RESUMEN
Intestinal epithelial cell (IEC) damage by T cells contributes to graft-versus-host disease, inflammatory bowel disease and immune checkpoint blockade-mediated colitis. But little is known about the target cell-intrinsic features that affect disease severity. Here we identified disruption of oxidative phosphorylation and an increase in succinate levels in the IECs from several distinct in vivo models of T cell-mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses, identified disruption of IEC-intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations. The relevance of IEC-intrinsic SDHA in mediating disease severity was confirmed by complementary chemical and genetic experimental approaches and validated in human clinical samples. These data identify a critical role for the alteration of the IEC-specific mitochondrial complex II component SDHA in the regulation of the severity of T cell-mediated intestinal diseases.
Asunto(s)
Colitis/enzimología , Colon/enzimología , Citotoxicidad Inmunológica , Complejo II de Transporte de Electrones/metabolismo , Células Epiteliales/enzimología , Enfermedad Injerto contra Huésped/enzimología , Mucosa Intestinal/enzimología , Mitocondrias/enzimología , Linfocitos T/inmunología , Animales , Estudios de Casos y Controles , Comunicación Celular , Células Cultivadas , Colitis/genética , Colitis/inmunología , Colitis/patología , Colon/inmunología , Colon/ultraestructura , Modelos Animales de Enfermedad , Complejo II de Transporte de Electrones/genética , Células Epiteliales/inmunología , Células Epiteliales/ultraestructura , Femenino , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Humanos , Inmunidad Mucosa , Mucosa Intestinal/inmunología , Mucosa Intestinal/ultraestructura , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/inmunología , Mitocondrias/ultraestructura , Fosforilación Oxidativa , Ácido Succínico/metabolismo , Linfocitos T/metabolismoRESUMEN
Whereas phosphorylation of serine, threonine, and tyrosine is exceedingly well characterized, the role of histidine phosphorylation in mammalian signaling is largely unexplored. Here we show that phosphoglycerate mutase family 5 (PGAM5) functions as a phosphohistidine phosphatase that specifically associates with and dephosphorylates the catalytic histidine on nucleoside diphosphate kinase B (NDPK-B). By dephosphorylating NDPK-B, PGAM5 negatively regulates CD4(+) T cells by inhibiting NDPK-B-mediated histidine phosphorylation and activation of the K(+) channel KCa3.1, which is required for TCR-stimulated Ca(2+) influx and cytokine production. Using recently developed monoclonal antibodies that specifically recognize phosphorylation of nitrogens at the N1 (1-pHis) or N3 (3-pHis) positions of the imidazole ring, we detect for the first time phosphoisoform-specific regulation of histidine-phosphorylated proteins in vivo, and we link these modifications to TCR signaling. These results represent an important step forward in studying the role of histidine phosphorylation in mammalian biology and disease.
Asunto(s)
Linfocitos T CD4-Positivos/enzimología , Activación de Linfocitos , Proteínas Mitocondriales/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Señalización del Calcio , Citocinas/metabolismo , Predisposición Genética a la Enfermedad , Enfermedad Injerto contra Huésped/enzimología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Células HEK293 , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Histidina , Humanos , Mediadores de Inflamación/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Células Jurkat , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/genética , Nucleósido-Difosfato Quinasa/metabolismo , Fenotipo , Fosfoproteínas Fosfatasas/deficiencia , Fosfoproteínas Fosfatasas/genética , Fosforilación , Interferencia de ARN , Receptores de Antígenos de Linfocitos T/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Chronic graft-versus-host disease (cGVHD) is an immune-mediated disorder characterized by chronic inflammation and fibrosis. Rho-associated coiled-coil-containing protein kinases (ROCKs) are key coordinators of tissue response to injury, regulating multiple functions, such as gene expression and cell migration, proliferation and survival. Relevant to cGVHD and autoimmunity, only the ROCK2 isoform drives a pro-inflammatory type 17 helper T (Th17) cell response. Moreover, ROCK2 inhibition shifts the Th17/regulatory T (Treg) cell balance toward Treg cells and restores immune homeostasis in animal models of autoimmunity and cGVHD. Furthermore, the selective inhibition of ROCK2 by belumosudil reduces fibrosis by downregulating both transforming growth factor-ß signaling and profibrotic gene expression, thereby impeding the creation of focal adhesions. Consistent with its anti-inflammatory and antifibrotic activities, belumosudil has demonstrated efficacy in clinical studies, resulting in an overall response rate of >70% in patients with cGVHD who failed 2 to 5 prior lines of systemic therapy. In summary, selective ROCK2 inhibition has emerged as a promising novel therapeutic approach for treating cGVHD and other immunologic diseases with unique mechanisms of action, targeting both immune imbalance and detrimental fibrotic responses.
Asunto(s)
Enfermedad Injerto contra Huésped/enzimología , Quinasas Asociadas a rho/inmunología , Acetamidas/farmacología , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Fibrosis , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/terapia , Humanos , Inmunomodulación , Modelos Inmunológicos , Inhibidores de Proteínas Quinasas/farmacología , Linfocitos T Reguladores/inmunología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Inflammasomes are multiprotein complexes that coordinate cellular inflammatory responses and mediate host defense. Following recognition of pathogens and danger signals, inflammasomes assemble and recruit and activate caspase-1, the cysteine protease that cleaves numerous downstream targets, including pro-IL-1ß and pro-IL-18 into their biologically active form. In this study, we sought to develop a biosensor that would allow us to monitor the initiation, progression, and resolution of inflammation in living animals. To this end, we inserted a known caspase-1 target sequence into a circularly permuted luciferase construct that becomes bioluminescent upon protease cleavage. This biosensor was activated in response to various inflammatory stimuli in human monocytic cell lines and murine bone marrow-derived macrophages. Next, we generated C57BL/6 transgenic mice constitutively expressing the caspase-1 biosensor. We were able to monitor the spatiotemporal dynamics of caspase-1 activation and onset of inflammation in individual animals in the context of a systemic bacterial infection, colitis, and acute graft-versus-host disease. These data established a model whereby the development and progression of inflammatory responses can be monitored in the context of these and other mouse models of disease.
Asunto(s)
Técnicas Biosensibles/métodos , Caspasa 1/análisis , Inflamación/etiología , Animales , Apoptosis , Colitis/enzimología , Progresión de la Enfermedad , Enfermedad Injerto contra Huésped/enzimología , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Infecciones Estafilocócicas/enzimología , Células THP-1RESUMEN
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the main therapeutic strategy for patients with both malignant and nonmalignant disorders. The therapeutic benefits of allo-HSCT in malignant disorders are primarily derived from the graft-versus-leukemia (GvL) effect, in which T cells in the donor graft recognize and eradicate residual malignant cells. However, the same donor T cells can also recognize normal host tissues as foreign, leading to the development of graft-versus-host disease (GvHD), which is difficult to separate from GvL and is the most frequent and serious complication following allo-HSCT. Inhibition of donor T cell toxicity helps in reducing GvHD but also restricts GvL activity. Therefore, developing a novel therapeutic strategy that selectively suppresses GvHD without affecting GvL is essential. Recent studies have shown that inhibition of histone deacetylases (HDACs) not only inhibits the growth of tumor cells but also regulates the cytotoxic activity of T cells. Here, we compile the known therapeutic potential of HDAC inhibitors in preventing several stages of GvHD pathogenesis. Furthermore, we will also review the current clinical features of HDAC inhibitors in preventing and treating GvHD as well as maintaining GvL.
Asunto(s)
Enfermedad Injerto contra Huésped/tratamiento farmacológico , Efecto Injerto vs Leucemia/efectos de los fármacos , Inhibidores de Histona Desacetilasas/uso terapéutico , Neoplasias/terapia , Ensayos Clínicos como Asunto , Enfermedad Injerto contra Huésped/enzimología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Humanos , Neoplasias/enzimología , Neoplasias/metabolismo , Linfocitos T Citotóxicos/efectos de los fármacos , Trasplante HomólogoRESUMEN
Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality following allotransplant. Activated donor effector T cells can differentiate into pathogenic T helper (Th)-17 cells and germinal center (GC)-promoting T follicular helper (Tfh) cells, resulting in cGVHD. Phosphoinositide-3-kinase-δ (PI3Kδ), a lipid kinase, is critical for activated T cell survival, proliferation, differentiation, and metabolism. We demonstrate PI3Kδ activity in donor T cells that become Tfh cells is required for cGVHD in a nonsclerodermatous multiorgan system disease model that includes bronchiolitis obliterans (BO), dependent upon GC B cells, Tfhs, and counterbalanced by T follicular regulatory cells, each requiring PI3Kδ signaling for function and survival. Although B cells rely on PI3Kδ pathway signaling and GC formation is disrupted resulting in a substantial decrease in Ig production, PI3Kδ kinase-dead mutant donor bone marrow-derived GC B cells still supported BO cGVHD generation. A PI3Kδ-specific inhibitor, compound GS-649443, that has superior potency to idelalisib while maintaining selectivity, reduced cGVHD in mice with active disease. In a Th1-dependent and Th17-associated scleroderma model, GS-649443 effectively treated mice with active cGVHD. These data provide a foundation for clinical trials of US Food and Drug Administration (FDA)-approved PI3Kδ inhibitors for cGVHD therapy in patients.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/enzimología , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Animales , Linfocitos B/inmunología , Trasplante de Médula Ósea/efectos adversos , Bronquiolitis Obliterante/tratamiento farmacológico , Bronquiolitis Obliterante/enzimología , Bronquiolitis Obliterante/etiología , Enfermedad Crónica , Fosfatidilinositol 3-Quinasa Clase I/deficiencia , Fosfatidilinositol 3-Quinasa Clase I/genética , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Esclerodermia Localizada/tratamiento farmacológico , Esclerodermia Localizada/enzimología , Esclerodermia Localizada/etiología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Bone marrow transplantation (BMT) of B10.D2 mice into sublethally irradiated BALB/c mice across minor histocompatibility loci is a well-established animal model for human sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) and systemic sclerosis (SSc). The p38 mitogen-activated protein kinase (MAPK) pathway is a key regulator of inflammation and cytokine production. Furthermore, the activation of p38 MAPK plays an important role in collagen production in SSc. We investigated the effects of p38 MAPK inhibitor, VX-702, on Scl-cGVHD mice. VX-702 was orally administered to Scl-cGVHD mice from day 7 to 35 after BMT. We compared skin fibrosis of Scl-cGVHD mice between the VX-702-treated group and control group. Allogeneic BMT increased the phosphorylation of p38 MAPK in the skin. The administration of VX-702 attenuated the skin fibrosis of Scl-cGVHD compared to the control mice. Immunohistochemical staining showed that VX-702 suppressed the infiltration of CD4+ T cells, CD8+ T cells, and CD11b+ cells into the dermis of Scl-cGVHD mice compared to the control mice. VX-702 attenuated the mRNA expression of extracellular matrix and fibrogenic cytokines, such as IL-6 and IL-13, in the skin of Scl-cGVHD mice. In addition, VX-702 directly inhibited collagen production from fibroblasts in vitro. VX-702 was shown to be a promising candidate for use in treating patients with Scl-cGVHD and SSc.
Asunto(s)
Enfermedad Injerto contra Huésped/enzimología , Enfermedad Injerto contra Huésped/prevención & control , Esclerodermia Localizada/enzimología , Esclerodermia Localizada/prevención & control , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Trasplante de Médula Ósea , Enfermedad Crónica , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/patología , Inflamación/patología , Masculino , Ratones Endogámicos BALB C , Compuestos de Fenilurea/administración & dosificación , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Esclerodermia Localizada/tratamiento farmacológico , Esclerodermia Localizada/patología , Piel/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Chronic graft-versus-host disease (cGVHD) remains a major complication following allogeneic bone marrow transplantation (BMT). The discovery of novel therapeutics is dependent on assessment in preclinical murine models of cGVHD. Rho-associated kinase 2 (ROCK2) recently was shown to be implicated in regulation of interleukin-21 (IL-21) and IL-17 secretion in mice and humans. Here, we report that the selective ROCK2 inhibitor KD025 effectively ameliorates cGVHD in multiple models: a full major histocompatibility complex (MHC) mismatch model of multiorgan system cGVHD with bronchiolitis obliterans syndrome and a minor MHC mismatch model of sclerodermatous GVHD. Treatment with KD025 resulted in normalization of pathogenic pulmonary function, which correlates with a marked reduction of antibody and collagen deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. Spleens of mice treated with KD025 had decreased frequency of T follicular helper cells and increased frequency of T follicular regulatory cells, accompanied by a reduction in signal transducer and activator of transcription 3 (STAT3) and concurrent increase in STAT5 phosphorylation. The critical role of STAT3 in this cGVHD model was confirmed by data showing that mice transplanted with inducible STAT3-deficient T cells had pulmonary function comparable to the healthy negative controls. The therapeutic potential of targeted ROCK2 inhibition in the clinic was solidified further by human data demonstrating the KD025 inhibits the secretion of IL-21, IL-17, and interferon γ along with decreasing phosphorylated STAT3 and reduced protein expression of interferon regulatory factor 4 and B-cell lymphoma 6 (BCL6) in human peripheral blood mononuclear cells purified from active cGVHD patients. Together these data highlight the potential of targeted ROCK2 inhibition for clinical cGVHD therapy.
Asunto(s)
Enfermedad Injerto contra Huésped/enzimología , Inhibidores de Proteínas Quinasas/uso terapéutico , Factor de Transcripción STAT3/fisiología , Quinasas Asociadas a rho/antagonistas & inhibidores , Animales , Bronquiolitis Obliterante/tratamiento farmacológico , Bronquiolitis Obliterante/etiología , Bronquiolitis Obliterante/fisiopatología , Enfermedad Crónica , Citocinas/biosíntesis , Citocinas/genética , Evaluación Preclínica de Medicamentos , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Humanos , Leucocitos Mononucleares/metabolismo , Pulmón/fisiopatología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Terapia Molecular Dirigida , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/biosíntesis , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-6/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-6/genética , Factor de Transcripción STAT3/deficiencia , Organismos Libres de Patógenos Específicos , Bazo/patología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/patología , Subgrupos de Linfocitos T/trasplante , Quinasas Asociadas a rho/fisiologíaRESUMEN
In this issue of Blood, Flynn et al1 provide key data that lend further support to the development of clinical trials of spleen tyrosine kinase (Syk) inhibition for more effective chronic graft-versus-host disease (cGVHD) treatment.
Asunto(s)
Linfocitos B/enzimología , Enfermedad Injerto contra Huésped/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Activación de Linfocitos/inmunología , Proteínas Tirosina Quinasas/metabolismo , Animales , Femenino , HumanosRESUMEN
Novel therapies for chronic graft-versus-host disease (cGVHD) are needed. Aberrant B-cell activation has been demonstrated in mice and humans with cGVHD. Having previously found that human cGVHD B cells are activated and primed for survival, we sought to further evaluate the role of the spleen tyrosine kinase (Syk) in cGVHD in multiple murine models and human peripheral blood cells. In a murine model of multiorgan system, nonsclerodermatous disease with bronchiolitis obliterans where cGVHD is dependent on antibody and germinal center (GC) B cells, we found that activation of Syk was necessary in donor B cells, but not T cells, for disease progression. Bone marrow-specific Syk deletion in vivo was effective in treating established cGVHD, as was a small-molecule inhibitor of Syk, fostamatinib, which normalized GC formation and decreased activated CD80/86(+) dendritic cells. In multiple distinct models of sclerodermatous cGVHD, clinical and pathological disease manifestations were not eliminated when mice were therapeutically treated with fostamatinib, though both clinical and immunologic effects could be observed in one of these scleroderma models. We further demonstrated that Syk inhibition was effective at inducing apoptosis of human cGVHD B cells. Together, these data demonstrate a therapeutic potential of targeting B-cell Syk signaling in cGVHD.
Asunto(s)
Linfocitos B/enzimología , Enfermedad Injerto contra Huésped/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Activación de Linfocitos/inmunología , Proteínas Tirosina Quinasas/metabolismo , Aminopiridinas , Animales , Linfocitos B/inmunología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Enfermedad Injerto contra Huésped/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Morfolinas , Oxazinas/farmacología , Piridinas/farmacología , Pirimidinas , Quinasa SykRESUMEN
The human gut microbiome is involved in vital biological functions, such as maintenance of immune homeostasis and modulation of intestinal development and enhanced metabolic capabilities. Disturbances of the intestinal microbiota have been associated with development and progression of inflammatory conditions, including graft-versus-host disease (GVHD). The fucosyltransferase 2 (FUT2) gene produces an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. FUT2 genotype has been shown to modify the gut microbiome. We hypothesized that FUT2 genotype influences risk of GVHD and bacterial translocation after allogeneic hematopoietic stem cell transplantation (HSCT). FUT2 genotype was determined in 150 consecutive patients receiving allogeneic HSCT at our center. We abstracted clinical characteristics and outcomes from the transplantation database. Cumulative risk of any acute GVHD varied by FUT2 genotype, with decreased risk in those with A/A genotype and increased risk in those with G/G genotype. In contrast, the cumulative incidence of bacteremia was increased in those with A/A genotype. We conclude that the FUT2 genotype influences risk of acute GVHD and bacteremia after HSCT. We hypothesize that the mechanisms involve altered intestinal surface glycosylation and microbial composition but this requires additional study.
Asunto(s)
Bacteriemia/genética , Fucosiltransferasas/genética , Microbioma Gastrointestinal , Genotipo , Enfermedad Injerto contra Huésped/genética , Trasplante de Células Madre Hematopoyéticas , Adolescente , Adulto , Aloinjertos , Bacteriemia/enzimología , Bacteriemia/etiología , Traslocación Bacteriana/genética , Niño , Preescolar , Bases de Datos Factuales , Femenino , Enfermedad Injerto contra Huésped/enzimología , Enfermedad Injerto contra Huésped/etiología , Humanos , Lactante , Masculino , Factores de Riesgo , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
Allogeneic hematopoietic cell transplantation (HCT) is the most effective therapy for hematopoietic malignancies through T-cell-mediated graft-vs-leukemia (GVL) effects but often leads to severe graft-vs-host disease (GVHD). Given that protein kinase Cθ (PKCθ), in cooperation with PKCα, is essential for T-cell signaling and function, we have evaluated PKCθ and PKCα as potential therapeutic targets in allogeneic HCT using genetic and pharmacologic approaches. We found that the ability of PKCα(-/-)/θ(-/-) donor T cells to induce GVHD was further reduced compared with PKCθ(-/-) T cells in relation with the relevance of both isoforms to allogeneic donor T-cell proliferation, cytokine production, and migration to GVHD target organs. Treatment with a specific inhibitor for both PKCθ and PKCα impaired donor T-cell proliferation, migration, and chemokine/cytokine production and significantly decreased GVHD in myeloablative preclinical murine models of allogeneic HCT. Moreover, pharmacologic inhibition of PKCθ and PKCα spared T-cell cytotoxic function and GVL effects. Our findings indicate that PKCα and θ contribute to T-cell activation with overlapping functions essential for GVHD induction while less critical to the GVL effect. Thus, targeting PKCα and PKCθ signaling with pharmacologic inhibitors presents a therapeutic option for GVHD prevention while largely preserving the GVL activity in patients receiving HCT.
Asunto(s)
Enfermedad Injerto contra Huésped/prevención & control , Efecto Injerto vs Leucemia/efectos de los fármacos , Isoenzimas/antagonistas & inhibidores , Proteína Quinasa C-alfa/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Animales , Separación Celular , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Enfermedad Injerto contra Huésped/enzimología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Leucemia/terapia , Activación de Linfocitos/efectos de los fármacos , Linfoma/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa C-theta , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Posttranscriptional modification of histones by methylation plays an important role in regulating Ag-driven T-cell responses. We have recently drawn correlations between allogeneic T-cell responses and the histone methyltransferase Ezh2, which catalyzes histone H3 lysine 27 trimethylation. The functional relevance of Ezh2 in T-cell alloimmunity remains unclear. Here, we identify a central role of Ezh2 in regulating allogeneic T-cell proliferation, differentiation, and function. Conditional loss of Ezh2 in donor T cells inhibited graft-versus-host disease (GVHD) in mice after allogeneic bone marrow (BM) transplantation. Although Ezh2-deficient T cells were initially activated to proliferate upon alloantigenic priming, their ability to undergo continual proliferation and expansion was defective during late stages of GVHD induction. This effect of Ezh2 ablation was largely independent of the proapoptotic molecule Bim. Unexpectedly, as a gene silencer, Ezh2 was required to promote the expression of transcription factors Tbx21 and Stat4. Loss of Ezh2 in T cells specifically impaired their differentiation into interferon (IFN)-γ-producing effector cells. However, Ezh2 ablation retained antileukemia activity in alloreactive T cells, leading to improved overall survival of the recipients. Our findings justify investigation of modulating Ezh2 as a therapeutic strategy for the treatment of GVHD and other T cell-mediated inflammatory disorders.
Asunto(s)
Epigénesis Genética , Enfermedad Injerto contra Huésped/enzimología , Complejo Represivo Polycomb 2/metabolismo , Linfocitos T/enzimología , Aloinjertos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Trasplante de Médula Ósea , Proteína Potenciadora del Homólogo Zeste 2 , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metilación , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Complejo Represivo Polycomb 2/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción STAT4/genética , Factor de Transcripción STAT4/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Linfocitos T/patologíaRESUMEN
We and others have previously shown that ICOS plays an important role in inducing acute graft-versus-host disease (GVHD) in murine models of allogeneic bone marrow transplantation. ICOS potentiates TCR-mediated PI3K activation and intracellular calcium mobilization. However, ICOS signal transduction pathways involved in GVHD remain unknown. In this study, we examined the contribution of ICOS-PI3K signaling in the pathogenic potential of T cells using a knock-in mouse strain, ICOS-YF, which selectively lost the ability to activate PI3K. We found that when total T cells were used as alloreactive T cells, ICOS-YF T cells caused less severe GVHD compared with ICOS wild-type T cells, but they induced much more aggressive disease than ICOS knockout T cells. This intermediate level of pathogenic capacity of ICOS-YF T cells was correlated with similar levels of IFN-γ-producing CD8 T cells that developed in the recipients of ICOS-WT or ICOS-YF T cells. We further evaluated the role of ICOS-PI3K signaling in CD4 versus CD8 T cell compartment using GVHD models that are exclusively driven by CD4 or CD8 T cells. Remarkably, ICOS-YF CD8 T cells caused disease similar to ICOS wild-type CD8 T cells, whereas ICOS-YF CD4 T cells behaved very similarly to their ICOS knockout counterparts. Consistent with their in vivo pathogenic potential, CD8 T cells responded to ICOS ligation in vitro by PI3K-independent calcium flux, T cell activation, and proliferation. Thus, in acute GVHD in mice, CD4 T cells heavily rely on ICOS-PI3K signaling pathways; in contrast, CD8 T cells can use PI3K-independent ICOS signaling pathways, possibly through calcium.
Asunto(s)
Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/metabolismo , Proteína Coestimuladora de Linfocitos T Inducibles/fisiología , Activación de Linfocitos/inmunología , Fosfatidilinositol 3-Quinasa/fisiología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Enfermedad Aguda , Animales , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Enfermedad Injerto contra Huésped/enzimología , Proteína Coestimuladora de Linfocitos T Inducibles/deficiencia , Activación de Linfocitos/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Transducción de Señal/genética , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/metabolismoRESUMEN
The fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation is associated with a high relapse rate for patients with acute myeloid leukemia (AML) even after allogeneic hematopoietic stem cell transplantation (HSCT). Sorafenib is a tyrosine kinase inhibitor, which inhibits the FLT3 tyrosine kinase and has shown encouraging activity in FLT3-ITD AML. We conducted a phase I trial of maintenance sorafenib after HSCT in patients with FLT3-ITD AML (ClinicalTrials.govNCT01398501). Patients received a variety of conditioning regimens and graft sources. A dose escalation 3 + 3 cohort design was used to define the maximum tolerated dose (MTD), with an additional 10 patients treated at the MTD. Sorafenib was initiated between days 45 and 120 after HSCT and continued for 12 28-day cycles. Twenty-two patients were enrolled (status at HSCT: first complete remission [CR1], n = 16; second complete remission [CR2], n = 3; refractory, n = 3). The MTD was established at 400 mg twice daily with 1 dose-limiting toxicity (DLT) observed (pericardial effusion). Two patients died of transplantation-related causes, both unrelated to sorafenib. Two patients stopped sorafenib after relapse and 5 stopped because of attributable toxicities after the DLT period. Median follow-up for surviving patients is 16.7 months after HSCT (range, 8.1 to 35.0). There was 1 case of grade II acute graft-versus-host disease (GVHD) after starting sorafenib and the 12-month cumulative incidence of chronic GVHD was 38% (90% confidence interval [CI], 21% to 56%). For all patients, 1-year progression-free survival (PFS) was 85% (90% CI, 66% to 94%) and 1-year overall survival (OS) was 95% (90% CI, 79% to 99%) after HSCT. For patients in CR1/CR2 before HSCT (n = 19), 1-year PFS was 95% (90% CI, 76% to 99%) and 1-year OS was 100%, with only 1 patient who relapsed. Sorafenib is safe after HSCT for FLT3-ITD AML and merits further investigation for the prevention of relapse.
Asunto(s)
Leucemia Mieloide Aguda , Quimioterapia de Mantención , Mutagénesis Insercional , Niacinamida/análogos & derivados , Compuestos de Fenilurea/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Tirosina Quinasa 3 Similar a fms , Adulto , Anciano , Aloinjertos , Supervivencia sin Enfermedad , Femenino , Enfermedad Injerto contra Huésped/enzimología , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/mortalidad , Enfermedad Injerto contra Huésped/terapia , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/terapia , Masculino , Persona de Mediana Edad , Niacinamida/administración & dosificación , Inducción de Remisión , Sorafenib , Tasa de Supervivencia , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
A putative involvement of the vasculature seems to play a critical role in the pathophysiology of graft-versus-host disease (GVHD). We aimed to characterize alterations of mesenteric resistance arteries in GVHD in a fully MHC-mismatched model of BALB/c mice conditioned with total body irradiation that underwent transplantation with bone marrow cells and splenocytes from syngeneic (BALB/c) or allogeneic (C57BL/6) donors. After 4 weeks, animals were sacrificed and mesenteric resistance arteries were studied in a pressurized myograph. The expression of endothelial (eNOS) and inducible nitric oxide (NO)-synthase (iNOS) was quantified and vessel wall ultrastructure was investigated with electron microscopy. The myograph study revealed an endothelial dysfunction in allogeneic-transplant recipients, whereas endothelium-independent vasodilation was similar to syngeneic-transplant recipients or untreated controls. The expression of eNOS was decreased and iNOS increased, possibly contributing to endothelial dysfunction. Additionally, arteries of allogeneic transplant recipients exhibited a geometry-independent increase in vessels strain. For both findings, electron microscopy provided a structural correlate by showing severe damage of the whole vessel wall in allogeneic-transplant recipient animals. Our study provides further data to prove, and is the first to characterize, functional and structural vascular alterations in the early course after allogeneic transplantation directly in an ex vivo setting and, therefore, strongly supports the hypothesis of a vascular form of GVHD.
Asunto(s)
Trasplante de Médula Ósea , Endotelio Vascular/fisiopatología , Enfermedad Injerto contra Huésped/fisiopatología , Arterias Mesentéricas/fisiopatología , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo II/genética , Animales , Modelos Animales de Enfermedad , Endotelio Vascular/enzimología , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Femenino , Expresión Génica , Enfermedad Injerto contra Huésped/enzimología , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Complejo Mayor de Histocompatibilidad , Arterias Mesentéricas/enzimología , Arterias Mesentéricas/inmunología , Arterias Mesentéricas/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Miografía , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Trasplante Homólogo , Trasplante Isogénico , Resistencia Vascular , Irradiación Corporal TotalRESUMEN
It is well known that allo-reactive T cells play a crucial role in graft-versus-leukemia and graft-versus-host disease after allogeneic hematopoietic stem cell transplantation (alloSCT). Allo-reactive CD4(+) T cells can mediate direct cytolysis, but may also stimulate production of IgG antibodies as helper cells. Immune complexes may subsequently be processed and presented by professional antigen presenting cells and stimulate induction of specific CD8(+) T cells. As such, proteins targeted in coordinated T- and B-cell responses may represent a class of immunodominant antigens in clinical responses after alloSCT. We previously identified LB-PTK2B-1T as HLA class II restricted polymorphic antigen in a patient treated with donor lymphocyte infusion for relapsed chronic myeloid leukemia after HLA-matched alloSCT. Since PTK2B has also been described as antibody target, we here investigated whether a coordinated T- and B-cell response against PTK2B was induced. Patient serum before and after alloSCT and donor lymphocyte infusion (DLI) was screened for antibodies, and we indeed observed development of a humoral immune response against PTK2B. Antibodies against PTK2B were only found after DLI and, in contrast to the CD4(+) T cells, recognized a monomorphic region of the protein. To our knowledge, this is the first description of a coordinated allo-reactive CD4(+) T-cell and auto-reactive antibody response against an autosomal antigen.
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Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Quinasa 2 de Adhesión Focal/inmunología , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas , Inmunoglobulina G/inmunología , Isoanticuerpos/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva , Aloinjertos , Linfocitos B/metabolismo , Linfocitos B/patología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Femenino , Quinasa 2 de Adhesión Focal/genética , Quinasa 2 de Adhesión Focal/metabolismo , Enfermedad Injerto contra Huésped/enzimología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/patología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunoglobulina G/sangre , Isoanticuerpos/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , MasculinoRESUMEN
OBJECTIVE: To explore the effects of fluvastatin on expression of p38 mitogen-activated protein kinase (p38MAPK) in renal tissue of chronic graft versus host disease (cGVHD) lupus nephritis in mice. METHODS: Mice were randomly divided into 4 groups of model, low-dose fluvastatin intervention (5 mg×kg(-1)×d(-1)), high-dose fluvastatin intervention (10 mg×kg(-1)×d(-1)) and normal control. The 24 h total amount of urine protein was evaluated by biuret reaction colorimetric assay. And the protein and mRNA expression levels of p38MAPK, p-p38MAPK and TGF-ß1 in renal tissue were detected by immunohistochemistry, Western blot and reverse transcription-polymerase chain reaction (RT-PCR) respectively. RESULTS: Compared with the normal control group, 24 h total amount of urine protein, protein and mRNA expression levels of p38MAPK, p-p38MAPK and TGF-ß1 increased significantly in the model group respectively ((7.84 ± 0.36) vs (1.15 ± 0.15) mg/24 h, 0.81 ± 0.03 vs 0.50 ± 0.01, 0.69 ± 0.02 vs 0.10 ± 0.01, 0.76 ± 0.02 vs 0.28 ± 0.02, 0.84 ± 0.04 vs 0.25 ± 0.02, 0.82 ± 0.04 vs 0.12 ± 0.02, all P < 0.01). Fluvastatin treatment suppressed markedly their increased expressions (P < 0.05). And high-dose fluvastatin treatment suppressed more significantly than low-dose group (P < 0.05). CONCLUSION: The renal protection effect of fluvastatin may be achieved by inhibiting the signal pathway of p38MAPK.
Asunto(s)
Ácidos Grasos Monoinsaturados/farmacología , Enfermedad Injerto contra Huésped/enzimología , Indoles/farmacología , Nefritis Lúpica/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Fluvastatina , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Nefritis Lúpica/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas , Ratones , Factor de Crecimiento Transformador beta1RESUMEN
Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism that plays an important role in the induction of immune tolerance. To evaluate the expression levels of IDO and interferon (IFN)-γ in patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) and to identify the correlation between IDO activity, IFN-γ, and acute graft-versus-host disease (aGVHD), we measured IDO mRNA expression in peripheral blood mononuclear cells in 89 allo-HSCT patients by reverse transcription-polymerase chain reaction. The IDO activity in plasma was also performed by reverse-phase high-performance liquid chromatography; plasma IFN-γ was detected by a standard enzyme-linked immunosorbent assay. IDO mRNA was detected in 55 of 74 patients (74.32%) with aGVHD. Of patients without aGVHD, only 2 of 26 expressed IDO mRNA (7.69%); none of 8 healthy volunteers was positive for IDO expression. Plasma IDO activity was much higher in aGVHD patients than in those without aGVHD (4.74 ± 3.35 vs 1.79 ± 1.02, respectively; P < .0001) or in healthy control subjects (4.74 ± 3.35 vs 1.77 ± .22; P < .0001). Patients with severe (grade III/IV) aGVHD had much higher IDO activity than those with mild (grade I/II) aGVHD (6.57 ± 3.34 vs 2.46 ± 1.41; P < .0001). Meanwhile, there was a significant increase in plasma IFN-γ level in aGVHD patients (P = .0043). IDO activity decreased after alleviation of aGVHD, whereas fluctuation of plasma IDO was also observed upon the recurrence of aGVHD. Plasma IDO activity was correlated with the level of plasma IFN-γ (r = .8288; P < .0001). Using receiver-operating characteristic curves analysis, the sensitivity and specificity for evaluation of aGVHD were determined. The area under the curve of IDO activity was higher than that of IFN-γ (.852 vs .694) with a sensitivity and specificity for IDO of 81% and 78%, respectively, whereas the sensitivity and specificity for IFN-γ were 41% and 93%, respectively. IDO mRNA was expressed in blood mononuclear cells of patients with aGVHD. Plasma IDO activity was elevated in aGVHD patients and was correlated with the severity of aGVHD. In combination with plasma IFN-γ, IDO activity may represent a potential biomarker for the diagnosis and evaluation of aGVHD after allo-HSCT. Intervention of the IDO pathway may also represent an alternative way to overcome steroid-resistant aGVHD.
Asunto(s)
Enfermedad Injerto contra Huésped/sangre , Trasplante de Células Madre Hematopoyéticas/métodos , Indolamina-Pirrol 2,3,-Dioxigenasa/sangre , Interferón gamma/sangre , Enfermedad Aguda , Adolescente , Adulto , Niño , Preescolar , Femenino , Enfermedad Injerto contra Huésped/enzimología , Enfermedad Injerto contra Huésped/inmunología , Humanos , Masculino , Persona de Mediana Edad , Trasplante Homólogo/métodos , Adulto JovenRESUMEN
The activity of acid sphingomyelinase (aSMase) was previously reported to be involved in glucocorticoid-induced cell death (GICD) of T lymphocytes. This mechanism in turn is believed to contribute to the therapeutic efficacy of glucocorticoids (GCs) in the treatment of inflammatory diseases. In this study, we reassessed the role of aSMase in GICD by using aSMase knockout mice. The absence of aSMase largely abolished the partial protection that effector memory CD4(+) T cells in wild-type mice possess against GICD. Reduced IL-2 secretion by aSMase-deficient CD4(+) T cells suggested that a lack of this important survival factor might be the cause of these cells' enhanced susceptibility to GICD. Indeed, addition of IL-2 restored the protection against GICD, whereas neutralization of IL-2 abrogated the otherwise protective effect seen in wild-type effector memory CD4(+) T cells. The therapeutic implications of the altered sensitivity of aSMase-deficient T cells to GICD were assessed in models of inflammatory disorders; namely, experimental autoimmune encephalomyelitis and acute graft-versus-host disease. Surprisingly, GC treatment was equally efficient in both models in terms of ameliorating the diseases, regardless of the genotype of the T cells. Thus, our data reveal a hitherto unrecognized contribution of aSMase to the sensitivity of effector memory CD4(+) T cells to GICD and call into question the traditionally attributed importance of GICD of T cells to the treatment of inflammatory diseases by GCs.