RESUMEN
Rationale: Primary graft dysfunction (PGD) is a severe form of acute lung injury, leading to increased early morbidity and mortality after lung transplant. Obesity is a major health problem, and recipient obesity is one of the most significant risk factors for developing PGD. Objectives: We hypothesized that T-regulatory cells (Tregs) are able to dampen early ischemia-reperfusion events and thereby decrease the risk of PGD, whereas that action is impaired in obese recipients. Methods: We evaluated Tregs, T cells, and inflammatory markers, plus clinical data, in 79 lung transplant recipients and 41 liver or kidney transplant recipients and studied two groups of mice on a high-fat diet (HFD), which did ("inflammatory" HFD) or did not ("healthy" HFD) develop low-grade inflammation with decreased Treg function. Measurements and Main Results: We identified increased levels of IL-18 as a previously unrecognized mechanism that impairs Tregs' suppressive function in obese individuals. IL-18 decreases levels of FOXP3, the key Treg transcription factor, decreases FOXP3 di- and oligomerization, and increases the ubiquitination and proteasomal degradation of FOXP3. IL-18-treated Tregs or Tregs from obese mice fail to control PGD, whereas IL-18 inhibition ameliorates lung inflammation. The IL-18-driven impairment in Tregs' suppressive function before transplant was associated with an increased risk and severity of PGD in clinical lung transplant recipients. Conclusions: Obesity-related IL-18 induces Treg dysfunction that may contribute to the pathogenesis of PGD. Evaluation of Tregs' suppressive function together with evaluation of IL-18 levels may serve as a screening tool to identify obese individuals with an increased risk of PGD before transplant.
Asunto(s)
Lesión Pulmonar Aguda/etiología , Interleucina-18/metabolismo , Trasplante de Pulmón/efectos adversos , Obesidad/complicaciones , Disfunción Primaria del Injerto/etiología , Daño por Reperfusión/etiología , Linfocitos T Reguladores/metabolismo , Lesión Pulmonar Aguda/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Obesos , Persona de Mediana Edad , Disfunción Primaria del Injerto/fisiopatología , Daño por Reperfusión/fisiopatologíaRESUMEN
Histone/protein deacetylases (HDACs) are frequently upregulated in human malignancies and have therefore become therapeutic targets in cancer therapy. However, inhibiting certain HDAC isoforms can have protolerogenic effects on the immune system, which could make it easier for tumor cells to evade the host immune system. Therefore, a better understanding of how each HDAC isoform affects immune biology is needed to develop targeted cancer therapy. Here, we studied the immune phenotype of HDAC5(-/-) mice on a C57BL/6 background. While HDAC5(-/-) mice replicate at expected Mendelian ratios and do not develop overt autoimmune disease, their T-regulatory (Treg) cells show reduced suppressive function in vitro and in vivo. Likewise, CD4(+) T-cells lacking HDAC5 convert poorly to Tregs under appropriately polarizing conditions. To test if this attenuated Treg formation and suppressive function translated into improved anticancer immunity, we inoculated HDAC5(-/-) mice and littermate controls with a lung adenocarcinoma cell line. Cumulatively, lack of HDAC5 did not lead to better anticancer immunity. We found that CD8(+) T cells missing HDAC5 had a reduced ability to produce the cytokine, IFN-γ, in vitro and in vivo, which may offset the benefit of weakened Treg function and formation. Taken together, targeting HDAC5 weakens suppressive function and de-novo induction of Tregs, but also reduces the ability of CD8(+) T cells to produce IFN-γ.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Línea Celular Tumoral , Citocinas/biosíntesis , Factores de Transcripción Forkhead/metabolismo , Eliminación de Gen , Marcación de Gen , Interferón gamma/biosíntesis , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Conventional T (Tcon) cells and Foxp3(+) T-regulatory (Treg) cells are thought to have differing metabolic requirements, but little is known of mitochondrial functions within these cell populations in vivo. In murine studies, we found that activation of both Tcon and Treg cells led to myocyte enhancer factor 2 (Mef2)-induced expression of genes important to oxidative phosphorylation (OXPHOS). Inhibition of OXPHOS impaired both Tcon and Treg cell function compared to wild-type cells but disproportionally affected Treg cells. Deletion of Pgc1α or Sirt3, which are key regulators of OXPHOS, abrogated Treg-dependent suppressive function and impaired allograft survival. Mef2 is inhibited by histone/protein deacetylase-9 (Hdac9), and Hdac9 deletion increased Treg suppressive function. Hdac9(-/-) Treg showed increased expression of Pgc1α and Sirt3, and improved mitochondrial respiration, compared to wild-type Treg cells. Our data show that key OXPHOS regulators are required for optimal Treg function and Treg-dependent allograft acceptance. These findings provide a novel approach to increase Treg function and give insights into the fundamental mechanisms by which mitochondrial energy metabolism regulates immune cell functions in vivo.
Asunto(s)
Metabolismo Energético/inmunología , Factores de Transcripción Forkhead/inmunología , Supervivencia de Injerto/inmunología , Mitocondrias/inmunología , Linfocitos T Reguladores/inmunología , Animales , Western Blotting , Metabolismo Energético/genética , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Supervivencia de Injerto/genética , Histona Desacetilasas/genética , Histona Desacetilasas/inmunología , Histona Desacetilasas/metabolismo , Factores de Transcripción MEF2/inmunología , Factores de Transcripción MEF2/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Fosforilación Oxidativa , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Especies Reactivas de Oxígeno/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/inmunología , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirtuina 3/genética , Sirtuina 3/inmunología , Sirtuina 3/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T Reguladores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismoRESUMEN
Protocols to use Foxp3+ T-regulatory (Treg) cells for cellular therapy, especially postallogeneic stem cell transplantation, are currently being developed and tested by various groups. Inhibitors of DNA methyltransferase (Dnmt) enzymes have been advocated as a means to promote and stabilize Foxp3 expression in Tregs undergoing expansion in vitro before their injection in vivo. We investigated the effects of conditionally deleting two Dnmt enzymes that co-immunoprecipitated with Foxp3 in Treg isolates. Deletion of Dnmt1, but not Dnmt3a, decreased the numbers and function of peripheral Tregs and impaired conversion of conventional T cells into Foxp3+ Tregs under polarizing conditions. Importantly, mice with conditional deletion of Dnmt1 in their Tregs died of autoimmunity by 3 to 4 weeks of age unless they were rescued by perinatal transfer of wild-type Tregs. Conditional Dnmt1 deletion did not affect methylation of CpG sites within Foxp3 but decreased global DNA methylation and altered Treg expression of several hundred pro-inflammatory and other genes. Hence, Dnmt1 is necessary for maintenance of the core gene program underlying Treg development and function, and its deletion within the Treg lineage leads to lethal autoimmunity. These data suggest that caution may be warranted when considering the use of DNMT inhibitors in development of Treg-based cellular therapies.
Asunto(s)
Enfermedades Autoinmunes/mortalidad , Enfermedades Autoinmunes/prevención & control , ADN (Citosina-5-)-Metiltransferasas/genética , Factores de Transcripción Forkhead/metabolismo , Linfocitos T Reguladores/fisiología , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Autoinmunidad/genética , Autoinmunidad/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/fisiología , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica/fisiología , Terapia Genética , Inmunoterapia Adoptiva/efectos adversos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/trasplanteRESUMEN
Histone deacetylases 1 and 2 play a major role in the transcriptional regulation of T-regulatory (Treg) cells via interactions with a myriad of coregulatory factors. Sin3a has been well established as a Hdac1/2 cofactor, while its role within Tregs has not been established. In this study, the effects of conditional deletion of Sin3a within Foxp3+ Tregs were evaluated. Developmental deletion of Sin3a from Foxp3+ Tregs resulted in the rapid onset of fatal autoimmunity. Treg numbers were greatly reduced, while residual Tregs had impaired suppressive function. Mice also showed effector T-cell activation, autoantibody production, and widespread tissue injury. Mechanistically, Sin3a deletion resulted in decreased transcription of Foxp3 with a complete lack of CNS2 CpG demethylation. In addition, Foxp3 protein stability was impaired with an increased ex-Treg population. Thus, Sin3a plays a critical role in the maintenance of Treg identity and function and is essential for the expression and stability of Foxp3.
Asunto(s)
Factores de Transcripción Forkhead , Complejo Correpresor Histona Desacetilasa y Sin3 , Linfocitos T Reguladores , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Animales , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3/genética , Ratones , Ratones Noqueados , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Regulación de la Expresión Génica , Autoinmunidad , Ratones Endogámicos C57BL , Activación de Linfocitos/inmunologíaRESUMEN
The Mads/Mef2 (Mef2a/b/c/d) family of transcription factors (TFs) regulates differentiation of muscle cells, neurons and hematopoietic cells. By functioning in physiological feedback loops, Mef2 TFs promote the transcription of their repressor, Hdac9, thereby providing temporal control of Mef2-driven differentiation. Disruption of this feedback is associated with the development of various pathologic states, including cancer. Beside their direct involvement in oncogenesis, Mef2 TFs indirectly control tumor progression by regulating antitumor immunity. We recently reported that in CD4+CD25+Foxp3+ T-regulatory (Treg) cells, Mef2d is required for the acquisition of an effector Treg (eTreg) phenotype and for the activation of an epigenetic program that suppresses the anti-tumor immune responses of conventional T and B cells. We now report that as with Mef2d, the deletion of Mef2c in Tregs switches off the expression of Il10 and Icos and leads to enhanced antitumor immunity in syngeneic models of lung cancer. Mechanistically, Mef2c does not directly bind the regulatory elements of Icos and Il10, but its loss-of-function in Tregs induces the expression of the transcriptional repressor, Hdac9. As a consequence, Mef2d, the more abundant member of the Mef2 family, is converted by Hdac9 into a transcriptional repressor on these loci. This leads to the impairment of Treg suppressive properties in vivo and to enhanced anti-cancer immunity. These data further highlight the central role played by the Mef2/Hdac9 axis in the regulation of CD4+Foxp3+ Treg function and adds a new level of complexity to the analysis and study of Treg biology.
Asunto(s)
Histona Desacetilasas/inmunología , Tolerancia Inmunológica , Neoplasias Pulmonares/inmunología , Neoplasias Experimentales/inmunología , Proteínas Represoras/inmunología , Linfocitos T Reguladores/inmunología , Animales , Histona Desacetilasas/genética , Inmunidad Celular , Neoplasias Pulmonares/genética , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neoplasias Experimentales/genética , Proteínas Represoras/genéticaRESUMEN
Foxp3+ T-regulatory (Treg) cells are capable of suppressing immune responses. Lysine acetylation is a key mechanism of post-translational control of various transcription factors, and when acetylated, Foxp3 is stabilized and transcriptionally active. Therefore, understanding the roles of various histone/protein deacetylases (HDAC) are key to promoting Treg-based immunotherapy. Several of the 11 classical HDAC enzymes are necessary for optimal Treg function while others are dispensable. We investigated the effect of HDAC10 in murine Tregs. HDAC10 deletion had no adverse effect on the health of mice, which retained normal CD4+ and CD8+ T cell function. However, HDAC10-/- Treg exhibited increased suppressive function in vitro and in vivo. C57BL/6 Rag1-/- mice adoptively transferred with HDAC10-/- but not wild Treg, were protected from developing colitis. HDAC10-/- but not wild-type mice receiving fully MHC-mismatched cardiac transplants became tolerant and showed long-term allograft survival (>100 d). We conclude that targeting of HDAC10 may be of therapeutic value for inflammatory disorders including colitis and also for transplantation.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Eliminación de Gen , Linfocitos T Reguladores/citología , Animales , Colitis/genética , Colitis/inmunología , Células HEK293 , Trasplante de Corazón/efectos adversos , Humanos , Ratones , Tolerancia al Trasplante/genéticaRESUMEN
BACKGROUND: Vascularized composite allotransplantation (VCA) is a novel and life-enhancing procedure to restore a patient's function and/or appearance. Current immunosuppression in VCA recipients is based on calcineurin inhibitor (CNI) therapy that can lead to severe complications, such that inducing immune tolerance is a major goal of VCA research. In contrast to CNI, rapamycin (RPM) is thought to be beneficial to the development of immune tolerance by suppressing T-effector cells (Teffs) and expanding T-regulatory (Treg) cells. However, we found high dose RPM monotherapy prolonged VCA survival by only a few days, leading us to explore the mechanisms responsible. METHODS: A mouse orthotopic forelimb transplantation model (BALB/c- > C57BL/6) was established using WT mice, as well as C57BL/6 recipients with conditional deletion of T-bet within their Treg cells. Events in untreated VCA recipients or those receiving RPM or FK506 therapy were analyzed by flow-cytometry, histopathology and real-time qPCR. RESULTS: Therapy with RPM (2 mg/kg/d, p < .005) or FK506 (2 mg/kg/d, p < .005) each prolonged VCA survival. In contrast to FK506, RPM increased the ratio of splenic Treg to Teff cells (p < .05) by suppressing Teff and expanding Treg cells. While the proportion of activated splenic CD4 + Foxp3- T cells expressing IFN-γ were similar in control and RPM-treated groups, RPM decreased the proportions ICOS+ and CD8+ IFN-γ + splenic T cells. However, RPM also downregulated CXCR3+ expression by Tregs, and forelimb allografts had reduced infiltration by CXCR3+ Treg cells. In addition, allograft recipients whose Tregs lacked T-bet underwent accelerated rejection compared to WT mice despite RPM therapy. CONCLUSIONS: We demonstrate that while RPM increased the ratio of Treg to Teff cells and suppressed CD8+ T cell allo-activation, it failed to prevent CD4 Teff cell activation and impaired CXCR3-dependent Treg graft homing, thereby limiting the efficacy of RPM in VCA recipients.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Miembro Anterior/inmunología , Inmunosupresores/uso terapéutico , Sirolimus/uso terapéutico , Tacrolimus/uso terapéutico , Alotrasplante Compuesto Vascularizado , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Miembro Anterior/trasplante , Técnicas de Silenciamiento del Gen , Humanos , Tolerancia Inmunológica , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores CXCR3/metabolismo , Proteínas de Dominio T Box/genéticaRESUMEN
Vascularized composite allotransplantation (VCA) allows tissue replacement after devastating loss but is currently limited in application and may be more widely performed if maintenance immunosuppression was not essential for graft acceptance. We tested whether peri-transplant costimulation blockade could prolong VCA survival and required donor bone-marrow cells, given that bone-marrow might promote graft immunogenicity or graft-versus-host disease. Peritransplant CD154 mAb/rapamycin (RPM) induced long-term orthotopic hindlimb VCA survival (BALB/c->C57BL/6), as did CTLA4Ig/RPM. Surprisingly, success of either protocol required a bone-marrow-associated, radiation-sensitive cell population, since long-bone removal or pre-transplant donor irradiation prevented long-term engraftment. Rejection also occurred if Rag1-/- donors were used, or if donors were treated with a CXCR4 inhibitor to mobilize donor BM cells pre-transplant. Donor bone-marrow contained a large population of Foxp3+ T-regulatory (Treg) cells, and donor Foxp3+ Treg depletion, by diphtheria toxin administration to DEREG donor mice whose Foxp3+ Treg cells expressed diphtheria toxin receptor, restored rejection with either protocol. Rejection also occurred if CXCR4 was deleted from donor Tregs pre-transplant. Hence, long-term VCA survival is possible across a full MHC disparity using peritransplant costimulation blockade-based approaches, but unexpectedly, the efficacy of costimulation blockade requires the presence of a radiation-sensitive, CXCR4+ Foxp3+ Treg population resident within donor BM.
Asunto(s)
Trasplante de Médula Ósea , Extremidades/trasplante , Supervivencia de Injerto/fisiología , Linfocitos T Reguladores/inmunología , Alotrasplante Compuesto Vascularizado/métodos , Abatacept/farmacología , Animales , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Ligando de CD40/inmunología , Toxina Diftérica/farmacología , Factores de Transcripción Forkhead/metabolismo , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Enfermedad Injerto contra Huésped/patología , Enfermedad Injerto contra Huésped/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Sirolimus/farmacologíaRESUMEN
Foxp3+ Tregs are key to immune homeostasis, but the contributions of various large, multiprotein complexes that regulate gene expression remain unexplored. We analyzed the role in Tregs of the evolutionarily conserved CoREST complex, consisting of a scaffolding protein, Rcor1 or Rcor2, plus Hdac1 or Hdac2 and Lsd1 enzymes. Rcor1, Rcor2, and Lsd1 were physically associated with Foxp3, and mice with conditional deletion of Rcor1 in Foxp3+ Tregs had decreased proportions of Tregs in peripheral lymphoid tissues and increased Treg expression of IL-2 and IFN-γ compared with what was found in WT cells. Mice with conditional deletion of the gene encoding Rcor1 in their Tregs had reduced suppression of homeostatic proliferation, inability to maintain long-term allograft survival despite costimulation blockade, and enhanced antitumor immunity in syngeneic models. Comparable findings were seen in WT mice treated with CoREST complex bivalent inhibitors, which also altered the phenotype of human Tregs and impaired their suppressive function. Our data point to the potential for therapeutic modulation of Treg functions by pharmacologic targeting of enzymatic components of the CoREST complex and contribute to an understanding of the biochemical and molecular mechanisms by which Foxp3 represses large gene sets and maintains the unique properties of this key immune cell.
Asunto(s)
Proteínas Co-Represoras/inmunología , Inmunidad Celular , Complejos Multiproteicos/inmunología , Neoplasias Experimentales/inmunología , Proteínas del Tejido Nervioso/inmunología , Linfocitos T Reguladores/inmunología , Animales , Línea Celular Tumoral , Proteínas Co-Represoras/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Complejos Multiproteicos/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Proteínas del Tejido Nervioso/genética , Linfocitos T Reguladores/patologíaRESUMEN
The transcription factor MEF2D is important in the regulation of differentiation and adaptive responses in many cell types. We found that among T cells, MEF2D gained new functions in Foxp3+ T regulatory (Treg) cells due to its interactions with the transcription factor Foxp3 and its release from canonical partners, like histone/protein deacetylases. Though not necessary for the generation and maintenance of Tregs, MEF2D was required for the expression of IL-10, CTLA4, and Icos, and for the acquisition of an effector Treg phenotype. At these loci, MEF2D acted both synergistically and additively to Foxp3, and downstream of Blimp1. Mice with the conditional deletion in Tregs of the gene encoding MEF2D were unable to maintain long-term allograft survival despite costimulation blockade, had enhanced antitumor immunity in syngeneic models, but displayed only minor evidence of autoimmunity when maintained under normal conditions. The role played by MEF2D in sustaining effector Foxp3+ Treg functions without abrogating their basal actions suggests its suitability for drug discovery efforts in cancer therapy.
Asunto(s)
Supervivencia de Injerto/inmunología , Trasplante de Corazón , Activación de Linfocitos , Neoplasias Experimentales/inmunología , Linfocitos T Reguladores/inmunología , Animales , Supervivencia de Injerto/genética , Células HEK293 , Humanos , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neoplasias Experimentales/genética , Linfocitos T Reguladores/patología , Trasplante IsogénicoRESUMEN
Functions of the GCN5-related N-acetyltransferase (GNAT) family of histone/protein acetyltransferases (HATs) in Foxp3+ T-regulatory (Treg) cells are unexplored, despite the general importance of these enzymes in cell biology. We now show that two prototypical GNAT family members, GCN5 (general control nonrepressed-protein 5, lysine acetyltransferase (KAT)2a) and p300/CBP-associated factor (p300/CBP-associated factor (PCAF), Kat2b) contribute to Treg functions through partially distinct and partially overlapping mechanisms. Deletion of Gcn5 or PCAF did not affect Treg development or suppressive function in vitro, but did affect inducible Treg (iTreg) development, and in vivo, abrogated Treg-dependent allograft survival. Contrasting effects were seen upon targeting of each HAT in all T cells; mice lacking GCN5 showed prolonged allograft survival, suggesting this HAT might be a target for epigenetic therapy in allograft recipients, whereas transplants in mice lacking PCAF underwent acute allograft rejection. PCAF deletion also enhanced anti-tumor immunity in immunocompetent mice. Dual deletion of GCN5 and PCAF led to decreased Treg stability and numbers in peripheral lymphoid tissues, and mice succumbed to severe autoimmunity by 3-4 weeks of life. These data indicate that HATs of the GNAT family have contributions to Treg function that cannot be replaced by the functions of previously characterized Treg HATs (CBP, p300, and Tip60), and may be useful targets in immuno-oncology.
RESUMEN
BACKGROUND: Vascularized composite allografts (VCA) are novel, life-enhancing forms of transplantation (Tx). However, host immune responses to the various VCA components, especially those involving skin, are complex and make selection of appropriate therapy challenging. Although the interplay between Foxp3+ T regulatory (Treg) cells and CD4 and CD8 effector T cells is of central importance in determining the acceptance or rejection of solid organ allografts, there is little information available concerning the contribution of Treg cells to VCA survival. In addition, the effects of therapeutic expansion in vivo of host Treg cell populations on VCA survival are unknown. METHODS: We established a fully major histocompatibility complex-disparate (BALB/c- > C57BL/6) murine orthotopic forelimb Tx model to explore the benefits of pre- and post-Tx IL-2/anti-IL-2 monoclonal antibody complex (IL-2C) administration to expand the host Treg cell population and thereby attempt to promote Treg cell-dependent VCA survival. RESULTS: Both strategies expanded the Treg cell population in vivo and prolonged VCA survival (P < 0.001), but IL-2C administration pre-Tx led to significantly longer survival compared with IL-2C administration post-Tx (P < 0.01). In addition, compared with post-Tx therapy, pre-Tx therapy resulted in an increased ratio of Treg cells to CD8+ T cells (P < 0.001), reduced proliferation of CD4 and CD8 effector T cells, and reduced production of IFN-γ. Optimal effects were seen when combined with rapamycin therapy, whereas the combination of IL-2C therapy plus calcineurin inhibitor was counterproductive. CONCLUSIONS: Our studies involving different IL-2C-mediated Treg cell expansion strategies demonstrate that pre-Tx IL-2C therapy may be a useful component for developing strategies to promote VCA survival.
Asunto(s)
Aloinjertos Compuestos , Miembro Anterior/trasplante , Supervivencia de Injerto/efectos de los fármacos , Interleucina-2/farmacología , Animales , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T Reguladores/inmunologíaRESUMEN
Although almost every known chemokine and chemokine receptor is expressed at some stage during development of allograft rejection, mechanistic studies indicate that the actual key effector mechanisms are rather few. Thus, in vivo studies have alleviated concerns regarding possible biological redundancy and the pleiotropic effects of these molecules, and have resulted in a focus on CXCR3, CCR5 and their respective ligands as key mediators of host alloresponses, especially in acute rejection. Data are also accruing regarding the importance of chemokine/chemokine receptor pathways in ischemia/reperfusion, chronic rejection and tolerance induction following co-stimulation blockade, providing new targets for immune monitoring and therapeutic intervention.
Asunto(s)
Quimiocinas/inmunología , Rechazo de Injerto/inmunología , Terapia de Inmunosupresión , Receptores de Quimiocina/inmunología , Quimiocinas/genética , Humanos , Polimorfismo Genético , Receptores CCR5/inmunología , Receptores de Quimiocina/genética , Daño por ReperfusiónRESUMEN
Determining protein acetylation by immunoprecipitation and immunoblotting can be challenging, especially if the tissue of interest is low in quantity, and when good quality acetylation site-specific antibodies are not available. Proximity ligation assays allow a sensitive and quantitative method to assess Foxp3 acetylation in regulatory T cells, with as little as 1.5 × 105 cells within two days turnaround time. This method is of potential use in other similar scenarios, when post-translational modifications of a protein of interest need to be determined with only a small amount of sample and in the absence of specific antibodies that can assess the post-translational modification in the protein of interest.
Asunto(s)
Bioensayo , Factores de Transcripción Forkhead/metabolismo , Separación Inmunomagnética/métodos , Procesamiento Proteico-Postraduccional , Linfocitos T Reguladores/metabolismo , Acetilación , Animales , Anticuerpos/química , Factores de Transcripción Forkhead/genética , Inmunoprecipitación , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Bazo/citología , Bazo/metabolismo , Linfocitos T Reguladores/citologíaRESUMEN
Current interest in Foxp3+ T-regulatory (Treg) cells as therapeutic targets in transplantation is largely focused on their harvesting pre-transplant, expansion and infusion post-transplantation. An alternate strategy of pharmacologic modulation of Treg function using histone/protein deacetylase inhibitors (HDACi) may allow more titratable and longer-term dosing. However, the effects of broadly acting HDACi vary, such that HDAC isoform-selective targeting is likely required. We report data from mice with constitutive or conditional deletion of HDAC11 within Foxp3+ Treg cells, and their use, along with small molecule HDAC11 inhibitors, in allograft models. Global HDAC11 deletion had no effect on health or development, and compared to WT controls, Foxp3+ Tregs lacking HDAC11 showed increased suppressive function, and increased expression of Foxp3 and TGF-ß. Likewise, compared to WT recipients, conditional deletion of HDAC11 within Tregs led to long-term survival of fully MHC-mismatched cardiac allografts, and prevented development of transplant arteriosclerosis in an MHC class II-mismatched allograft model. The translational significance of HDAC11 targeting was shown by the ability of an HDAC11i to promote long-term allograft allografts in fully MHC-disparate strains. These data are powerful stimuli for the further development and testing of HDAC11-selective pharmacologic inhibitors, and may ultimately provide new therapies for transplantation and autoimmune diseases.
Asunto(s)
Factores de Transcripción Forkhead/inmunología , Histona Desacetilasas/inmunología , Histonas/inmunología , Linfocitos T Reguladores/inmunología , Aloinjertos , Animales , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica/métodos , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/genética , Supervivencia de Injerto/inmunología , Células HEK293 , Trasplante de Corazón/métodos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Linfocitos T Reguladores/metabolismoRESUMEN
Immune cells function in diverse metabolic environments. Tissues with low glucose and high lactate concentrations, such as the intestinal tract or ischemic tissues, frequently require immune responses to be more pro-tolerant, avoiding unwanted reactions against self-antigens or commensal bacteria. T-regulatory cells (Tregs) maintain peripheral tolerance, but how Tregs function in low-glucose, lactate-rich environments is unknown. We report that the Treg transcription factor Foxp3 reprograms T cell metabolism by suppressing Myc and glycolysis, enhancing oxidative phosphorylation, and increasing nicotinamide adenine dinucleotide oxidation. These adaptations allow Tregs a metabolic advantage in low-glucose, lactate-rich environments; they resist lactate-mediated suppression of T cell function and proliferation. This metabolic phenotype may explain how Tregs promote peripheral immune tolerance during tissue injury but also how cancer cells evade immune destruction in the tumor microenvironment. Understanding Treg metabolism may therefore lead to novel approaches for selective immune modulation in cancer and autoimmune diseases.
Asunto(s)
Microambiente Celular/inmunología , Reprogramación Celular/inmunología , Factores de Transcripción Forkhead/inmunología , Glucosa/inmunología , Ácido Láctico/inmunología , Linfocitos T Reguladores/inmunología , Animales , Línea Celular , Microambiente Celular/genética , Reprogramación Celular/genética , Factores de Transcripción Forkhead/genética , Glucosa/genética , Glucólisis/genética , Glucólisis/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fosforilación Oxidativa , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunologíaRESUMEN
BACKGROUND: Given the shortage of liver donors and the development of techniques for partial liver transplantation, we compared chemokine expression and inflammatory cell infiltration of partial versus whole grafts in a mouse syngeneic liver transplant model. METHODS: Orthotopic liver transplantation, using whole or partial murine liver grafts, was performed following cold preservation in ViaSpan solution for periods of one to eight hours. RESULTS: Partial grafts showed more severe cold ischemia/reperfusion injury and greater inflammatory cell infiltration than whole grafts, and was accompanied by the marked intrahepatic upregulation of multiple chemokines. Quantitative analysis showed that compared with expression in whole grafts harvested after the same period of cold ischemia, partial grafts had eightfold more T-cell activation gene (TCA)-3 (CCL1) chemokine messenger RNA (mRNA) expression (P<0.01) and sixfold more inducible protein (IP)-10 chemokine (CCL10) mRNA expression (P<0.01), as well as increased expression of the chemokine receptors CCR8 (receptor for TCA3) and CXCR3 (receptor for IP-10; P<0.01). Blockade of TCA3 by neutralizing monoclonal antibody significantly decreased intragraft IP-10 expression (P<0.05) but not tumor necrosis factor-alpha or interleukin-6 expression in partial grafts, and significantly decreased cold ischemia/reperfusion injury (P<0.05) and associated neutrophil and T-cell infiltration (P<0.01). CONCLUSIONS: These data demonstrate that the chemokine TCA3/CCL1 is important to the pathogenesis of ischemic injury of experimental partial liver grafts, and that its therapeutic targeting within such grafts can overcome the deleterious effects of prolonged cold preservation and restore liver function to the level achieved using whole liver grafts.
Asunto(s)
Quimiocinas CC/antagonistas & inhibidores , Isquemia Fría , Trasplante de Hígado , Preservación de Órganos , Daño por Reperfusión/prevención & control , Animales , Anticuerpos Monoclonales/farmacología , Quimiocina CCL1 , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Criopreservación , Masculino , Ratones , Infiltración Neutrófila/efectos de los fármacos , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptores CCR8 , Receptores CXCR3 , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Linfocitos T/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Foxp3+ T-regulatory (Treg) cells are known to suppress protective host immune responses to a wide variety of solid tumors, but their therapeutic targeting is largely restricted to their transient depletion or "secondary" modulation, e.g. using anti-CTLA-4 monoclonal antibody. Our ongoing studies of the post-translational modifications that regulate Foxp3 demonstrated that the histone/protein acetyltransferase, Tip60, plays a dominant role in promoting acetylation, dimerization and function in Treg cells. We now show that the ubiquitin-specific protease, Usp7, controls Treg function largely by stabilizing the expression and promoting the multimerization of Tip60 and Foxp3. Genetic or pharmacologic targeting of Usp7 impairs Foxp3+ Treg suppressive functions, while conventional T cell responses remain intact. As a result, pharmacologic inhibitors of Usp7 can limit tumor growth in immunocompetent mice, and promote the efficacy of antitumor vaccines and immune checkpoint therapy with anti-PD1 monoclonal antibody in murine models. Hence, pharmacologic therapy with Usp7 inhibitors may have an important role in future cancer immunotherapy.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Histona Acetiltransferasas/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Transactivadores/metabolismo , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Animales , Autoinmunidad/genética , Autoinmunidad/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Expresión Génica , Inmunidad , Activación de Linfocitos/inmunología , Lisina Acetiltransferasa 5 , Ratones , Ratones Noqueados , Ratones Transgénicos , Neoplasias/patología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Carga Tumoral , Peptidasa Específica de Ubiquitina 7 , Proteasas Ubiquitina-Específicas/genéticaRESUMEN
Treg dysfunction is associated with a variety of inflammatory diseases. Treg populations are defined by expression of the oligomeric transcription factor FOXP3 and inability to produce IL-2, a cytokine required for T cell maintenance and survival. FOXP3 activity is regulated post-translationally by histone/protein acetyltransferases and histone/protein deacetylases (HDACs). Here, we determined that HDAC3 mediates both the development and function of the two main Treg subsets, thymus-derived Tregs and induced Tregs (iTregs). We determined that HDAC3 and FOXP3 physically interact and that HDAC3 expression markedly reduces Il2 promoter activity. In murine models, conditional deletion of Hdac3 during thymic Treg development restored Treg production of IL-2 and blocked the suppressive function of Tregs. HDAC3-deficient mice died from autoimmunity by 4-6 weeks of age; however, injection of WT FOXP3+ Tregs prolonged survival. Adoptive transfer of Hdac3-deficient Tregs, unlike WT Tregs, did not control T cell proliferation in naive mice and did not prevent allograft rejection or colitis. HDAC3 also regulated the development of iTregs, as HDAC3-deficient conventional T cells were not converted into iTregs under polarizing conditions and produced large amounts of IL-2, IL-6, and IL-17. We conclude that HDAC3 is essential for the normal development and suppressive functions of thymic and peripheral FOXP3+ Tregs.