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1.
Cytotherapy ; 14(10): 1164-70, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22574720

ABSTRACT

BACKGROUND AIMS: Apoptosis of radiosensitive cells in the bone marrow and gut is a serious, at times life-threatening, complication arising from radiation exposure. METHODS: We investigated whether adoptive transfer of allogeneic bone marrow-derived mesenchymal stromal cells (MSC) could exert cytoprotective and life-sparing effects in a mouse model of sublethal total body irradiation (TBI). RESULTS: We demonstrated that a single intraperitoneal injection of C57Bl/6 MSC given to major histocompatibility complex (MHC)-mismatched Balb/c mice within 24 h of sublethal TBI significantly reduced mortality in a dose-dependent manner. Histologic analysis and Ki67 immunostaining of jejunum sections collected 3 and 6 days post-TBI indicated that MSC protected the gastrointestinal epithelium from TBI-induced damage and significantly accelerated recovery of the gut by stimulating proliferation of the crypt cell pool. Using interleukin-6(-/-) (IL-6) MSC, we demonstrated that IL-6 expressed by MSC played a role in gastrointestinal epithelium regeneration. CONCLUSIONS: Our results suggest that allogeneic MHC-mismatched MSC may be exploited to reduce gastrointestinal complications and mortality arising from ionizing radiation exposure.


Subject(s)
Adoptive Transfer , Interleukin-6/metabolism , Intestinal Mucosa/physiopathology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Regeneration/radiation effects , Whole-Body Irradiation , Animals , Bone Marrow Cells/cytology , Intestinal Mucosa/radiation effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Transplantation, Homologous
2.
Cancer Res ; 72(5): 1210-20, 2012 Mar 01.
Article in English | MEDLINE | ID: mdl-22241086

ABSTRACT

We have previously shown that interleukin (IL)-2 receptor-expressing lymphoid cells stimulated with a chimeric protein linking IL-2 to the ectodomain of TGF-ß receptor II (also known as FIST) become resistant to TGF-ß-mediated suppression and produce significant amounts of proinflammatory cytokines. In this study, we have characterized the antigen presentation properties of FIST-stimulated B cells (hereafter inducible B effector cells, iBEC). FIST converts naïve splenic B cells to B effector cells characterized by potent antigen presentation properties and production of TNFα and IFNγ. iBECs display hyperphosphorylation of STAT3 and STAT5 downstream of the IL-2 receptor and upregulation of T-bet expression. iBECs maintain B-cell identity based on the expression of PAX5 and CD19 and overexpress Smad7, which confers resistance to TGF-ß-mediated suppression of B-cell activation. iBEC antitumor immunity was determined by a mouse model of lymphoma-expressing ovalbumin (E.G7-OVA) as a specific tumor antigen. OVA-pulsed iBECs function as antigen-presenting cells (APC) in vitro by inducing the activation of OVA-specific CD4(+) and CD8(+) T cells, respectively, and in vivo by conferring complete protective immunity against E.G7-OVA tumor challenge. In addition, OVA-pulsed iBECs promote tumor regression in immunocompetent C57Bl/6 mice bearing E.G7-OVA tumors. In conclusion, iBECs represent an entirely novel B cell-derived APC for immune therapy of cancer.


Subject(s)
Antigen-Presenting Cells/immunology , B-Lymphocyte Subsets/immunology , Interleukin-2/immunology , Lymphocyte Activation , Neoplasms, Experimental/immunology , Animals , Cytotoxicity, Immunologic , Female , Gene Knockout Techniques , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Recombinant Fusion Proteins/immunology
3.
Mol Cancer ; 10: 121, 2011 Sep 24.
Article in English | MEDLINE | ID: mdl-21943176

ABSTRACT

BACKGROUND: The CCL2 chemokine is involved in promoting cancer angiogenesis, proliferation and metastasis by malignancies that express CCR2 receptor. Thus the CCL2/CCR2 axis is an attractive molecular target for anticancer drug development. METHODS: We have generated a novel fusion protein using GMCSF and an N-terminal truncated version of MCP1/CCL2 (6-76) [hereafter GMME1] and investigated its utility as a CCR2-specific tumoricidal agent. RESULTS: We found that distinct to full length CCL2 or its N-truncated derivative (CCL2 5-76), GMME1 bound to CCR2 on mouse lymphoma EG7, human multiple myeloma cell line U266, or murine and human medulloblastoma cell lines, and led to their death by apoptosis. We demonstrated that GMME1 specifically blocked CCR2-associated STAT3 phosphorylation and up-regulated pro-apoptotic BAX. Furthermore, GMME1 significantly inhibited EG7 tumor growth in C57BL/6 mice, and induced apoptosis of primary myeloma cells from patients. CONCLUSION: Our data demonstrate that GMME1 is a fusokine with a potent, CCR2 receptor-mediated pro-apoptotic effect on tumor cells and could be exploited as a novel biological therapy for CCR2+ malignancies including lymphoid and central nervous system malignancies.


Subject(s)
Antineoplastic Agents/pharmacology , Chemokine CCL2/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Receptors, CCR2/metabolism , Recombinant Fusion Proteins/pharmacology , Animals , Antigens, CD/metabolism , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Cell Line, Tumor , Female , Humans , Lymphoma , Medulloblastoma , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/therapeutic use , Tumor Burden , Xenograft Model Antitumor Assays
4.
J Immunol ; 187(6): 3133-44, 2011 Sep 15.
Article in English | MEDLINE | ID: mdl-21844388

ABSTRACT

Innate CD8 T cells are found in mutant mouse models, but whether they are produced in a normal thymus remains controversial. Using the RAG2p-GFP mouse model, we found that ∼10% of TCRαß(+) CD4(-)CD8(+) thymocytes were innate polyclonal T cells (GFP(+)CD44(hi)). Relative to conventional T cells, innate CD8 thymocytes displayed increased cell surface amounts of B7-H1, CD2, CD5, CD38, IL-2Rß, and IL-4Rα and downmodulation of TCRß. Moreover, they overexpressed several transcripts, including T-bet, Id3, Klf2, and, most of all, Eomes. Innate CD8 thymocytes were positively selected, mainly by nonhematopoietic MHCIa(+) cells. They rapidly produced high levels of IFN-γ upon stimulation and readily proliferated in response to IL-2 and IL-4. Furthermore, low numbers of innate CD8 thymocytes were sufficient to help conventional CD8 T cells expand and secrete cytokine following Ag recognition. This helper effect depended on CD44-mediated interactions between innate and conventional CD8 T cells. We concluded that innate TCRαß(+) CD8 T cells represent a sizeable proportion of normal thymocytes whose development and function differ in many ways from those of conventional CD8 T cells.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Immunity, Innate/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocyte Subsets/immunology , Animals , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Cytokines/biosynthesis , Cytokines/immunology , Enzyme-Linked Immunosorbent Assay , Gene Expression Profiling , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phenotype , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/metabolism
5.
Mol Ther ; 19(11): 2072-83, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21847101

ABSTRACT

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.


Subject(s)
Acute Kidney Injury/therapy , Erythropoietin/genetics , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Acute Kidney Injury/chemically induced , Animals , Bone Marrow Cells/cytology , Cell Survival/drug effects , Cisplatin , Culture Media, Conditioned/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Kidney/drug effects , Kidney/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Survival Analysis , Transduction, Genetic , Transplantation, Homologous
6.
J Immunol ; 186(12): 6933-44, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21551368

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Neovascularization, Pathologic/drug therapy , Receptors, Interleukin-2/metabolism , STAT1 Transcription Factor/metabolism , Transforming Growth Factor beta/antagonists & inhibitors , Animals , Interleukin-2 Receptor beta Subunit , Killer Cells, Natural/immunology , Mice , Neoplasms/drug therapy , Neoplasms/immunology , Neoplasms/pathology , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/therapeutic use , Tumor Burden/drug effects
7.
Biomaterials ; 32(1): 295-305, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20864158

ABSTRACT

Gene therapy for hemophilia B and other hereditary plasma protein deficiencies showed great promise in pre-clinical and early clinical trials. However, safety concerns about in vivo delivery of viral vectors and poor post-transplant survival of ex vivo modified cells remain key hurdles for clinical translation of gene therapy. We here describe a 3D scaffold system based on porous hydroxyapatite-PLGA composites coated with biomineralized collagen 1. When combined with autologous gene-engineered factor IX (hFIX) positive mesenchymal stem cells (MSCs) and implanted in hemophilic mice, these scaffolds supported long-term engraftment and systemic protein delivery by MSCs in vivo. Optimization of the scaffolds at the macro-, micro- and nanoscales provided efficient cell delivery capacity, MSC self-renewal and osteogenesis respectively, concurrent with sustained delivery of hFIX. In conclusion, the use of gene-enhanced MSC-seeded scaffolds may be of practical use for treatment of hemophilia B and other plasma protein deficiencies.


Subject(s)
Genetic Therapy/methods , Hemophilia B/therapy , Mesenchymal Stem Cells/metabolism , Tissue Scaffolds/chemistry , Animals , Calcium Phosphates/pharmacology , Cell Lineage/drug effects , Cell Proliferation/drug effects , Ceramics/pharmacology , Factor IX/genetics , Factor IX/therapeutic use , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/ultrastructure , Mice , Nanoparticles/ultrastructure , Particle Size , Porosity/drug effects
8.
J Immunol ; 185(12): 7358-66, 2010 Dec 15.
Article in English | MEDLINE | ID: mdl-21076067

ABSTRACT

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.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Immunity, Cellular/drug effects , Interleukins/immunology , Mammary Neoplasms, Experimental/immunology , Melanoma/immunology , Recombinant Fusion Proteins/pharmacology , Adoptive Transfer , Animals , Antigen Presentation/drug effects , Antigen Presentation/genetics , Antigen Presentation/immunology , Antigens, Differentiation/genetics , Antigens, Differentiation/immunology , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cell Movement/immunology , Cytokines/genetics , Cytokines/immunology , Dendritic Cells/transplantation , Female , Immunity, Cellular/genetics , Immunity, Cellular/immunology , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/therapy , Melanoma/genetics , Melanoma/therapy , Mice , Mice, Inbred BALB C , Mice, Knockout , Transplantation, Autologous
9.
Cancer Res ; 70(20): 7742-7, 2010 Oct 15.
Article in English | MEDLINE | ID: mdl-20924101

ABSTRACT

It is unknown whether mesenchymal stromal cells (MSC) can regulate immune responses targeting tumor autoantigens of low immunogenicity. We tested here whether immunization with MSC could break immune tolerance towards the ErbB-2/HER-2/neu tumor antigen and the effects of priming with IFN-γ and tumor necrosis factor-α (TNF-α) on this process. BALB/c- and C57BL/6-derived MSC were lentivirally transduced to express a kinase-inactive rat neu mutant (MSC/Neu). Immunization of BALB/c mice with nontreated or IFN-γ-primed allogeneic or syngeneic MSC/Neu induced similar levels of anti-neu antibody titers; however, only syngeneic MSC/Neu induced protective neu-specific CD8(+) T cell responses. Compared to immunization with nontreated or IFN-γ-primed syngeneic MSC/Neu, the number of circulating neu-specific CD8(+) T cells and titers of anti-neu antibodies were observed to be decreased after immunizations with IFN-γ- plus TNF-α-primed MSC/Neu. In addition, syngeneic MSC/Neu seemed more efficient than IFN-γ-primed MSC/Neu at inducing a protective therapeutic antitumor immune response resulting in the regression of transplanted neu-expressing mammary tumor cells. In vitro antigen-presenting cell assays performed with paraformaldehyde-fixed or live MSC showed that priming with IFN-γ plus TNF-α, compared to priming with IFN-γ alone, increased antigen presentation as well as the production of immunosuppressive factors. These data suggest that whereas MSC could effectively serve as antigen-presenting cells to induce immune responses aimed at tumor autoantigens, these functions are critically regulated by IFN-γ and TNF-α.


Subject(s)
Breast Neoplasms/immunology , Interferon-gamma/therapeutic use , Mammary Neoplasms, Experimental/immunology , Mesenchymal Stem Cells/immunology , Receptor, ErbB-2/biosynthesis , Tumor Necrosis Factor-alpha/therapeutic use , Animals , Breast Neoplasms/therapy , Cancer Vaccines/therapeutic use , Female , Humans , Mammary Neoplasms, Experimental/pathology , Mammary Tumor Virus, Mouse/genetics , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Promoter Regions, Genetic , Rats , Stromal Cells/immunology , Stromal Cells/pathology
10.
J Immunol ; 185(11): 7014-25, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-20971926

ABSTRACT

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.


Subject(s)
Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Integrins/biosynthesis , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Neovascularization, Physiologic/immunology , Receptors, Immunologic/biosynthesis , Animals , Cell Communication/immunology , Cell Proliferation , Cells, Cultured , Chemotaxis, Leukocyte/immunology , Cytotoxicity Tests, Immunologic , Endothelium, Vascular/cytology , Integrins/physiology , Interleukin-2/physiology , Lectins, C-Type , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Receptors, Immunologic/physiology , Tumor Necrosis Factor-alpha/physiology
11.
Am J Physiol Renal Physiol ; 299(6): F1288-98, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20844023

ABSTRACT

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.


Subject(s)
Acute Kidney Injury/therapy , Cisplatin/adverse effects , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/physiology , Acute Kidney Injury/chemically induced , Acute Kidney Injury/pathology , Animals , Cell Proliferation/drug effects , Cells, Cultured , Culture Media, Conditioned/pharmacology , Cytokines/blood , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Middle Aged , Proto-Oncogene Proteins c-akt/metabolism , Stromal Cells/physiology
12.
Mol Ther ; 18(7): 1293-301, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20389285

ABSTRACT

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.


Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Interleukin-21 Receptor alpha Subunit/metabolism , Interleukins/metabolism , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/physiology , Animals , Apoptosis/drug effects , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Cell Line , Cells, Cultured , Cytokines/metabolism , Female , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Interleukins/genetics , Macrophages/drug effects , Macrophages/metabolism , Melanoma, Experimental/metabolism , Melanoma, Experimental/therapy , Mice , Mice, Inbred C57BL , Recombinant Fusion Proteins/genetics , Signal Transduction/drug effects , Signal Transduction/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism
13.
Nat Med ; 15(9): 1038-45, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19668193

ABSTRACT

We have previously shown that a granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-15 (IL-15) 'fusokine' (GIFT15) exerts immune suppression via aberrant signaling through the IL-15 receptor on lymphomyeloid cells. We show here that ex vivo GIFT15 treatment of mouse splenocytes generates suppressive regulatory cells of B cell ontogeny (hereafter called GIFT15 B(reg) cells). Arising from CD19+ B cells, GIFT15 B(reg) cells express major histocompatibility complex class I (MHCI) and MHCII, surface IgM and IgD, and secrete IL-10, akin to previously described B10 and T2-MZP B(reg) cells, but lose expression of the transcription factor PAX5, coupled to upregulation of CD138 and reciprocal suppression of CD19. Mice with experimental autoimmune encephalomyelitis went into complete remission after intravenous infusion of GIFT15 B(reg) cells paralleled by suppressed neuroinflammation. The clinical effect was abolished when GIFT15 B(reg) cells were derived from mmicroMT (lacking B cells), MHCII-knockout, signal transducer and activator of transcription-6 (STAT-6)-knockout, IL-10-knockout or allogeneic splenocytes, consistent with a pivotal role for MHCII and IL-10 by sygeneic B cells for the observed therapeutic effect. We propose that autologous GIFT15 B(reg) cells may serve as a new treatment for autoimmune ailments.


Subject(s)
B-Lymphocyte Subsets/drug effects , B-Lymphocyte Subsets/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Interleukin-15/pharmacology , Recombinant Fusion Proteins/pharmacology , Animals , B-Lymphocyte Subsets/transplantation , Cytokines , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/therapy , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class II/metabolism , Immune Tolerance/drug effects , In Vitro Techniques , Interferon-gamma/biosynthesis , Interleukin-10/biosynthesis , Mice , Mice, Knockout , Mice, Transgenic , Recombinant Proteins , STAT6 Transcription Factor/deficiency , STAT6 Transcription Factor/genetics , STAT6 Transcription Factor/immunology , Transplantation, Homologous , Transplantation, Isogeneic
14.
Mol Ther ; 17(10): 1799-803, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19602999

ABSTRACT

The use of allogeneic "universal donor" mesenchymal stromal cells (MSCs) may be a substantial clinical convenience for treatment of autoimmune ailments such as multiple sclerosis. We therefore tested whether allogeneic MSCs can be exploited for treatment of experimental autoimmune encephalomyelitis (EAE) in mice with otherwise intact immune system. Administration of allogeneic Balb/c-derived MSCs to C57Bl/6 mice with pre-established EAE led to a significant decrease in disease score over time comparable to that achieved with syngeneic MSCs, and was correlated with a significant blunting of immune cell infiltration to the spinal cord and reduced circulating levels of interferon-gamma (IFN-gamma) and interleukin-17. Pretreatment of allogeneic MSCs with IFN-gamma increased the expression levels of CCL2 as well as major histocompatibility complex I (MHCI) and MHCII, but also led to complete loss of suppressive activity in vivo that correlated with immune rejection. In conclusion, allogeneic MSCs can suppress the manifestations of EAE, yet retain the potential for alloimmunization.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/therapy , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/physiology , Transplantation, Homologous/methods , Animals , Chemokine CCL2/metabolism , Encephalomyelitis, Autoimmune, Experimental/metabolism , Genes, MHC Class I/physiology , Genes, MHC Class II/physiology , Interferon-gamma/metabolism , Interleukin-17/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL
15.
J Immunol ; 182(10): 5994-6002, 2009 May 15.
Article in English | MEDLINE | ID: mdl-19414750

ABSTRACT

The administration of ex vivo culture-expanded mesenchymal stromal cells (MSCs) has been shown to reverse symptomatic neuroinflammation observed in experimental autoimmune encephalomyelitis (EAE). The mechanism by which this therapeutic effect occurs remains unknown. In an effort to decipher MSC mode of action, we found that MSC conditioned medium inhibits EAE-derived CD4 T cell activation by suppressing STAT3 phosphorylation via MSC-derived CCL2. Further analysis demonstrates that the effect is dependent on MSC-driven matrix metalloproteinase proteolytic processing of CCL2 to an antagonistic derivative. We also show that antagonistic CCL2 suppresses phosphorylation of AKT and leads to a reciprocal increased phosphorylation of ERK associated with an up-regulation of B7.H1 in CD4 T cells derived from EAE mice. CD4 T cell infiltration of the spinal cord of MSC-treated group was robustly decreased along with reduced plasma levels of IL-17 and TNF-alpha levels and in vitro from restimulated splenocytes. The key role of MSC-derived CCL2 was confirmed by the observed loss of function of CCL2(-/-) MSCs in EAE mice. In summary, this is the first report of MSCs modulating EAE biology via the paracrine conversion of CCL2 from agonist to antagonist of CD4 Th17 cell function.


Subject(s)
Chemokine CCL2/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Mesenchymal Stem Cells/immunology , Stromal Cells/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Blotting, Western , Chemokine CCL2/metabolism , Encephalomyelitis, Autoimmune, Experimental/metabolism , Female , Flow Cytometry , Interleukin-17/immunology , Interleukin-17/metabolism , Mesenchymal Stem Cells/metabolism , Mice , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Stromal Cells/metabolism , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Helper-Inducer/metabolism
16.
J Immunol ; 182(5): 2620-7, 2009 Mar 01.
Article in English | MEDLINE | ID: mdl-19234156

ABSTRACT

We describe the generation of a fusion cytokine consisting of GM-CSF in tandem with N-terminal-truncated MCP-1 (6-76), hereafter GMME1. Treatment of activated T cells with recombinant GMME1 protein leads to proinflammatory cytokine reduction and apoptosis via a CCR2-restricted pathway. Similarly, cell death is triggered in macrophages cultured with GMME1, while an inhibition of Ab production from plasma cells is observed. Treatment of CD4 T cells derived from experimental autoimmune encephalomyelitis mice with GMME1 leads to p38 hyperphosphorylation, inhibition of p44/42, AKT and STAT3 phosphorylation, and caspase-3 activation. GMME1 administration to experimental autoimmune encephalomyelitis mice suppresses symptomatic disease and correlates with decreased levels of inflammatory cytokines including IL-17, MOG-specific Ab titers, and blockade of CD4 and CD8 T cell infiltration in spinal cords. We propose that GMME1 defines a new class of agents for the treatment of autoimmune ailments by selectively targeting lymphomyeloid cells expressing CCR2.


Subject(s)
Chemokine CCL2/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Lymphocytes/immunology , Myeloid Cells/immunology , Receptors, CCR2/antagonists & inhibitors , Receptors, CCR2/genetics , Recombinant Fusion Proteins/physiology , Amino Acid Sequence , Animals , Apoptosis/genetics , Apoptosis/immunology , Cells, Cultured , Chemokine CCL2/administration & dosage , Chemokine CCL2/therapeutic use , Coculture Techniques , Encephalomyelitis, Autoimmune, Experimental/pathology , Encephalomyelitis, Autoimmune, Experimental/therapy , Female , Granulocyte-Macrophage Colony-Stimulating Factor/administration & dosage , Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use , HeLa Cells , Humans , Lymphocytes/metabolism , Lymphocytes/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Myeloid Cells/metabolism , Myeloid Cells/pathology , Receptors, CCR2/biosynthesis , Receptors, CCR2/deficiency , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/chemical synthesis
17.
Mol Ther ; 17(2): 369-72, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19088705

ABSTRACT

Autologous bone marrow mesenchymal stromal cells (MSCs) have been successfully used for the delivery of erythropoietin (EPO) in murine models of anemia and myocardial infarction. For clinical applications where a transient effect would be adequate, such as myocardial infarction, the use of EPO-engineered universal donor allogeneic MSCs would be a substantial convenience. We thus investigated whether MSCs from C57BL/6 mice would permit robust transient EPO delivery in normal BALB/c allorecipients. Implantation of MSCs overexpressing murine EPO led to increases in hematocrit in syngeneic and allogeneic mice, but the latter eventually developed severe anemia due to acquired neutralizing anti-EPO antibodies. As MSCs constitutively produce the CCL2 chemokine which may behave as an adjuvant to the anti-EPO immune response, experiments were performed using EPO-engineered MSCs derived from CCL2(-/-) mice and similar results were obtained. In conclusion, MHC-mismatched MSCs can break the tolerance to autoantigens and lead to the development of pathogenic autoantibodies.


Subject(s)
Anemia/etiology , Antibodies/immunology , Erythropoietin/immunology , Erythropoietin/metabolism , Mesenchymal Stem Cell Transplantation/methods , Stromal Cells/cytology , Animals , Cell Line , Enzyme-Linked Immunosorbent Assay , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Stromal Cells/physiology , Transplantation, Homologous
18.
Blood ; 112(13): 4991-8, 2008 Dec 15.
Article in English | MEDLINE | ID: mdl-18812467

ABSTRACT

We demonstrate that the secretome of mesenchymal stromal cells (MSCs) suppresses plasma cell (PC) immunoglobulin (Ig) production, induces plasmablast proliferation, and leads to interleukin-10-mediated blockade in vitro. We found that these effects are the result of MSC-derived CC chemokine ligands CCL2 and CCL7. More specifically, MSCs further processed these CC chemokines by the activity of matrix metalloproteinases (MMPs), leading to the generation of proteolytically processed antagonistic CCL2 variant. Neutralizing CCL2 or inhibiting MMP enzymatic activity abolished the PC-suppressive effect of MSCs. We also observed that MMP-processed CCL2 suppresses signal transducer and activator of transcription 3 (STAT3) activation in PC. As a result, the transcription factor PAX5 is induced, thus explaining the inhibition of Ig synthesis. The absence of inhibitory effects by MSC on the humoral response of CCR2(-/-) mice to xenoantigen suggests that MMP-cleaved CCL2/CCR2 interaction as well as downstream phosphatase activity is necessary for antagonistic effect. We tested syngeneic MSCs in hemophilic B6 mice with predeveloped antihuman factor VIII (hFVIII) antibodies and demonstrated a robust decrease in hFVIII-specific IgG levels. Thus, MSCs may play a role in modulating Ig production by PCs via MMP processing of CCL2 and may represent an appealing cell therapy approach for pathologic humoral responses.


Subject(s)
Chemokine CCL2/immunology , Immunoglobulins/biosynthesis , Mesenchymal Stem Cells/immunology , PAX5 Transcription Factor/genetics , Plasma Cells/metabolism , STAT3 Transcription Factor/metabolism , Stromal Cells/immunology , Animals , Chemokine CCL7/immunology , Matrix Metalloproteinases/metabolism , Mice , Plasma Cells/immunology , Transcriptional Activation/immunology
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