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1.
Am J Transplant ; 19(6): 1652-1662, 2019 06.
Article in English | MEDLINE | ID: mdl-30549427

ABSTRACT

Stem cell-based approaches have the potential to address the organ shortage in transplantation. Whereas both embryonic stem cells and induced pluripotent stem cells have been utilized as cellular sources for differentiation and lineage specification, their relative ability to be recognized by immune effector cells is unclear. We determined the expression of immune recognition molecules on hepatocyte-like cells (HLC) generated from murine embryonic stem cells and induced pluripotent stem cells, compared to adult hepatocytes, and we evaluated the impact on recognition by natural killer (NK) cells. We report that HLC lack MHC class I expression, and that embryonic stem cell-derived HLC have higher expression of the NK cell activating ligands Rae1, H60, and Mult1 than induced pluripotent stem cell-derived HLC and adult hepatocytes. Moreover, the lack of MHC class I renders embryonic stem cell-derived HLC, and induced pluripotent stem cell-derived HLC, susceptible to killing by syngeneic and allogeneic NK cells. Both embryonic stem cell-derived HLC, and induced pluripotent stem cell-derived HLC, are killed by NK cells at higher levels than adult hepatocytes. Finally, we demonstrate that the NK cell activation receptor, NKG2D, plays a key role in NK cell cytotoxicity of embryonic stem cell-derived HLC, but not induced pluripotent stem cell-derived HLC.


Subject(s)
Embryonic Stem Cells/immunology , Hepatocytes/immunology , Hepatocytes/transplantation , Induced Pluripotent Stem Cells/immunology , NK Cell Lectin-Like Receptor Subfamily K/immunology , Allografts , Animals , Cell Differentiation , Cell Transplantation/methods , Cytotoxicity, Immunologic , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Female , Gene Expression Profiling , Hepatocytes/cytology , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Isografts , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Ligands , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Mice, Transgenic , Models, Animal , NK Cell Lectin-Like Receptor Subfamily K/deficiency , NK Cell Lectin-Like Receptor Subfamily K/genetics
2.
Am J Transplant ; 19(5): 1305-1314, 2019 05.
Article in English | MEDLINE | ID: mdl-30549430

ABSTRACT

Posttransplant lymphoproliferative disorder (PTLD) is a serious complication of organ transplantation that often manifests as Epstein-Barr virus (EBV)-associated B cell lymphomas. Current treatments for PTLD have limited efficacy and can be associated with graft rejection or systemic toxicities. The mTOR inhibitor, rapamycin, suppresses tumor growth of EBV+ B cell lymphoma cells in vitro and in vivo; however, the efficacy is limited and clinical benefits of mTOR inhibitors for PTLD are variable. Here, we show constitutive activation of multiple nodes within the PI3K/Akt/mTOR pathway in EBV+ PTLD-derived cell lines. Inhibition of either PI3K or Akt, with specific inhibitors CAL-101 and MK-2206, respectively, diminished growth of EBV+ B cell lines from PTLD patients in a dose-dependent manner. Importantly, rapamycin combined with CAL-101 or MK-2206 had a synergistic effect in suppressing cell growth as determined by IC50 isobolographic analysis and Loewe indices. Moreover, these combinations were significantly more effective than rapamycin alone in inhibiting tumor xenograft growth in NOD-SCID mice. Finally, both CAL-101 and MK-2206 also prolonged survival of heterotopic cardiac allografts in C57BL/6 mice. Thus, combination therapy with rapamycin and a PI3K inhibitor, or an Akt inhibitor, can be an efficacious treatment for EBV-associated PTLD, while simultaneously promoting allograft survival.


Subject(s)
Epstein-Barr Virus Infections/prevention & control , Graft Survival , Lymphoma, B-Cell/prevention & control , Lymphoproliferative Disorders/prevention & control , Phosphoinositide-3 Kinase Inhibitors/administration & dosage , Postoperative Complications/prevention & control , Allografts , Animals , B-Lymphocytes , Female , Graft Rejection , Heart Transplantation/adverse effects , Heart Transplantation/methods , Heterocyclic Compounds, 3-Ring/administration & dosage , Humans , Inhibitory Concentration 50 , Lymphoma, B-Cell/virology , Lymphoproliferative Disorders/virology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Neoplasm Transplantation , Organ Transplantation , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Purines/administration & dosage , Quinazolinones/administration & dosage , Sirolimus/administration & dosage , TOR Serine-Threonine Kinases/antagonists & inhibitors
3.
Transplantation ; 101(3): 524-530, 2017 03.
Article in English | MEDLINE | ID: mdl-27893611

ABSTRACT

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate the posttranscriptional expression of target genes and are important regulators in immune responses. Previous studies demonstrated that the miRNA, miR-182 was significantly increased during allograft rejection. Further, the transcription factor Forkhead box (FOX) protein 1, (FOXO1) was shown to be a target of miR-182. The aim of this study is to further examine the role of miR-182 in alloimmune responses. METHODS: Transplantation of BALB/c cardiac allografts was performed in C57BL/6, miR-182, B6.129S-H2 (MHC II and CD4 T cell-deficient) and B6.129S2-Tap1 (MHC I and CD8 T cell-deficient) mice, with or without CTLA-4Ig administration. T cell phenotype, FOXO1 protein levels and graft infiltrating lymphocytes were determined in C57BL/6 or miR-182 mice by flow cytometric analysis, Western blot, and immunohistochemistry, respectively. RESULTS: We now show that T cells, mainly CD4 are the main cellular source of miR-182 during allograft rejection. In the absence of miR-182, CTLA-4Ig treatment significantly increased allograft survival (31.5 days C57BL/6 vs 60 days miR-182; P < 0.01). Further, CTLA4-Ig treatment inhibits miR-182 expression, increases FOXO1 levels, and reduces the percentage of CD4CD44 T cells after transplantation. Fewer T cells infiltrate the cardiac allografts, and memory T cells are significantly decreased in allograft recipients deficient in miR-182 with CTLA4-Ig treatment (P < 0.01). CONCLUSIONS: Our findings suggest that miR-182 contributes to the T-cell responses to alloantigen especially under costimulation blockade. Therapeutics that target specific miRNAs may prove beneficial in transplantation.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Graft Survival , Heart Transplantation , MicroRNAs/metabolism , Myocardium/metabolism , Abatacept/pharmacology , Allografts , Animals , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , Cells, Cultured , Chemotaxis, Leukocyte , Forkhead Box Protein O1/metabolism , Graft Survival/drug effects , Immunologic Memory , Immunosuppressive Agents/pharmacology , Interferon-gamma/immunology , Interferon-gamma/metabolism , Isoantigens/immunology , Lymphocyte Activation , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/genetics , Myocardium/immunology , Myocardium/pathology , Time Factors
4.
J Clin Invest ; 124(12): 5159-74, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25401476

ABSTRACT

Angioplasty and stenting is the primary treatment for flow-limiting atherosclerosis; however, this strategy is limited by pathological vascular remodeling. Using a systems approach, we identified a role for the network hub gene glutathione peroxidase-1 (GPX1) in pathological remodeling following human blood vessel stenting. Constitutive deletion of Gpx1 in atherosclerotic mice recapitulated this phenotype of increased vascular smooth muscle cell (VSMC) proliferation and plaque formation. In an independent patient cohort, gene variant pair analysis identified an interaction of GPX1 with the orphan protooncogene receptor tyrosine kinase ROS1. A meta-analysis of the only genome-wide association studies of human neointima-induced in-stent stenosis confirmed the association of the ROS1 variant with pathological remodeling. Decreased GPX1 expression in atherosclerotic mice led to reductive stress via a time-dependent increase in glutathione, corresponding to phosphorylation of the ROS1 kinase activation site Y2274. Loss of GPX1 function was associated with both oxidative and reductive stress, the latter driving ROS1 activity via s-glutathiolation of critical residues of the ROS1 tyrosine phosphatase SHP-2. ROS1 inhibition with crizotinib and deglutathiolation of SHP-2 abolished GPX1-mediated increases in VSMC proliferation while leaving endothelialization intact. Our results indicate that GPX1-dependent alterations in oxido-reductive stress promote ROS1 activation and mediate vascular remodeling.


Subject(s)
Atherosclerosis/enzymology , Muscle Proteins/metabolism , Muscle, Smooth, Vascular/enzymology , Myocytes, Smooth Muscle/enzymology , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Vascular Remodeling , Amino Acid Substitution , Animals , Atherosclerosis/genetics , Atherosclerosis/pathology , Cells, Cultured , Crizotinib , Female , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/genetics , Glutathione Peroxidase/biosynthesis , Glutathione Peroxidase/genetics , Humans , Male , Mice , Mice, Knockout , Muscle Proteins/antagonists & inhibitors , Muscle Proteins/genetics , Muscle, Smooth, Vascular/pathology , Mutation, Missense , Myocytes, Smooth Muscle/pathology , Oxidation-Reduction , Oxidative Stress/drug effects , Oxidative Stress/genetics , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Pyrazoles/pharmacology , Pyridines/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/genetics , Glutathione Peroxidase GPX1
5.
J Cell Biol ; 192(1): 171-88, 2011 Jan 10.
Article in English | MEDLINE | ID: mdl-21220513

ABSTRACT

We present a novel cell-signaling paradigm in which bone morphogenetic protein 2 (BMP-2) consecutively and interdependently activates the wingless (Wnt)-ß-catenin (ßC) and Wnt-planar cell polarity (PCP) signaling pathways to facilitate vascular smooth muscle motility while simultaneously suppressing growth. We show that BMP-2, in a phospho-Akt-dependent manner, induces ßC transcriptional activity to produce fibronectin, which then activates integrin-linked kinase 1 (ILK-1) via α4-integrins. ILK-1 then induces the Wnt-PCP pathway by binding a proline-rich motif in disheveled (Dvl) and consequently activating RhoA-Rac1-mediated motility. Transfection of a Dvl mutant that binds ßC without activating RhoA-Rac1 not only prevents BMP-2-mediated vascular smooth muscle cell motility but promotes proliferation in association with persistent ßC activity. Interfering with the Dvl-dependent Wnt-PCP activation in a murine stented aortic graft injury model promotes extensive neointima formation, as shown by optical coherence tomography and histopathology. We speculate that, in response to injury, factors that subvert BMP-2-mediated tandem activation of Wnt-ßC and Wnt-PCP pathways contribute to obliterative vascular disease in both the systemic and pulmonary circulations.


Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Cell Movement/drug effects , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Signal Transduction/drug effects , Wnt Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Aorta/drug effects , Aorta/transplantation , Axin Protein , Becaplermin , Bone Morphogenetic Protein Receptors, Type II/metabolism , Cell Proliferation/drug effects , Dishevelled Proteins , Enzyme Activation/drug effects , Fibronectins/metabolism , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Mice , Models, Biological , Mutant Proteins/metabolism , Myocytes, Smooth Muscle/enzymology , Neointima/pathology , Phosphoproteins/metabolism , Platelet-Derived Growth Factor/pharmacology , Protein Binding/drug effects , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-sis , Repressor Proteins/metabolism , beta Catenin/metabolism , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolism
6.
J Immunol ; 176(1): 128-37, 2006 Jan 01.
Article in English | MEDLINE | ID: mdl-16365403

ABSTRACT

STATs play key roles in immune function. We examined the role of STAT5a/b in allograft rejection. STAT5a/b-deficient mice showed a 4-fold increased survival time of heart allografts (p < 0.01). Unlike wild type, purified STAT5a/b-/- T cells transferred to Rag1-/- recipients failed to mediate heart allograft rejection until supplemented with STAT5a/b-/- B cells. In vitro, STAT5a/b-/- T cells did not proliferate in response to Con A or alloantigens but entered apoptosis within 48 h (95%). Activated STAT5a/b-/- T cells showed increased expression of proapoptotic (caspases, DNA repair genes, TNF/TNFR-associated factor family genes) and decreased antiapoptotic mRNAs in microarrays, while Western blots confirmed reduced antiapoptotic Bcl-2 and elevated proapoptotic Bax protein expression. Interestingly, at 24 h postactivation, STAT5a/b+/+ and STAT5a/b-/- T cells produced similar levels of IL-2, IL-4, IL-10, and IFN-gamma mRNA; ELISPOT assay showed an equivalent number of IL-4- and IFN-gamma-producing T cells in both STAT5a/b+/+ and STAT5a/b-/- splenic populations. Sera from STAT5a/b+/+ and STAT5a/b-/- rejectors had donor-specific IgM, IgG1, IgG2a, and IgG2b Ab, while STAT5a/b deficiency had no impact on B cell survival or proliferation in response to LPS. Compared with allografts from STAT5a/b+/+ recipients, heart allografts from STAT5a/b-/- recipients had markedly reduced infiltration by CD4 and CD8 T cells but increased infiltration by B cells and dense endothelial deposition of C4d, a marker of humoral rejection. Thus, activated STAT5a/b-/- T cells produce cytokines prior to entering apoptosis, thereby promoting differentiation of B cells yielding donor-specific IgM and IgG Ab that mediate allograft rejection.


Subject(s)
Apoptosis/immunology , B-Lymphocytes/immunology , Graft Rejection/immunology , Heart Transplantation/immunology , STAT5 Transcription Factor/immunology , T-Lymphocytes/immunology , Adoptive Transfer , Animals , Blotting, Western , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , In Situ Nick-End Labeling , Mice , Mice, Knockout , Oligonucleotide Array Sequence Analysis , STAT5 Transcription Factor/deficiency , Transplantation, Homologous/immunology
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