RESUMEN
BACKGROUND: Regulatory T cell (Treg) therapy is a promising approach to amelioration of allograft rejection and promotion of organ transplant tolerance. However, the fate of infused Treg, and how this relates to their therapeutic efficacy using different immunosuppressive regimens is poorly understood. Our aim was to analyze the tissue distribution, persistence, replicative activity and phenotypic stability of autologous, donor antigen alloreactive Treg (darTreg) in anti-thymocyte globulin (ATG)-lymphodepleted, heart-allografted cynomolgus monkeys. METHODS: darTreg were expanded ex vivo from flow-sorted, circulating Treg using activated donor B cells and infused posttransplant into recipients of major histocompatibility complex-mismatched heart allografts. Fluorochrome-labeled darTreg were identified and characterized in peripheral blood, lymphoid, and nonlymphoid tissues and the graft by flow cytometric analysis. RESULTS: darTreg selectively suppressed autologous T cell responses to donor antigens in vitro. However, following their adoptive transfer after transplantation, graft survival was not prolonged. Early (within 2 wk posttransplant; under ATG, tacrolimus, and anti-IL-6R) or delayed (6-8 wk posttransplant; under rapamycin) darTreg infusion resulted in a rapid decline in transferred darTreg in peripheral blood. Following their early or delayed infusion, labeled cells were evident in lymphoid and nonlymphoid organs and the graft at low percentages (<4% CD4+ T cells). Notably, infused darTreg showed reduced expression of immunoregulatory molecules (Foxp3 and CTLA4), Helios, the proliferative marker Ki67 and antiapoptotic Bcl2, compared with preinfusion darTreg and endogenous CD4+CD25hi Treg. CONCLUSIONS: Lack of therapeutic efficacy of infused darTreg in lymphodepleted heart graft recipients appears to reflect loss of a regulatory signature and proliferative and survival capacity shortly after infusion.
Asunto(s)
Traslado Adoptivo , Suero Antilinfocítico/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Proliferación Celular , Rechazo de Injerto/prevención & control , Supervivencia de Injerto , Trasplante de Corazón , Activación de Linfocitos , Depleción Linfocítica , Linfocitos T Reguladores/trasplante , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Rechazo de Injerto/inmunología , Rechazo de Injerto/metabolismo , Trasplante de Corazón/efectos adversos , Macaca fascicularis , Masculino , Fenotipo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factores de TiempoRESUMEN
Liver interstitial dendritic cells (DCs) have been implicated in the control of ischemia-reperfusion injury (IRI) and host immune responses following liver transplantation. Mechanisms underlying these regulatory functions of hepatic DCs remain unclear. We have shown recently that the transmembrane immunoadaptor DNAX-activating protein of 12 kDa (DAP12) negatively regulates mouse liver DC maturation and proinflammatory and immune stimulatory functions. Here, we used PCR analysis and flow cytometry to characterize expression of DAP12 and its associated triggering receptor, triggering receptor expressed on myeloid cells 2 (TREM2), by mouse and human liver DCs and other immune cells compared with DCs in other tissues. We also examined the roles of DAP12 and TREM2 and their expression by liver DCs in the regulation of liver IRI. Injury was induced in DAP12-/- , TREM2-/- , or wild-type (WT) mice by 1 hour of 70% clamping and quantified following 6 hours of reperfusion. Both DAP12 and TREM2 were coexpressed at comparatively high levels by liver DCs. Mouse liver DCs lacking DAP12 or TREM2 displayed enhanced levels of nuclear factor κB and costimulatory molecule expression. Unlike normal WT liver DCs, DAP12-/- liver DC failed to inhibit proliferative responses of activated T cells. In vivo, DAP12-/- and TREM2-/- mice exhibited enhanced IRI accompanied by augmented liver DC activation. Elevated alanine aminotransferase levels and tissue injury were markedly reduced by infusion of WT but not DAP12-/- DC. Conclusion: Our data reveal a close association between DAP12 and TREM2 expression by liver DC and suggest that, by negatively regulating liver DC stimulatory function, DAP12 promotes their control of hepatic inflammatory responses; the DAP12/TREM2 signaling complex may represent a therapeutic target for control of acute liver injury/liver inflammatory disorders.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Hígado/irrigación sanguínea , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/fisiología , Receptores Inmunológicos/fisiología , Daño por Reperfusión/etiología , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Animales , Células Dendríticas/metabolismo , Humanos , Hígado/citología , Masculino , Glicoproteínas de Membrana/biosíntesis , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Receptores Inmunológicos/biosíntesisRESUMEN
Although a key role of cross-dressing has been established in immunity to viral infection and more recently in the instigation of transplant rejection, its role in tolerance is unclear. We investigated the role of intragraft dendritic cells (DCs) and cross-dressing in mouse major histocompatibility complex (MHC)-mismatched liver transplant tolerance that occurs without therapeutic immunosuppression. Although donor interstitial DCs diminished rapidly after transplantation, they were replaced in the liver by host DCs that peaked on postoperative day (POD) 7 and persisted indefinitely. Approximately 60% of these recipient DCs displayed donor MHC class I, indicating cross-dressing. By contrast, only a very minor fraction (0%-2%) of cross-dressed DCs (CD-DCs) was evident in the spleen. CD-DCs sorted from liver grafts expressed much higher levels of T cell inhibitory programed death ligand 1 (PD-L1) and high levels of interleukin-10 compared with non-CD-DCs (nCD-DCs) isolated from the graft. Concomitantly, high incidences of programed death protein 1 (PD-1)hi T cell immunoglobulin and mucin domain containing 3 (TIM-3)+ exhausted graft-infiltrating CD8+ T cells were observed. Unlike nCD-DCs, the CD-DCs failed to stimulate proliferation of allogeneic T cells but markedly suppressed antidonor host T cell proliferation. CD-DCs were much less evident in allografts from DNAX-activating protein of 12 kDa (DAP12)-/- donors that were rejected acutely. CONCLUSION: These findings suggest that graft-infiltrating PD-L1hi CD-DCs may play a key role in the regulation of alloimmunity and in the induction of liver transplant tolerance. (Hepatology 2018;67:1499-1515).
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Células Dendríticas/inmunología , Supervivencia de Injerto/inmunología , Hígado/inmunología , Tolerancia al Trasplante/inmunología , Animales , Citometría de Flujo , Microscopía Intravital , Trasplante de Hígado/efectos adversos , Complejo Mayor de Histocompatibilidad/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Trasplante HomólogoRESUMEN
BACKGROUND: Little is known about how new-generation adenosine triphosphate-competitive mechanistic target of rapamycin (mTOR) kinase inhibitors affect immunity and allograft rejection. METHODS: mTOR complex (C) 1 and 2 signaling in dendritic cells and T cells was analyzed by Western blotting, whereas immune cell populations in normal and heart allograft recipient mice were analyzed by flow cytometry. Alloreactive T cell proliferation was quantified in mixed leukocyte reaction; intracellular cytokine production and serum antidonor IgG levels were determined by flow analysis and immunofluorescence staining used to detect IgG in allografts. RESULTS: The novel target of rapamycin kinase inhibitor AZD2014 impaired dendritic cell differentiation and T cell proliferation in vitro and depressed immune cells and allospecific T cell responses in vivo. A 9-day course of AZD2014 (10 mg/kg, intraperitoneally, twice daily) or rapamycin (RAPA) (1 mg/kg, intraperitoneally, daily) prolonged median heart allograft survival time significantly (25 days for AZD2014, 100 days for RAPA, 9.5 days for control). Like RAPA, AZD2014 suppressed graft mononuclear cell infiltration, increased regulatory T cell to effector memory T cell ratios and reduced T follicular helper and B cells 7 days posttransplant. By 21 days (10 days after drug withdrawal), however, T follicular helper and B cells and donor-specific IgG1 and IgG2c antibody titers were significantly lower in RAPA-treated compared with AZD2014-treated mice. Elevated regulatory T cell to effector memory T cell ratios were maintained after RAPA, but not AZD2014 withdrawal. CONCLUSIONS: Immunomodulatory effects of AZD2014, unlike those of RAPA, were not sustained after drug withdrawal, possibly reflecting distinct pharmacokinetics or/and inhibitory effects of AZD2014 on mTORC2.
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Adenosina Trifosfato/química , Rechazo de Injerto , Trasplante de Corazón , Sistema Inmunológico/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Morfolinas/farmacología , Animales , Benzamidas , Proliferación Celular , Células Dendríticas/citología , Supervivencia de Injerto/efectos de los fármacos , Inmunoglobulina G/química , Inmunosupresores/farmacología , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas , Sirolimus/farmacología , Linfocitos T/citología , Trasplante HomólogoRESUMEN
BACKGROUND: Auxiliary partial liver transplantation (APLT) in humans is a therapeutic modality used especially to treat liver failure in children or congenital metabolic disease. Animal models of APLT have helped to explore therapeutic options. Though many groups have suggested improvements, standardizing the surgical procedure has been challenging. Additionally, the question of whether graft livers are reconstituted by recipient-derived cells after transplantation has been controversial. The aim of this study was to improve experimental APLT in rats and to assess cell recruitment in the liver grafts. METHODS: To inhibit recipient liver regeneration and to promote graft regeneration, we treated recipients with retrorsine and added arterial anastomosis. Using green fluorescence protein transgenic rats as recipients, we examined liver resident cell recruitment within graft livers by immunofluorescence costaining. RESULTS: In the improved APLT model, we achieved well-regenerated grafts that could maintain regeneration for at least 4 weeks. Regarding the cell recruitment, there was no evidence of recipient-derived hepatocyte, cholangiocyte, or hepatic stellate cell recruitment into the graft. Macrophages/monocytes, however, were consistently recruited into the graft and increased over time, which might be related to inflammatory responses. Very few endothelial cells showed colocalization of markers. CONCLUSIONS: We have successfully established an improved rat APLT model with arterial anastomosis as a standard technique. Using this model, we have characterized cell recruitment into the regenerating grafts.
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Movimiento Celular , Proliferación Celular , Regeneración Hepática , Trasplante de Hígado/métodos , Hígado/cirugía , Animales , Linaje de la Célula , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Hígado/fisiopatología , Regeneración Hepática/efectos de los fármacos , Masculino , Modelos Animales , Alcaloides de Pirrolicidina/farmacología , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Ratas Transgénicas , Factores de TiempoAsunto(s)
Rechazo de Injerto/inmunología , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Células Mieloides/inmunología , Traslado Adoptivo , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Enfermedad Injerto contra Huésped/metabolismo , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Células Mieloides/clasificación , Células Mieloides/metabolismo , Células Mieloides/trasplante , FenotipoRESUMEN
The surgically demanding mouse orthotopic liver transplant model was first described in 1991. It has proved to be a powerful research tool for the investigation of liver biology, tissue injury, the regulation of alloimmunity and tolerance induction, and the pathogenesis of specific liver diseases. Liver transplantation in mice has unique advantages over transplantation of the liver in larger species, such as the rat or pig, because the mouse genome is well characterized and there is much greater availability of both genetically modified animals and research reagents. Liver transplant experiments using various transgenic or gene knockout mice have provided valuable mechanistic insights into the immunobiology and pathobiology of the liver and the regulation of graft rejection and tolerance over the past 25 years. The molecular pathways identified in the regulation of tissue injury and promotion of liver transplant tolerance provide new potential targets for therapeutic intervention to control adverse inflammatory responses/immune-mediated events in the hepatic environment and systemically. In conclusion, orthotopic liver transplantation in the mouse is a valuable model for gaining improved insights into liver biology, immunopathology, and allograft tolerance that may result in therapeutic innovation in the liver and in the treatment of other diseases.
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Rechazo de Injerto/inmunología , Trasplante de Hígado/veterinaria , Hígado/inmunología , Daño por Reperfusión/veterinaria , Tolerancia al Trasplante , Animales , Rechazo de Injerto/veterinaria , Células Madre Hematopoyéticas/inmunología , Hígado/fisiopatología , Hígado/cirugía , Hepatopatías/etiología , Trasplante de Hígado/métodos , Ratones , Modelos Animales , Daño por Reperfusión/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Memory T cells (Tmem), particularly those resistant to costimulation blockade (CB), are a major barrier to transplant tolerance. The transcription factor Eomesodermin (Eomes) is critical for Tmem development and maintenance, but its expression by alloactivated T cells has not been examined in nonhuman primates. METHODS: We evaluated Eomes and coinhibitory cytotoxic T lymphocyte antigen-4 (CTLA4) expression by alloactivated rhesus monkey T cells in the presence of CTLA4 immunoglobulin, both in vitro and in renal allograft recipients treated with CTLA4Ig, with or without regulatory dendritic cell (DCreg) infusion. RESULTS: In normal monkeys, CD8+ T cells expressed significantly more Eomes than CD4+ T cells. By contrast, CD8+ T cells displayed minimal CTLA4. Among T cell subsets, central Tmem (Tcm) expressed the highest levels of Eomes. Notably, Eomes(lo)CTLA4(hi) cells displayed higher levels of CD25 and Foxp3 than Eomes(hi)CTLA4(lo) CD8+ T cells. After allostimulation, distinct proliferating Eomes(lo)CTLA4(hi) and Eomes(hi)CTLA4(lo) CD8+ T cell populations were identified, with a high proportion of Tcm being Eomes(lo)CTLA4(hi). CB with CTLA4Ig during allostimulation of CD8+ T cells reduced CTLA4 but not Eomes expression, significantly reducing Eomes(lo)CTLA4(hi) cells. After transplantation with CB and rapamycin, donor-reactive Eomes(lo)CTLA4(hi) CD8+ T cells were reduced. However, in monkeys also given DCreg, absolute numbers of these cells were elevated significantly. CONCLUSIONS: Low Eomes and high CTLA4 expression by donor-reactive CD8+ Tmem is associated with prolonged renal allograft survival induced by DCreg infusion in CTLA4Ig-treated monkeys. Prolonged allograft survival associated with DCreg infusion may be related to maintenance of donor-reactive Eomes(lo)CTLA4(hi) Tcm.
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Abatacept/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Antígeno CTLA-4/metabolismo , Células Dendríticas/trasplante , Supervivencia de Injerto/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Inmunosupresores/farmacología , Trasplante de Riñón , Activación de Linfocitos/efectos de los fármacos , Proteínas de Dominio T Box/metabolismo , Aloinjertos , Animales , Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Antígeno CTLA-4/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunofenotipificación , Trasplante de Riñón/efectos adversos , Macaca mulatta , Masculino , Fenotipo , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Sirolimus/farmacología , Proteínas de Dominio T Box/inmunología , Factores de TiempoAsunto(s)
Inmunosupresores/uso terapéutico , Trasplante de Riñón/efectos adversos , Complejos Multiproteicos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Descubrimiento de Drogas , Humanos , Inmunosupresores/efectos adversos , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Terapia Molecular Dirigida , Complejos Multiproteicos/metabolismo , Inhibidores de Proteínas Quinasas/efectos adversos , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Resultado del TratamientoRESUMEN
UNLABELLED: Plasmacytoid dendritic cells (pDC) constitute the body's principal source of type I interferon (IFN) and are comparatively abundant in the liver. Among various cytokines implicated in liver ischemia and reperfusion (I/R) injury, type I IFNs have been described recently as playing an essential role in its pathogenesis. Moreover, type I IFNs have been shown to up-regulate hepatocyte expression of IFN regulatory factor 1 (IRF-1), a key transcription factor that regulates apoptosis and induces liver damage after I/R. Our aim was to ascertain the capacity of IFN-α released by liver pDC to induce liver damage through hepatic IRF-1 up-regulation after I/R injury. Our findings show that liver pDC mature and produce IFN-α in response to liver I/R. Liver pDC isolated after I/R induced elevated levels of IRF-1 production by hepatocytes compared with liver pDC isolated from sham-operated mice. Notably, hepatic IRF-1 expression was reduced significantly by neutralizing IFN-α. In vivo, IFN-α neutralization protected the liver from I/R injury by reducing hepatocyte apoptosis. This was associated with impaired expression of IRF-1 and proapoptotic molecules such as Fas ligand, its receptor (Fas) and death receptor 5, which are regulated by IRF-1. Furthermore, pDC-depleted mice failed to up-regulate hepatic IFN-α and displayed less liver injury associated with reduced levels of hepatic interleukin (IL)-6, tumor necrosis factor-α, and hepatocyte apoptosis after I/R compared with controls. CONCLUSION: these data support the hypothesis that IFN-α derived from liver pDC plays a key role in the pathogenesis of liver I/R injury by enhancing apoptosis as a consequence of induction of hepatocyte IRF-1 expression.
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Células Dendríticas/inmunología , Factor 1 Regulador del Interferón/inmunología , Interferón-alfa/inmunología , Hepatopatías/inmunología , Daño por Reperfusión/inmunología , Animales , Apoptosis/inmunología , Células Cultivadas , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Hepatocitos/citología , Hepatocitos/inmunología , Hepatocitos/metabolismo , Factor 1 Regulador del Interferón/metabolismo , Interferón-alfa/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Hepatopatías/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión/patología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
UNLABELLED: Hepatic innate immune cells, in particular, interstitial dendritic cells (DCs), regulate inflammatory responses and may promote inherent liver tolerogenicity. After tissue injury, adenosine triphosphate (ATP) is released and acts as a damage-associated molecular pattern that activates innate immune cells by pattern recognition receptors. CD39 (ectonucleoside triphosphate diphosphohydrolase-1) rapidly hydrolyzes extracellular ATP to maintain physiological levels. We hypothesized that CD39 expression on liver DCs might contribute to regulation of their innate immune functions. Mouse liver conventional myeloid DCs (mDCs) were hyporesponsive to ATP, compared with their splenic counterparts. This disparity was ascribed to more efficient hydrolysis of ATP by higher expression of CD39 on liver mDCs. Human liver mDCs expressed greater levels of CD39 than those from peripheral blood. The comparatively high expression of CD39 on liver mDCs correlated strongly with both ATP hydrolysis and adenosine production. Notably, CD39(-/-) mouse liver mDCs exhibited a more mature phenotype, greater responsiveness to Toll-like receptor 4 ligation, and stronger proinflammatory and immunostimulatory activity than wild-type (WT) liver mDCs. To investigate the role of CD39 on liver mDCs in vivo, we performed orthotopic liver transplantation with extended cold preservation using CD39(-/-) or WT donor mouse livers. Compared to WT liver grafts, CD39(-/-) grafts exhibited enhanced interstitial DC activation, elevated proinflammatory cytokine levels, and more-severe tissue injury. Moreover, portal venous delivery of WT, but not CD39(-/-) liver mDCs, to donor livers immediately post-transplant exerted a protective effect against graft injury in CD39(-/-) to CD39(-/-) liver transplantation. CONCLUSIONS: These data reveal that CD39 expression on conventional liver mDCs limits their proinflammatory activity and confers protective properties on these important innate immune cells against liver transplant ischemia/reperfusion injury.
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Antígenos CD/biosíntesis , Apirasa/biosíntesis , Células Dendríticas/metabolismo , Trasplante de Hígado , Hígado/inmunología , Daño por Reperfusión/prevención & control , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Humanos , Inmunidad Innata , Hígado/efectos de los fármacos , Masculino , Ratones , Daño por Reperfusión/metabolismo , Inmunología del TrasplanteRESUMEN
OBJECTIVE: Recent evidence suggests that functional deficiency in regulatory T cells (Tregs), an innate immunomodulator, exacerbates brain damage after cerebral ischemia. We therefore evaluated the effect of Treg transfer in rodent models of ischemic stroke and further investigated the mechanism underlying Treg-afforded neuroprotection. METHODS: We examined the therapeutic potential of Tregs and the mechanisms of neuroprotection in vivo in 2 rodent models of ischemic stroke and in vitro in Treg-neutrophil cocultures using a combined approach including cell-specific depletion, gene knockout mice, and bone marrow chimeras. RESULTS: Systemic administration of purified Tregs at 2, 6, or even 24 hours after middle cerebral artery occlusion resulted in a marked reduction of brain infarct and prolonged improvement of neurological functions lasting out to 4 weeks. Treg-afforded neuroprotection was accompanied by attenuated blood-brain barrier (BBB) disruption during early stages of ischemia, decreased cerebral inflammation, and reduced infiltration of peripheral inflammatory cells into the lesioned brain. Surprisingly, Tregs exerted early neuroprotection without penetrating into the brain parenchyma or inhibiting the activation of residential microglia. Rather, both in vivo and in vitro studies demonstrated that Tregs suppressed peripheral neutrophil-derived matrix metallopeptidase-9 production, thus preventing proteolytic damage of the BBB. In addition to its potent central neuroprotection, Treg treatment was shown to ameliorate poststroke lymphopenia, suggesting a beneficial effect on immune status. INTERPRETATION: Our study suggests that Treg adoptive therapy is a novel and potent cell-based therapy targeting poststroke inflammatory dysregulation and neurovascular disruption.