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

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.

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Nitric oxide plays a critical role in suppression of T-cell proliferation by mesenchymal stem cells

Blood ◽

10.1182/blood-2006-02-002246 ◽

2006 ◽

Vol 109 (1) ◽

pp. 228-234 ◽

Cited By ~ 596

Author(s):

Kazuya Sato ◽

Katsutoshi Ozaki ◽

Iekuni Oh ◽

Akiko Meguro ◽

Keiko Hatanaka ◽

...

Keyword(s):

Nitric Oxide ◽

Stem Cells ◽

Cell Proliferation ◽

T Cells ◽

Mesenchymal Stem Cells ◽

T Cell ◽

Molecular Mechanisms ◽

Critical Role ◽

Specific Inhibitor ◽

T Cell Proliferation

Abstract The molecular mechanisms by which mesenchymal stem cells (MSCs) suppress T-cell proliferation are poorly understood, and whether a soluble factor plays a major role remains controversial. Here we demonstrate that the T-cell–receptor complex is not a target for the suppression, suggesting that downstream signals mediate the suppression. We found that Stat5 phosphorylation in T cells is suppressed in the presence of MSCs and that nitric oxide (NO) is involved in the suppression of Stat5 phosphorylation and T-cell proliferation. The induction of inducible NO synthase (NOS) was readily detected in MSCs but not T cells, and a specific inhibitor of NOS reversed the suppression of Stat5 phosphorylation and T-cell proliferation. This production of NO in the presence of MSCs was mediated by CD4 or CD8 T cells but not by CD19 B cells. Furthermore, inhibitors of prostaglandin synthase or NOS restored the proliferation of T cells, whereas an inhibitor of indoleamine 2,3-dioxygenase and a transforming growth factor–β–neutralizing antibody had no effect. Finally, MSCs from inducible NOS−/− mice had a reduced ability to suppress T-cell proliferation. Taken together, these results suggest that NO produced by MSCs is one of the major mediators of T-cell suppression by MSCs.

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Allergen-Induced C5a/C5aR1 Axis Activation in Pulmonary CD11b+ cDCs Promotes Pulmonary Tolerance through Downregulation of CD40

Cells ◽

10.3390/cells9020300 ◽

2020 ◽

Vol 9 (2) ◽

pp. 300 ◽

Cited By ~ 4

Author(s):

Konstantina Antoniou ◽

Fanny Ender ◽

Tillman Vollbrandt ◽

Yves Laumonnier ◽

Franziska Rathmann ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

T Cell Proliferation ◽

C5a Receptor ◽

The Impact

Activation of the C5/C5a/C5a receptor 1 (C5aR1) axis during allergen sensitization protects from maladaptive T cell activation. To explore the underlying regulatory mechanisms, we analyzed the impact of C5aR1 activation on pulmonary CD11b+ conventional dendritic cells (cDCs) in the context of house-dust-mite (HDM) exposure. BALB/c mice were intratracheally immunized with an HDM/ovalbumin (OVA) mixture. After 24 h, we detected two CD11b+ cDC populations that could be distinguished on the basis of C5aR1 expression. C5aR1− but not C5aR1+ cDCs strongly induced T cell proliferation of OVA-reactive transgenic CD4+ T cells after re-exposure to antigen in vitro. C5aR1− cDCs expressed higher levels of MHC-II and CD40 than their C5aR1+ counterparts, which correlated directly with a higher frequency of interactions with cognate CD4+ T cells. Priming of OVA-specific T cells by C5aR1+ cDCs could be markedly increased by in vitro blockade of C5aR1 and this was associated with increased CD40 expression. Simultaneous blockade of C5aR1 and CD40L on C5aR1+ cDCs decreased T cell proliferation. Finally, pulsing with OVA-induced C5 production and its cleavage into C5a by both populations of CD11b+ cDCs. Thus, we propose a model in which allergen-induced autocrine C5a generation and subsequent C5aR1 activation in pulmonary CD11b+ cDCs promotes tolerance towards aeroallergens through downregulation of CD40.

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Chimeric Regulatory T cells Enhance HSC Engraftment in An Antigen-Specific Fashion.

Blood ◽

10.1182/blood.v114.22.2424.2424 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2424-2424

Author(s):

Yiming Huang ◽

Larry D Bozulic ◽

Thomas Miller ◽

Hong Xu ◽

Yujie Wen ◽

...

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Third Party ◽

T Cell Proliferation ◽

Hematopoietic Stem ◽

Mixed Chimeras

Abstract Abstract 2424 Poster Board II-401 We previously reported that CD8+TCR- facilitating cells (FC) induce the generation of chimeric regulatory T cells (Treg) in vivo. Transplantation of a mixture of CD8+/TCR- FC and hematopoietic stem cells (HSC) into ablated recipients results in chimerism and tolerance. Treg harvested from the spleen of chimeras (chimeric Treg) potently increase long-term donor chimerism in secondary NOD recipient mice. Here, we evaluated whether chimeric Treg enhance engraftment of hematopoietic stem cells (HSC) in an antigen-specific manner. To prepare mixed chimeras (B6 → NOD), NOD recipients were conditioned with 950 cGy TBI and transplanted with 10,000 B6 HSC and 1,000 NOD HSC plus 45,000 CD8+TCR- B6 FC. At 5 weeks, CD8-CD4+CD25bright chimeric Treg were sorted from spleens of the mixed chimeras (B6 → NOD). 100,000 chimeric Treg were then mixed with 10,000 B6 HSC (donor-specific) + 10,000 B10.BR HSC (third-party) and transplanted into conditioned NOD recipients in competitive repopulation assays. NOD mice given HSC plus nonchimeric naïve B6 Treg or HSC alone served as controls. Two of the four animals that received HSC alone engrafted and exhibited an average of 6.7% donor B6 chimerism at 30 days, 11.2% at 60 days, and 10.6% at 90 days. Three of five animals given HSC plus naïve B6 Treg engrafted with 21.3% donor B6 chimerism at 30 days, 28.8% at 60 days, and 28.9% at 90 days. In contrast, eight of nine recipients of HSC + chimeric Treg engrafted. These animals exhibited a significantly higher level of donor B6 chimerism, ranging from 56.3% at 30 days, 75.4% at 60 days to 85% at 90 days (P = 0.034). None of the recipients engrafted with the MHC-disparate third-party B10.BR HSC. We then assessed the suppressive function of chimeric Tregin vitro by using MLR suppressor cell assays. CD8-/CD4+/CD25bright Treg were sorted from chimeric spleens 5 wks to 12 wks after HSC + FC transplantation. As shown in the Figure 1, Treg from naïve B6 mice resulted in 1.9 fold; 1.3 fold and 1.1 fold inhibition of proliferation at 1:1, 1:0.25, 1:0.125 responder/Treg ratios (n = 3). In contrast, chimeric Treg potently suppressed T cell proliferation by 10.5 fold; 3.2 fold; and 1.7 fold at responder/Treg ratios of 1:1, 1:0.25, 1:0.125 (n = 4). Chimeric Treg significantly suppressed T cell proliferation at responder/Treg ratios of 1:1 and 1:0.25 compared with naïve B6 Treg (P < 0.05). NOD responder splenocytes remained hypoproliferative in response to B6 stimulator and chimeric Treg compared with stimulator plus B6 Treg, suggesting that chimeric Treg are significantly more potent than naïve B6 Treg in suppressing effector T cell proliferation in vitro. These data show that chimeric Treg enhance donor B6 HSC engraftment but not third-party B10.BR HSC, demonstrating that chimeric Treg function in vivo in an antigen-specific fashion. These data also show that the mechanism of FC function in vivo is associated with the establishment of an antigen-specific regulatory feedback loop. Figure 1 Figure 1. Disclosures: Bozulic: Regenerex: Employment. Ildstad:Regenerex: Equity Ownership.

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Alloreactive CD8+ Effector T Cells Proliferate and Persist Upon Chronic Exposure to Alloantigens through Reactivation of Stem Cell Transcriptional Programs.

Blood ◽

10.1182/blood.v114.22.2444.2444 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2444-2444

Author(s):

Koji Kato ◽

Shuaiying Cui ◽

Rork Kuick ◽

Shin Mineishi ◽

Elizabeth Hexner ◽

...

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Chronic Exposure ◽

Research Funding ◽

Effector T Cells ◽

T Cell Proliferation ◽

Effector T Cell

Abstract Abstract 2444 Poster Board II-421 Alloreactive effector T cells are the central to graft-versus-host disease (GVHD), a life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT). In GVHD host antigens are never cleared and alloreactive effector T cells are continuously generated over a period of several months or longer, but their suppression and control have proven to be difficult in practice. Using mouse models of GVHD directed against minor histocompatibility antigens (miHAs), we demonstrate that alloreactive effector T cells proliferate and persist upon chronic exposure to alloantigens via reactivation of stem cell transcriptional programs normally expressed in embryonic stem cells and neural stem cells. Many activated stem cell genes in effector T cells were distinct from those in memory T cells and were maintained at high levels upon T cell receptor activation, suggesting a specific role in chronically activated effector T cells. One of these genes, Ezh2, encodes a chromatin modifying enzyme essential to the proliferation, survival and differentiation of stem cells, was upregulated in CD8+ effector T cells upon antigenic stimulation and downregulated when the antigen was withdrawn. Pharmacologically inactivation of EZH2 with 3-Deazaneplanocin A inhibited effector T cell proliferation and survival. Silencing Ezh2 independently validated that Ezh2 was important for regulating effector T cell proliferation and expression of many stem cell genes. To further evaluate whether alloreactive CD8+ effector T cells obtained stem cell-like properties, e.g. the ability to self-renew to continually generate effector cells, we adoptively transferred highly purified miHA H60-specific (H60+) CD8+ effector T cells into secondary allogeneic and congenic recipients, respectively. As compared to congenic recipients, allogeneic recipients had 80-fold more proliferating H60+CD8+ effector T cells. These donor H60+CD8+ effector T cells expressed high levels of CD122, CD69, CXCR3, PD1, IFNγ and Granzyme B, required miHA H60 stimulation to sustain their replication with effector function and expression of stem cell genes, and caused severe GVHD in secondary allogeneic recipients. These results indicate that stem cell transcriptional programs expressed in embryonic and neural stem cells may play important roles in effector T cells. Among these stem cell genes, Ezh2 emerges as an important therapeutic target in modulating alloreactive T cell-mediated GVHD. Disclosures: Zhang: University of Michigan Comprehensive Cancer Center: Research Funding; Damon Runyon Cancer Research Foundation: Research Funding.

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Mesenchymal Stem Cells Inhibit Th17 Differentiation through PGE2 Production.

Blood ◽

10.1182/blood.v114.22.3633.3633 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3633-3633

Author(s):

Raine Tatara ◽

Katsutoshi Ozaki ◽

Lekuni Oh ◽

Keiko Hatanaka ◽

Akiko Meguro ◽

...

Keyword(s):

Nitric Oxide ◽

Stem Cells ◽

Cell Proliferation ◽

T Cells ◽

Mesenchymal Stem Cells ◽

T Cell ◽

T Cell Proliferation ◽

Th17 Differentiation ◽

Treg Differentiation ◽

Immunomodulatory Function

Abstract Abstract 3633 Poster Board III-569 Mesenchymal stem cells (MSCs) possess an immunomodulatory function and show promise as a cell therapy for graft-versus-host disease (GVHD). In a phase II study in Europe, injections of MSCs caused 60-70% overall response rate, with longer survival of complete responder. In contrast to its clinical efficacy, the molecular mechanism(s) underlying immunomodulation by MSCs has not been fully established. Prostaglandin E2 (PGE2), tumor growth factor-b1 (TGF-b1), and indoleamine-2,3-dioxygenase have been reported to mediate the immunomodulatory function of MSCs, and we reported evidence that nitric oxide is also a mediator (Blood 2007, 109, 228). Th17 is a recently recognized differentiation category, in which CD4 cells produce IL-17. It has been reported that Th17 is crucial for experimental autoimmune encephalomyelitis (a model of the human disease, multiple sclerosis) and is also thought to be important in other autoimmune diseases. Regulatory T cells (Treg) are another newly recognized differentiation category, in which CD4 T cells have high levels of Foxp3 expression and suppress T cell proliferation. It has been reported that Th17 and Treg can be induced by incubation with TGF-b1 and IL-6 or IL-21, and TGF-b1 and IL-2, respectively, and that these two differentiations are in a reciprocal relationship. Whereas the role of Th17 in GVHD is still controversial, Treg has been reported to suppress GVHD in a mouse model. To elucidate the molecular mechanism(s) of the immunomodulatory function of MSCs, we herein sought to identify the effects of MSCs on these relatively new differentiations. MSCs inhibit Th17 differentiation even in conditions in which growth is not completely inhibited. Interestingly, an inhibitor of prostaglandin production, indomethacin, and an inhibitor of indoleamine 2,3-dioxygenase, 1-methyltryptophan, partially restore Th17 differentiation, whereas inhibitors of nitric oxide synthase do not. These results suggest that PGE2 and depletion of tryptophan, but not nitric oxide, mediate inhibitory effects of MSCs on Th17. Additionally, we found that MSCs produced PGE2 when co-cultured with CD4 T cells in Th17 differentiation condition and PGE2 per se suppresses Th17 differentiation. Thus, our results suggest that MSCs block Th17 differentiation through PGE2 prodction. In contrast to Th17 differentiation, Treg differentiation was not significantly inhibited by MSCs. However, MSCs still inhibited proliferation of T cells under these conditions, and T cell proliferation was restored by the addition of indomethacin. These results suggest that MSCs inhibit proliferation but not Treg differentiation through PGE2 production. The mechanism by which PGE2 differentially regulates these differentiations is unknown and remains an area for further investigation. Disclosures: Ozawa: Alexion: Research Funding.

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Striking Clinical, Molecular and Immunological Outcomes in Non-Human Primate Hematopoietic Stem Cell Transplantation Using a Novel OX40L Blockade Agent, KY1005, Combined with Rapamycin

Blood ◽

10.1182/blood.v128.22.3341.3341 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3341-3341

Author(s):

Victor Tkachev ◽

Scott N. Furlan ◽

Ben Watkins ◽

Betty Zheng ◽

Daniel Hunt ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Cd8 T Cell ◽

Effector Memory ◽

T Cell Proliferation ◽

Hematopoietic Stem ◽

Free Survival ◽

The Impact

Abstract While calcineurin inhibition (CNI)-based strategies remain the mainstay for GVHD prevention, CNI are notoriously antagonistic to immune tolerance induction. Rapamycin (Rapa) has been shown to be more pro-tolerogenic; however, the best agents to combine with Rapa are still undetermined, and it remains a second-line GVHD prevention strategy without clear superiority over CNI. Finding tolerogenic partners for Rapa, therefore, represents a critical unmet need in the field. Of the possible partners for Rapa, the OX40/OX40L pathway represents an important target. OX40 is a costimulatory receptor expressed on activated human T cells, which, upon interaction with OX40L delivers activation signals to conventional T cells (Tconv) promoting their proliferation, survival and clonal expansion. Notably, these same OX40/OX40L signals may either inhibit or promote Treg functions, depending on context, suggesting that blockade of this pathway may simultaneously control Tconv activation while permitting Treg homeostasis. During GVHD in non-human primates (NHP), we found OX40L upregulation on myeloid dendritic cells and OX40 upregulation on activated T cells in recipients treated with multiple immunosuppressive agents, including Rapa (Fig 1). These data provided strong rationale for testing KY1005, a novel human monoclonal antibody that binds to OX40L and blocks its interaction with OX40, as a potential partner with Rapa. We tested the outcomes of prophylactic blockade of this pathway on NHP GVHD, using KY1005 alone and in combination with Rapa. These experiments utilized our previously published NHP GVHD model, in which GVHD is studied after T cell-replete haplo-identical HCT. KY1005 was dosed at 10mg/kg weekly from days -2ˆ+54 and Rapa was continued through Day +100. Prophylaxis with KY1005 alone provided initial evidence for its in vivo activity, with control of CD4>CD8 T cell proliferation and mitigation of the expansion of CD4>CD8 T effector/memory cells. Consistent with the partial control of T cell activation, these recipients demonstrated improved GVHD-free survival versus unprophylaxed controls, but disease ultimately broke through (Median Survival Time (MST) = 19.5 days with KY1005 (n=4) compared to 8 days in unprophylaxed recipients (n= 10, Fig 2)). We next investigated the impact of OX40L blockade + Rapa. We have published that Rapa as a monotherapy minimally controlled both immunologic and clinical disease, with an MST = 14 days (n=6). Combined prophylaxis was striking: recipients given KY1005+Rapa (n=5) maintained robust health throughout the entire experiment (MST >100d), and demonstrated high levels of donor T cell chimerism (86 +/- 3% at Day 100), rapid hematopoietic reconstitution, and had a terminal GVHD Grade of 0, compared to a Grade of III-IV in both KY1005- and Rapa-monotherapy cohorts. Immunologic analysis demonstrated synergistic control of both CD4 and CD8 T cell proliferation, restoring it to the level observed during autologous immune reconstitution, and resulting in a concomitant abrogation of CD4 and CD8 memory/effector expansion while preserving T cells with a na•ve phenotype. In striking contrast to the inhibition of Tconv activation by KY1005+Rapa, recipients of dual therapy demonstrated intact Treg reconstitution post-HCT, which resulted in a favorable Treg:Tconv ratio of 5.4 vs 1.4:100 in KY1005+Rapa treated compared to untreated recipients (p < 0.05). Transcriptomic analysis confirmed the unique immunologic state conferred by KY1005+Rapa on purified T cells, with gene arrays from these recipients demonstrating separation from all other transplant cohorts in Principal Component space (Figure 3A) and Class Neighbor Analysis identifying unique expression modules that tracked with KY1005 + Rapa prophylaxis (Figure 3B red and blue boxes). These results underscore the critical role of OX40/OX40L signaling in the development of GVHD and demonstrate the striking control of GVHD in KY1005+Rapa recipients. They represent the first demonstration of uniform, long-term GVHD-free survival in the primate model of high-risk haplo-identical HCT, and the first therapeutic strategy that simultaneously controls Tconv activation while supporting Treg homeostasis in this model. They suggest that OX40L blockade + Rapa is a novel, evidence-based combinatorial strategy to control GVHD that is an exceptional candidate regimen for clinical translation. Disclosures Tkachev: Kymab Ltd: Patents & Royalties: US Patent 9,382,325, Research Funding. Casson:Kymab Ltd: Employment. Kirby:Kymab Ltd: Employment, Patents & Royalties: US Patent 9,382,325. Bland-Ward:Kymab Ltd: Employment, Patents & Royalties: US Patent 9,382,325. Kean:Juno Therapeutics, Inc: Research Funding.

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Human Mesenchymal Stem Cells Exert Immunosuppressive Capacities by Induction of Regulatory T Cells

Blood ◽

10.1182/blood.v112.11.2414.2414 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2414-2414

Author(s):

Kirsten Canté-Barrett ◽

Jessica M.E. van den Oever ◽

Willem E. Fibbe

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Fold Increase ◽

T Cell Proliferation ◽

Soluble Factors ◽

Cell Divisions ◽

And Function ◽

Cells Cultured

Abstract It is widely accepted that Mesenchymal Stem Cells (MSCs) exhibit immunosuppressive capacities in vitro and in vivo. In an effort to understand the mechanism of suppression, co-cultures of MSCs with several types of immune cells have been studied. MSCs inhibit B- and T cell proliferation and inhibit the generation, maturation and function of dendritic cells. In the present study, we evaluated the effect of MSCs on the expansion and function of regulatory T cells (T regs). Human bone-marrow derived MSCs were cultured and expanded in low-glucose DMEM containing 10% FCS and frozen until used in experiments. Freshly isolated, MACS selected human CD4+CD25+ T regs were cultured in IMDM, supplemented with 10% pooled human serum and 300 units/ml IL-2, in the presence or absence of irradiated MSCs (60 Gy) (ratio MSCs: T regs = 1:5). Results are derived from 3 to 15 independent experiments. In the presence of IL-2 and MSCs, the percentage of FOXP3+ CD4+CD25+ T cells increased from 26.8% ±2.2 (no MSCs) to 42.3% ±2.4 (with MSCs) over a period of 5 days, representing a 1.6 (±0.1)-fold induction. Moreover, a distinct CD4+CD25+ population with high FOXP3 expression appeared after 5 days of culture in the presence of MSCs (23.3% ±2.5 in the presence of MSCs versus 7.2% ±1.0 in the absence of MSCs). This CD4+CD25+FOXP3hi population was not observed after co-culturing MSCs and CD4+CD25− T cells (1.7% ±0.6). To show that the MSC-induced T regs were functionally suppressive, freshly isolated CFSE-labeled CD4+ T cells were stimulated with PHA (0.8 μg/ml). After 3 days, about 40% of the T cells had undergone one or more cell divisions as measured by CFSE dilution. Addition of MSC-induced T regs (in a 1:1 ratio) resulted in a 50% reduction of the proliferation of CFSE-labeled T cells (down to 15–20% of cells undergoing one or more cell divisions). Control CD4+CD25+ T cells cultured in the absence of MSCs did not suppress T cell proliferation. These results indicate that MSC-induced CD4+CD25+ FOXP3hi cells exert regulatory function. To study whether the induction of T regs by MSCs was dependent on cell-cell contact, co-culture experiments were performed in transwells where MSCs were physically separated from T cells. CD4+CD25+ T cells co-cultured with MSCs in transwells showed a 1.4 (±0.1)-fold increase in the percentage of FOXP3hi cells, in comparison with T cells cultured in the absence of MSCs. Similarly, direct co-cultures of MSCs and T cells resulted in a 1.8 (±0.2)-fold increase in the percentage of FOXP3hi T regs. In addition, medium derived from co-cultures of MSCs and CD4+CD25+ T cells and added to freshly isolated CD4+CD25+ T cells resulted in a 2.6 (±0.6) fold increase in the percentage of CD4+CD25+FOXP3hi T regs, implicating that FOXP3hi induction by MSCs was mediated by soluble factors. Since T Cell Receptor-stimulated naïve T cells (as well as natural T regs, generated in the thymus) require both IL-2 and TGF-β to become induced T regs, we analyzed the involvement of TGF-β. Addition of the pharmacological inhibitor of the TGF-β receptor (SB431542) only marginally reduced FOXP3 induction in the presence of MSCs, suggesting that MSC-mediated expression of high FOXP3 levels requires alternate or additional cytokines. In conclusion, we show that MSCs promote the induction of CD4+CD25+ T cells that express high levels of FOXP3 and these MSC-induced T regs suppress proliferation of PHA-stimulated CD4+ T cells. These effects are mediated by soluble factors produced during the co-culture of MSCs and T cells. The cytokines involved are presently unknown, but likely do not involve TGF-β.

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Mesenchymal STEM CELLS Favor Tumor Growth By Generating Granulocyte-like Myeloid Derived Suppressor CELLS in CML Patients

Blood ◽

10.1182/blood.v126.23.4018.4018 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4018-4018

Author(s):

Cesarina Giallongo ◽

Nunziatina L Parrinello ◽

Daniele Tibullo ◽

Claudia Bellofiore ◽

Piera La Cava ◽

...

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

T Cells ◽

Mesenchymal Stem Cells ◽

T Cell ◽

Peripheral Blood ◽

Suppressor Cells ◽

T Cell Proliferation ◽

Myeloid Derived Suppressor Cells ◽

Healthy Donors

Abstract INTRODUCTION. The complex interplay between cancer cells and immune system allows neoplastic cells to evade immune surveillance and expand. Recently, our and another group have demonstrated that a subpopulation of myeloid cells, defined as "granulocytic myeloid-derived suppressor cells" (G-MDSC), plays an important role for immune escape in chronic myeloid leukemia (CML) patients by reducing T cell activation. The aim of this study was to evaluate the influence of Mesenchymal stem cells (MSC) on generation of MDSCs by comparing CML MSCs (n=10) with healthy donors (HD) MSC (n=8). METHODS. G-MDSC (CD11b+CD33+CD14-HLADR- cells) were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis by cytofluorimetric analysis. Immuno-suppressive activity was tested through incubation of G-MDSC with autologous CFSE-labeled T cells and stimulation with phytohaemagglutinin (PHA). Controls included a positive T cell proliferation control (T cells plus PHA) and a negative one (T cells only). After three days, T cell proliferation was analyzed by flow cytometry. For G-MDSC generation, human peripheral blood mononucleated cells (PBMC) from HD were cultured alone and with MSC of CML (n=10) or HD (n=8) (1:100 ratio). After one week, G-MDSC were isolated using anti-CD66b magnetic microbeads and the phenotype was confirmed by cytofluorimetric analysis. Expression of ARG1, NOS2, PTGS2, TNFα, TGFβ, IL6, IL10, IL1β was also evaluated using real time PCR. RESULTS. Percentage of cells with a G-MDSC phenotype was greater in PB obtained from CML patients than HD (82.5±9.6% vs 56,2±5.4%, p<0.0001). G-MDSC were able to inhibit T cell proliferation compared to positive control (25±5% vs 48±7.6%, p=0.0057). To investigate if CML MSC may be involved in G-MDSC generation, we incubated HD PBMC with CML or HD MSC for one week. After magnetic isolation, we found that only CML MSC-educated G-MDSC acquired immune-suppressive ability, inhibiting T cell proliferation compared to G-MDSCs control (isolated from PBMC cultured in medium alone) (32±12% vs 63±5.9%, p=0.003). On the contrary, HD MSC-educated G-MDSC did not show any suppressive effect. We also found that CML MSC-educated G-MDSC expressed higher level of the following immune modulatory factors: TNFα (20.8±19.3, p=0.006), IL1β (47.3±25.2, p=0.001), PTGS2 (20.7±10.9, p=0.002) and IL6 (33.8±13.9, p=0.004) compared to HD MSC-educated G-MDSCs (arbitrarily 2-ΔΔCt value: 1). MSC WE also observed ane an up-regulation of PTGS2 (19±4.4, p=0.04), TGFβ (6±3, p=0.01) and IL6 (5±2.8, p=0.04) in CML MSCs at time 0 with a great variability among the patients (calculated value of 2-ΔΔCt in HD MSC was 1). After 48 h of co-culture with PBMC, CML MSC showed statistically significant up-regulation of ARG1 (23.5±11.9, p=0.02), TGFβ (4.8±3, p=0.04), IL10 (5.6±2.8, p=0.03) and IL6 (54.3±23, p=0.02) expression, suggesting that multiple mechanisms are involved in MDSC induction by CML MSC. CONCLUSION. Our work demonstrates that CML MSCs are able to activate MDSCs favoring cancer immune evasion in CML patients. Disclosures Palumbo: Novartis: Honoraria, Other: Advisory Board.

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Inhibition of Allogeneic and Autologous T Cell Proliferation by Adipose-Derived Mesenchymal Stem Cells of Ankylosing Spondylitis Patients

Stem Cells International ◽

10.1155/2021/6637328 ◽

2021 ◽

Vol 2021 ◽

pp. 1-17

Author(s):

Ewa Kuca-Warnawin ◽

Magdalena Plebańczyk ◽

Krzysztof Bonek ◽

Ewa Kontny

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

T Cells ◽

Mesenchymal Stem Cells ◽

Ankylosing Spondylitis ◽

T Cell ◽

Cd8 T Cells ◽

T Cell Response ◽

T Cell Proliferation

Background. In ankylosing spondylitis (AS), accompanied by chronic inflammation, T cell expansion plays a pathogenic role; the immunoregulatory properties of bone marrow-derived mesenchymal stem cells (BM-MSCs) are impaired, while functional characteristics of their adipose tissue-derived counterparts are (ASCs) unknown. Methods. We evaluated the antiproliferative activity of AS/ASCs, obtained from 20 patients, towards allogeneic and autologous T lymphocytes, using ASCs from healthy donors (HD/ASCs) as the reference cell lines. The PHA-activated peripheral blood mononuclear cells (PBMCs) were cocultured in cell-cell contact and transwell conditions with untreated or TNF + IFNγ- (TI-) licensed ASCs, then analyzed by flow cytometry to identify proliferating and nonproliferating CD4+ and CD8+ T cells. The concentrations of kynurenines, prostaglandin E2 (PGE2), and IL-10 were measured in culture supernatants. Results. In an allogeneic system, HD/ASCs and AS/ASCs similarly decreased the proliferation of CD4+ and CD8+ T cells and acted mainly via soluble factors. The concentrations of kynurenines and PGE2 inversely correlated with T cell proliferation, and selective inhibitors of these factors synthesis significantly restored T cell response. AS/ASCs exerted a similar antiproliferative impact also on autologous T cells. Conclusion. We report for the first time that despite chronic in vivo exposure to inflammatory conditions, AS/ASCs retain the normal capability to restrain expansion of allogeneic and autologous CD4+ and CD8+ T cells, act primarily via kynurenines and PGE2, and thus may have potential therapeutic value. Some distinctions between the antiproliferative effects of AS/ASCs and HD/ASCs suggest in vivo licensing of AS/ASCs.

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A role for heme oxygenase-1 in the immunosuppressive effect of adult rat and human mesenchymal stem cells

Blood ◽

10.1182/blood-2007-02-075481 ◽

2007 ◽

Vol 110 (10) ◽

pp. 3691-3694 ◽

Cited By ~ 232

Author(s):

Dominique Chabannes ◽

Marcelo Hill ◽

Emmanuel Merieau ◽

Julien Rossignol ◽

Régis Brion ◽

...

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

T Cell ◽

Heme Oxygenase ◽

T Cell Proliferation ◽

Heme Oxygenase 1 ◽

Human Mscs ◽

Adult Rat

Abstract Mesenchymal stem cells (MSCs) display immunomodulatory properties mediated by various factors, including inducible nitric oxide synthase (iNOS). Since heme oxygenase-1 (HO-1) is a potent immunosuppressive enzyme, we tested the hypothesis that HO-1 could mediate the immunosuppressive effects of MSCs. We generated adult rat MSCs that inhibited T-cell proliferation in vitro. These MSCs expressed both HO-1 and iNOS. In vitro, whereas neither HO-1 nor iNOS inhibition alone could interfere with the immunosuppressive properties of rat MSCs, simultaneous inhibition of both enzymes restored T-cell proliferation. In vivo, injection of MSCs significantly delayed heart allograft rejection, and inhibition of either HO-1 or iNOS totally reversed the protective activity of MSCs, inducing rejection. Adult human MSCs also expressed HO-1; in these cells, HO-1 inhibition was sufficient to completely block their immunosuppressive capacity. In conclusion, we show, for the first time, that HO-1 mediates the immunosuppressive properties of rat and human MSCs.

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