (n = 3, with a total of 25 mice per group). Control group in grey, were untreated mice (n = 6 mice). *P < 0.05 by Mann Whitney t‐test.

4. D

   Kaplan Meier plot showing percent survival of GVHD mice after transplantation of an equal amount (12 × 10⁶ cells) of mitocepted PBMC or control PBMC cells (n = 3, with a total of 25 mice per group) (P = 0.0218 by log‐rank test).

5. E

   FACS analysis of human Treg (CD4⁺ CD25⁺ FoxP3⁺) cell engraftment in mouse spleen following transplantation of PBMC‐MitoT or control PBMC cells (n = 3, with a total of 25 and 27 mice per group, respectively).

6. F

   FACS analysis of human pro‐inflammatory T helper1 (CD4⁺ IFN‐γ⁺, left panel) and T cytotoxic (CD8⁺ IFN‐γ⁺, right panel) cell engraftment in mouse spleen following transplantation of mitocepted or control PBMC cells (n = 3, with a total of 25 and 27 mice per group, respectively).

7. G

   Representative histopathologic images of GVHD in liver and small intestine sections obtained from PBMC‐MitoT, PBMC and non‐transplanted control samples, stained with Masson's trichrome staining. Asterisk showed increased periportal collagen deposits in the liver and increased piknotic cells in the small intestine crypts. Arrowheads represents a reduced number of Paneth cells.

8. H, I

   Semiquantitative scoring system for pathologic damage in the liver and small intestine of mitocepted PBMC (n = 8 independent mice) or control PBMC transplanted mice (n = 5 independent mice).

9. J

   Quantification of Paneth cells per crypt, from at least 50 crypts per PBMC‐MitoT (n = 8 mice), PBMC transplanted (n = 5 mice) or non‐transplanted control (n = 3) mouse.

10. K

    Representative histopathologic images of GVHD in lung sections obtained from PBMC‐MitoT, PBMC and non‐transplanted control, stained with Masson's trichrome staining.

11. L

    Semiquantitative scoring system for pathologic damage in lung of mitocepted PBMC (n = 8 independent mice) or control PBMC transplanted mice (n = 5 independent mice).

12. M

    FACS analysis of total human CD4⁺ cell engraftment in mouse lungs following transplantation of mitocepted PBMC or control PBMC cells (n = 3, with a total of 25 and 27 mice per group, respectively).

Data information: (E–M) represent mice from three independent in vivo experiments. In (E–F, H–J and L–M), graphs show mean ± SEM and statistical analysis by Student's t‐test.

Figure EV5. Efficient ex vivo MitoT in inflammatory T cells isolated from a SLE mouse model

1. A–E

   Representative gating strategy used for the in vivo analyses. Representative FACS plots of (B) Tregs (FoxP3⁺ CD25⁺), (C) T cytotoxic (CD8⁺ IFNg⁺), (D) Thelper 1 (CD4⁺ IFNg⁺) and (E) Thelper 17 (CD4⁺ IL17⁺) populations cell engraftment in mouse spleen following transplantation of mitocepted or control PBMC cells.

2. F

   Representative FACS plot, out of 3 independent experiments, on MRL/MpJ/Fas mouse CD45⁺ CD3⁺ cells from spleen or lymph nodes co‐cultured for 24 h with MitoGreen labeled UC‐MSCs.

3. G

   FACS analysis of ex vivo MitoT from labeled UC‐MSCs to MRL/MpJ/Fas (lupic) or MRL/MpJ (control) mouse CD45⁺ CD3⁺ cells from spleen or lymph nodes (n = 3 biological replicates). Graph shows mean ± SEM and statistical analysis by Student's t‐test.