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1.
Methods Mol Biol ; 2614: 17-36, 2023.
Article in English | MEDLINE | ID: mdl-36587116

ABSTRACT

Flow cytometry is an essential tool for studying the tumor-immune microenvironment. It allows us to quickly quantify and identify multiple cell types in a heterogeneous sample. This chapter provides an overview of the flow cytometry instrumentation and a discussion of the appropriate considerations and steps in building a reproducible flow cytometry staining panel. We present an updated lymphoid tissue and solid tumor-infiltrating leucocyte flow cytometry staining protocol and an example of flow cytometry data analysis.


Subject(s)
Neoplasms , Humans , Flow Cytometry/methods , Tumor Microenvironment , Leukocytes
2.
Leuk Lymphoma ; 62(10): 2352-2359, 2021 10.
Article in English | MEDLINE | ID: mdl-34020575

ABSTRACT

Diagnosis and minimal residual disease (MRD) monitoring of chronic lymphocytic leukemia (CLL) by flow cytometry currently requires multiple antibody panels. We added CD23 and CD200 to the EuroFlowTM lymphoid screening tube (LST) to create a 10-color modified LST (mLST) capable of diagnosing typical CLL in a single tube. We then explored if the mLST could be used for MRD by comparing its performance to the European Research Initiative on CLL (ERIC) panel using spiked cryopreserved and fresh patient samples. Over 1 year of use in our clinical laboratory, the mLST diagnosed CLL without further immunophenotyping in 56% of samples with an abnormal clone. There was good agreement in MRD results between the mLST and ERIC panels. Therefore, the mLST can streamline CLL diagnosis by reducing technician time and the number of panels required. It may have the potential to screen for MRD in laboratories without access to dedicated panels (ERIC).


Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell , Flow Cytometry , Humans , Immunophenotyping , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Multilocus Sequence Typing , Neoplasm, Residual
3.
Cell Rep ; 33(13): 108560, 2020 12 29.
Article in English | MEDLINE | ID: mdl-33378678

ABSTRACT

The role of RNA binding proteins in regulating the phagocytic and cytokine-releasing functions of microglia is unknown. Here, we show that microglia deficient for the QUAKING (QKI) RNA binding protein have increased proinflammatory cytokine release and defects in processing phagocytosed cargo. Splicing analysis reveals a role for QKI in regulating microexon networks of the Rho GTPase pathway. We show an increase in RhoA activation and proinflammatory cytokines in QKI-deficient microglia that are repressed by treating with a Rock kinase inhibitor. During the cuprizone diet, mice with QKI-deficient microglia are inefficient at supporting central nervous system (CNS) remyelination and cause the recruited oligodendrocyte precursor cells to undergo apoptosis. Furthermore, the expression of QKI in microglia is downregulated in preactive, chronic active, and remyelinating white matter lesions of multiple sclerosis (MS) patients. Overall, our findings identify QKI as an alternative splicing regulator governing a network of Rho GTPase microexons with implications for CNS remyelination and MS patients.


Subject(s)
Alternative Splicing , Gene Expression Regulation , Microglia/physiology , RNA-Binding Proteins/physiology , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism , Animals , Cells, Cultured , Central Nervous System/metabolism , Cytokines/metabolism , Female , Homeostasis , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microglia/cytology , Multiple Sclerosis/genetics , Phagocytosis , RNA/metabolism , RNA-Seq , Remyelination , Signal Transduction/drug effects , rho-Associated Kinases/metabolism
4.
Toxicol Sci ; 170(1): 45-56, 2019 07 01.
Article in English | MEDLINE | ID: mdl-30912803

ABSTRACT

Tungsten is an emerging environmental toxicant associated with several pediatric leukemia clusters, although a causal association has not been established. Our previous work demonstrated that tungsten exposure resulted in an accumulation of pre-B cells in the bone marrow, the same cell type that accumulates in pediatric acute lymphoblastic leukemia (ALL). To better understand the relevant molecular mechanisms, we performed RNA-sequencing on flow sorted pre-B cells from control and tungsten-exposed mice. Tungsten decreased the expression of multiple genes critical for B cell development, including members of the interleukin-7 receptor (IL-7R) and pre-B cell receptor signaling pathways, such as Jak1, Stat5a, Pax5, Syk, and Ikzf3. These results were confirmed in an in vitro model of B cell differentiation, where tungsten arrested differentiation at the pro-B cell stage and inhibited proliferation. These changes were associated with decreased expression of multiple genes in the IL-7R signaling pathway and decreased percentage of IL-7R, phosphorylated STAT5 double-positive cells. Supplementation with IL-7 or overexpression of Pax5, the transcription factor downstream of IL-7R, rescued the tungsten-induced differentiation block. Together, these data support the hypothesis that IL-7R/Pax5 signaling axis is critical to tungsten-mediated effects on pre-B cell development. Importantly, many of these molecules are modulated in ALL.


Subject(s)
B-Lymphocytes/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , PAX5 Transcription Factor/metabolism , Receptors, Interleukin-7/metabolism , Tungsten Compounds/toxicity , Animals , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Down-Regulation , Gene Expression/drug effects , Male , Mice, Inbred C57BL , PAX5 Transcription Factor/genetics , Receptors, Interleukin-7/genetics , Signal Transduction/drug effects , Signal Transduction/genetics
5.
Nat Commun ; 8: 14638, 2017 03 09.
Article in English | MEDLINE | ID: mdl-28276425

ABSTRACT

Tyrosine kinase signalling within cancer cells is central to the establishment of an immunosuppressive microenvironment. Although tyrosine kinase inhibitors act, in part, to augment adaptive immunity, the increased heterogeneity and functional redundancy of the tyrosine kinome is a hurdle to achieving durable responses to immunotherapies. We previously identified the Shc1 (ShcA) scaffold, a central regulator of tyrosine kinase signalling, as essential for promoting breast cancer immune suppression. Herein we show that the ShcA pathway simultaneously activates STAT3 immunosuppressive signals and impairs STAT1-driven immune surveillance in breast cancer cells. Impaired Y239/Y240-ShcA phosphorylation selectively reduces STAT3 activation in breast tumours, profoundly sensitizing them to immune checkpoint inhibitors and tumour vaccines. Finally, the ability of diminished tyrosine kinase signalling to initiate STAT1-driven immune surveillance can be overcome by compensatory STAT3 hyperactivation in breast tumours. Our data indicate that inhibition of pY239/240-ShcA-dependent STAT3 signalling may represent an attractive therapeutic strategy to sensitize breast tumours to multiple immunotherapies.


Subject(s)
Breast Neoplasms/immunology , Immunologic Surveillance , Mammary Neoplasms, Experimental/immunology , STAT1 Transcription Factor/immunology , STAT3 Transcription Factor/immunology , Src Homology 2 Domain-Containing, Transforming Protein 1/metabolism , Animals , Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Agents, Immunological/therapeutic use , Breast Neoplasms/genetics , Breast Neoplasms/therapy , Cancer Vaccines/immunology , Cancer Vaccines/therapeutic use , Cell Line, Tumor , Computational Biology , Costimulatory and Inhibitory T-Cell Receptors/antagonists & inhibitors , Costimulatory and Inhibitory T-Cell Receptors/immunology , Datasets as Topic , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/immunology , Humans , Interferon-gamma/immunology , Interferon-gamma/metabolism , Mammary Neoplasms, Experimental/genetics , Mice, Transgenic , Primary Cell Culture , STAT1 Transcription Factor/metabolism , STAT3 Transcription Factor/metabolism , Sequence Analysis, RNA , Signal Transduction/genetics , Signal Transduction/immunology , Src Homology 2 Domain-Containing, Transforming Protein 1/genetics , Src Homology 2 Domain-Containing, Transforming Protein 1/immunology , Treatment Outcome , Xenograft Model Antitumor Assays
6.
Methods Mol Biol ; 1458: 95-110, 2016.
Article in English | MEDLINE | ID: mdl-27581017

ABSTRACT

Flow cytometry is an essential tool for studying the tumor microenvironment. It allows us to quickly quantify and identify multiple cell types in a heterogeneous sample. A brief overview of flow cytometry instrumentation and the appropriate considerations and steps in building a good flow cytometry staining panel are discussed. In addition, a lymphoid tissue and solid tumor leukocyte infiltrate flow cytometry staining protocol and an example of flow cytometry data analysis are presented.


Subject(s)
Flow Cytometry , Neoplasms/metabolism , Neoplasms/pathology , Tumor Microenvironment , Biomarkers , Flow Cytometry/methods , Gene Expression , Genes, Reporter , Humans , Immunophenotyping
7.
Am J Physiol Renal Physiol ; 308(12): F1474-83, 2015 Jun 15.
Article in English | MEDLINE | ID: mdl-25904702

ABSTRACT

The efficacy of cell therapy for many diseases can be limited by the poor survival of implanted cells in an environment of tissue injury. Melatonin has been reported to have antioxidative and antiapoptotic effects. Adipose tissue-derived mesenchymal stromal cells (ASCs), cells easily obtained in high amounts and with minimal discomfort, have shown great promise in cell therapy applications, such as in acute kidney injury. We hypothesized that melatonin pretreatment of human ASCs (hASCs) would improve their renoprotective and prosurvival effects. We therefore investigated the action of melatonin on hASCs, as well as the effect of the resulting hASCs-conditioned media (CM) on human kidney cells exposed to oxidative and apoptotic injury-provoking doses of cisplatin. Our results demonstrated that pretreatment of hASCs with melatonin, 100 µM for 3 h, significantly increased their proliferation and their expression of prosurvival P-Erk1/2 and P-Akt, and of antioxidative enzymes catalase and heme oxygenase (HO)-1. In addition, the CM from hASCs pretreated with melatonin provoked a significantly higher proliferation and migration of HK-2 human kidney epithelial cells. Furthermore, this CM exerted significantly higher prosurvival and antiapoptotic actions on HK-2 cells exposed to cisplatin in vitro. Western blot analysis showed higher expression of P-Erk1/2, Bcl-2, SOD-1, and HO-1 in the HK-2 cells exposed to cisplatin in the presence of CM from melatonin-pretreated hASCs. In sum, our study revealed that in vitro pretreatment of hASCs with melatonin may significantly enhance their survival and their therapeutic effectiveness on injured tissue.


Subject(s)
Adipose Tissue/cytology , Cell Proliferation/drug effects , Kidney/drug effects , Melatonin/pharmacology , Mesenchymal Stem Cells/drug effects , Acute Kidney Injury/drug therapy , Cell Line , Culture Media, Conditioned/metabolism , Heme Oxygenase-1/metabolism , Humans , Melatonin/administration & dosage , Mesenchymal Stem Cells/cytology
8.
Cytotherapy ; 16(2): 191-202, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24438900

ABSTRACT

BACKGROUND AIMS: Mesenchymal 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. METHODS: MSCs 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. RESULTS: Unprimed 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. CONCLUSIONS: Unprimed 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.


Subject(s)
Bone Marrow Cells/cytology , Mesenchymal Stem Cells/immunology , T-Lymphocytes/immunology , Adult , Aged , Antigens, CD/metabolism , Cell Proliferation , Cells, Cultured , Coculture Techniques , Cytokines/metabolism , Female , Humans , Immunosuppression Therapy , Inflammation Mediators/metabolism , Lymphocyte Activation , Male , Middle Aged
9.
Toxicol Sci ; 131(2): 434-46, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23152188

ABSTRACT

High environmental tungsten levels were identified near the site of a childhood pre-B acute lymphoblastic leukemia cluster; however, a causal link between tungsten and leukemogenesis has not been established. The major site of tungsten deposition is bone, the site of B-cell development. In addition, our in vitro data suggest that developing B lymphocytes are susceptible to tungsten-induced DNA damage and growth inhibition. To extend these results, we assessed whether tungsten exposure altered B-cell development and induced DNA damage in vivo. Wild-type mice were exposed to tungsten in their drinking water for up to 16 weeks. Tungsten concentration in bone was analyzed by inductively coupled plasma mass spectrometry and correlated with B-cell development and DNA damage within the bone marrow. Tungsten exposure resulted in a rapid deposition within the bone following 1 week, and tungsten continued to accumulate thereafter albeit at a decreased rate. Flow cytometric analyses revealed a transient increase in mature IgD(+) B cells in the first 8 weeks of treatment, in animals of the highest and intermediate exposure groups. Following 16 weeks of exposure, all tungsten groups had a significantly greater percentage of cells in the late pro-/large pre-B developmental stages. DNA damage was increased in both whole marrow and isolated B cells, most notably at the lowest tungsten concentration tested. These findings confirm an immunological effect of tungsten exposure and suggest that tungsten could act as a tumor promoter, providing leukemic "hits" in multiple forms to developing B lymphocytes within the bone marrow.


Subject(s)
B-Lymphocytes/drug effects , Bone Marrow/drug effects , DNA Damage , Tungsten/toxicity , Animals , B-Lymphocytes/ultrastructure , Blotting, Western , Cell Lineage , Comet Assay , Flow Cytometry , Male , Mice
10.
PLoS One ; 7(8): e42840, 2012.
Article in English | MEDLINE | ID: mdl-22912749

ABSTRACT

Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100-400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNS(Red) transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules.


Subject(s)
Amino Acid Transport Systems, Neutral/metabolism , Cystine/metabolism , Cystinosis/metabolism , Cystinosis/pathology , Exosomes/metabolism , Mesenchymal Stem Cells/cytology , Amino Acid Transport Systems, Neutral/genetics , Animals , Cystinosis/genetics , Cystinosis/surgery , Fibroblasts/metabolism , Humans , Lysosomes/metabolism , Mesenchymal Stem Cell Transplantation , Mice , Mutation , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism
11.
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
12.
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
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.
J Immunol ; 183(3): 1759-66, 2009 Aug 01.
Article in English | MEDLINE | ID: mdl-19592643

ABSTRACT

CCR2 is a chemokine receptor widely expressed by lymphomyeloid cells involved in maladaptive autoimmune ailments. Therefore CCR2 is of great interest as a biological target for immune suppression due to its direct implication in autoimmune diseases such as rheumatoid arthritis. We have generated a novel fusion protein using GM-CSF and an N-terminal truncated version of MCP-1/CCL2 (6-76, GMME1) and investigated its utility as a CCR2-specific immune suppressor. Using BRET studies, we found that distinct to CCL2, GMME1 binding to CCR2 led to altered conformational changes in the CCR2 homodimer and did not induce the recruitment of beta-arrestin 2 to the receptor. However, CCR2-dependent calcium mobilization, BAX induction and caspase-3 activation followed by cell death was observed. Using Th17 cells harvested from DBA/1 mice ill with bovine collagen-induced arthritis, we demonstrate that GMME1 is capable of blocking their production of IL-17 in vitro. Upon its delivery to mice symptomatic with inflammatory arthritis, a robust clinical recovery occurred with decreased paw thickness to normal levels and a significant reduction in anti-collagen Ab titer and rheumatoid factor titer, as well as reduction of proinflammatory cytokines levels both intraarticular and systemic. Our data demonstrate that GMME1 is a powerful synthetic suppressor cytokine that coopts CCR2-dependent cellular signaling and blunts the effects of CCR2-expressing lymphomyeloid cells causative of autoimmune arthritis.


Subject(s)
Arthritis, Experimental/drug therapy , Chemokine CCL2/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Protein Engineering/methods , Receptors, CCR2/metabolism , Recombinant Fusion Proteins/pharmacology , Animals , Arthritis, Experimental/prevention & control , Cells, Cultured , Disease Models, Animal , Inflammation/drug therapy , Inflammation/prevention & control , Interleukin-17 , Mice , Protein Binding , Receptors, CCR2/chemistry , Recombinant Fusion Proteins/therapeutic use , T-Lymphocyte Subsets
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 Exp Med ; 202(5): 575-81, 2005 Sep 05.
Article in English | MEDLINE | ID: mdl-16129703

ABSTRACT

Fas (CD95), a member of the tumor necrosis factor-receptor superfamily, has been studied extensively as a death-inducing receptor in the immune system. However, Fas is also widely expressed in a number of other tissues, including in neurons. Here, we report that defects in the Fas/Fas ligand system unexpectedly render mice highly susceptible to neural degeneration in a model of Parkinson's disease. We found that Fas-deficient lymphoproliferative mice develop a dramatic phenotype resembling clinical Parkinson's disease, characterized by extensive nigrostriatal degeneration accompanied by tremor, hypokinesia, and loss of motor coordination, when treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at a dose that causes no neural degeneration or behavioral impairment in WT mice. Mice with generalized lymphoproliferative disease, which express a mutated Fas ligand, display an intermediate phenotype between that of lymphoproliferative and WT mice. Moreover, Fas engagement directly protects neuronal cells from MPTP/1-methyl-4-phenylpyridinium ion toxicity in vitro. Our data show that decreased Fas expression renders dopaminergic neurons highly susceptible to degeneration in response to a Parkinson-causing neurotoxin. These findings constitute the first evidence for a neuroprotective role for Fas in vivo.


Subject(s)
Lymphoproliferative Disorders/metabolism , MPTP Poisoning/metabolism , Neurons/metabolism , Parkinson Disease/metabolism , Signal Transduction/physiology , Up-Regulation , fas Receptor/metabolism , Aged , Animals , Brain/pathology , Chromatography, High Pressure Liquid , Female , Flow Cytometry , Humans , Immunohistochemistry , Male , Mice , Mice, Mutant Strains , Middle Aged
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