Skip to main content
Download PDF

Human MuStem cells repress T-cell proliferation and cytotoxicity through both paracrine and contact-dependent pathways

Stem Cell Research & Therapy volume 13, Article number: 7 (2022) Cite this article

Abstract

Background

Muscular dystrophies (MDs) are inherited diseases in which a dysregulation of the immune response exacerbates disease severity and are characterized by infiltration of various immune cell types leading to muscle inflammation, fiber necrosis and fibrosis. Immunosuppressive properties have been attributed to mesenchymal stem cells (MSCs) that regulate the phenotype and function of different immune cells. However, such properties were poorly considered until now for adult stem cells with myogenic potential and advanced as possible therapeutic candidates for MDs. In the present study, we investigated the immunoregulatory potential of human MuStem (hMuStem) cells, for which we previously demonstrated that they can survive in injured muscle and robustly counteract adverse tissue remodeling.

Methods

The impact of hMuStem cells or their secretome on the proliferative and phenotypic properties of T-cells was explored by co-culture experiments with either peripheral blood mononucleated cells or CD3-sorted T-cells. A comparative study was produced with the bone marrow (BM)-MSCs. The expression profile of immune cell-related markers on hMuStem cells was determined by flow cytometry while their secretory profile was examined by ELISA assays. Finally, the paracrine and cell contact-dependent effects of hMuStem cells on the T-cell-mediated cytotoxic response were analyzed through IFN-γ expression and lysis activity.

Results

Here, we show that hMuStem cells have an immunosuppressive phenotype and can inhibit the proliferation and the cytotoxic response of T-cells as well as promote the generation of regulatory T-cells through direct contact and via soluble factors. These effects are associated, in part, with the production of mediators including heme-oxygenase-1, leukemia inhibitory factor and intracellular cell adhesion molecule-1, all of which are produced at significantly higher levels by hMuStem cells than BM-MSCs. While the production of prostaglandin E2 is involved in the suppression of T-cell proliferation by both hMuStem cells and BM-MSCs, the participation of inducible nitric oxide synthase activity appears to be specific to hMuStem cell-mediated one.

Conclusions

Together, our findings demonstrate that hMuStem cells are potent immunoregulatory cells. Combined with their myogenic potential, the attribution of these properties reinforces the positioning of hMuStem cells as candidate therapeutic agents for the treatment of MDs.

Background

Muscular dystrophy (MD) is a heterogeneous group of more than 50 genetically distinct neuromuscular diseases characterized by local or generalized fiber degeneration [1]. While clinical presentation and severity can vary considerably, the progressive loss of muscle mass and function, leading to muscle weakness, reduced motility and premature death, is common to all forms [1]. As a consequence of molecular defects mainly affecting cytoskeletal or extracellular matrix proteins, muscle fiber membrane damage is common and results in profound disruption of homeostasis, as well as inflammation and fibrosis [2]. Duchenne muscular dystrophy (DMD) is the most common and devastating form of MD, affecting 1 in 3500–5500 male newborns [3, 4]. DMD is caused by mutations in the gene encoding dystrophin, leading to a lack of a functional protein essential for maintenance of muscle fiber integrity [5].

The presence of muscle inflammation and a significant increase in the proportion of immune cells in dystrophic muscle reveals an essential functional role of the immune system in the pathogenesis of MD [6, 7]. This association was initially supported by studies demonstrating that glucocorticosteroid-mediated immunosuppression limits clinical signs and delays the course of DMD in patients and animal models [8, 9]. The diversity of leukocytes that infiltrate dystrophic muscle, including neutrophils, eosinophils, macrophages, helper CD4+ T-lymphocytes and cytotoxic CD8+ T-lymphocytes (CTLs), further supports an immunological component [10,11,12]. In X-chromosome-linked muscular dystrophy (mdx) mice, one of the most commonly used animal models of DMD [13], depletion of myeloid cells at an early age markedly decreases the proportion of necrotic fibers [11, 12]. T-cells are among the first cells to infiltrate mdx mouse muscle, prior to the onset of necrosis, indicating an important role as effectors in early disease pathogenesis [14]. In young mdx mice, selective T-cell inhibition markedly diminishes the extent of inflammatory cell infiltrate and reduces both muscle necrosis and fibrosis, underscoring the importance of the adaptive immune system in DMD. Furthermore, disease severity is reduced in mdx mice after pharmacological inhibition or genetic ablation of the inflammatory cytokines interferon-γ (IFN-γ) and tumor-necrosis factor-α (TNF-α) [15, 16]. Finally, accumulation of regulatory T cells (Tregs) has been demonstrated in infiltrates in muscle tissue from DMD patients [17], mdx mice [17, 18] and dysferlin-deficient mice [19]. These findings highlight the therapeutic importance of regulating the infiltration and accumulation of immune cells in dystrophic muscle, both of which contribute to disease progression and severity.

Over the last two decades, muscle repair potential has been attributed to tissue-resident stem cells capable of forming new muscle fibers in response to acute injury. These cells include side population (SP) cells [20, 21], CD133+ cells [22, 23], mesoangioblasts (Mabs) [24,25,26], bone-marrow- and adipose tissue-derived-mesenchymal stem cells (BM-MSCs and AD-MSCs, respectively) [27, 28], PW1+/Pax7+ interstitial cells (PICs) [29] and muscle-derived stem cells (MDSCs) [30, 31]. The newly discovered potential of these adult stem cells has opened novel therapeutic avenues for MDs in addition to strategies targeting dystrophin restoration including exon skipping with antisense oligonucleotides, vector-mediated gene therapy and CRISPR/Cas9-mediated gene editing [32]. In particular, it constitutes a promising means of overcoming the limited efficacy of myoblast transplantation. Moreover, most if not all MSCs are immunoprivileged. MSCs lack human leukocyte antigen type-II (HLA-II) and co-stimulatory molecules CD80 and CD86, which are required for T-lymphocyte activation, but express HLA-I on their surface [33]. An ability to escape immune recognition has been described for human AD-MSCs in immunocompetent animal models of DMD and allows long-term integration of these cells into muscle tissue [28, 34]. MSCs are also potent immunoregulatory cells and appear to be capable of regulating both innate and adaptive immune responses in vitro and in vivo [35,36,37,38]. Moreover, MSCs control the phenotype and immunological responses of T-cell subsets (helper CD4+, CTLs, Treg cells) [38,39,40,41,42], B-cells [43], natural killer (NK) cells [44, 45], dendritic cells (DC) [46, 47] and macrophages [48, 49]. In vitro, MSCs suppress T-cell proliferation in response to mitogens, alloantigens and activating antibodies [39, 50] and promote the generation of Tregs [38, 51]. In vivo, MSCs enhance long-term allograft acceptance and tolerance [40, 52] and exert therapeutic effects in experimental models of inflammatory and autoimmune disorders [53, 54]. These immunomodulatory/immune-dampening properties of MSCs involve direct contact with target cells [55, 56], but are largely mediated by the release or synthesis of numerous factors [57], the best described of which are indoleamine 2,3-dioxygenase (IDO) [58], prostaglandin E2 (PGE2) [59, 60], inducible nitric oxide synthase (iNOS) [61] and heme oxygenase-1 (HO-1) [62].

In the past few years, we have characterized a type of MDSC isolated from tissue samples from healthy dogs and humans. Based on their phenotype and plasticity, we have shown that these cells, referred as MuStem cells, correspond to early myogenic-committed progenitors with a mesenchymal perivascular profile. Importantly, these oligopotent stem cells display an interesting potential for skeletal and cardiac muscle repair [63,64,65,66]. After allogeneic transplantation into golden retriever muscular dystrophy (GRMD) dogs, a clinically relevant animal model of DMD, MuStem cells contribute to muscle fiber formation, induce long-term muscle fiber regeneration and limit the progression of muscle damage and fibrosis [63, 67]. Similarly, human MuStem (hMuStem) cells show in vivo a robust capacity for muscle regeneration after delivery into injured skeletal muscle in immunodeficient mice [64] and efficiently counteract adverse tissue remodeling, primarily by limiting fibrosis, in an immunodeficient rat model of myocardial infarction [66]. While these findings have positioned hMuStem cells as an attractive tool for muscle regenerative medicine, their immunoregulatory properties, which are pivotal for tissue remodeling, have not been investigated to date.

In the present study, we investigated whether hMuStem cells can exert immunosuppressive effects by regulating the immune response. We characterized the immunophenotype of these cells and compared it with that of BM-MSCs. Moreover, we studied the effect of hMuStem cells on T-cell proliferation and cytotoxicity in co-culture experiments. These original data provide new insights into the modes of action of hMuStem cells and reinforce their potential as an effective therapeutic agent for the treatment of MDs.

Methods

Human skeletal muscle tissue

Tissue samples were obtained from Paravertebralis muscle biopsies collected from patients aged 12–19 years. Patients were free of known muscle disease and had undergone surgery for acute scoliosis at the Department of Pediatric surgery of the Centre Hospitalier Universitaire (CHU) de Nantes (France). Written informed consent was obtained from all patients. All protocols were approved by the Clinical Research Department of the CHU (Nantes, France), according to the rules of the French Regulatory Health Authorities (Approval Number: MESR/DC-2010-1199). The biological sample bank was created in compliance with national guidelines regarding the use of human tissue for research (Approval Number: CPP/29/10).

Isolation and culture of human MuStem cells

Human MuStem cells were independently isolated from muscle biopsies from 5 patients and cultured, as previously described [64, 66]. For pro-inflammatory stimulation, cells were expanded until they reached approximately 60–70% confluence, and then the medium was changed for growth medium containing 50 ng/mL of both TNF-α and IFN-γ (Miltenyi, Bergisch Gladbach, Germany) and cultured for 24 h.

Isolation and culture of human bone marrow-derived mesenchymal stem cells

Human BM-MSCs were collected from 4 patients aged 5, 11, 19 and 60 years who underwent hip replacement surgery. The patients were informed and provided written informed consent prior to collection of tissue samples as approved by the French Ministry of Higher Education and Research (DC-2010-1185). BM-MSCs were cultured, characterized at the phenotypic level and tested for their tri-lineage differentiation potential as previously described [68, 69]. For pro-inflammatory stimulation, cells were expanded until they reached approximately 60–70% confluence, and then the medium was changed for fresh medium containing 50 ng/mL of both TNF-α and IFN-γ (Miltenyi) and cultured for 24 h.

Immunosuppression assay

Three sets of independent experiments were conducted with hMuStem cells that were successively co-cultured with allogeneic human PBMCs, human CD3+ lymphocytes/allogeneic PBMCs and human CD8+ T-cell clones. A detailed description of these immunosuppression assays is provided in Additional file 5: Methods.

Cytotoxicity assay

Meso 34 NanoLuc cells were seeded at 5 × 103 cells/well in 96-well plates [70, 71]. After 3 h, 1 × 104 clonally-derived CD8+ T-cells previously co-cultured with either hMuStem cells, BM-MSCs or their respective secretome were added. To determine their cytotoxic effects on Meso 34 NanoLuc cells, 45 μL of medium was collected after 24 h and light emission was measured at 480 nm immediately after addition of 5 μL of 30 μM coelenterazine using a Mithras LB 940 microplate analyzer (Berthold Technologies, Baden Württemberg, Germany), which measures nanoluciferase activity released in the supernatant following cell lysis.

Flow cytometry

Human MuStem cells, BM-MSCs, PBMCs or MUC-1-specific CD8+ T-cells were resuspended in cold phosphate buffered saline (PBS)/2% human serum and 1 × 105 cells were incubated (30 min, 4 °C) in darkness with fluorochrome-conjugated Ab at a saturating concentration. The Abs used are listed in Additional file 4: Table S1. Isotype-matched Ab and fluorescence minus one-control samples were used as negative controls for gating and analyses. Where applicable, 7-amino-actinomycin D (7-AAD; BD Biosciences, Franklin Lakes, NJ, USA) was added to evaluate cell viability. For immunophenotyping of hMuStem cells, samples were acquired using a FACS Aria flow cytometer (BD Biosciences), and for analyses of PBMCs and CD8+ T-cells a FACS Canto II (BD FACSDivaTM Software) was used. For each experiment and labeling, at least 15 × 103 viable cells were considered. All the collected data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).

Immunocytochemistry

Human macrophages, obtained from the Clinical Transfer Facility of the CHU de Nantes (CICBT0503, Nantes, France), were seeded at the density of 1.5 × 105 cells/cm2 in 12-well plates and cultured in RPMI 1640 (Thermo Fischer Scientific, Illkirch, France) containing 10% human serum (EFS, Nantes, France), 1% glutamin (Sigma-Aldrich, Saint Quentin-Fallavier, France), 1000 IU/mL M-CSF (Miltenyi) and 1% 10,000 IU/mL penicillin, 10 mg/mL streptomycin, 25 µg/mL fungizone (amphotericin B) (PSF; Sigma-Aldrich). After 72 h, they were activated with 100 ng/mL of lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Sigma-Aldrich) for 24 h. RAW 264.7 murine macrophage cell line (ATCC TIB-71, Manassas, VA, USA) was provided by the cell bank of the IECM Lab (Nantes, France). It was seeded at the density of 2.2 × 104 cells/cm2 in 12-well plates and cultured in RPMI 1640 (Gibco) containing 10% fetal calf serum (FCS), 1% glutamin (Sigma-Aldrich), 1% PSF (Sigma-Aldrich) during 96 h. Human MuStem cells, BM-MSCs, human macrophages or RAW cells were fixed in cold methanol (15 min, − 20 °C) and treated with 0.3% triton X-100 (30 min, 4 °C). After incubation (1 h, RT) in blocking buffer (5% goat serum in PBS), cells were incubated overnight with iNOS Ab (1:100, sc-651 clone, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and counterstained (15 min, 37 °C) with DAPI fluorescent cell-permeable DNA probe (Life Technologies Ltd, Paisley, UK). The number of positive cells was determined using Fiji image analysis software [72]. For each condition, more than 300 cells were counted.

Reverse transcription and real-time semi-quantitative PCR

Total RNA was extracted using the RNeasy mini or micro kit following the manufacturer’s instructions (Qiagen, Santa Clara, CA, USA), quantified using a NanoDrop spectrophotometer (Labtech, Wilmington, DE, USA) after DNase treatment (Ambion, Austin, TX, USA) and converted to cDNA by reverse transcription as described previously [64]. Oligonucleotide primers used for semiquantitative RT-PCR analysis of gene expression were designed using Oligo Primer Analysis Software v.7 (Molecular Biology Insights Inc., Colorado Springs, CO, USA) and are listed in Additional file 4: Table S2. Data were normalized to mRNA levels of the housekeeping gene RPS18 and were calculated using the 2–∆Ct method.

ELISA assay

Supernatant of CTLs, hMuStem cells, or BM-MSCs (cultured under either basal or TNF-α/IFN-γ-stimulated conditions) was collected, centrifuged to remove any cell fragments and stored at − 20 °C. The presence of secreted proteins, specifically granzyme B, hepatocyte growth factor (HGF), VEGF, PGE2, IL-6, IL-8, IL-10, LIF and Gal-1 was measured by ELISA (granzyme B, VEGF, IL-6, IL-8, IL-10, LIF, Gal-1: DuoSet ELISA R&D Systems; PGE2: Enzo Life Sciences; IL35: Wuhan Fine Biological Technology, Tebu bio-SAS, Le Perray En Yvelynes, France), according to the manufacturer’s instructions.

Statistical analysis

All data are reported as the mean ± SEM. Lymphocyte proliferation was compared between co-cultures containing different proportions of MuStem cells using a Kruskal–Wallis test followed by Dunn’s multiple comparisons between pairs. Expression and secretion of regulatory molecules by hMuStem cells was compared with that of BM-MSCs using the Mann–Whitney U test. Expression and secretion of regulatory molecules by hMuStem cells expanded under stimulated or unstimulated conditions was compared using the Wilcoxon matched-pairs signed rank test. A value of p < 0.05 was considered statistically significant.

Results

hMuStem cells display a poorly immunogenic and immunosuppressive phenotype

To determine whether the hMuStem cells display an immunosuppressive phenotype similar to BM-MSCs [33, 73, 74], we assessed the expression of HLA-I and -II, as well as molecules involved in T-cell interaction. Human MuStem cells from 5 independent donors were expanded in vitro and analyzed at passage 5 (P5) using fluorescence-activated cell sorting (FACS). All hMuStem cells shared a typical expression pattern of myogenic progenitors with a signature of perivascular MSCs (Additional file 2: Figure S1), in agreement with our previous findings [64, 66, 75]. Given that stimulation with pro-inflammatory cytokines modifies the phenotype of MSCs [73], hMuStem cells were cultured either in basal media or in presence of TNF-α and IFN-γ. While hMuStem cells constitutively expressed HLA-I molecules (HLA-ABC, 100%) and HLA-DP (29.2% ± 23.9%; range 9.5–59.0%), they were uniformly negative for HLA-DQ and HLA-DR (Fig. 1A; Table 1). Moreover, they did not express HLA-G1 or HLA-E, which are involved in immune tolerance and NK cell inhibition, respectively. Human MuStem cell stimulation with TNF-α/IFN-γ increased HLA-ABC expression (relative mean fluorescence intensity [rMFI]: 21.2 ± 10.3 vs. 13.3 ± 7.3 in basal conditions) and resulted in HLA-DP expression in 88.3% ± 33.2% of cells. Stimulation with TNF-α/IFN-γ had no effect on expression levels of HLA-DQ, HLA-DR or HLA-G1. Notably, HLA-E expression was induced in all hMuStem cells in response to stimulation with TNF-α/IFN-γ.

Fig. 1
figure 1

Phenotypic profile of human MuStem cells focused on immune-cell-related markers. Cell events were first selected upon their size and granularity (forward scatter vs. side scatter density plot), while the FSC-A/FSC-H plot on gated events allowed identification of single cells. Flow cytometry comparison of: a HLA class I (HLA-ABC, HLA-G1 and HLA-E) and class II (HLA-DQ, -DR and -DP) molecules; b co-stimulatory molecules CD80, CD86 and CD40; c programmed death ligand (PDL)-1 and -2; d TIGIT receptor CD112 and CD155; and e intracellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) in hMuStem cells cultured in basal condition (unstimulated) or after stimulation with TNF-α/IFN-γ (pro-inflammatory condition). When an expression is detected, mean ± SEM of positive cells and rMFI are reported on the top right corner. Results of one representative cell batch out of five independent batches are presented

Full size image
Table 1 Expression levels of immune cell-related markers measured in human MuStem cells
Full size table

We next examined the expression pattern of molecules involved in the regulation of T-lymphocyte activity. All hMuStem cells were homogeneously negative for the co-stimulatory molecules CD80, CD86 and CD40, which are required for efficient T-cell activation, even after stimulation with TNF-α/IFN-γ (Fig. 1B). Human MuStem cells did not express the inhibitory lymphocyte molecule corresponding to programmed death ligand-1 (PDL-1), but homogeneous low-level PDL-2 expression was detected under basal conditions (Fig. 1C). Interestingly, stimulation with TNF-α/IFN-γ induced uniform PDL-1 expression by hMuStem cells and significantly enhanced the intensity of PDL-2 expression (rMFI: 3.7 ± 1.1 vs. 2.3 ± 0.3 in basal conditions), as previously described for BM-MSCs [76]. We next investigated the expression of CD112 and CD155, two molecules expressed by antigen-presenting cells and known to mediate inhibition of lymphocytes by their interaction with TIGIT [77]. While CD112 expression was not detected in either basal or stimulated conditions, hMuStem cells showed constitutive CD155 expression, which was unaffected by stimulation with TNF-α/IFN-γ (Fig. 1D). Finally, in basal conditions hMuStem cells expressed two molecules known to induce lymphocyte adhesion: intracellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (37.4% ± 11.2% and 8.3% ± 4.0%, respectively) (Fig. 1E). Stimulation with TNF-α/IFN-γ significantly increased the expression of both ICAM-1 and VCAM-1 (100% positivity for both molecules), and the first in a much higher extent as appreciated by a strongly shifted rMFI (Fig. 1E).

Together, these data suggest that hMuStem cells are poorly immunogenic cells considering the expression of HLA, co-stimulatory and inhibitory molecules and that they interact with T-cells such as BM-MSCs.

hMuStem cells inhibit T-lymphocyte proliferation and induce Treg-like cells in peripheral blood mononuclear cells

Based on the phenotypic features of hMuStem cells, we sought to determine whether they could exert immunosuppressive effects on T cells. PHA-stimulated allogeneic peripheral blood mononuclear cells (PBMCs) previously stained with cell trace violet (CTV) were co-cultured for 3 days with hMuStem cells (n = 5, independent donors) or BM-MSCs, used as reference (n = 4, independent donors), at a ratio of 1:10 PBMCs. Compared with PBMC-only cultures, hMuStem cells resulted in significant inhibition of both CD4+ and CD8+ T-cell proliferation, as evidenced by proportions of proliferating cells of 54.5% ± 28.0% and 48.1% ± 29.9%, respectively (p < 0.001; Fig. 2A). Similar results were obtained for BM-MSCs with 48.9% ± 13.0% and 46.3% ± 21.1% of CD4+ and CD8+ T cells, respectively.

Fig. 2
figure 2

Effect of human MuStem cells and bone marrow-derived mesenchymal stem cells on proliferation and phenotype of T-cells in peripheral blood mononucleated cells. a Representative profile (left panel) and quantification (right panel) of the proliferation of Cell Trace Violet (CTV)-labeled CD4+ and CD8+ T-cells in peripheral blood mononucleated cells (PBMCs) cultured alone or with unstimulated hMuStem cells (n = 5, independent batches) or bone marrow-derived mesenchymal stem cells (BM-MSCs; n = 4, independent batches). PBMCs were stimulated with phyto-hemagglutinin (PHA) to induce T-cell proliferation. The suppressive capacity of hMuStem cells or BM-MSCs was determined by tracking cell division of CTV-labeled PBMCs. b Representative FACS plots with gating of CD25+ cells in CD4+ and CD8+ T-cell fractions of PBMCs cultured alone or under PHA stimulation and with hMuStem cells or BM-MSCs. Percentages are expressed as the percentage of PBMCs cultured alone. c Representative FACS plots with gating of IL-10+, FoxP3+, IFN-γ+ and IL-17+ cells in CD4+ T-cell fractions of PBMCs cultured under PHA stimulation only and with hMuStem cells or BM-MSCs. Percentages are expressed as the percentage of PBMCs cultured alone. Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, **p < 0.001, ****p < 0.0001; Mann–Whitney U test)

Full size image

Expression of the T-cell activation marker CD25 decreased when hMuStem cells were co-cultured with PBMCs (66.0% ± 7.2% and 67.7% ± 7.6% for CD4+ and CD8+ T-cells, p < 0.01; Fig. 2B). While a similar proportion of CD4+/CD25+ cells (59.2% ± 8.2% with respect to PBMC-only culture) was observed in BM-MSC + PBMC co-cultures, those of CD8+/CD25+ cells was lower (48.2% ± 8.5%), suggesting a stronger effect of BM-MSCs than hMuStem cells on CD8+ T-cell activation (p < 0.001).

To further investigate the effects of hMuStem cells on T-cells, we characterized the expression profile of the T-cell subset regulatory factors IL-10 and FoxP3 and the pro-inflammatory factors IFN-γ and IL-17 in CD4+ T-cells collected from hMuStem cell + PBMC and BM-MSC + PBMC co-cultures. Remarkably, the proportion of CD4+/IL-10+ cells and CD4+/FoxP3+ cells in hMuStem cell + PBMC co-cultures was significantly higher than that observed in PBMC-only cultures (p < 0.05; Fig. 2C). However, compared with hMuStem cells, BM-MSCs were more efficient at generating CD4+/FoxP3+ cells. Moreover, FACS analysis revealed that in hMuStem cell + PBMC and BM-MSC + PBMC co-cultures, the proportions of CD4+/IFN-γ+ cells and CD4+/IL-17+ T-cells were similar to those seen in PBMC-only cultures, indicating that hMuStem cells, like BM-MSCs, did not significantly affect the proportions of pro-inflammatory lymphocytes. Overall, these results demonstrate that hMuStem cells can significantly reduce proliferation and activation of CD4+ and CD8+ T-cells and exert an immunosuppressive effect by promoting a Treg cell-like phenotype in CD4+ T-lymphocytes without affecting their pro-inflammatory capacity.

hMuStem cells express classical MSC immunomodulatory mediators

To investigate the mechanisms underlying the suppressive activity of hMuStem cells, we examined the expression of a panel of well-described regulatory mediators including metabolic enzymes, pleiotropic hormones and interleukins using flow cytometry, immunocytochemistry and enzyme-linked immunosorbent assay (ELISA). In 4 out of 5 hMuStem cell batches, more than 95% of cells were positive for the heme oxygenase 1 (HO-1), whereas 26.2% HO-1+ cells were detected in the remaining batch (Fig. 3A). Remarkably, in BM-MSC batches the proportion of HO-1+ cells ranged from 11.6 to 50.2% (i.e., markedly lower than detected in hMuStem cells). Moreover, stimulation with TNF-α/IFN-γ induced a more than twofold increase in the proportion of HO-1+ cells in the hMuStem cell batch exhibiting initially non-homogenous expression for the metabolic enzyme, whereas no clear change was observed in stimulated BM-MSCs. Immunocytochemistry analysis revealed that hMuStem cells and BM-MSCs were both uniformly positive for the expression of iNOS (Fig. 3B). IDO-1 gene expression was detected in both hMuStem cells and BM-MSCs, and increased significantly (by a factor of 141,000 and 36,000 in hMuStem cells and BM-MSCs, respectively) upon stimulation with TNF-α/IFN-γ (Fig. 3C).

Fig. 3
figure 3

Expression and secretion of immunoregulatory mediators in cultured human MuStem cells and bone marrow-derived mesenchymal stem cells. a Flow cytometry comparison of heme oxygenase-1 (HO-1) expression in hMuStem cells and BM-MSCs. b Fluorescent immunolabeling of inducible nitric oxide synthase (iNOS) in hMuStem cells and BM-MSCs. Lipopolysaccharide (LPS)-human monocyte-derived activated macrophages for 24 h and RAW 264.7 cell line were used as positive (C+) and negative (C−) controls, respectively. Nuclei were counterstained with DAPI (blue). Scale bars, 100 µm. c Representative RT-PCR profile of indoleamine 2,3-dioxygenase-1 (IDO-1) gene obtained for hMuStem cells and BM-MSCs. For each sample, the level of IDO-1 expression was quantified using the average mRNA level of IDO-1 obtained in unstimulated BM-MSCs as a reference. LPS-human monocyte-derived activated macrophages for 24 h and water were used as positive (C+) and negative (C−) controls, respectively. d Interleukin, growth factor, enzyme and carbohydrate-binding protein secretion profile of hMuStem cells and BM-MSCs. ELISA assays were performed using culture supernatant collected 24 h after medium change. Results are expressed as individual values and normalized as concentration relative to 1 million cultured cells (ng or pg/106 cells). Each experiment was performed on at least 5 and 4 independent batches of hMuStem cells and BM-MSCs, respectively. Stimulation corresponds to a 24-h treatment with 50 ng/mL of TNF-α and IFN-γ. *p < 0.05, **p < 0.01, **p < 0.001, ****p < 0.0001; Wilcoxon matched-pairs signed rank test (unstimulated vs. stimulated) or Mann–Whitney U test (hMuStem cells vs. BM-MSCs). US, unstimulated; S, TNF-α/IFN-γ-stimulated

Full size image

Human MuStem cells and BM-MSCs showed similar levels of IL-6 expression (10.4 ± 2.5 and 13.7 ± 6.8 ng/106 cells, respectively). Stimulation with TNF-α/IFN-γ resulted in a significant increase of IL-6 secretion in some hMuStem cell batches (p < 0.05, Fig. 3D; Table 2). This increase was greater than that observed in BM-MSCs. IL-8 production was observed in all hMuStem cells (13.2 ± 14.8 ng/106 cells) and increased significantly in response to stimulation with TNF-α/IFN-γ (640.2 ± 426.3 ng/106 cells; p < 0.05). A similar profile was observed in BM-MSCs, with, however, a less pronounced impact of the stimulation. Neither IL-10 nor IL-35 were detected in hMuStem cell or BM-MSC culture supernatant. Moreover, in hMuStem cell batches secretion of leukemia inhibitory factor (LIF) was 90-fold higher than in BM-MSCs (p < 0.01). LIF secretion was unchanged by stimulation with TNF-α/IFN-γ in both hMuStem cells and BM-MSCs (Fig. 3C). Levels of PGE2 secretion were similar in hMuStem cells and BM-MSCs (6.9 ± 3.3 and 10.1 ± 8.5 pg/106 cells, respectively), although greater variability was observed in BM-MSC batches. Following stimulation with TNF-α/IFN-γ, increased PGE2 secretion was observed for all batches of both cell types. In MuStem cells, stimulation resulted in a twofold increase (p < 0.05). Analysis of vascular endothelial growth factor (VEGF) secretion revealed significantly lower levels in hMuStem cells than BM-MSCs, both in basal conditions and following stimulation with TNF-α/IFN-γ (p < 0.05). Interestingly, stimulation induced a twofold decrease in VEGF secretion in hMuStem cells, whereas in BM-MSCs a variable response was observed. Finally, variable levels of Galectin-1 (Gal-1) secretion were detected in the different batches of hMuStem cells and BM-MSCs. In hMuStem cells, stimulation with TNF-α/IFN-γ resulted in a twofold decrease in Gal-1 levels compared with basal conditions (p < 0.05). Levels of secreted Gal-1 were lower in stimulated hMuStem cells than stimulated BM-MSCs (p < 0.05). Overall, our findings indicate that hMuStem cells, like BM-MSCs, produce inflammation-modulated soluble factors that exert immunomodulatory effects, including PGE2, IL-6, LIF, VEGF and Gal-1. The secretion profile of hMuStem cells was clearly impacted by pro-inflammatory conditions, as evidenced by concomitant increases in the secretion of PGE2, IL-6 and LIF and decreases in levels of VEGF and Gal-1. Human MuStem cells are distinguished from BM-MSCs by higher levels of HO-1 expression and LIF secretion and lower levels of VEGF secretion in basal conditions, and by decreased secretion of VEGF and Gal-1 in pro-inflammatory conditions.

Table 2 Secretory profile of immunoregulatory mediator by human MuStem cells and bone marrow-derived mesenchymal stem cells cultured in basal or pro-inflammatory conditions
Full size table

hMuStem cells suppress T-cell proliferation through PGE2 secretion and iNOS activity

To clarify the mechanism underlying the immunosuppressive effects of hMuStem cells on lymphocyte proliferation, we performed a series of inhibitory assays. Indomethacin, a selective inhibitor of cyclooxygenase-2, which mediates PGE2 synthesis, was added to co-cultures of hMuStem cells + PHA-stimulated PBMCs, after first verifying that indomethacin does not interfere with PBMC proliferation in PBMC-only cultures. BM-MSCs were also considered in the experiment. In indomethacin-treated co-cultures of hMuStem cells + stimulated PBMCs, the proportions of proliferative CD4+ and CD8+ T-lymphocytes were significantly higher than those observed in untreated co-cultures (42.9% ± 8.8% vs. 24.2% ± 5.9% and 50.4% ± 14.6% vs. 17.0% ± 5.6%, respectively; p < 0.001 in both cases; Fig. 4A), revealing partial but significant restoration of both CD4+ and CD8+ T-cell proliferation. Similar results were obtained in BM-MSC + stimulated PBMC co-cultures. Next, we examined the effect of the addition of L-NMMA, which suppresses NO production, in co-cultures of hMuStem cells + PHA-stimulated PBMCs and of BM-MSCs + PHA-stimulated PBMCs. In L-NMMA-treated co-cultures of hMuStem cells + PBMCs, the proportions of proliferative CD4+ and CD8+ T-lymphocytes were significantly higher than those observed in untreated co-cultures (73.3% ± 30.5% vs. 54.4% ± 27.2% and 72.8% ± 40.7% vs. 50.4% ± 38.8%, respectively; p < 0.001 in both cases; Fig. 4B). These results reflect a 21.4% ± 9.6%, and 24.3% ± 16.4% restoration of PBMC proliferation, respectively (p < 0.05; Fig. 4B) Importantly, the proportions of proliferating CD4+ and CD8+ T-lymphocytes were unchanged in the L-NMMA-treated BM-MSC + PBMC co-cultures, despite the fact that iNOS was expressed by BM-MSCs.

Fig. 4
figure 4

Involvement of prostaglandin E2 secretion and inducible nitric oxide synthase activity in human MuStem cell-mediated T-cell inhibition. Quantification of the proliferation of Cell Trace Violet (CTV)-labeled CD4+ and CD8+ T cells in co-cultures of naïve hMuStem cells + PBMCs or naïve BM-MSCs + PBMCs a with or without indomethacin, a prostaglandin E2 (PGE2) inhibitor; and b with or without NG-monomethyl-L-arginine (L-NMMA), an inducible nitric oxide synthase (iNOS) inhibitor. Experiments were performed on 5 and 4 independent batches of hMuStem cells and BM-MSCs, respectively. Data are presented as the mean ± SEM (*p < 0.05, ***p < 0.001; Mann–Whitney U test)

Full size image

Taken together, these findings point to PGE2 as a mediator of the immunosuppressive effects of hMuStem cells on T-lymphocyte proliferation, as already described for BM-MSCs, and also indicate a role of iNOS that appears not to be shared with BM-MSCs.

hMuStem cells directly inhibit CD3 + T-lymphocyte proliferation in a dose-dependent manner

To further characterize the impact of hMuStem cells on T-lymphocyte regulation, we investigated whether hMuStem cell-induced inhibition of T-cell proliferation is mediated directly by hMuStem cells, or also involves the regulation of other cells within PBMCs. CD3-sorted lymphocytes stimulated with allogeneic PBMCs in MLR were co-cultured with hMuStem cells (n = 4, independent donors) at T-cell:hMuStem cell ratios of 16:1 to 1:2. The addition of hMuStem cells at ratios of 16:1 and 4:1 resulted in no change in the proliferation of MLR-stimulated CD3+ lymphocytes relative to that observed in T-cells alone (Additional file 3: Figure S2). By contrast, the addition of hMuStem cells at ratios of 1:1 and 1:2 dramatically inhibited the proliferation of MLR-stimulated CD3+ lymphocytes (88.1% ± 12.6% and 99.0% ± 1.20%, respectively), demonstrating that hMuStem cells strongly suppress T-cell proliferation in a dose-dependent manner (p < 0.03). Importantly, these findings show that hMuStem cells can act directly on T-cells independently of the participation of other immune cell types.

hMuStem cells inhibit the cytotoxic response of CD8+ T-lymphocytes through cell–cell contact and paracrine activity

Having established that hMuStem cells can inhibit T-cell proliferation and promote a Treg cell-like phenotype, we next sought to determine their impact on the cytotoxic activity of T-lymphocytes. To this end, we examined IFN-γ expression and the cytotoxic response of a CD8+ cytotoxic T-lymphocyte (CTL) clone that specifically recognizes the HLA-A2/MUC1 peptide complex after contact with a luciferase-expressing mesothelioma cell line expressing this complex. First, the CTL clone was co-cultured for 6 h either directly with hMuStem cells or BM-MSCs at ratio of 1:10 (stem cells:CTLs) or with the corresponding secretome. After contact with the specific mesothelioma cell line, the proportion of IFN-γ+ cells was determined in the different conditions (Fig. 5A). In the co-cultures of CTLs with either hMuStem cells (panel c) or their secretome (panel d), the percentage of cells expressing IFN-γ was 47.2% ± 8.4% and 58.4% ± 4.8% of that observed with CTLs cultured alone (native CTLs; panel b), respectively (Fig. 5B). While a trend toward a reduction in the proportion of IFN-γ+ cells was observed in presence of hMuStem cell secretome compared to native CTLs, a significant decrease was observed only for CTLs co-cultured directly with hMuStem cells (p < 0.05), suggesting that suppression of CTL activation by hMuStem cells relies on a direct contact. In comparison, when BM-MSCs (panel e) or their secretome (panel f) were added to CTLs, the proportion of cells expressing IFN-γ corresponded to 79.4% ± 18.7% and 112.4% ± 19.1% of that observed in native condition, revealing an absence of any inhibitory action from BM-MSC on CTL activation. Given that hMuStem cells and BM-MSCs enhance the expression and secretion of regulatory molecules in pro-inflammatory conditions, we next repeated the cytotoxicity assay using TNF-α/IFN-γ-stimulated cells. Compared to CTLs co-cultured with unstimulated hMuStem cells, no changes were observed in CTLs cultured in presence of stimulated hMuStem cells (panel g). However, a significant decrease of IFN-γ+ cells was noted with stimulated hMuStem cell secretome (representing decreases of 2.0-fold and 2.4-fold, respectively; p < 0.05; panel h). In presence of stimulated BM-MSCs (panel i), the percentage of IFN-γ+ cells was significantly reduced (43.6% ± 16.9% of that observed in CTLs cultured alone) showing the ability of BM-MSCs to potently suppress the activation of CTL through direct contact (p < 0.05). The percentage of IFN-γ+ cells was lower when CTLs were co-cultured with stimulated BM-MSC secretome as compared to the unstimulated BM-MSC secretome (70.7% ± 15.2% vs 112.4% ± 19.1% of native CTLs; p < 0.05; panel j). Altogether, those results show that, in contrast to hMuStem cells, BM-MSCs require a TNF-α/IFN-γ stimulation to suppress CTL activation through both direct cell contact and the secretion of soluble factors.

Fig. 5
figure 5

Effect of human MuStem cells and human bone-marrow mesenchymal stem cells on the cytotoxic response of CD8+ T-lymphocytes. a Flow cytometry comparison of IFN-γ expression on CD8+ cytotoxic T-lymphocytes (CTLs) cultured either alone or with a MUC-1-expressing mesothelioma cell line. Native CTLs correspond to CTLs cultured in standard medium. Conditioned-CTLs correspond to CTLs co-cultured with hMuStem cells or BM-MSCs (unstimulated or TNF-α/IFN-γ-stimulated) or with their corresponding secretome (collected in both conditions). Percentage of IFN-γ+ CD8+ cells from one representative experiment are reported in the right upper corner of each corresponding plot. b Percentage of IFN-γ+ cells in CTLs co-cultured with hMuStem cells or BM-MSCs (unstimulated or TNF-α/IFN-γ-stimulated) or with their corresponding secretome. c Quantification of specific lysis of CTLs co-cultured with hMuStem cells or BM-MSCs (unstimulated or TNF-α/IFN-γ-stimulated) or with their corresponding secretome. d Quantification of granzyme B secretion by CTLs cultured with a MUC-1 expressing mesothelioma cell line. CTLs were cultured in basal conditions (native CTLs) or conditioned with either hMuStem cells and BM-MSCs (unstimulated or TNF-α/IFN-γ-stimulated) or their corresponding secretome. Experiments were performed on 5 and 4 independent batches of hMuStem cells and BM-MSCs, respectively. Data are presented as mean ± SEM (significant difference compared to native CTLs: ▴p < 0.05, ▴▴p < 0.01; significant difference between two conditions; *p < 0.05, **p < 0.01; Mann–Whitney U test or Wilcoxon matched-pairs signed rank test (unstimulated vs. stimulated condition)

Full size image

Luciferase activity in supernatant of co-cultures revealed specific lysis in CTLs co-cultured with hMuStem cells and those co-cultured with hMuStem cell secretome, corresponding to 46.4% ± 16.7% and 58.4% ± 23.9% with respect to the native condition (Fig. 5C). These results reveal lower cytotoxic activity in conditioned CTLs than native CTLs (p < 0.01 and p < 0.05, respectively), although this reduction was significantly more pronounced when CTLs were in direct contact with hMuStem cells (p < 0.05). In CTLs co-cultured with BM-MSCs or their secretome, lytic activity corresponded to 30.1% ± 34.3% and 49.1% ± 44.6% of native condition, respectively, revealing a lower intensity in both conditions (p < 0.05). Thus, inhibition of CTL cytotoxic activity induced by hMuStem cells and BM-MSCs appears to be mediated by cell–cell contact-dependent mechanisms as well as secreted factors. Interestingly, no statistical differences in the intensity of inhibition of CTL-mediated lysis were observed either between unstimulated and TNF-α/IFN-γ stimulated conditions or between hMuStem cells and BM-MSCs. In line with this view, secretion of granzyme B by CTLs was markedly reduced when they were co-cultured with either hMuStem cells or their secretome (Fig. 5D). Similar results were observed in co-cultures performed with BM-MSCs and their secretome. Also, TNF-α/IFN-γ stimulation did not alter the degree of inhibition of granzyme B secretion in both cultures done with hMuStem cells and BM-MSCs. Overall, these findings demonstrate that naïve hMuStem cells, unlike BM-MSCs that require a pro-inflammatory stimulation, reduce the cytotoxic response of CTLs, through mechanisms that involve both cell–cell contact and soluble factors.

Discussion

In this study, we provide original and compelling data demonstrating the capacity of hMuStem cells to control T-cell function, highlighting the potent immunosuppressive properties of these cells. Our data indicate that hMuStem cells exhibit a poorly immunogenic phenotype and also interact with T-cells to regulate their functions. We show that hMuStem cells can (1) induce the generation of Treg cells; (2) inhibit CD4+ and CD8+ T-lymphocyte proliferation in part by regulating PGE2 secretion and iNOS activity; and (3) repress the CTL response both through direct cell–cell contact and paracrine factors.

The present study provides the first evidence that hMuStem cells and BM-MSCs share a similar immunophenotype. Indeed, in basal conditions, hMuStem cells are positive for HLA-I molecules and are negative for HLA-II (i.e., HLA-DQ, HLA-DR) and for co-stimulatory molecules, including CD80, CD86 and CD40, that are required for complete T-cell activation. We found that 10–60% of hMuStem cells were HLA-DP+. Our data suggest that hMuStem cells, like BM-MSCs, have a poorly immunogenic profile. Moreover, we found that stimulation of hMuStem cells with TNF-α/IFN-γ upregulated HLA-I molecules and uniformed HLA-DP expression. These observations distinguish hMuStem cells from BM-MSCs, in which HLA-DR is upregulated in response to pro-inflammatory stimulation [74]. The phenotype described here for hMuStem cells is also consistent with the characterization of Mabs, for which immunomodulatory properties have been described [78]. Mabs constitutively express HLA-ABC and low levels of HLA-DR in the resting state, and express neither the co-stimulatory molecules CD40, CD80 or CD86 nor the inhibitory molecules PDL-1 or cytotoxic T-lymphocyte antigen-4. They also observed increased expression of HLA-DR, but not HLA-ABC after IFN-γ stimulation. BM-MSCs exert their immunosuppressive function through the expression of a wide number of membrane molecules, including PDL-1 and PDL-2, which are ligands of PD-1 expressed by T-lymphocytes [76, 79, 80]. PD-1 signal transduction pathways in T-cells mediate the inhibition of proliferation, hyporesponsiveness and apoptosis [81]. We found that hMuStem cells did not express PDL-1, but were homogeneously positive for PDL-2, which negatively regulates T-cell responses and plays a pivotal role in immune tolerance [82]. Moreover, stimulation with TNF-α/IFN-γ induced the expression of PDL-1 while up-regulating PDL-2, indicating that hMuStem cells have a favorable phenotypic profile for the modulation of T-cell activity.

Human MuStem cells express the main lymphocyte adhesion molecules ICAM-1 and VCAM-1, which are crucial for leukocyte adhesion and for the formation and stabilization of immunological synapses between T-cells and APCs [83]. ICAM-1 and VCAM-1 are also critical for MSC-mediated immunosuppression [84]. ICAM-1 signaling increases the immunosuppressive capacity of MSCs by enhancing PGE2 secretion and IDO expression [85]. Moreover, VCAM-1-positive MSCs exert a greater suppressive effect than VCAM-1-negative MSCs by inhibiting the Th1 response and inducing the generation of Treg cells [86]. We found that in basal conditions hMuStem cells, like BM-MSCs, expressed low levels of ICAM-1 and VCAM-1, both of which increased after exposure to pro-inflammatory cytokines (TNF-α/IFN-γ), as evidenced by detection of 100% of positive cells and a 27-fold increase in ICAM-1 expression. This expression profile could explain the immunoregulatory properties of hMuStem cells, their capacity to physically interact with T-cells and thereby reinforce inhibitory molecule-mediated signaling (i.e., via PDL-1, PDL-2, CD155), and the proximity of T-cells to secreted immunosuppressive mediators.

Human MuStem cells inhibited T-cell proliferation when co-cultured with a mix of PBMCs or isolated activated T cells, revealing a direct inhibitory effect of hMuStem cells (i.e., without the involvement of other immune cell partners). These observations are consistent with the findings of multiple studies showing that human MSCs directly suppress T-cell proliferation in a dose-dependent manner [39, 87]. Human Mabs have also been shown to potently and dose-dependently suppress CD4+ and CD8+ T-cell proliferation in vitro, exerting a significant inhibitory effect at Mabs:PBMC ratios of between 1:1 and 1:4 [88]. Our investigation of the underlying mechanisms revealed the involvement of PGE2, as previously reported for both MSCs [89] and Mabs [78]. We also uncovered a role of iNOS activity in the inhibitory activity of hMuStem cells, but not BM-MSCs, highlighting a major difference between these two cell types. We found that BM-MSCs were uniformly positive for iNOS expression, in contrast to the findings of several studies demonstrating that iNOS is not expressed by human MSCs but is expressed in MSCs derived from other species including mouse, rat, hamster and rabbit [90].

Another key finding of our study is the capacity of hMuStem cells to specifically interact with CTLs and suppress their cytotoxic response. Indeed, after 24 h of contact with hMuStem cells, CTL clones showed a decrease in their ability to express IFN-γ, secrete granzyme B and lyse target cells following recognition of a specific HLA-I/peptide signal. These findings indicate that hMuStem cells inhibit the activation and function of fully differentiated IL-2-activated clonally-derived CTLs. Importantly, this global inhibitory effect is constitutive for hMuStem cells whereas it is for BM-MSCs. In line with this finding, an inhibitory effect was observed when BM-MSCs were co-cultured with PBMCs, but only when they were added prior to CTL activation or during the early phase of CTL activation and in a dose-dependent manner [91]. This finding could suggest that the lack of effect observed here on IFN-γ expression could be related to a lesser action of BM-MSCs compared to hMuStem cells. In addition, it would be interesting to further explore this ability of hMuStem cells by studying their effect on the activity of NK cells, which are constitutively cytotoxic. This would be all the more interesting as studies of the inhibitory effects of BM-MSCs on NK cells have produced conflicting findings, ranging from no effects [91] to reduced cytotoxic activity of NK cells [44].

The co-culture of PBMCs or CTLs with hMuStem cell secretome instead of hMuStem cells produced similar, albeit milder, effects, suggesting that the inhibitory effect of hMuStem cells involves paracrine activity but also requires direct cell–cell contact. A role of direct contact is supported by the constitutive and high-level expression of CD155 and ICAM-1, respectively, observed in all hMuStem cells. Pro-inflammatory stimulation is known to increase both the secretion of immunomodulatory mediators and the expression of the majority of molecules involved in interactions with lymphocytes. Notably, CTLs previously cultured with stimulated hMuStem cells showed lytic capacities and IFN-γ expression levels similar to those of CTLs cultured with naïve hMuStem cells. This may be explained by the fact that the co-culture environment already provides an inflammatory signal to the hMuStem cells due to the presence of IFN-γ produced by CTLs. An alternative hypothesis is that the membrane molecules that induce CTL inhibition are not modulated by IFN-γ or TNF-α, as described for CD155. For further comparison between hMuStem cells and BM-MSCs, it would be informative to determine why TNF-α/IFN-γ stimulation is instead required for BM-MSCs to reduce IFN-γ expression by CTLs. The secretome of stimulated hMuStem cells did not further decrease granzyme B secretion or the lytic capacity of CTLs relative to naïve hMuStem cells but did result in lower IFN-γ expression in CTLs after activation by the HLA-I/peptide complex. This indicates that prior stimulation of hMuStem cells leads to a greater paracrine effect on IFN-γ expression but not on specific lytic activity of CTLs. Of note, a similar impact with a more marked intensity was observed for the secretome of stimulated BM-MSCs. This result suggests that the hMuStem cells use distinct paracrine mechanisms of action to inhibit IFN-γ expression and to suppress the lytic capacity of CTLs. Consistent with this view, IFN-γ production appears not to be directly correlated with cytotoxic function [92]. Together, these findings highlight the diversity of modes of action used by hMuStem cells to suppress the cytotoxic response of CTLs, a key characteristic that appears to distinguish hMuStem cells from BM-MSCs.

Here, we found that such as BM-MSCs [51, 60], hMuStem cells induced the generation of Treg-like cells corresponding to Tr-1 and CD4+FoxP3+ cells. However, in our experimental conditions, the capacity of hMuStem cells to induce Treg cells was significant but modest, resulting in the generation of few Tr1 and CD4+FoxP3+ cells. Therefore, it will be of interest to further elucidate the immunoregulatory mechanisms of hMuStem cells by studying the regulatory function of Tr1 and CD4+FoxP3+ cells generated at the end of co-culture experiments. This type of functional study is warranted given that Treg cells play a critical role in the pathogenesis of MDs, as evidenced by the direct relationship between the increase in Treg cell number and the reduction in both inflammation and fiber necrosis in dystrophic mice [17, 18].

Finally, our findings provide evidence that LIF secretion clearly distinguishes hMuStem cells from BM-MSCs. LIF has been proposed to play a key role in the generation of Treg cells [93, 94]. Indeed, specific blockade of the glycoprotein cytokine LIF in co-cultures of human BM-, Wharton’s jelly- and adipose tissue-derived MSCs + PBMCs resulted in restoration of CD3+ lymphocyte proliferation by up to 91% and a decrease in CD4+CD25+Foxp3+ cells. Given the inhibitory effect of hMuStem cells on T-cell proliferation and their capacity to generate Tregs described here, it would be informative to further examine the role of LIF in the immunosuppressive effect of hMuStem cells.

Conclusion

Our findings show that hMuStem cells exert potent immunosuppressive properties on T-cells, inhibiting their proliferation and cytotoxic response and inducing the generation of Tregs. Human MuStem cells may modulate immune cell activity in addition to directly contribute to muscle fiber formation/regeneration, thereby exerting positive effects on the pathophysiology of MDs through two complementary modes of action. Human MuStem cells could therefore constitute a particularly efficient therapeutic tool for clinical application in the context of MDs.

Availability of data and materials

The datasets used and/or analyzed during the current study to support the conclusions are included in the article and in the corresponding additional files. They can be made available from the corresponding author on reasonable request.

Abbreviations

7-AAD:

7-Amino-actinomycin D

α-MEM:

Alpha-minimum essential medium

Ab:

Antibody

APC:

Antigen-presenting cell

AD-MSC:

Adipose tissue-derived mesenchymal stem cell

bFGF:

Basic fibroblast growth factor

BM-MSC:

Bone marrow-derived mesenchymal stem cell

CTV:

Cell trace violet

DC:

Dendritic cell

DMD:

Duchenne muscular dystrophy

EGF:

Epidermal growth factor

FCS:

Fetal calf serum

Gal-1:

Galectin-1

GM:

Growth medium

GRMD:

Golden retriever muscular dystrophy

HGF:

Hepatocyte growth factor

HLA:

Human leukocyte antigen

HO-1:

Heme oxygenase-1

IDO:

Indoleamine 2,3-dioxygenase

IFN:

Interferon

IMDM:

Iscove’s modified Dulbecco’s medium

iNOS:

Inducible nitrite oxide synthase

IL:

Interleukin

LIF:

Leukemia inhibitory factor

L-NMMA:

NG-monomethyl-L-arginine

Mabs:

Mesoangioblasts

MD:

Muscular dystrophy

MDSC:

Muscle-derived stem cell

mdx :

X-chromosome-linked muscular dystrophy

MLR:

Mixed lymphocyte reaction

mRNA:

Messenger ribonucleic acid

MSC::

Mesenchymal stem cell

NEAA::

Non-essential amino-acid

NK::

Natural killer

NO::

Nitric oxide

PBMC::

Peripheral blood mononuclear cell

PBS::

Phosphate buffered saline

PGE2::

Prostaglandin E2

PHA::

Phyto-hemagglutinin

PICs::

PW1+ Pax7+ interstitial cells

PMA::

Phorbol myristate acetate

PSF::

Penicillin–streptomycin-fungizone

RT-PCR::

Reverse transcription-polymerase chain reaction

SCID::

Severe combined-immunodeficiency

SP::

Side population

TNF::

Tumor necrosis factor

Treg::

Regulatory T

VEGF::

Vascular endothelial growth factor

References

  1. Emery AEH. The muscular dystrophies. Lancet. 2002;359:687–95.

    Article  CAS  PubMed  Google Scholar 

  2. Matsumura K, Campbell KP. Deficiency of dystrophin-associated proteins: a common mechanism leading to muscle cell necrosis in severe childhood muscular dystrophies. Neuromuscul Disord. 1993;3:109–18.

    Article  CAS  PubMed  Google Scholar 

  3. Emery AEH. Population frequencies of inherited neuromuscular diseases—a world survey. Neuromuscul Disord Neuromuscul Disord. 1991;1:19–29.

    Article  CAS  PubMed  Google Scholar 

  4. Moat SJ, Bradley DM, Salmon R, Clarke A, Hartley L. Newborn bloodspot screening for Duchenne Muscular Dystrophy: 21 years experience in Wales (UK). Eur J Hum Genet. 2013;21:1049–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hoffman EP, Brown RH, Kunkel LM. Dystrophin: the protein product of the duchenne muscular dystrophy locus. Cell. 1987;51:919–28.

    Article  CAS  PubMed  Google Scholar 

  6. Arahata K, Engel AG. Monoclonal antibody analysis of mononuclear cells in myopathies, I: quantitation of subsets according to diagnosis and sites of accumulation and demonstration and counts of muscle fibers invaded by T cells. Ann Neurol Ann Neurol. 1984;16:193–208.

    Article  CAS  PubMed  Google Scholar 

  7. McDouall RM, Dunn MJ, Dubowitz V. Nature of the mononuclear infiltrate and the mechanism of muscle damage in juvenile dermatomyositis and Duchenne muscular dystrophy. J Neurol Sci J Neurol Sci. 1990;99:199–217.

    CAS  PubMed  Google Scholar 

  8. Wehling-Henricks M, Lee JJ, Tidball JG. Prednisolone decreases cellular adhesion molecules required for inflammatory cell infiltration in dystrophin-deficient skeletal muscle. Neuromuscul Disord Neuromuscul Disord. 2004;14:483–90.

    Article  PubMed  Google Scholar 

  9. Balaban B, Matthews DJ, Clayton GH, Carry T. Corticosteroid treatment and functional improvement in Duchenne muscular dystrophy long-term effect. Am J Phys Med Rehabil. 2005;84:843–50.

    Article  PubMed  Google Scholar 

  10. Cai B, Spencer MJ, Nakamura G, Tseng-Ong L, Tidball JG. Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors. Am J Pathol. 2000;156:1789–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wehling M, Spencer MJ, Tidball JG. A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice. J Cell Biol J Cell Biol. 2001;155:123–31.

    Article  CAS  PubMed  Google Scholar 

  12. Hodgetts S, Radley H, Davies M, Grounds MD. Reduced necrosis of dystrophic muscle by depletion of host neutrophils, or blocking TNFα function with Etanercept in mdx mice. Neuromuscul Disord Neuromuscul Disord. 2006;16:591–602.

    Article  PubMed  Google Scholar 

  13. Bulfield G, Siller WG, Wight PAL, Moore KJ. X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci USA. 1984;81:1189–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lozanoska-Ochser B, Benedetti A, Rizzo G, Marrocco V, Di Maggio R, Fiore P, et al. Targeting early PKCθ-dependent T-cell infiltration of dystrophic muscle reduces disease severity in a mouse model of muscular dystrophy. J Pathol. 2018;244:323–33.

    Article  CAS  PubMed  Google Scholar 

  15. Villalta SA, Deng B, Rinaldi C, Wehling-Henricks M, Tidball JG. IFN-γ promotes muscle damage in the mdx mouse model of duchenne muscular dystrophy by suppressing M2 macrophage activation and inhibiting muscle cell proliferation. J Immunol. 2011;187:5419–28.

    Article  CAS  PubMed  Google Scholar 

  16. Spencer MJ, Marino MW, Winckler WM. Altered pathological progression of diaphragm and quadriceps muscle in TNF-deficient, dystrophin-deficient mice. Neuromuscul Disord Neuromuscul Disord. 2000;10:612–9.

    Article  CAS  PubMed  Google Scholar 

  17. Villalta SA, Rosenthal W, Martinez L, Kaur A, Sparwasser T, Tidball JG, et al. Regulatory T cells suppress muscle inflammation and injury in muscular dystrophy. Sci Transl Med. 2014;6:258ra142.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Eghtesad S, Jhunjhunwala S, Little SR, Clemens PR. Rapamycin ameliorates dystrophic phenotype in mdx mouse skeletal muscle. Mol Med Mol Med. 2011;17:917–24.

    Article  CAS  PubMed  Google Scholar 

  19. Tabebordbar M, Wang ET, Wagers AJ. Skeletal muscle degenerative diseases and strategies for therapeutic muscle repair. Annu Rev Pathol Mech Dis. 2013;8:441–75.

    Article  CAS  Google Scholar 

  20. Gussoni E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, Flint AF, et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature. 1999;401:390–4.

    Article  CAS  PubMed  Google Scholar 

  21. Jackson KA, Mi T, Goodell MA. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci USA. 1999;96:14482–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Torrente Y, Belicchi M, Sampaolesi M, Pisati F, Meregalli M, D’Antona G, et al. Human circulating AC133+ stem cells restore dystrophin expression and ameliorate function in dystrophic skeletal muscle. J Clin Invest. 2004;114:182–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Benchaouir R, Meregalli M, Farini A, D’Antona G, Belicchi M, Goyenvalle A, et al. Restoration of human dystrophin following transplantation of exon-skipping-engineered dmd patient stem cells into dystrophic mice. Cell Stem Cell Cell Stem Cell. 2007;1:646–57.

    Article  CAS  PubMed  Google Scholar 

  24. Minasi MG, Riminucci M, De Angelis L, Borello U, Berarducci B, Innocenzi A, et al. The meso-angioblast: a multipotent, self-renewing cell that originates from the dorsal aorta and differentiates into most mesodermal tissues. Development. 2002;129:2773–83.

    Article  CAS  PubMed  Google Scholar 

  25. Sampaolesi M, Torrente Y, Innocenzi A, Tonlorenzi R, D’Antona G, Pellegrino MA, et al. Cell therapy of α-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science. 2003;301:487–92.

    Article  CAS  PubMed  Google Scholar 

  26. Sampaolesi M, Blot S, D’Antona G, Granger N, Tonlorenzi R, Innocenzi A, et al. Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature. 2006;444:574–9.

    Article  CAS  PubMed  Google Scholar 

  27. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–7.

    Article  CAS  PubMed  Google Scholar 

  28. Rodriguez AM, Pisani D, Dechesne CA, Turc-Carel C, Kurzenne JY, Wdziekonski B, et al. Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse. J Exp Med. 2005;201:1397–405.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Mitchell KJ, Pannérec A, Cadot B, Parlakian A, Besson V, Gomes ER, et al. Identification and characterization of a non-satellite cell muscle resident progenitor during postnatal development. Nat Cell Biol. 2010;12:257–66.

    Article  CAS  PubMed  Google Scholar 

  30. Qu Z, Balkir L, Van Deutekom JCT, Robbins PD, Pruchnic R, Huard J. Development of approaches to improve cell survival in myoblast transfer therapy. J Cell Biol. 1998;142:1257–67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Qu-Petersen Z, Deasy B, Jankowski R, Ikezawa M, Cummins J, Pruchnic R, et al. Identification of a novel population of muscle stem cells in mice: Potential for muscle regeneration. J Cell Biol. 2002;157:851–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sun C, Shen L, Zhang Z, Xie X. Therapeutic strategies for duchenne muscular dystrophy: an update. Genes (Basel). 2020;11:1–25.

    Article  Google Scholar 

  33. Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringdén O. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol. 2003;31:890–6.

    Article  PubMed  CAS  Google Scholar 

  34. Vieira NM, Valadares M, Zucconi E, Secco M, Bueno Junior CR, Brandalise V, et al. Human adipose-derived mesenchymal stromal cells injected systemically Into GRMD dogs without immunosuppression are able to reach the host muscle and express human dystrophin. Cell Transplant Cell Transplant. 2012;21:1407–17.

    Article  CAS  PubMed  Google Scholar 

  35. Le Blanc K, Mougiakakos D. Multipotent mesenchymal stromal cells and the innate immune system. Nat Rev Immunol. 2012;12:383–96.

    Article  PubMed  CAS  Google Scholar 

  36. Djouad F, Plence P, Bony C, Tropel P, Apparailly F, Sany J, et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood. 2003;102:3837–44.

    Article  CAS  PubMed  Google Scholar 

  37. Cao W, Cao K, Cao J, Wang Y, Shi Y. Mesenchymal stem cells and adaptive immune responses. Immunol Lett. 2015;168:147–53.

    Article  CAS  PubMed  Google Scholar 

  38. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005;105:1815–22.

    Article  CAS  PubMed  Google Scholar 

  39. Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood. 2002;99:3838–43.

    Article  PubMed  Google Scholar 

  40. Bartholomew A, Sturgeon C, Siatskas M, Ferrer K, McIntosh K, Patil S, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol. 2002;30:42–8.

    Article  PubMed  Google Scholar 

  41. Najar M, Raicevic G, Fayyad-Kazan H, Bron D, Toungouz M, Lagneaux L. Mesenchymal stromal cells and immunomodulation: a gathering of regulatory immune cells. Cytotherapy. 2016;18:160–71.

    Article  CAS  PubMed  Google Scholar 

  42. Luz-Crawford P, Kurte M, Bravo-Alegría J, Contreras R, Nova-Lamperti E, Tejedor G, et al. Mesenchymal stem cells generate a CD4+CD25+Foxp3 + regulatory T cell population during the differentiation process of Th1 and Th17 cells. Stem Cell Res Ther. 2013;4:65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Corcione A, Benvenuto F, Ferretti E, Giunti D, Cappiello V, Cazzanti F, et al. Human mesenchymal stem cells modulate B-cell functions. Blood. 2006;107:367–72.

    Article  CAS  PubMed  Google Scholar 

  44. Spaggiari GM, Capobianco A, Becchetti S, Mingari MC, Moretta L. Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood. 2006;107:1484–90.

    Article  CAS  PubMed  Google Scholar 

  45. Sotiropoulou PA, Perez SA, Gritzapis AD, Baxevanis CN, Papamichail M. Interactions between human mesenchymal stem cells and natural killer cells. Stem Cells. 2006;24:74–85.

    Article  PubMed  Google Scholar 

  46. Jiang XX, Zhang Y, Liu B, Zhang SX, Wu Y, Yu XD, et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood. 2005;105:4120–6.

    Article  CAS  PubMed  Google Scholar 

  47. Djouad F, Charbonnier L-M, Bouffi C, Louis-Plence P, Bony C, Apparailly F, et al. Mesenchymal stem cells inhibit the differentiation of dendritic cells through an interleukin-6-dependent mechanism. Stem Cells. 2007;25:2025–32.

    Article  CAS  PubMed  Google Scholar 

  48. Németh K, Leelahavanichkul A, Yuen PST, Mayer B, Parmelee A, Doi K, et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E 2-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med. 2009;15:42–9.

    Article  PubMed  CAS  Google Scholar 

  49. Kim J, Hematti P. Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages. Exp Hematol. 2009;37:1445–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Rasmusson I, Ringdén O, Sundberg B, Le Blanc K. Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms. Exp Cell Res. 2005;305:33–41.

    Article  CAS  PubMed  Google Scholar 

  51. Selmani Z, Naji A, Zidi I, Favier B, Gaiffe E, Obert L, et al. Human leukocyte antigen-g5 secretion by human mesenchymal stem cells is required to suppress T lymphocyte and natural killer function and to induce CD4 + CD25 high FOXP3 + regulatory T cells. Stem Cells. 2008;26:212–22.

    Article  CAS  PubMed  Google Scholar 

  52. Casiraghi F, Perico N, Remuzzi G. Mesenchymal stromal cells for tolerance induction in organ transplantation. Hum Immunol. 2018;79:304–13.

    Article  PubMed  Google Scholar 

  53. Djouad F, Bouffi C, Ghannam S, Noël D, Jorgensen C. Mesenchymal stem cells: innovative therapeutic tools for rheumatic diseases. Nat Rev Rheumatol. 2009;5:392–9.

    Article  CAS  PubMed  Google Scholar 

  54. Luz-Crawford P, Ipseiz N, Espinosa-Carrasco G, Caicedo A, Tejedor G, Toupet K, et al. PPARβ/δ directs the therapeutic potential of mesenchymal stem cells in arthritis. Ann Rheum Dis. 2016;75:2166–74.

    Article  CAS  PubMed  Google Scholar 

  55. Ren G, Zhang L, Zhao X, Xu G, Zhang Y, Roberts AI, et al. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell. 2008;2:141–50.

    Article  CAS  PubMed  Google Scholar 

  56. Quaedackers ME, Baan CC, Weimar W, Hoogduijn MJ. Cell contact interaction between adipose-derived stromal cells and allo-activated T lymphocytes. Eur J Immunol. 2009;39:3436–46.

    Article  CAS  PubMed  Google Scholar 

  57. Caplan AI, Correa D. The MSC: an injury drugstore. Cell Stem Cell. 2011;9:11–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Meisel R, Zibert A, Laryea M, Göbel U, Däubener W, Dilloo D. Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation. Blood. 2004;103:4619–21.

    Article  CAS  PubMed  Google Scholar 

  59. Spaggiari GM, Abdelrazik H, Becchetti F, Moretta L. MSCs inhibit monocyte-derived DC maturation and function by selectively interfering with the generation of immature DCs: central role of MSC-derived prostaglandin E2. Blood. 2009;113:6576–83.

    Article  CAS  PubMed  Google Scholar 

  60. English K, Ryan JM, Tobin L, Murphy MJ, Barry FP, Mahon BP. Cell contact, prostaglandin E2 and transforming growth factor beta 1 play non-redundant roles in human mesenchymal stem cell induction of CD4+CD25Highforkhead box P3+ regulatory T cells. Clin Exp Immunol. 2009;156:149–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Sato K, Ozaki K, Oh I, Meguro A, Hatanaka K, Nagai T, et al. Nitric oxide plays a critical role in suppression of T-cell proliferation by mesenchymal stem cells. Blood. 2007;109:228–34.

    Article  CAS  PubMed  Google Scholar 

  62. Mougiakakos D, Jitschin R, Johansson CC, Okita R, Kiessling R, Le Blanc K. The impact of inflammatory licensing on heme oxygenase-1-mediated induction of regulatory T cells by human mesenchymal stem cells. Blood. 2011;117:4826–35.

    Article  CAS  PubMed  Google Scholar 

  63. Rouger K, Larcher T, Dubreil L, Deschamps J-Y, Le Guiner C, Jouvion G, et al. Systemic delivery of allogenic muscle stem cells induces long-term muscle repair and clinical efficacy in duchenne muscular dystrophy dogs. Am J Pathol. 2011;179:2501–18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Lorant J, Saury C, Schleder C, Robriquet F, Lieubeau B, Négroni E, et al. Skeletal muscle regenerative potential of human MuStem cells following transplantation into injured mice muscle. Mol Ther. 2018;26:618–33.

    Article  CAS  PubMed  Google Scholar 

  65. Lorant J, Larcher T, Jaulin N, Hedan B, Lardenois A, Leroux I, et al. Vascular delivery of allogeneic MuStem cells in dystrophic dogs requires only short-term immunosuppression to avoid host immunity and generate clinical/tissue benefits. Cell Transplant. 2018;27:1096–110.

    Article  PubMed  PubMed Central  Google Scholar 

  66. Rannou A, Toumaniantz G, Larcher T, Leroux I, Ledevin M, Hivonnait A, et al. Human MuStem cell grafting into infarcted rat heart attenuates adverse tissue remodeling and preserves cardiac function. Mol Ther Methods Clin Dev. 2020;18:446–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Robriquet F, Lardenois A, Babarit C, Larcher T, Dubreil L, Leroux I, et al. Differential gene expression profiling of dystrophic dog muscle after MuStem cell transplantation. PLoS ONE. 2015;10:1–24.

    Article  CAS  Google Scholar 

  68. Djouad F, Bony C, Häupl T, Uzé G, Lahlou N, Louis-Plence P, et al. Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells. Arthritis Res Ther BioMed Central. 2005;7:1304–15.

    Article  CAS  Google Scholar 

  69. Maria ATJ, Toupet K, Maumus M, Fonteneau G, Le Quellec A, Jorgensen C, et al. Human adipose mesenchymal stem cells as potent anti-fibrosis therapy for systemic sclerosis. J Autoimmun. 2016;70:31–9.

    Article  CAS  PubMed  Google Scholar 

  70. Roulois D, Vignard V, Gueugnon F, Labarrière N, Grégoire M, Fonteneau JF. Recognition of pleural mesothelioma by mucin-1(950–958)/human leukocyte antigen A*0201-specific CD8+ T-cells. Eur Respir J. 2011;38:1117–26.

    Article  CAS  PubMed  Google Scholar 

  71. Blondy T, D’Almeida SM, Briolay T, Tabiasco J, Meiller C, Chéné AL, et al. Involvement of the M-CSF/IL-34/CSF-1R pathway in malignant pleural mesothelioma. J Immunother Cancer. 2020;8:e000182.

    Article  PubMed  PubMed Central  Google Scholar 

  72. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82.

    Article  CAS  PubMed  Google Scholar 

  73. Najar M, Raicevic G, Kazan HF, de Bruyn C, Bron D, Toungouz M, et al. Immune-related antigens, surface molecules and regulatory factors in human-derived mesenchymal stromal cells: the expression and impact of inflammatory priming. Stem Cell Rev Rep. 2012;8:1188–98.

    Article  CAS  PubMed  Google Scholar 

  74. Majumdar MK, Keane-Moore M, Buyaner D, Hardy WB, Moorman MA, McIntosh KR, et al. Characterization and functionality of cell surface molecules on human mesenchymal stem cells. J Biomed Sci. 2003;10:228–41.

    Article  CAS  PubMed  Google Scholar 

  75. Saury C, Lardenois A, Schleder C, Leroux I, Lieubeau B, David L, et al. Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches. Stem Cell Res Ther. 2018;9:1–20.

    Article  CAS  Google Scholar 

  76. Li H, Wang W, Wang G, Hou Y, Xu F, Liu R, et al. Interferon-γ and tumor necrosis factor-α promote the ability of human placenta-derived mesenchymal stromal cells to express programmed death ligand-2 and induce the differentiation of CD4+interleukin-10+ and CD8+interleukin-10+Treg subsets. Cytotherapy. 2015;17:1560–71.

    Article  CAS  PubMed  Google Scholar 

  77. Anderson AC, Joller N, Kuchroo VK. Functions in immune regulation. Immunity. 2017;44:989–1004.

    Article  CAS  Google Scholar 

  78. English K, Tonlorenzi R, Cossu G, Wood KJ. Mesoangioblasts suppress T cell proliferation through IDO and PGE-2-dependent pathways. Stem Cells Dev. 2013;22:512–23.

    Article  CAS  PubMed  Google Scholar 

  79. Luz-Crawford P, Noël D, Fernandez X, Khoury M, Figueroa F, Carrión F, et al. Mesenchymal stem cells repress Th17 molecular program through the PD-1 pathway. PLoS ONE. 2012;7:e45272.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Davies LC, Heldring N, Kadri N, Le Blanc K. Mesenchymal stromal cell secretion of programmed death-1 ligands regulates T cell mediated immunosuppression. Stem Cells. 2017;35:766–76.

    Article  CAS  PubMed  Google Scholar 

  81. Wang G, Zhang S, Wang F, Li G, Zhang L, Luan X. Expression and biological function of programmed death ligands in human placenta mesenchymal stem cells. Cell Biol Int. 2013;37:137–48.

    Article  CAS  PubMed  Google Scholar 

  82. Okazaki T, Honjo T. The PD-1-PD-L pathway in immunological tolerance. Trends Immunol. 2006;27:195–201.

    Article  CAS  PubMed  Google Scholar 

  83. Bromley SK, Burack WR, Johnsonn KG, Somersalo K, Sims TN, Sumen C, et al. The immunological synapse. Annu Rev Immunol. 2001;19:375–96.

    Article  CAS  PubMed  Google Scholar 

  84. Ren G, Zhao X, Zhang L, Zhang J, L’Huillier A, Ling W, et al. Inflammatory cytokine-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in mesenchymal stem cells are critical for immunosuppression. J Immunol. 2010;184:2321–8.

    Article  CAS  PubMed  Google Scholar 

  85. Espagnolle N, Balguerie A, Arnaud E, Sensebé L, Varin A. CD54-mediated interaction with pro-inflammatory macrophages increases the immunosuppressive function of human mesenchymal stromal cells. Stem Cell Rep. 2017;8:961–76.

    Article  CAS  Google Scholar 

  86. Yang ZX, Han ZB, Ji YR, Wang YW, Liang L, Chi Y, et al. CD106 identifies a subpopulation of mesenchymal stem cells with unique immunomodulatory properties. PLoS ONE. 2013;8:1–12.

    Google Scholar 

  87. Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol. 2003;57:11–20.

    Article  PubMed  Google Scholar 

  88. English K. Mechanisms of mesenchymal stromal cell immunomodulation. Immunol Cell Biol. 2013;91:19–26.

    Article  CAS  PubMed  Google Scholar 

  89. Najar M, Raicevic G, Boufker HI, Kazan HF, De BC, Meuleman N, et al. Mesenchymal stromal cells use PGE2 to modulate activation and proliferation of lymphocyte subsets: combined comparison of adipose tissue, Wharton’s Jelly and bone marrow sources. Cell Immunol. 2010;264:171–9.

    Article  CAS  PubMed  Google Scholar 

  90. Ren G, Su J, Zhang L, Zhao X, Ling W, L’Huillie A, et al. Species variation in the mechanisms of mesenchymal stem cell-mediated immunosuppression. Stem Cells. 2009;27:1954–62.

    Article  CAS  PubMed  Google Scholar 

  91. Rasmusson I, Ringdén O, Sundberg B, Le Blanc K. Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells. Transplantation. 2003;76:1208–13.

    Article  PubMed  Google Scholar 

  92. Einarsdottir T, Lockhart E, Flynn JAL. Cytotoxicity and secretion of gamma interferon are carried out by distinct CD8 T cells during Mycobacterium tuberculosis infection. Infect Immun. 2009;77:4621–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Nasef A, Mazurier C, Bouchet S, François S, Chapel A, Thierry D, et al. Leukemia inhibitory factor: role in human mesenchymal stem cells mediated immunosuppression. Cell Immunol. 2008;253:16–22.

    Article  CAS  PubMed  Google Scholar 

  94. Najar M, Raicevic G, Boufker HI, Fayyad-Kazan H, De Bruyn C, Meuleman N, et al. Adipose-tissue-derived and Wharton’s jelly-derived mesenchymal stromal cells suppress lymphocyte responses by secreting leukemia inhibitory factor. Tissue Eng Part A. 2010;16:3537–46.

    Article  CAS  PubMed  Google Scholar 

  95. Fonteneau JF, Larsson M, Somersan S, Sanders C, Münz C, Kwok WW, et al. Generation of high quantities of viral and tumor-specific human CD4 + and CD8 + T-cell clones using peptide pulsed mature dendritic cells. J Immunol Methods. 2001;258:111–26.

    Article  CAS  PubMed  Google Scholar 

  96. Gueugnon F, Leclercq S, Blanquart C, Sagan C, Cellerin L, Padieu M, et al. Identification of novel markers for the diagnosis of malignant pleural mesothelioma. Am J Pathol. 2011;178:1033–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Anne Fernandez (Institut de Génétique Humaine, CNRS UMR 9002; Université de Montpellier, France) and Cédric Menard (INSERM UMR1236, Rennes, France) for helpful advice and discussions to improve the experimental design.

Funding

This work was supported by a grant from the “Fonds Européen de Développement Régional” (FEDER; No. 38436 and No. PL0003686). It was performed in the context of the IHU-CESTI project that received French government financial support managed by the ANR (Agence Nationale de la Recherche) through the investment of the future program ANR-10-IBHU-005. The IHU-CESTI project was also supported by grants from the Région Pays de la Loire and Nantes Métropole. MC was recipient of a Ph.D grant from the French Ministry of Higher Education, Research and Innovation.

Author information

Author notes

  1. Marine Charrier, Judith Lorant, Farida Djouad and Karl Rouger have contributed equally to this work

Authors and Affiliations

  1. INRAE, Oniris, PAnTher, UMR 703, Oniris - Site de La Chantrerie, 101, Route de Gachet, CS. 40706, 44307, Nantes, France

    Marine Charrier, Judith Lorant, Cindy Schleder, Isabelle Leroux, Candice Babarit & Karl Rouger

  2. L’institut du Thorax, INSERM, CNRS, UNIV Nantes, 44007, Nantes, France

    Marine Charrier & Guillaume Lamirault

  3. Université de Nantes, Nantes, France

    Marine Charrier

  4. INSERM U1183 IRMB, Hôpital Saint Eloi, CHRU Montpellier, Université de Montpellier, 80, Rue Augustin Fliche, 34295, Montpellier, France

    Rafael Contreras-Lopez, Gautier Téjédor & Farida Djouad

  5. Laboratorio de Immunología Celular y Molecular, Facultad de Medicina, Universidad de los Andes, Las Condes, Chile

    Rafael Contreras-Lopez & Patricia Luz-Crawford

  6. CNRS, INSERM, CRCINA, Université de Nantes, 44000, Nantes, France

    Christophe Blanquart, Sophie Deshayes & Jean-François Fonteneau

  7. INRAE, Oniris, IECM, 44307, Nantes, France

    Blandine Lieubeau

  8. Service de Chirurgie Infantile, Centre Hospitalier Universitaire (CHU) de Nantes, 44093, Nantes, France

    Antoine Hamel

  9. Laboratoire d’Explorations Fonctionnelles, Centre de Référence Maladies Neuromusculaires AOC, CHU Nantes, 44093, Nantes, France

    Armelle Magot & Yann Péréon

  10. Biotherapy Division, Macopharma, 59420, Mouvaux, France

    Sabrina Viau & Bruno Delorme

  11. IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Santiago, Chile

    Patricia Luz-Crawford

Authors
  1. Marine Charrier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Judith Lorant
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rafael Contreras-Lopez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gautier Téjédor
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christophe Blanquart
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Blandine Lieubeau
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Cindy Schleder
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Isabelle Leroux
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sophie Deshayes
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jean-François Fonteneau
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Candice Babarit
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Antoine Hamel
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Armelle Magot
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Yann Péréon
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Sabrina Viau
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Bruno Delorme
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Patricia Luz-Crawford
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Guillaume Lamirault
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Farida Djouad
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Karl Rouger
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

MC conceived and designed the experiments, collected and/or assembled the data, participated to data analysis, interpretation and manuscript writing. JL performed the co-culture experiments and immunolabeling, ELISA assays and participated to data analysis and interpretation; RCL and CB performed the co-culture and flow cytometry experiments, and participated to data analysis and interpretation; GT, SD and JFF contributed to the in vitro and flow cytometry experiments, data analysis; BL performed the flow cytometry experiments, data analysis and interpretation; CS isolated and expanded the MuStem cell batches, ELISA assays, data analysis; IL isolated and expanded the MuStem cell batches; CB performed the dosage analysis; AH, AM and YP contributed to the study material; SV, BD performed some of the flow cytometry experiments, data analysis; PLC, GL and FD conceived the experimental design and contributed significantly to the data analysis and interpretation as well as to the writing of the manuscript; KR conceived and designed the study, participated to the data interpretation and wrote the manuscript. KR is the guarantor of this work, has full access to all study data and takes responsibility for the integrity of the data. The authors have agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Farida Djouad or Karl Rouger.

Ethics declarations

Ethics approval and consent to participate

Human skeletal muscle tissue: Human MuStem cells were isolated from Paravertebralis muscle biopsies collected from patients aged 12–19 years. Patients were free of known muscle disease and had undergone surgery for acute scoliosis at the Department of Pediatric surgery of the Centre Hospitalier Universitaire (CHU) de Nantes (France). Written informed consent was obtained from all patients. All protocols were approved by the Clinical Research Department of the CHU (Nantes, France), according to the rules of the French Regulatory Health Authorities (approval number: MESR/DC-2010-1199). The biological sample bank was created in compliance with national guidelines regarding the use of human tissue for research (approval number: CPP/29/10). Isolation and culture of human bone marrow-derived mesenchymal stem cells: Human BM-MSCs were collected from patients undergoing hip replacement surgery. The patients were informed and provided written informed consent prior to collection of tissue samples as approved by the French Ministry of Higher Education and Research (DC-2010–1185).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no financial and non-financial competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Methods S1.

 Immunosuppression assay.

Additional file 2: Table S1.

List of antibodies used for flow cytometry analysis.

Additional file 3: Table S2.

Primers used for RT-qPCR analysis.

Additional file 4: Figure S1.

Expression profile for cell lineage-specific surface markers by human MuStem cell.

Additional file 5: Figure S2.

Dose effect of human MuStem cells on the proliferation of CD3+ lymphocytes cultured with irradiated peripheral blood mononucleated cells.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Charrier, M., Lorant, J., Contreras-Lopez, R. et al. Human MuStem cells repress T-cell proliferation and cytotoxicity through both paracrine and contact-dependent pathways. Stem Cell Res Ther 13, 7 (2022). https://doi.org/10.1186/s13287-021-02681-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13287-021-02681-3

Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512