Novel TGF-beta antagonist inhibits tumor growth and angiogenesis by inducing IL-2 receptor-driven STAT1 activation.

Carcinoma derived TGF-ß acts as a potent pro-oncogenic factor and suppresses antitumor immunity. To antagonize TGF-ß-mediated effects in tandem with a proinflammatory immune stimulus, we generated a chimeric protein borne of the fusion of IL-2 and the soluble extracellular domain of TGF-ßR II (FIST). FIST acts as a decoy receptor trapping active TGF-ß in solution and interacts with IL-2-responsive lymphoid cells, inducing a distinctive hyperactivation of STAT1 downstream of IL-2R, which in turn promotes SMAD7 overexpression. Consequently, FIST-stimulated lymphoid cells are resistant to TGF-ß-mediated suppression and produce significant amounts of proinflammatory cytokines. STAT1 hyperactivation further induces significant secretion of angiostatic CXCL10. Moreover, FIST upregulates T-bet expression in NK cells promoting a potent Th1-mediated antitumor response. As a result, FIST stimulation completely inhibits pancreatic cancer (PANC02) and melanoma (B16) tumor growth in immunocompetent C57BL/6 mice. In addition, melanoma cells expressing FIST fail to form tumors in CD8(-/-), CD4(-/-), B cell-deficient (µMT), and beige mice, but not in NOD-SCID and Rag2/γc knockout mice, consistent with the pivotal role of FIST-responsive, cancer-killing NK cells in vivo. In summary, FIST constitutes a novel strategy of treating cancer that targets both the host's angiogenic and innate immune response to malignant cells.

Induction of cardiac angiogenesis requires killer cell lectin-like receptor 1 and α4ß7 integrin expression by NK cells.

Recent findings indicate that NK cells are involved in cardiac repair following myocardial infarction. The aim of this study is to investigate the role NK cells in infarct angiogenesis and cardiac remodeling. In normal C57BL/6 mice, myelomonocytic inflammatory cells invaded infarcted heart within 24 h followed by a lymphoid/NK cell infiltrate by day 6, accompanied by substantial expression of IL-2, TNF-α, and CCL2. In contrast, NOD SCID mice had virtually no lymphoid cells infiltrating the heart and did not upregulate IL-2 levels. In vitro and in vivo, IL-2-activated NK cells promoted TNF-α-stimulated endothelial cell proliferation, enhanced angiogenesis and reduced fibrosis within the infarcted myocardium. Adoptive transfer of IL-2-activated NK cells to NOD SCID mice improved post-myocardial infarction angiogenesis. RNA silencing technology and neutralizing Abs demonstrated that this process involved α4ß7 integrin/VCAM-1 and killer cell lectin-like receptor 1/N-cadherin-specific binding. In this study, we show that IL-2-activated NK cells reduce myocardial collagen deposition along with an increase in neovascularization following acute cardiac ischemia through specific interaction with endothelial cells. These data define a potential role of activated NK cells in cardiac angiogenesis and open new perspectives for the treatment of ischemic diseases.

A dendritic cell population generated by a fusion of GM-CSF and IL-21 induces tumor-antigen-specific immunity.

We have previously shown that the fusion of GM-CSF and IL-21 (GIFT-21) possesses a potent immune stimulatory effect on myeloid cells. In this study, we define the effect of GIFT-21 on naive murine monocytes (GIFT-21 dendritic cells [DCs]), which express increased levels of Gr-1, CD45R, MHC class I, CD80, CD86, and CXCR4 and suppress CD11c and MHC class II. Compared with conventional dendritic cells, GIFT-21 DCs produced substantially more CCL2, IL-6, TNF-α, and IFN-α and induced significantly greater production of IFN-γ by CD8(+) T cells in MHC class I-restricted Ag presentation assays. B16 melanoma and D2F2 Neu breast cancer growth was inhibited in mice treated with Ag-naive GIFT-21 DCs. This effect was lost in CD8(-/-) and CCR2(-/-) mice and when mice were treated with ß(2)-microglobulin-deficient GIFT-21 DCs, indicating that GIFT-21 DCs migrated to and sampled from the tumors to present tumor Ags to CCL2 recruited CD8(+) T cells via MHC class I. We propose that autologous GIFT-21 DCs may serve as a cell therapy platform for the treatment of cancer.

Erythropoietin gene-enhanced marrow mesenchymal stromal cells decrease cisplatin-induced kidney injury and improve survival of allogeneic mice.

Bone marrow-derived mesenchymal stromal cells (MSCs) are promising for regenerative medicine applications, such as for renoprotection and repair in acute kidney injury (AKI). Erythropoietin (Epo) can also exert cytoprotective effects on various tissues including the kidney. We hypothesized that MSCs gene-enhanced to secrete Epo may produce a significant beneficial effect in AKI. Mouse Epo-secreting MSCs were generated, tested in vitro, and then implanted by intraperitoneal injection in allogeneic mice previously administered cisplatin to induce AKI. Epo-MSCs significantly improved survival of implanted mice as compared to controls (67% survival versus 33% with Vehicle only). Also, Epo-MSCs led to significantly better kidney function as shown by lower levels of blood urea nitrogen (72 ± 9.5 mg/dl versus 131 ± 9.20 mg/dl) and creatinine (74 ± 17 µmol/l versus 148±19.4 µmol/l). Recipient mice also showed significantly decreased amylase and alanine aminotransferase blood concentrations. Kidney sections revealed significantly less apoptotic cells and more proliferating cells. Furthermore, PCR revealed the presence of implanted cells in recipient kidneys, with Epo-MSCs leading to significantly increased expression of Epo and of phosphorylated-Akt (Ser473) (P-Akt) in these kidneys. In conclusion, our study demonstrates that Epo gene-enhanced MSCs exert significant tissue protective effects in allogeneic mice with AKI, and supports the potential use of gene-enhanced cells as universal donors in acute injury.

Bone marrow mesenchymal stromal cell therapy for external urethral sphincter restoration in a rat model of stress urinary incontinence.

OBJECTIVE: To assess the effect of intra-sphincteric injections of bone marrow mesenchymal stromal cells (MSCs) on Valsalva leak point pressure (VLPP) changes in an animal model of stress urinary incontinence (SUI). MATERIALS AND METHODS: Twenty-four female Sprague-Dawley rats underwent bilateral pudendal nerve section to induce SUI. Six rats were SUI controls, 6 received periurethral injection of Plasma-Lyte (SUI placebo control) and 12 were given periurethral injection of PKH26-labeled MSCs. Four weeks after injection, conscious cystometry was undertaken in animals and VLPP recorded. All groups were sacrificed, and frozen urethra sections were submitted to pathology and immunohistochemistry assessment. RESULTS: Rat MSCs were positive for the cell surface antigens CD44, CD73, CD90, and RT1A, and negative for CD31, CD45, and RT1B, confirming their stem cell phenotype. In vitro, differentiated MSCs expressed α-smooth muscle actin (SMA) and desmin, markers of smooth and striated muscles in vivo. Immunohistochemistry of rat urethras revealed PKH26-labeled MSCs in situ and at the injection site. LPP was significantly improved in animals injected with MSCs. Mean LPP was 24.28 ± 1.47 cmH(2) O in rats implanted with MSCs and 16.21 ± 1.26 cmH(2) O in SUI controls (P<0.001). Atrophic urethras with implanted MSCs were positively stained for myosin heavy chain and desmin. CONCLUSION: Rat MSCs have the ability to differentiate and skew their phenotype towards smooth and striated muscles, as demonstrated by SMA up-regulation and desmin expression. Periurethral injection of MSCs in an animal model of SUI restored the damaged external urethral sphincter and significantly improved VLPP.

Cytokine modulation of TLR expression and activation in mesenchymal stromal cells leads to a proinflammatory phenotype.

Bone marrow-derived mesenchymal stromal cells (MSC) possess an immune plasticity manifested by either an immunosuppressive or, when activated with IFN-gamma, an APC phenotype. Herein, TLR expression by MSC and their immune regulatory role were investigated. We observed that human MSC and macrophages expressed TLR3 and TLR4 at comparable levels and TLR-mediated activation of MSC resulted in the production of inflammatory mediators such as IL-1beta, IL-6, IL-8/CXCL8, and CCL5. IFN-alpha or IFN-gamma priming up-regulated production of these inflammatory mediators and expression of IFNB, inducible NO synthase (iNOS), and TRAIL upon TLR activation in MSC and macrophages, but failed to induce IL-12 and TNF-alpha production in MSC. Nonetheless, TLR activation in MSC resulted in the formation of an inflammatory site attracting innate immune cells, as evaluated by human neutrophil chemotaxis assays and by the analysis of immune effectors retrieved from Matrigel-embedded MSC injected into mice after in vitro preactivation with cytokines and/or TLR ligands. Hence, TLR-activated MSC are capable of recruiting immune inflammatory cells. In addition, IFN priming combined with TLR activation may increase immune responses induced by Ag-presenting MSC through presentation of Ag in an inflammatory context, a mechanism that could be applied in a cell-based vaccine.

A fusion of GMCSF and IL-21 initiates hypersignaling through the IL-21Ralpha chain with immune activating and tumoricidal effects in vivo.

We hypothesized that fusing granulocyte-macrophage colony-stimulation factor (GMCSF) and interleukin (IL)-21 as a single bifunctional cytokine (hereafter GIFT-21) would lead to synergistic anticancer immune effects because of their respective roles in mediating inflammation. Mechanistic analysis of GIFT-21 found that it leads to IL-21Ralpha-dependent STAT3 hyperactivation while also contemporaneously behaving as a dominant-negative inhibitor of GMCSF-driven STAT5 activation. GIFT-21's aberrant interactions with its cognate receptors on macrophages resulted in production of 30-fold greater amounts of IL-6, TNF-alpha, and MCP-1 when compared to controls. Furthermore, GIFT-21 treatment of primary B and T lymphocytes leads to STAT1-dependent apoptosis of IL-21Ralpha(+) lymphocytes. B16 melanoma cells gene-enhanced to produce GIFT-21 were immune rejected by syngeneic C57Bl/6 mice comparable to the effect of IL-21 alone. However, a significant GIFT-21-driven survival advantage was seen when NOD-SCID mice were implanted with GIFT-21-secreting B16 cells, consistent with a meaningful role of macrophages in tumor rejection. Because GIFT-21 leads to apoptosis of IL-21Ralpha(+) lymphocytes, we tested its cytolytic effect on IL-21Ralpha(+) EL-4 lymphoma tumors implanted in C57Bl/6 mice and could demonstrate a significant increase in survival. These data indicate that GIFT-21 is a novel IL-21Ralpha agonist that co-opts IL-21Ralpha-dependent signaling in a manner permissive for targeted cancer immunotherapy.

Human marrow-derived mesenchymal stromal cells decrease cisplatin renotoxicity in vitro and in vivo and enhance survival of mice post-intraperitoneal injection.

Acute kidney injury (AKI) can occur from the toxic side-effects of chemotherapeutic agents such as cisplatin. Bone marrow-derived mesenchymal stromal cells (MSCs) have demonstrated wide therapeutic potential often due to beneficial factors they secrete. The goal of this investigation was to evaluate in vitro the effect of human MSCs (hMSCs) secretome on cisplatin-treated human kidney cells, and in vivo the consequence of hMSCs intraperitoneal (ip) implantation in mice with AKI. Our results revealed that hMSCs-conditioned media improved survival of HK-2 human proximal tubular cells exposed to cisplatin in vitro. This enhanced survival was linked to increased expression of phosphorylated Akt (Ser473) and was reduced by a VEGF-neutralizing antibody. In vivo testing of these hMSCs established that ip administration in NOD-SCID mice decreased cisplatin-induced kidney function impairment, as demonstrated by lower blood urea nitrogen levels and higher survival. In addition, blood phosphorous and amylase levels were also significantly decreased. Moreover, hMSCs reduced the plasma levels of several inflammatory cytokines/chemokines. Immunohistochemical examination of kidneys showed less apoptotic and more proliferating cells. Furthermore, PCR indicated the presence of hMSCs in mouse kidneys, which also showed enhanced expression of phosphorylated Akt. In conclusion, our study reveals that hMSCs can exert prosurvival effects on renal cells in vitro and in vivo, suggests a paracrine contribution for kidney protective abilities of hMSCs delivered ip, and supports their clinical potential in AKI.

Airway delivery of mesenchymal stem cells prevents arrested alveolar growth in neonatal lung injury in rats.

RATIONALE: Bronchopulmonary dysplasia (BPD) and emphysema are characterized by arrested alveolar development or loss of alveoli; both are significant global health problems and currently lack effective therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) prevent adult lung injury, but their therapeutic potential in neonatal lung disease is unknown. OBJECTIVES: We hypothesized that intratracheal delivery of BMSCs would prevent alveolar destruction in experimental BPD. METHODS: In vitro, BMSC differentiation and migration were assessed using co-culture assays and a modified Boyden chamber. In vivo, the therapeutic potential of BMSCs was assessed in a chronic hyperoxia-induced model of BPD in newborn rats. MEASUREMENTS AND MAIN RESULTS: In vitro, BMSCs developed immunophenotypic and ultrastructural characteristics of type II alveolar epithelial cells (AEC2) (surfactant protein C expression and lamellar bodies) when co-cultured with lung tissue, but not with culture medium alone or liver. Migration assays revealed preferential attraction of BMSCs toward oxygen-damaged lung versus normal lung. In vivo, chronic hyperoxia in newborn rats led to air space enlargement and loss of lung capillaries, and this was associated with a decrease in circulating and resident lung BMSCs. Intratracheal delivery of BMSCs on Postnatal Day 4 improved survival and exercise tolerance while attenuating alveolar and lung vascular injury and pulmonary hypertension. Engrafted BMSCs coexpressed the AEC2-specific marker surfactant protein C. However, engraftment was disproportionately low for cell replacement to account for the therapeutic benefit, suggesting a paracrine-mediated mechanism. In vitro, BMSC-derived conditioned medium prevented O(2)-induced AEC2 apoptosis, accelerated AEC2 wound healing, and enhanced endothelial cord formation. CONCLUSIONS: BMSCs prevent arrested alveolar and vascular growth in part through paracrine activity. Stem cell-based therapies may offer new therapeutic avenues for lung diseases that currently lack efficient treatments.

Inhibiting myostatin with follistatin improves the success of myoblast transplantation in dystrophic mice.

Duchenne muscular dystrophy is a recessive disease due to a mutation in the dystrophin gene. Myoblast transplantation permits to introduce the dystrophin gene in dystrophic muscle fibers. However, the success of this approach is reduced by the short duration of the regeneration following the transplantation, which reduces the number of hybrid fibers. Our aim was to verify whether the success of the myoblast transplantation is enhanced by blocking the myostatin signal with an antagonist, follistatin. Three different approaches were studied to overexpress follistatin in the muscles of mdx mice transplanted with myoblasts. First, transgenic follistatin/mdx mice were generated; second, a follistatin plasmid was electroporated in mdx muscles, and finally, follistatin was induced in mdx mice muscles by a treatment with a histone deacetylase inhibitor. The three approaches improved the success of the myoblast transplantation. Moreover, fiber hypertrophy was also observed in all muscles, demonstrating that myostatin inhibition by follistatin is a good method to improve myoblast transplantation and muscle function. Myostatin inhibition by follistatin in combination with myoblast transplantation is thus a promising novel therapeutic approach for the treatment of muscle wasting in diseases such as Duchenne muscular dystrophy.

1,25-dihydroxyvitamin D3 increases the transplantation success of human muscle precursor cells in SCID mice.

Human muscle precursor cell (hMPC) transplantation is a potential therapy for severe muscle trauma or myopathies. Some previous studies demonstrated that 1,25-dihydroxyvitamin-D3 (1,25-D3) acted directly on myoblasts, regulating their proliferation and fusion. 1,25-D3 is also involved in apoptosis modulation of other cell types and may thus contribute to protect the transplanted hMPCs. We have therefore investigated whether 1,25-D3 could improve the hMPC graft success. The 1,25-D3 effects on hMPC proliferation, fusion, and survival were initially monitored in vitro. hMPCs were also grafted in the tibialis anterior of SCID mice treated or not with 1,25-D3 to determine its in vivo effect. Graft success, proliferation, and viability of transplanted hMPCs were evaluated. 1,25-D3 enhanced proliferation and fusion of hMPCs in vitro and in vivo. However, 1,25-D3 did not protect hMPCs from various proapoptotic factors (in vitro) or during the early posttransplantation period. 1,25-D3 enhanced hMPC graft success because the number of muscle fibers expressing human dystrophin was significantly increased in the TA sections of 1,25-D3-treated mice (166.75 +/- 20.64) compared to the control mice (97.5 +/- 16.58). This result could be partly attributed to the improvement of the proliferation and differentiation of hMPCs in the presence of 1,25-D3. Thus, 1,25-D3 administration could improve the clinical potential of hMPC transplantation currently developed for muscle trauma or myopathies.

Exercise improves the success of myoblast transplantation in mdx mice.

Transplantation of normal muscle precursor cells is a potential approach to restore dystrophin expression within dystrophin [deficient] mdx mice, a model of Duchenne Muscular Dystrophy. This study aims to evaluate whether exercise could improve graft success and hybrid fiber distribution within mdx muscle. eGFP(+) Muscle precursor cells were transplanted into tibialis anterior muscles of mdx mice using a single injection trajectory. During the following weeks, muscle fiber breaks were induced by making mdx mice swim. To evaluate fiber damage, Evans blue solution was injected intraperitoneally to mice 16h before their sacrifice. Tibialis anterior muscles were then harvested and eGFP, dystrophin and Evans blue labeling were analyzed by fluorescent microscopy. Twenty minutes of exercise (i.e., swimming) were used to induce damage in about 30% of TA muscle fibers. Graft success, evaluated as the percentage of hybrid fibers which are eGFP(+), was improved by 1.9-fold after swimming 3 times per week during 4 weeks and by 1.8-fold after daily swimming. Hybrid muscle fiber transversal and longitudinal distribution were also increased after repeated physical efforts. Exercise induced fiber breaks, which improved MPC recruitment and fusion and increased long-term graft success and also transverse and longitudinal distribution of hybrid fibers.

Induction of tolerance across fully mismatched barriers by a nonmyeloablative treatment excluding antibodies or irradiation use.

A mixed-chimerism approach is a major goal to circumvent sustained immunosuppression, but most of the proposed protocols need antibody treatment or host irradiation. Another promising experience involves busulfan combined with cyclophosphamide treatment. Additionally, recent publications demonstrated that, differing from busulfan, treosulfan administration does not present severe organ or hemato toxicities. Currently, Duchenne muscular dystrophy (DMD) patients are treated with chronic immunosuppression for muscle precursor cell transplantation (MT). We have developed a safe tolerance approach within this cellular allotransplantation therapy background. Thus, we have conditioned, prior to a donor BALB/c MT, the dystrophic mouse model C57Bl10J mdx/mdx, with our treatment based on a donor-specific transfusion, then a treosulfan treatment combined with single cyclophosphamide dose, and finally a donor bone marrow transplantation (TTCB). A first MT was performed in all mixed chimeric mice resulting from the TTCB treatment in the left tibialis anterior (TA) muscles. A second MT from the same donor strain was performed 100 days later in the right TA without any additional therapy. Results show that all treated mice developed permanent mixed chimerism. Long-lasting donor-positive fibers were present in both TAs of the mice, which received MT after the TTCB treatment. Only a basal level of infiltration was observed around donor fibers and mixed chimeric mice rejected third-party haplotype skin grafts. Thus, mixed chimerism development with this TTCB conditioning regimen promotes donor-specific stable tolerance, avoiding costimulatory blockade antibodies or irradiation use and side effects of sustained immunosuppressive treatments. This protocol could be eventually applied for MT to DMD patients or others tissue transplantations.

Improved success of myoblast transplantation in mdx mice by blocking the myostatin signal.

BACKGROUND: : Duchenne muscular dystrophy (DMD) is caused by a dystrophin gene mutation. Transplantation of normal myoblasts results in long-term restoration of dystrophin. However, the success of this approach is compromised by the limited time of regeneration following muscle damage. Myostatin is known to be responsible for limiting skeletal muscle regeneration. Our purpose is to verify whether blocking the myostatin signal in mdx host mice or in normal myoblasts transplanted in mdx host mice would increase the extent of muscle repair and thus allow the formation of more dystrophin-positive fibers. METHODS: : Transgenic mdx mice carrying a dominant negative form of myostatin receptor (dnActRIIB) were used to test the fiber resistance to damage and to act as a host for normal myoblast transplantation. Myoblasts obtained from nondystrophic transgenic mice carrying the dominant negative myostatin receptor were also transplanted in nontransgenic mdx mice. RESULTS: : Transgenic mdx mice carrying the dnActRIIB gene have bigger muscles than mdx mice with the normal gene of ActRIIB. Their fiber resistance to exercise-induced damage was also greatly improved. Moreover, the success of normal myoblast transplantation was significantly enhanced in mdx/dnActRIIB mice. Finally, nondystrophic dnActRIIB myoblasts formed more abundant and bigger dystrophin positive fibers when transplanted in mdx mice. CONCLUSIONS: : Blocking the myostatin signal in mdx mice allowed the size of muscle fibers to increase, the fiber resistance to damage induced by exercise to increase, and the success of normal myoblast transplantation to improve. The transplantation in mdx mice of dnActRIIB myoblasts formed more abundant and larger dystrophin positive fibers.

Real-time imaging of myoblast transplantation using the human sodium iodide symporter.

The quantification of the graft success is a key element to evaluate the efficiency of cellular therapies for several pathologies such as Duchenne muscular dystrophy. This study describes an approach to evaluate the success of myoblast transplantation (i.e., survival of the transplanted cells and the muscle fibers formed) by real-time imaging. C2C12 myoblasts were first transfected with a plasmid containing the human sodium iodide symporter (hNIS) gene. Specific uptake of the radioactive sodium pertechnetate (Na99mTcO4) by the hNIS-positive myoblasts was demonstrated in vitro, while only background level of Na99mTcO4 was observed within the control cells. The cells were then transplanted into the tibialis anterior (TA) muscle of mdx (X-linked dystrophic) mice. Following intraperitoneal administration of Na99mTcO4, scintigraphies were performed to detect hNIS-dependent Na99mTcO4 uptake within the TA. This approach permitted to evaluate the progression of the transplantation and the graft success without having to biopsy the animals during the follow-up period.

Myoblast survival enhancement and transplantation success improvement by heat-shock treatment in mdx mice.

BACKGROUND: Duchenne muscular dystrophy is a disease caused by the incapacity to synthesize dystrophin, which is implicated in the maintenance of the sarcolemma integrity. Myoblast transplantation is a potential treatment of this disease. However, most of the transplanted cells die very rapidly after their injection. Heat-shock proteins (HSPs) are over-expressed when cells undergo various types of stresses. Our goal was thus to investigate whether the expression of HSPs (HSP70 in particular) could protect myoblasts from death after intramuscular injection. METHODS: HSP70 expression was induced by warming the cells at 42 degrees C for 60 minutes. HSP70 over-expression was quantified by Western blot analysis. The in vitro effect of HSPs on cell survival was evaluated by fluorescence-activated cell sorter analysis using the Hoescht/propidium iodide-labeling technique, and their in vivo effects were investigated by transplanting TnI-LacZ myoblasts labeled with [methyl-14C] thymidine. RESULTS: Western blots indicated a sevenfold over-expression of the HSP70 after the heat-shock treatment. In vitro, the heat-shock treatment protected 18% of the cells from staurosporine- (1 microM) induced apoptosis. HSPs also protected 10% of the cells from death induced by either tumor necrosis factor-alpha (30 ng/mL) or glucose oxydase (0.1 U/mL). In vivo, the treatment improved the cell survival by twofold 5 days after the graft and increased by fourfold the long-term graft success. CONCLUSIONS: The heat-shock treatment is a practical approach for improving the success of myoblast transplantation; in fact, using this kind of treatment, there is no need to genetically modify the cells before their transplantation.