Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation

Background aimsMesenchymal stromal cells (MSCs) suppress T-cell proliferation, especially after activation with inflammatory cytokines. We compared the dynamic action of unprimed and interferon (IFN)-γ plus tumor necrosis factor (TNF)-α–pretreated human bone marrow–derived MSCs on resting or activated T cells.MethodsMSCs were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) at high MSC-to-PBMC ratios in the absence or presence of concomitant CD3/CD28-induced T-cell activation. The kinetic effects of MSCs on cytokine production and T-cell proliferation, cell cycle and apoptosis were assessed.ResultsUnprimed MSCs increased the early production of IFN-γ and interleukin (IL)-2 by CD3/CD28-activated PBMCs before suppressing T-cell proliferation. In non-activated PBMC co-cultures, low levels of IL-2 and IL-10 synthesis were observed with MSCs in addition to low levels of CD69 expression by T cells and no T-cell proliferation. MSCs also decreased apoptosis in resting and activated T cells and inhibited the transition of these cells into the sub-G0/G1 and the S phases. With inhibition of indoleamine 2,3 dioxygenase, MSCs increased CD3/CD28-induced T-cell proliferation. After priming with IFN-γ plus TNF-α, MSCs were less potent at increasing cytokine production by CD3/CD28-activated PBMCs and more effective at inhibiting T-cell proliferation but had preserved anti-apoptotic functions.ConclusionsUnprimed MSCs induce a transient increase in IFN-γ and IL-2 synthesis by activated T cells. Pre-treatment of MSCs with IFN-γ plus TNF-α may increase their effectiveness and safety in vivo.     

Impact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease

Background aimsGraft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects.MethodsThe ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rγ(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed.ResultsInjection of 200 × 10⁶ human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM₁ antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 × 10⁶ MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 × 10⁶ human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 × 10⁶ MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs.ConclusionsInjection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models.     

Immunophenotypic characteristics of multipotent mesenchymal stromal cells that affect the efficacy of their use in the prevention of acute graft vs host disease

BACKGROUNDMultipotent mesenchymal stromal cells (MSCs) are widely used in the clinic due to their unique properties, namely, their ability to differentiate in all mesenchymal directions and their immunomodulatory activity. Healthy donor MSCs were used to prevent the development of acute graft vs host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT). The administration of MSCs to patients was not always effective. The MSCs obtained from different donors have individual characteristics. The differences between MSC samples may affect their clinical efficacy.AIMTo study the differences between effective and ineffective MSCs.METHODSMSCs derived from the bone marrow of a hematopoietic stem cells donor were injected intravenously into allo-BMT recipients for GVHD prophylaxis at the moment of blood cell reconstitution. Aliquots of 52 MSC samples that were administered to patients were examined, and the same cells were cultured in the presence of peripheral blood mononuclear cells (PBMCs) from a third-party donor or treated with the pro-inflammatory cytokines IL-1β, IFN and TNF. Flow cytometry revealed the immunophenotype of the nontreated MSCs, the MSCs cocultured with PBMCs for 4 d and the MSCs exposed to cytokines. The proportions of CD25-, CD146-, CD69-, HLA-DR- and PD-1-positive CD4+ and CD8+ cells and the distribution of various effector and memory cell subpopulations in the PBMCs cocultured with the MSCs were also determined.RESULTSDifferences in the immunophenotypes of effective and ineffective MSCs were observed. In the effective samples, the mean fluorescence intensity (MFI) of HLA-ABC, HLA-DR, CD105, and CD146 was significantly higher. After MSCs were treated with IFN or cocultured with PBMCs, the HLA-ABC, HLA-DR, CD90 and CD54 MFI showed a stronger increase in the effective MSCs, which indicated an increase in the immunomodulatory activity of these cells. When PBMCs were cocultured with effective MSCs, the proportions of CD4+ and CD8+central memory cells significantly decreased, and the proportion of CD8+CD146+ lymphocytes increased more than in the subpopulations of lymphocytes cocultured with MSC samples that were ineffective in the prevention of GVHD; in addition, the proportion of CD8+effector memory lymphocytes decreased in the PBMCs cocultured with the effective MSC samples but increased in the PBMCs cocultured with the ineffective MSC samples. The proportion of CD4+CD146+ lymphocytes increased only when cocultured with the inefficient samples.CONCLUSIONFor the first time, differences were observed between MSC samples that were effective for GVHD prophylaxis and those that were ineffective. Thus, it was shown that the immunomodulatory activity of MSCs depends on the individual characteristics of the MSC population.     

Monocytic MDSC mobilization promotes tumor recurrence after liver transplantation via CXCL10/TLR4/MMP14 signaling

Tumor recurrence is the major obstacle for pushing the envelope of liver transplantation for hepatocellular carcinoma (HCC) patients. The inflammatory cascades activated by acute liver graft injury promote tumor recurrence. We aimed to explore the role and mechanism of myeloid-derived suppressor cell (MDSC) mobilization induced by liver graft injury on tumor recurrence. By analyzing 331 HCC patients who received liver transplantation, the patients with graft weight ratio (GWR, the weight of liver graft divided by the estimated standard liver weight of recipient) <60% had higher tumor recurrence than GWR ≥60% ones. MDSCs and CXCL10/TLR4 levels were significantly increased in patients with GWR <60% or tumor recurrence. These findings were further validated in our rat orthotopic liver transplantation model. In CXCL10^−/− and TLR4^−/− mice of hepatic ischemia/reperfusion injury plus major hepatectomy (IRH) model, monocytic MDSCs, instead of granulocytic MDSCs, were significantly decreased. Importantly, CXCL10 deficiency reduced the accumulation of TLR4⁺ monocytic MDSCs, and CXCL10 increased MDSC mobilization in the presence of TLR4. Moreover, MMP14 was identified as the key molecule bridging CXCL10/TLR4 signaling and MDSC mobilization. Knockout or inhibition of CXCL10/TLR4 signaling significantly reduced the tumor growth with decreased monocytic MDSCs and MMP14 in the mouse tumor recurrent model. Our data indicated that monocytic MDSCs were mobilized and recruited to liver graft during acute phase injury, and to promote HCC recurrence after transplantation. Targeting MDSC mobilization via CXCL10/TLR4/MMP14 signaling may represent the therapeutic potential in decreasing post-transplant liver tumor recurrence.Subject terms: Liver cancer, Experimental models of disease     

Ex vivo immunological evaluation of stable mixed chimeric patients after matched related donor allogeneic transplantation in sickle cell disease

Background aimsAllogeneic hematopoietic stem cell transplantation is curative for sickle cell disease, and the use of matched related donors, non-myeloablative conditioning and sirolimus immunosuppression results in stable mixed chimerism without graft-versus-host disease (GVHD). However, the time to terminate sirolimus while maintaining mixed chimerism is unclear.MethodsIn this study, we developed a two-way mixed lymphocyte reaction (MLR) to evaluate ex vivo immunoreaction in mixed chimeric patients.ResultsIn co-culture of peripheral blood mononuclear cells (PBMCs) from two healthy controls (without irradiation), we detected proliferation at various ratios of PBMC mixtures (1:9 to 9:1) as well as various concentrations of sirolimus, suggesting that two-way MLR is applicable to patients (having >10% chimerism) undergoing sirolimus treatment. In two-way MLR using PBMCs (including donor and recipient cells) from mixed chimeric patients (n = 28), greater ex vivo proliferation was observed <6 months compared with >6 months post-transplant and healthy control PBMC monoculture. Robust ex vivo proliferation was observed in a patient with acute GVHD, and persistent ex vivo proliferation (until 2 years) was observed in a patient with decreasing donor chimerism.ConclusionsIn summary, we demonstrated that in two-way MLR, ex vivo immunoreaction decreases to low levels ~6 months post-transplant. These findings suggest a rationale to continue immunosuppression for 6 months.     

Myeloid-Derived Suppressor Cells Modulate Immune Responses Independently of NADPH Oxidase in the Ovarian Tumor Microenvironment in Mice

The phagocyte NADPH oxidase generates superoxide anion and downstream reactive oxidant intermediates in response to infectious threat, and is a critical mediator of antimicrobial host defense and inflammatory responses. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are recruited by cancer cells, accumulate locally and systemically in advanced cancer, and can abrogate anti-tumor immunity. Prior studies have implicated the phagocyte NADPH oxidase as being an important component promoting MDSC accumulation and immunosuppression in cancer. We therefore used engineered NADPH oxidase-deficient (p47^phox−/−) mice to delineate the role of this enzyme complex in MDSC accumulation and function in a syngeneic mouse model of epithelial ovarian cancer. We found that the presence of NADPH oxidase did not affect tumor progression. The accumulation of MDSCs locally and systemically was similar in tumor-bearing wild-type (WT) and p47^phox−/− mice. Although MDSCs from tumor-bearing WT mice had functional NADPH oxidase, the suppressive effect of MDSCs on ex vivo stimulated T cell proliferation was NADPH oxidase-independent. In contrast to other tumor-bearing mouse models, our results show that MDSC accumulation and immunosuppression in syngeneic epithelial ovarian cancer is NADPH oxidase-independent. We speculate that factors inherent to the tumor, tumor microenvironment, or both determine the specific requirement for NADPH oxidase in MDSC accumulation and function.     

International Society for Cell & Gene Therapy Stem Cell Engineering Committee: Cellular therapies for the treatment of graft-versus-host-disease after hematopoietic stem cell transplant

Background aimsAllogeneic hematopoietic stem cell transplant is a curative approach for many malignant and non-malignant hematologic conditions. Despite advances in its prevention and treatment, the morbidity and mortality related to graft-versus-host disease (GVHD) remains. The mechanisms by which currently used pharmacologic agents impair the activation and proliferation of potentially alloreactive T cells reveal pathways essential for the detrimental activities of these cell populations. Importantly, these same pathways can be important in mediating the graft-versus-leukemia effect in recipients transplanted for malignant disease. This knowledge informs potential roles for cellular therapies such as mesenchymal stromal cells and regulatory T cells in preventing or treating GVHD. This article reviews the current state of adoptive cellular therapies focused on GVHD treatment.MethodsWe conducted a search for scientific literature in PubMed® and ongoing clinical trials in clinicaltrial.gov with the keywords “Graft-versus-Host Disease (GVHD),” “Cellular Therapies,” “Regulatory T cells (Tregs),” “Mesenchymal Stromal (Stem) Cells (MSCs),” “Natural Killer (NK) Cells,” “Myeloid-derived suppressor cells (MDSCs),” and “Regulatory B-Cells (B-regs).” All the published and available clinical studies were included.ResultsAlthough most of the existing clinical data focus on cellular therapies for GVHD prevention, there are observational and interventional clinical studies that explore the potential for cellular therapies to be safe modalities for GVHD treatment while maintaining the graft-versus-leukemia effect in the context of malignant diseases. However, there are multiple challenges that limit the broader use of these approaches in the clinical scenario.ConclusionsThere are many ongoing clinical trials to date with the promise to expand our actual knowledge on the role of cellular therapies for GVHD treatment in an attempt to improve GVHD-related outcomes in the near future.     

Mesenchymal stromal cell supported umbilical cord blood ex vivo expansion enhances regulatory T cells and reduces graft versus host disease

Background aimsDouble cord blood transplantation (DCBT) may shorten neutrophil and platelet recovery times compared with standard umbilical cord blood transplantation. However, DCBT may be associated with a higher incidence of graft versus host disease (GVHD). In this study, we explored the effect of ex vivo expansion of a single cord blood unit (CBU) in a DCBT setting on GVHD and engraftment.MethodsPost-thaw cryopreserved CBUs from cord blood banks, hereinafter termed “banked” CBUs, were co-cultured with confluent bone marrow mesenchymal stromal cells (MSCs) supplemented with a cytokine cocktail comprising 100 ng/mL stem cell factor, 50 ng/mL flt3-ligand, 100 ng/mL thrombopoietin and 20 ng/mL insulin-like growth factor binding protein 2 for 12 days.ResultsWhen DCBT of one unexpanded and one expanded CBU was performed in non-obese diabetic/severe combined immunodeficient-IL2Rgamma^null (NOD/SCID-IL2γ^?/?, NSG) mice, the expanded CBU significantly boosted in vivo hematopoiesis of the unexpanded CBU. The median survival of NSG mice was significantly improved from 63.4% (range, 60.0–66.7%) for mice receiving only unexpanded units to 86.5% (range, 80.0–92.9%) for mice receiving an expanded unit (P < 0.001). The difference in survival appeared to be due to a lower incidence of GVHD in the mice receiving expanded cells. This effect on GVHD was mediated by a significant increase in regulatory T cells seen in the presence of MSC co-culture.ConclusionsMSC-supported ex vivo expansion of “banked” CBU boosted unexpanded CBU hematopoiesis in vivo, increased regulatory T cell content and decreased the incidence of GVHD.     

Deregulation of Apoptotic Factors Bcl-xL and Bax Confers Apoptotic Resistance to Myeloid-derived Suppressor Cells and Contributes to Their Persistence in Cancer

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cells that accumulate in response to tumor progression. Compelling data from mouse models and human cancer patients showed that tumor-induced inflammatory mediators induce MDSC differentiation. However, the mechanisms underlying MDSC persistence is largely unknown. Here, we demonstrated that tumor-induced MDSCs exhibit significantly decreased spontaneous apoptosis as compared with myeloid cells with the same phenotypes from tumor-free mice. Consistent with the decreased apoptosis, cell surface Fas receptor decreased significantly in tumor-induced MDSCs. Screening for changes of key apoptosis mediators downstream the Fas receptor revealed that expression levels of IRF8 and Bax are diminished, whereas expression of Bcl-xL is increased in tumor-induced MDSCs. We further determined that IRF8 binds directly to Bax and Bcl-x promoter in primary myeloid cells in vivo, and IRF8-deficient MDSC-like cells also exhibit increased Bcl-xL and decreased Bax expression. Analysis of CD69 and CD25 levels revealed that cytotoxic T lymphocytes (CTLs) are partially activated in tumor-bearing hosts. Strikingly, FasL but not perforin and granzymes were selectively activated in CTLs in the tumor-bearing host. ABT-737 significantly increased the sensitivity of MDSCs to Fas-mediated apoptosis in vitro. More importantly, ABT-737 therapy increased MDSC spontaneous apoptosis and decreased MDSC accumulation in tumor-bearing mice. Our data thus determined that MDSCs use down-regulation of IRF8 to alter Bax and Bcl-xL expression to deregulate the Fas-mediated apoptosis pathway to evade elimination by host CTLs. Therefore, targeting Bcl-xL is potentially effective in suppression of MDSC persistence in cancer therapy.     

Swertianolin ameliorates immune dysfunction in sepsis via blocking the immunosuppressive function of myeloid-derived suppressor cells

In this study, we studied the long-term proliferation trajectory of myeloid-derived suppressor cells (MDSCs) in murine sepsis model and investigated whether swertianolin could modulate the immunosuppressive function of MDSCs. A murine sepsis model was established by cecal ligation and perforation (CLP), according to the Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) guidelines. The bone marrow and spleen of the mice were collected at 24 h, 72 h, 7 and 15 d after sepsis induction. The proportions of monocytic- MDSCs (M-MDSCs; CD11b⁺LY6G^–LY6C^hi) and granulocytic-MDSCs (G-MDSC, CD11b⁺ Ly6G⁺ Ly6C^low) were analyzed by flow cytometry. Then, we have investigated whether swertianolin could modulate the immunosuppressive function of MDSCs in in vitro experiments. G-MDSCs and M-MDSCs increased acutely after sepsis with high levels sustained over a long period of time. G-MDSCs were the main subtype identified in the murine model of sepsis with polymicrobial peritonitis. Furthermore, it was found that swertianolin reduced significantly interleukin-10 (IL-10), nitric oxide (NO), reactive oxygen species (ROS), and arginase production in MDSCs, while reducing MDSC proliferation and promoting MDSC differentiation into dendritic cells. Swertianolin also improved T-cell activity by blocking the immunosuppressive effect of MDSCs. Both subsets of MDSCs significantly increased in the bone marrow and spleen of the mice with sepsis, with GMDSCs being the main subtype identified. Swertianolin effectively regulated the functions of MDSCs and reduced immune suppression.Key words: Sepsis, myeloid-derived suppressor cells (MDSCs), immunosuppression, swertianolin     

CD1d is a novel cell-surface marker for human monocytic myeloid-derived suppressor cells with T cell suppression activity in peripheral blood after allogeneic hematopoietic stem cell transplantation

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that regulate immune responses in cancer and various pathological conditions. However, the phenotypic and functional heterogeneity of human MDSCs represents a major hurdle for the development of therapeutic strategies targeting or regulating MDSCs in tumor progression, inflammation, and graft-versus-host disease (GVHD). We previously shown that circulating HLA-DR^-CD14⁺ monocytic MDSCs are a major contributor to clinical outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we identified, using high-throughput screening, a set of surface markers that are strongly expressed in HLA-DR^-CD14⁺ monocytic MDSCs isolated from the peripheral blood (PB) of patients receiving allo-HSCT. Subsequent experiments showed the consistent dominant expression of CD1d in monocytic MDSCs of allo-HSCT PB in comparison with granulocytic MDSCs. In addition, CD1d-expressing cells isolated from PB of allo-HSCT patients showed the suppressive activity of T cell proliferation and higher expression of MyD88 and IDO compared with CD1d^? cells. Our results suggest that CD1d could be a valuable marker for further therapeutic evaluation of human monocytic MDSCs for immune-related diseases, including GVHD.     

Circulating myeloid-derived suppressor cells: An independent prognostic factor in patients with breast cancer

Evading immune destruction is a hallmark of cancer. Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid immune cells, are thought to foster the establishment of an immunosuppressive tumor microenvironment, but it remains unclear how. This study aims to determine the levels of circulating MDSCs and their subpopulations and test their immunosuppressive functions in patients with breast cancer (BC). We analyzed the fractions of MDSCs in freshly isolated peripheral blood mononuclear cells of patients with BC and healthy donors using flow cytometry. Circulating MDSCs were further phenotyped using fluorescently labeled antihuman monoclonal antibodies. Coculture experiments revealed the effects of MDSCs on CD3⁺ T cell response. Moreover, we correlated circulating MDSC levels with clinicopathological features of patients with BC. We show that the fraction of HLA-DR ^– CD33 ⁺ MDSCs in peripheral blood is about 10-fold higher in patients with BC than in healthy control individuals. The levels of all MDSC subpopulations, including monocytic and granulocytic MDSCs, are significantly elevated. Coculture experiments of purified HLA-DR ^– CD33 ⁺ MDSCs and CD3 ⁺ T cells demonstrate that T cell proliferation is more effectively inhibited by BC patient-derived MDSCs than by healthy control MDSCs. Moreover, increased circulating MDSC levels robustly associate with advanced BC stage and positive lymph node status. By being more abundant and more effective T cell suppressors, BC patient-derived circulating MDSCs exert a dual immunosuppressive effect. Our findings pave the way to develop novel diagnostic and immunotherapeutic strategies, aimed at detecting and inhibiting MDSCs in patients with BC.     

Identification of myeloid derived suppressor cells in dogs with naturally occurring cancer

Dogs with naturally occurring cancer represent an important large animal model for drug development and testing novel immunotherapies. However, poorly defined immunophenotypes of canine leukocytes have limited the study of tumor immunology in dogs. The accumulation of myeloid derived suppressor cells (MDSCs) is known to be a key mechanism of immune suppression in tumor-bearing mice and in human patients. We sought to identify MDSCs in the blood of dogs with cancer. Peripheral blood mononuclear cells (PBMCs) from dogs with advanced or early stage cancer and from age-matched healthy controls were analyzed by flow cytometry and microscopy. Suppressive function was tested in T cell proliferation and cytokine elaboration assays. Semi-quantitative RT-PCR was used to identify potential mechanisms responsible for immunosuppression. PBMCs from dogs with advanced or metastatic cancer exhibited a significantly higher percentage of CD11b(+)CD14(-)MHCII(-) cells compared to dogs diagnosed with early stage non-metastatic tumors and healthy dogs. These CD11b(+) CD14(-)MHCII(-) cells constitute a subpopulation of activated granulocytes that co-purify with PBMCs, display polymorphonuclear granulocyte morphology, and demonstrate a potent ability to suppress proliferation and IFN-γ production in T cells from normal and tumor-bearing donors. Furthermore, these cells expressed hallmark suppressive factors of human MDSC including ARG1, iNOS2, TGF-β and IL-10. In summary our data demonstrate that MDSCs accumulate in the blood of dogs with advanced cancer and can be measured using this three-marker immunophenotype, thereby enabling prospective studies that can monitor MDSC burden.     

Rapid ex vivo expansion of highly enriched human invariant natural killer T cells via single antigenic stimulation for cell therapy to prevent graft-versus-host disease

Background aims

CD1d-restricted invariant natural killer (iNK) T cells are rare regulatory T cells that may contribute to the immune-regulation in allogeneic stem cell transplantation (ASCT). Here, we sought to develop an effective strategy to expand human iNK T cells for use in cell therapy to prevent graft-versus-host disease (GVHD) in ASCT.

Identification of alpha-enolase as a potential immunogenic molecule during allogeneic transplantation of human adipose-derived mesenchymal stromal cells

Background aimsGiven their low immunogenicity, immunoregulatory effects and multiple differentiation capacity, mesenchymal stromal cells (MSCs) have the potential to be used for “off-the-shelf” cell therapy to treat various diseases. However, the allorejection of MSCs indicates that they are not fully immune-privileged. In this study, the authors investigated the immunogenicity of human adipose-derived MSCs (Ad-MSCs) and identified potential immunogenic molecules.MethodsTo evaluate the immunogenicity of human Ad-MSCs in vivo, cells were transplanted into humanized mice (hu-mice), then T-cell infiltration and clearance of human Ad-MSCs were observed by immunofluorescence and bioluminescence imaging. One-way mixed lymphocyte reaction and flow cytometry were performed to evaluate the immunogenicity of human Ad-MSCs in vitro. High-throughput T-cell receptor (TCR) repertoire sequencing and mass spectrometry were applied to identified potential immunogenic molecules.ResultsThe authors observed that allogeneic Ad-MSCs recruited human T cells and caused faster clearance in hu-mice than non-humanized NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice. The proliferation and activation of T cells were significantly enhanced during in vitro co-culture with human Ad-MSCs. In addition, the level of HLA-II expression on human Ad-MSCs was dramatically increased after co-culture with human peripheral blood mononuclear cells (PBMCs). High-throughput sequencing was applied to analyze the TCR repertoire of the Ad-MSC-recruited T cells to identify dominant TCR CDR3 sequences. Using synthesized TCR CDR3 peptides, the authors identified several potential immunogenic candidates, including alpha-enolase (ENO1). The ENO1 expression level of Ad-MSCs significantly increased after co-culture with PBMCs, whereas ENO1 inhibitor (ENOblock) treatment decreased the expression level of ENO1 and Ad-MSC-induced proliferation of T cells.ConclusionsThe authors’ findings improve the understanding of the immunogenicity of human Ad-MSCs and provide a theoretical basis for the safe clinical application of allogeneic MSC therapy.     

Optimization of the therapeutic efficacy of human umbilical cord blood–mesenchymal stromal cells in an NSG mouse xenograft model of graft-versus-host disease

Background aimsAlthough in vitro studies have demonstrated the immunosuppressive capacity of mesenchymal stromal cells (MSCs), most in vivo studies on graft-versus-host disease (GVHD) have focused on prevention, and the therapeutic effect of MSCs is controversial. Moreover, optimal time intervals for infusing MSCs have not been established.MethodsWe attempted to evaluate whether human umbilical cord blood–MSCs (hUCB-MSCs) could either prevent or treat GVHD in an NSG mouse xenograft model by injection of MSCs before or after in vivo clearance. Mice were infused with either a single dose or multiple doses of 5 × 10⁵ hUCB-MSCs (3- or 7-day intervals) before or after GVHD onset.ResultsBefore onset, hUCB-MSCs significantly improved the survival rate only when repeatedly injected at 3-day intervals. In contrast, single or repeated injections after GVHD onset significantly increased the survival rate and effectively attenuated tissue damage and inflammation. Furthermore, the levels of prostaglandin E₂ and transforming growth factor-β1 increased significantly, whereas the level of interferon-γ decreased significantly in all MSC treatment groups.ConclusionsThese data establish the optimal time intervals for preventing GVHD and show that hUCB-MSCs effectively attenuated symptoms and improved survival rate when administered after the onset of GVDH.     

ADRB3 induces mobilization and inhibits differentiation of both breast cancer cells and myeloid-derived suppressor cells

Metastatic tumors are mainly composed of neoplastic cells escaping from the primary tumor and inflammatory cells egressing from bone marrow. Cancer cell and inflammatory cell are remained in the state of immaturity during migration to distant organs. Here, we show that ADRB3 is crucial in cell mobilization and differentiation. Immunohistochemistry revealed ADRB3 expression is significantly more frequent in breast cancer tissues than in adjacent noncancerous tissues (92.1% vs. 31.5%). Expression of ADRB3 correlated with malignant degree, TNM stage and poor prognosis. Moreover, ADRB3 expression was markedly high in activated disseminated tumor cells, myeloid-derived suppressor cells (MDSCs), lymphocytes and neutrophil extracellular traps of patients. Importantly, ADRB3 promoted the expansion of MDSC through stimulation of bone marrow mobilization and inhibiting of the differentiation of immature myeloid cells. Furthermore, ADRB3 promoted MCF-7 cells proliferation and inhibited transdifferentiation into adipocyte-like cell by activating mTOR pathway. Ultimately, the MDSC-deficient phenotype of ADRB3 ^-/- PyMT mice was associated with impairment of mammary tumorigenesis and reduction in pulmonary metastasis. Collectively, ADRB3 promotes metastasis by inducing mobilization and inhibiting differentiation of both breast cancer cells and MDSCs.Subject terms: Breast cancer, Breast cancer     

A simple method for identifying bone marrow mesenchymal stromal cells with a high immunosuppressive potential

Background aimsThe beneficial activity of mesenchymal stromal cells (MSC) in allogeneic hematopietic stem cell transplantation requires correct use in terms of cell dose and timing of infusion and the identification of biomarkers for selection. The immunosuppressive bone marrow (BM)-derived MSC (BM-MSC) functions have been associated with the production of soluble HLA-G molecules (sHLA-G) via interleukin (IL)-10. We have established a reliable method for evaluating BM-MSC HLA-G expression without the influence of peripheral blood mononuclear cells (PBMC).MethodsThirteen BM-MSC from donors were activated with recombinant IL-10 or co-cultured with 10 different phytohemagglutinin (PHA)-treated PBMC (PHA-PBMC). Membrane-bound and sHLA-G expression was evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively; lymphoproliferation was measured by (methyl-³H)thymidine.ResultsThe results demonstrated the ability of IL-10 to stimulate both membrane-bound and sHLA-G production by BM-MSC. The levels of HLA-G expression induced by IL-10 in BM-MSC were associated with the inhibition of PHA-PBMC proliferation (sHLA-G, P = 0.0008, r = 0.9308; membrane HLA-G, P = 0.0005, r = 0.9502).ConclusionsWe propose the evaluation of sHLA-G production in IL-10-treated BM-MSC cultures as a possible marker of immunoregulatory function.     

Umbilical cord-derived mesenchymal stem cells promote myeloid-derived suppressor cell enrichment by secreting CXCL1 to prevent graft-versus-host disease after hematopoietic stem cell transplantation

Background aimsHuman mesenchymal stem cells (MSCs) from various tissues have emerged as attractive candidates for the prevention and treatment of graft-versus-host disease (GVHD). However, the molecular machinery that defines and channels the behavior of these cells remains poorly understood.MethodsIn this study, the authors compared the efficacy of four tissue-derived MSC types in controlling GVHD in a murine model and investigated their immunomodulatory effects.ResultsHuman umbilical cord-derived mesenchymal stem cells (hUCMSCs) effectively decreased the incidence and severity of GVHD, which was mediated by the enrichment of myeloid-derived suppressor cells in GVHD target tissues. RNA sequencing results showed that hUCMSCs highly expressed CXCL1.ConclusionsThese results suggest a novel prophylactic application of hUCMSCs for controlling GVHD after allogeneic hematopoietic stem cell transplantation.