RESUMEN
T-cell activation releases inositol 1,4,5-trisphosphate (IP3), inducing cytoplasmic calcium (Ca2+) influx. In turn, inositol 1,4,5-trisphosphate 3-kinase B (Itpkb) phosphorylates IP3 to negatively regulate and thereby tightly control Ca2+ fluxes that are essential for mature T-cell activation and differentiation and protection from cell death. Itpkb pathway inhibition increases intracellular Ca2+, induces apoptosis of activated T cells, and can control T-cell-mediated autoimmunity. In this study, we employed genetic and pharmacological approaches to inhibit Itpkb signaling as a means of controlling graft-versus-host disease (GVHD). Murine-induced, Itpkb-deleted (Itpkb-/-) T cells attenuated acute GVHD in 2 models without eliminating A20-luciferase B-cell lymphoma graft-versus-leukemia (GVL). A highly potent, selective inhibitor, GNF362, ameliorated acute GVHD without impairing GVL against 2 acute myeloid leukemia lines (MLL-AF9-eGFP and C1498-luciferase). Compared with FK506, GNF362 more selectively deleted donor alloreactive vs nominal antigen-responsive T cells. Consistent with these data and as compared with FK506, GNF362 had favorable acute GVHD and GVL properties against MLL-AF9-eGFP cells. In chronic GVHD preclinical models that have a pathophysiology distinct from acute GVHD, Itpkb-/- donor T cells reduced active chronic GVHD in a multiorgan system model of bronchiolitis obliterans (BO), driven by germinal center reactions and resulting in target organ fibrosis. GNF362 treatment reduced active chronic GVHD in both BO and scleroderma models. Thus, intact Itpkb signaling is essential to drive acute GVHD pathogenesis and sustain active chronic GVHD, pointing toward a novel clinical application to prevent acute or treat chronic GVHD.
Asunto(s)
Enfermedad Injerto contra Huésped/prevención & control , Efecto Injerto vs Leucemia , Leucemia Experimental/complicaciones , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Tacrolimus/farmacología , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped/metabolismo , Enfermedad Injerto contra Huésped/patología , Inmunosupresores/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiologíaRESUMEN
MicroRNA-155 (miR-155) is a small noncoding RNA critical for the regulation of inflammation as well as innate and adaptive immune responses. MiR-155 has been shown to be dysregulated in both donor and recipient immune cells during acute graft-versus-host disease (aGVHD). We previously reported that miR-155 is upregulated in donor T cells of mice and humans with aGVHD and that mice receiving miR-155-deficient (miR155-/-) splenocytes had markedly reduced aGVHD. However, molecular mechanisms by which miR-155 modulates T cell function in aGVHD have not been fully investigated. We identify that miR-155 expression in both donor CD8+ T cells and conventional CD4+ CD25- T cells is pivotal for aGVHD pathogenesis. Using murine aGVHD transplant experiments, we show that miR-155 strongly impacts alloreactive T cell expansion through multiple distinct mechanisms, modulating proliferation in CD8+ donor T cells and promoting exhaustion in donor CD4+ T cells in both the spleen and colon. Additionally, miR-155 drives a proinflammatory Th1 phenotype in donor T cells in these two sites, and miR-155-/- donor T cells are polarized toward an IL-4-producing Th2 phenotype. We further demonstrate that miR-155 expression in donor T cells regulates CCR5 and CXCR4 chemokine-dependent migration. Notably, we show that miR-155 expression is crucial for donor T cell infiltration into multiple target organs. These findings provide further understanding of the role of miR-155 in modulating aGVHD through T cell expansion, effector cytokine production, and migration.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Movimiento Celular/genética , Enfermedad Injerto contra Huésped/genética , MicroARNs/genética , Enfermedad Aguda , Animales , Proliferación Celular/genética , Colon/metabolismo , Subunidad alfa del Receptor de Interleucina-2/genética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Receptores CCR5/genética , Receptores CXCR4/genética , Bazo/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
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
The common γ chain (CD132) is a subunit of the interleukin (IL) receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Because levels of several of these cytokines were shown to be increased in the serum of patients developing acute and chronic graft-versus-host disease (GVHD), we reasoned that inhibition of CD132 could have a profound effect on GVHD. We observed that anti-CD132 monoclonal antibody (mAb) reduced acute GVHD potently with respect to survival, production of tumor necrosis factor, interferon-γ, and IL-6, and GVHD histopathology. Anti-CD132 mAb afforded protection from GVHD partly via inhibition of granzyme B production in CD8 T cells, whereas exposure of CD8 T cells to IL-2, IL-7, IL-15, and IL-21 increased granzyme B production. Also, T cells exposed to anti-CD132 mAb displayed a more naive phenotype in microarray-based analyses and showed reduced Janus kinase 3 (JAK3) phosphorylation upon activation. Consistent with a role of JAK3 in GVHD, Jak3(-/-) T cells caused less severe GVHD. Additionally, anti-CD132 mAb treatment of established chronic GVHD reversed liver and lung fibrosis, and pulmonary dysfunction characteristic of bronchiolitis obliterans. We conclude that acute GVHD and chronic GVHD, caused by T cells activated by common γ-chain cytokines, each represent therapeutic targets for anti-CD132 mAb immunomodulation.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Trasplante de Médula Ósea/efectos adversos , Citocinas/metabolismo , Enfermedad Injerto contra Huésped/prevención & control , Subunidad gamma Común de Receptores de Interleucina/antagonistas & inhibidores , Enfermedad Aguda , Animales , Western Blotting , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Células Cultivadas , Enfermedad Crónica , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/mortalidad , Humanos , Janus Quinasa 3/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Members of the B7 family have been shown to be important for regulating immune responses by providing either positive or negative costimulatory signals. The function of B7-H3 has been controversial. We show that B7-H3 is upregulated in graft-versus-host disease (GVHD) target organs, including the colon, liver, and lung. Infusion of allogeneic donor T cells into B7-H3(-/-) vs wild-type (WT) recipients resulted in increased GVHD lethality associated with increased T-cell proliferation, colonic inflammatory cytokines, and destruction of epithelial barriers. Allogeneic B7-H3(-/-) vs WT donor T cells also had increased T-cell proliferation and GVHD lethality associated with increased proliferation and cytokine secretion in the spleen, intraepithelial lymphocyte inflammatory cytokines, and intestinal permeability. Both resting and activated regulatory T cells (Tregs) lack B7-H3 messenger RNA. Consistent with these data, GVHD was augmented in recipients of B7-H3(-/-) Treg-depleted grafts. In two delayed lymphocyte infusion (DLI) models, T cells lacking B7-H3 are capable of providing graft-versus-leukemia (GVL) effects. We conclude that B7-H3 is responsible for providing a negative costimulatory signal. Our studies provide support for developing and testing new therapies directed toward the B7-H3 pathway, including approaches to augment host B7-H3 early after bone marrow transplantation to prevent GVHD and to develop potent antagonistic antibodies later after transplant to facilitate DLI-mediated GVL without GVHD complications.
Asunto(s)
Antígenos B7/inmunología , Trasplante de Médula Ósea/efectos adversos , Enfermedad Injerto contra Huésped/inmunología , Linfocitos T/inmunología , Aloinjertos , Animales , Citometría de Flujo , Humanos , Inmunohistoquímica , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
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
Interleukin (IL)-33 binding to the receptor suppression of tumorigenicity 2 (ST2) produces pro-inflammatory and anti-inflammatory effects. Increased levels of soluble ST2 (sST2) are a biomarker for steroid-refractory graft-versus-host disease (GVHD) and mortality. However, whether sST2 has a role as an immune modulator or only as a biomarker during GVHD was unclear. We show increased IL-33 production by nonhematopoietic cells in the gastrointestinal (GI) tract in mice post-conditioning and patients during GVHD. Exogenous IL-33 administration during the peak inflammatory response worsened GVHD. Conversely, GVHD lethality and tumor necrosis factor-α production was significantly reduced in il33(-/-) recipients. ST2 was upregulated on murine and human alloreactive T cells and sST2 increased as experimental GVHD progressed. Concordantly, st2(-/-) vs wild-type (WT) donor T cells had a marked reduction in GVHD lethality and GI histopathology. Alloantigen-induced IL-18 receptor upregulation was lower in st2(-/-) T cells, and linked to reduced interferon-γ production by st2(-/-) vs WT T cells during GVHD. Blockade of IL-33/ST2 interactions during allogeneic-hematopoietic cell transplantation by exogenous ST2-Fc infusions had a marked reduction in GVHD lethality, indicating a role of ST2 as a decoy receptor modulating GVHD. Together, these studies point to the IL-33/ST2 axis as a novel and potent target for GVHD therapy.
Asunto(s)
Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/metabolismo , Interleucinas/metabolismo , Receptores de Superficie Celular/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Enfermedad Aguda , Animales , Análisis por Conglomerados , Modelos Animales de Enfermedad , Expresión Génica , Perfilación de la Expresión Génica , Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/mortalidad , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Interferón gamma/biosíntesis , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/genética , Mucosa Intestinal/metabolismo , Intestinos/patología , Intestinos/efectos de la radiación , Isoantígenos/inmunología , Ratones , Ratones Noqueados , Receptores de Superficie Celular/genética , Índice de Severidad de la Enfermedad , Donantes de Tejidos , Acondicionamiento Pretrasplante , Trasplante HomólogoRESUMEN
Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Having shown that germinal center (GC) formation and immunoglobulin deposition are required for multiorgan system cGVHD and associated bronchiolitis obliterans syndrome (BOS) in a murine model, we hypothesized that T follicular helper (Tfh) cells are necessary for cGVHD by supporting GC formation and maintenance. We show that increased frequency of Tfh cells correlated with increased GC B cells, cGVHD, and BOS. Although administering a highly depletionary anti-CD20 monoclonal antibody (mAb) to mice with established cGVHD resulted in peripheral B-cell depletion, B cells remained in the lung, and BOS was not reversed. BOS could be treated by eliminating production of interleukin-21 (IL-21) by donor T cells or IL-21 receptor (IL-21R) signaling of donor B cells. Development of BOS was dependent upon T cells expressing the chemokine receptor CXCR5 to facilitate T-cell trafficking to secondary lymphoid organ follicles. Blocking mAbs for IL-21/IL-21R, inducible T-cell costimulator (ICOS)/ICOS ligand, and CD40L/CD40 hindered GC formation and cGVHD. These data provide novel insights into cGVHD pathogenesis, indicate a role for Tfh cells in these processes, and suggest a new line of therapy using mAbs targeting Tfh cells to reverse cGVHD.
Asunto(s)
Linfocitos B/inmunología , Bronquiolitis Obliterante/inmunología , Centro Germinal/inmunología , Enfermedad Injerto contra Huésped/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD20/inmunología , Antígenos CD20/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Bronquiolitis Obliterante/genética , Bronquiolitis Obliterante/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Antígenos CD40/inmunología , Antígenos CD40/metabolismo , Ligando de CD40/inmunología , Ligando de CD40/metabolismo , Enfermedad Crónica , Citometría de Flujo , Centro Germinal/efectos de los fármacos , Centro Germinal/metabolismo , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/metabolismo , Ligando Coestimulador de Linfocitos T Inducibles/inmunología , Ligando Coestimulador de Linfocitos T Inducibles/metabolismo , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Interleucinas/genética , Interleucinas/inmunología , Interleucinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Receptores CXCR5/genética , Receptores CXCR5/inmunología , Receptores CXCR5/metabolismo , Receptores de Interleucina-21/genética , Receptores de Interleucina-21/inmunología , Receptores de Interleucina-21/metabolismo , Síndrome , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, play an important role in the maintenance of peripheral tolerance. We explored the role of PD-1 ligands in regulating graft-versus-host disease (GVHD). Both PD-L1 and PD-L2 expression were upregulated in the spleen, liver, colon, and ileum of GVHD mice. Whereas PD-L2 expression was limited to hematopoietic cells, hematopoietic and endothelial cells expressed PD-L1. PD-1/PD-L1, but not PD-1/PD-L2, blockade markedly accelerated GVHD-induced lethality. Chimera studies suggest that PD-L1 expression on host parenchymal cells is more critical than hematopoietic cells in regulating acute GVHD. Rapid mortality onset in PD-L1-deficient hosts was associated with increased gut T-cell homing and loss of intestinal epithelial integrity, along with increased donor T-cell proliferation, activation, Th1 cytokine production, and reduced apoptosis. Bioenergetics profile analysis of proliferating alloreactive donor T-cells demonstrated increased aerobic glycolysis and oxidative phosphorylation in PD-L1-deficient hosts. Donor T-cells exhibited a hyperpolarized mitochondrial membrane potential, increased superoxide production, and increased expression of a glucose transporter in PD-L1-deficient hosts. Taken together, these data provide new insight into the differential roles of host PD-L1 and PD-L2 and their associated cellular and metabolic mechanisms controlling acute GVHD.
Asunto(s)
Antígeno B7-H1/metabolismo , Trasplante de Médula Ósea , Enfermedad Injerto contra Huésped/metabolismo , Tolerancia Periférica/inmunología , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Tolerancia al Trasplante/inmunología , Enfermedad Aguda , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Proliferación Celular , Regulación de la Expresión Génica , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/inmunología , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Glucólisis , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/mortalidad , Enfermedad Injerto contra Huésped/patología , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Intestinos/patología , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Ratones , Fosforilación Oxidativa , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Proteína 2 Ligando de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Bazo/inmunología , Bazo/metabolismo , Bazo/patología , Análisis de SupervivenciaRESUMEN
Graft-versus-host disease (GVHD) is a critical complication after allogeneic bone marrow transplantation. During GVHD, donor T cells are activated by host antigen-presenting cells and differentiate into T-effector cells (Teffs) that migrate to GVHD target organs. However, local environmental factors influencing Teff differentiation and migration are largely unknown. Vitamin A metabolism within the intestine produces retinoic acid, which contributes to intestinal homeostasis and tolerance induction. Here, we show that the expression and function of vitamin A-metabolizing enzymes were increased in the intestine and mesenteric lymph nodes in mice with active GVHD. Moreover, transgenic donor T cells expressing a retinoic acid receptor (RAR) response element luciferase reporter responded to increased vitamin A metabolites in GVHD-affected organs. Increasing RAR signaling accelerated GVHD lethality, whereas donor T cells expressing a dominant-negative RARα (dnRARα) showed markedly diminished lethality. The dnRARα transgenic T cells showed reduced Th1 differentiation and α4ß7 and CCR9 expression associated with poor intestinal migration, low GVHD pathology, and reduced intestinal permeability, primarily via CD4(+) T cells. The inhibition of RAR signaling augmented donor-induced Treg generation and expansion in vivo, while preserving graft-versus-leukemia effects. Together, these results suggested that reagents blunting donor T-cell RAR signaling may possess therapeutic anti-GVHD properties.
Asunto(s)
Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/metabolismo , Intestinos/inmunología , Receptores de Ácido Retinoico/metabolismo , Transducción de Señal , Linfocitos T/citología , Linfocitos T/inmunología , Animales , Trasplante de Médula Ósea/efectos adversos , Diferenciación Celular/inmunología , Enfermedad Injerto contra Huésped/mortalidad , Ratones , Receptores Mensajeros de Linfocitos/metabolismo , Receptor alfa de Ácido Retinoico , Transducción de Señal/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
Chronic GVHD (cGVHD) poses a significant risk for HSCT patients. Preclinical development of new therapeutic modalities has been hindered by models with pathologic findings that may not simulate the development of human cGVHD. Previously, we have demonstrated that cGVHD induced by allogeneic HSCT after a conditioning regimen of cyclophosphamide and total-body radiation results in pulmonary dysfunction and airway obliteration, which leads to bronchiolitis obliterans (BO), which is pathognomonic for cGVHD of the lung. We now report cGVHD manifestations in a wide spectrum of target organs, including those with mucosal surfaces. Fibrosis was demonstrated in the lung and liver and was associated with CD4(+) T cells and B220(+) B-cell infiltration and alloantibody deposition. Donor bone marrow obtained from mice incapable of secreting IgG alloantibody resulted in less BO and cGVHD. Robust germinal center reactions were present at the time of cGVHD disease initiation. Blockade of germinal center formation with a lymphotoxin-receptor-immunoglobulin fusion protein suppressed cGVHD and BO. We conclude that cGVHD is caused in part by alloantibody secretion, which is associated with fibrosis and cGVHD manifestations including BO, and that treatment with a lymphotoxin-ß receptor-immunoglobulin fusion protein could be beneficial for cGVHD prevention and therapy.
Asunto(s)
Linfocitos B/inmunología , Bronquiolitis Obliterante/inmunología , Centro Germinal/inmunología , Enfermedad Injerto contra Huésped/inmunología , Isoanticuerpos/inmunología , Animales , Linfocitos B/metabolismo , Donantes de Sangre , Bronquiolitis Obliterante/prevención & control , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Enfermedad Crónica , Femenino , Técnica del Anticuerpo Fluorescente , Centro Germinal/efectos de los fármacos , Enfermedad Injerto contra Huésped/patología , Enfermedad Injerto contra Huésped/prevención & control , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Antígenos Comunes de Leucocito/inmunología , Antígenos Comunes de Leucocito/metabolismo , Cirrosis Hepática/inmunología , Cirrosis Hepática/metabolismo , Receptor beta de Linfotoxina/genética , Receptor beta de Linfotoxina/inmunología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
T-cell immunoglobulin mucin-3 (Tim-3) is expressed on pathogenic T cells, and its ligand galectin-9 (gal-9) is up-regulated in inflamed tissues. When Tim-3(+) T cells encounter high gal-9 levels, they are deleted. Tim-3 is up-regulated on activated T cells during GVHD. Inhibition of Tim-3/gal-9 binding by infusion of a Tim-3-Ig fusion protein or Tim-3(-/-) donor T cells increased T-cell proliferation and GVHD lethality. When the Tim-3/gal-9 pathway engagement was augmented using gal-9 transgenic recipients, GVHD lethality was slowed. Together, these data indicate a potential for modulating this pathway to reduce disease by increasing Tim-3 or gal-9 engagement. Paradoxically, when Tim-3/gal-9 was inhibited in the absence of donor T-regulatory cells (Tregs), GVHD was inhibited. GVHD reduction was associated with decreased colonic inflammatory cytokines as well as epithelial barrier destruction. CD25-depleted Tim-3(-/-) donor T cells underwent increased activation-induced cell death because of increased IFN-γ production. To our knowledge, these studies are the first to show that although the absence of Tim-3/gal-9 pathway interactions augments systemic GVHD, concurrent donor Treg depletion paradoxically and surprisingly inhibits GVHD. Thus, although donor Tregs typically inhibit GVHD, under some conditions, such Tregs actually may contribute to GVHD by reducing activation-induced T-cell death.
Asunto(s)
Trasplante de Médula Ósea/efectos adversos , Galectinas/metabolismo , Enfermedad Injerto contra Huésped/inmunología , Receptores Virales/metabolismo , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Enfermedad Aguda , Animales , Muerte Celular/inmunología , División Celular/inmunología , Galectinas/genética , Galectinas/inmunología , Enfermedad Injerto contra Huésped/metabolismo , Enfermedad Injerto contra Huésped/mortalidad , Receptor 2 Celular del Virus de la Hepatitis A , Interferón gamma/antagonistas & inhibidores , Interferón gamma/sangre , Intestinos/inmunología , Intestinos/patología , Depleción Linfocítica/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Virales/genética , Receptores Virales/inmunología , Linfocitos T Reguladores/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic stem cell transplant (alloHSCT), underscoring the need to further elucidate its mechanisms and develop novel treatments. Based on recent observations that microRNA-155 (miR-155) is up-regulated during T-cell activation, we hypothesized that miR-155 is involved in the modulation of aGVHD. Here we show that miR-155 expression was up-regulated in T cells from mice developing aGVHD after alloHSCT. Mice receiving miR-155-deficient donor lymphocytes had markedly reduced lethal aGVHD, whereas lethal aGVHD developed rapidly in mice recipients of miR-155 overexpressing T cells. Blocking miR-155 expression using a synthetic anti-miR-155 after alloHSCT decreased aGVHD severity and prolonged survival in mice. Finally, miR-155 up-regulation was shown in specimens from patients with pathologic evidence of intestinal aGVHD. Altogether, our data indicate a role for miR-155 in the regulation of GVHD and point to miR-155 as a novel target for therapeutic intervention in this disease.
Asunto(s)
Enfermedad Injerto contra Huésped/genética , MicroARNs/fisiología , Enfermedad Aguda , Animales , Células Cultivadas , Femenino , Regulación de la Expresión Génica/genética , Terapia Genética , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/metabolismo , Humanos , Activación de Linfocitos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Bazo/citología , Bazo/metabolismo , Bazo/trasplante , Linfocitos T/metabolismoRESUMEN
The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Trasplante de Médula Ósea , Rechazo de Injerto/prevención & control , Enfermedad Injerto contra Huésped/prevención & control , Inmunosupresores/uso terapéutico , Glicoles de Propileno/uso terapéutico , Esfingosina/análogos & derivados , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/patología , Ligando de CD40/antagonistas & inhibidores , Ligando de CD40/inmunología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Clorhidrato de Fingolimod , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Ratones , Esfingosina/uso terapéutico , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patología , Timocitos/efectos de los fármacos , Timocitos/inmunología , Timocitos/patología , Timo/efectos de los fármacos , Timo/inmunología , Quimera por Trasplante , Trasplante Homólogo , Irradiación Corporal TotalRESUMEN
Myeloablative conditioning before bone marrow transplantation (BMT) results in thymic epithelial cell (TEC) injury, T-cell immune deficiency, and susceptibility to opportunistic infections. Conditioning regimen-induced TEC damage directly contributes to slow thymopoietic recovery after BMT. Keratinocyte growth factor (KGF) is a TEC mitogen that stimulates proliferation and, when given before conditioning, reduces TEC injury. Some TEC subsets are refractory to KGF and functional T-cell responses are not fully restored in KGF-treated BM transplant recipients. Therefore, we investigated whether the addition of a pharmacologic inhibitor, PFT-beta, to transiently inhibit p53 during radiotherapy could spare TECs from radiation-induced damage in congenic and allogeneic BMTs. Combined before BMT KGF + PFT-beta administration additively restored numbers of cortical and medullary TECs and improved thymic function after BMT, resulting in higher numbers of donor-derived, naive peripheral CD4(+) and CD8(+) T cells. Radiation conditioning caused a loss of T-cell zone fibroblastic reticular cells (FRCs) and CCL21 expression in lymphoid stroma. KGF + PFT-beta treatment restored both FRC and CCL21 expression, findings that correlated with improved T-cell reconstitution and an enhanced immune response against Listeria monocytogenes infection. Thus, transient p53 inhibition combined with KGF represents a novel and potentially translatable approach to promote rapid and durable thymic and peripheral T-cell recovery after BMT.
Asunto(s)
Trasplante de Médula Ósea , Células Epiteliales/efectos de los fármacos , Factor 7 de Crecimiento de Fibroblastos/farmacología , Linfocitos T/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Inmunidad Adaptativa/efectos de los fármacos , Inmunidad Adaptativa/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Benzotiazoles/farmacología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/efectos de la radiación , Quimiocina CCL21/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Listeria monocytogenes/inmunología , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/microbiología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/microbiología , Linfocitos T/citología , Linfocitos T/metabolismo , Timo/citología , Factores de Tiempo , Tolueno/análogos & derivados , Tolueno/farmacología , Proteína p53 Supresora de Tumor/antagonistas & inhibidoresRESUMEN
Myeloid-derived suppressor cells (MDSCs) are a well-defined population of cells that accumulate in the tissue of tumor-bearing animals and are known to inhibit immune responses. Within 4 days, bone marrow cells cultured in granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor resulted in the generation of CD11b(+)Ly6G(lo)Ly6C(+) MDSCs, the majority of which are interleukin-4Rα (IL-4Rα(+)) and F4/80(+). Such MDSCs potently inhibited in vitro allogeneic T-cell responses. Suppression was dependent on L-arginine depletion by arginase-1 activity. Exogenous IL-13 produced an MDSC subset (MDSC-IL-13) that was more potently suppressive and resulted in arginase-1 up-regulation. Suppression was reversed with an arginase inhibitor or on the addition of excess L-arginine to the culture. Although both MDSCs and MDSC-IL-13 inhibited graft-versus-host disease (GVHD) lethality, MDSC-IL-13 were more effective. MDSC-IL-13 migrated to sites of allopriming. GVHD inhibition was associated with limited donor T-cell proliferation, activation, and proinflammatory cytokine production. GVHD inhibition was reduced when arginase-1-deficient MDSC-IL-13 were used. MDSC-IL-13 did not reduce the graft-versus-leukemia effect of donor T cells. In vivo administration of a pegylated form of human arginase-1 (PEG-arg1) resulted in L-arginine depletion and significant GVHD reduction. MDSC-IL-13 and pegylated form of human arginase-1 represent novel strategies to prevent GVHD that can be clinically translated.
Asunto(s)
Arginasa/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Enfermedad Injerto contra Huésped/prevención & control , Interleucina-13/farmacología , Animales , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Células Cultivadas , Enfermedad Injerto contra Huésped/enzimología , Factor Estimulante de Colonias de Granulocitos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Immunoblotting , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/fisiología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Regulación hacia ArribaRESUMEN
Overexpression of a constitutively active form of Stat5b (Stat5b-CA) increases regulatory T cells (Tregs). We show that Stat5b-CA transgenic (TG) CD4(+) T cells had a markedly reduced graft-versus-host disease (GVHD) capacity versus wild-type (WT) T cells. Stat5b-CA TG versus WT CD4(+) T cells had a higher proportion of Tregs, which were superior in suppressing alloresponses mediated by CD4(+)CD25(-) effector T cells (Teffs). By day 5 after transplantation, Stat5b-CA TG Tregs had expanded approximately 3-fold more than WT Tregs. Purified Stat5b-CA TG Tregs added to WT CD4(+)CD25(-) Teffs were superior on a per-cell basis for inhibiting GVHD versus WT Tregs. Surprisingly, rigorously Treg-depleted Stat5b-CA TG versus WT CD4(+)CD25(-) Teffs caused less GVHD lethality associated with diminished Teff proinflammatory and increased Th2 anti-inflammatory cytokine responses. Reduced GVHD by Stat5b-CA TG versus WT Teffs could not be explained by conversion into Tregs in day 10 posttransplantation spleen or small intestine. In addition, Stat5b-CA TG Teffs retained a graft-versus-leukemia response. These results indicate a major role for Stat5 in Treg expansion and potency along with a lesser but significant role in Teff activation and suggest a strategy of pharmacologic Stat5b up-regulation as a means of decreasing GVHD while retaining a graft-versus-leukemia effect.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Enfermedad Injerto contra Huésped/prevención & control , Factor de Transcripción STAT5/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Apoptosis/inmunología , Trasplante de Médula Ósea/inmunología , Linfocitos T CD4-Positivos/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Expresión Génica , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/metabolismo , Efecto Injerto vs Leucemia/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT5/genéticaAsunto(s)
Dermatitis Atópica/inmunología , Dermatitis Atópica/microbiología , Microbiota/inmunología , Piel/inmunología , Piel/microbiología , Adolescente , Adulto , Factores de Edad , Estudios de Casos y Controles , Niño , Preescolar , Dermatitis Atópica/patología , Femenino , Humanos , Masculino , Microbiota/genética , Persona de Mediana Edad , Piel/patología , Adulto JovenRESUMEN
BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with increased susceptibility to recurrent skin infections. OBJECTIVE: We sought to determine why a subset of patients with AD have an increased risk of disseminated viral skin infections. METHODS: Human subjects with AD with a history of eczema herpeticum (EH) and various control groups were enrolled. Vaccinia virus (VV) expression was measured by means of PCR and immunofluorescent staining in skin biopsy specimens from each study group after incubation with VV. Transgenic mice with a constitutively active signal transducer and activator of transcription 6 gene (STAT6) were characterized for response to VV skin inoculation. Genotyping for 10 STAT6 single nucleotide polymorphisms (SNPs) was performed in a white patient sample (n = 444). RESULTS: VV gene and protein expression were significantly increased in the skin of patients with EH compared with other subject groups after incubation with VV in vitro. Antibody neutralization of IL-4 and IL-13 resulted in lower VV replication in patients with a history of EH. Mice that expressed a constitutively active STAT6 gene compared with wild-type mice had increased mortality and satellite lesion formation after VV skin inoculation. Significant associations were observed between STAT6 SNPs and EH (rs3024975, rs841718, rs167769, and rs703817) and IFN-γ production. The strongest association was observed for a 2-SNP haplotype (patients with AD with a history of EH vs patients with AD without a history of EH, 24.9% vs 9.2%; P = 5.17 × 10(-6)). CONCLUSION: The STAT6 gene increases viral replication in the skin of patients with AD with a history of EH. Further genetic association studies and functional investigations are warranted.
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Dermatitis Atópica/complicaciones , Dermatitis Atópica/genética , Erupción Variceliforme de Kaposi/complicaciones , Erupción Variceliforme de Kaposi/genética , Factor de Transcripción STAT6/genética , Enfermedades Cutáneas Virales/complicaciones , Adulto , Animales , Dermatitis Atópica/virología , Técnica del Anticuerpo Fluorescente , Predisposición Genética a la Enfermedad/genética , Humanos , Erupción Variceliforme de Kaposi/virología , Ratones , Ratones Transgénicos , Polimorfismo de Nucleótido Simple , Enfermedades Cutáneas Virales/genética , Vacuna contra Viruela/efectos adversos , Vaccinia/complicaciones , Vaccinia/genética , Virus VacciniaRESUMEN
BACKGROUND: The basis for increased susceptibility of patients with atopic dermatitis (AD) to develop disseminated viral skin infections such as eczema herpeticum (AD with a history of eczema herpeticum, ADEH(+)) is poorly understood. OBJECTIVE: We sought to determine whether subjects with AD prone to disseminated viral skin infections have defects in their IFN responses. METHODS: GeneChip profiling was used to identify differences in gene expression of PBMCs from patients with ADEH(+) compared with patients with AD without a history of eczema herpeticum (ADEH(-)) and nonatopic controls. Key differences in protein expression were verified by enzyme-linked immunosorbent spot assay and/or ELISA. Clinical relevance was further demonstrated by a mouse model of disseminated viral skin infection and genetic association analysis for genetic variants in IFNG and IFNGR1 and ADEH among 435 cases and controls. RESULTS: We demonstrate by global gene expression analysis selective transcriptomic changes within the IFN superfamily of PBMCs from subjects with ADEH(+) reflecting low IFN-γ and IFN-γ receptor gene expression. IFN-γ protein production was also significantly lower in patients with ADEH(+) (n = 24) compared with patients with ADEH(-) (n = 20) and nonatopic controls (n = 20). IFN-γ receptor knockout mice developed disseminated viral skin infection after epicutaneous challenge with vaccinia virus. Genetic variants in IFNG and IFNGR1 single nucleotide polymorphisms (SNPs) were significantly associated with ADEH (112 cases, 166 controls) and IFN-γ production: a 2-SNP (A-G) IFNGR1 haplotype (rs10457655 and rs7749390) showed the strongest association with a reduced risk of ADEH+ (13.2% ADEH(+) vs 25.5% ADEH(-); P = .00057). CONCLUSION: Patients with ADEH(+) have reduced IFN-γ production, and IFNG and IFNGR1 SNPs are significantly associated with ADEH(+) and may contribute to an impaired immune response to herpes simplex virus.