RESUMEN
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.
Asunto(s)
Células Madre Embrionarias/inmunología , Hepatocitos/inmunología , Hepatocitos/trasplante , Células Madre Pluripotentes Inducidas/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Aloinjertos , Animales , Diferenciación Celular , Trasplante de Células/métodos , Citotoxicidad Inmunológica , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Perfilación de la Expresión Génica , Hepatocitos/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Isoinjertos , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Modelos Animales , Subfamilia K de Receptores Similares a Lectina de Células NK/deficiencia , Subfamilia K de Receptores Similares a Lectina de Células NK/genéticaRESUMEN
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.
Asunto(s)
Infecciones por Virus de Epstein-Barr/prevención & control , Supervivencia de Injerto , Linfoma de Células B/prevención & control , Trastornos Linfoproliferativos/prevención & control , Inhibidores de las Quinasa Fosfoinosítidos-3/administración & dosificación , Complicaciones Posoperatorias/prevención & control , Aloinjertos , Animales , Linfocitos B , Femenino , Rechazo de Injerto , Trasplante de Corazón/efectos adversos , Trasplante de Corazón/métodos , Compuestos Heterocíclicos con 3 Anillos/administración & dosificación , Humanos , Concentración 50 Inhibidora , Linfoma de Células B/virología , Trastornos Linfoproliferativos/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Trasplante de Órganos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Purinas/administración & dosificación , Quinazolinonas/administración & dosificación , Sirolimus/administración & dosificación , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Supervivencia de Injerto , Trasplante de Corazón , MicroARNs/metabolismo , Miocardio/metabolismo , Abatacept/farmacología , Aloinjertos , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Quimiotaxis de Leucocito , Proteína Forkhead Box O1/metabolismo , Supervivencia de Injerto/efectos de los fármacos , Memoria Inmunológica , Inmunosupresores/farmacología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Isoantígenos/inmunología , Activación de Linfocitos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Miocardio/inmunología , Miocardio/patología , Factores de TiempoRESUMEN
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.
Asunto(s)
Aterosclerosis/enzimología , Proteínas Musculares/metabolismo , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Remodelación Vascular , Sustitución de Aminoácidos , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Células Cultivadas , Crizotinib , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Glutatión Peroxidasa/biosíntesis , Glutatión Peroxidasa/genética , Humanos , Masculino , Ratones , Ratones Noqueados , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , Músculo Liso Vascular/patología , Mutación Missense , Miocitos del Músculo Liso/patología , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Pirazoles/farmacología , Piridinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Glutatión Peroxidasa GPX1RESUMEN
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.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Movimiento Celular/efectos de los fármacos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Wnt/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/trasplante , Proteína Axina , Becaplermina , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Proliferación Celular/efectos de los fármacos , Proteínas Dishevelled , Activación Enzimática/efectos de los fármacos , Fibronectinas/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Ratones , Modelos Biológicos , Proteínas Mutantes/metabolismo , Miocitos del Músculo Liso/enzimología , Neointima/patología , Fosfoproteínas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-sis , Proteínas Represoras/metabolismo , beta Catenina/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
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.