RESUMEN
The role of B cells in anti-tumour immunity is still debated and, accordingly, immunotherapies have focused on targeting T and natural killer cells to inhibit tumour growth1,2. Here, using high-throughput flow cytometry as well as bulk and single-cell RNA-sequencing and B-cell-receptor-sequencing analysis of B cells temporally during B16F10 melanoma growth, we identified a subset of B cells that expands specifically in the draining lymph node over time in tumour-bearing mice. The expanding B cell subset expresses the cell surface molecule T cell immunoglobulin and mucin domain 1 (TIM-1, encoded by Havcr1) and a unique transcriptional signature, including multiple co-inhibitory molecules such as PD-1, TIM-3, TIGIT and LAG-3. Although conditional deletion of these co-inhibitory molecules on B cells had little or no effect on tumour burden, selective deletion of Havcr1 in B cells both substantially inhibited tumour growth and enhanced effector T cell responses. Loss of TIM-1 enhanced the type 1 interferon response in B cells, which augmented B cell activation and increased antigen presentation and co-stimulation, resulting in increased expansion of tumour-specific effector T cells. Our results demonstrate that manipulation of TIM-1-expressing B cells enables engagement of the second arm of adaptive immunity to promote anti-tumour immunity and inhibit tumour growth.
Asunto(s)
Linfocitos B , Melanoma , Animales , Ratones , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Activación de Linfocitos , Melanoma/inmunología , Melanoma/patología , Melanoma/prevención & control , Linfocitos T/citología , Linfocitos T/inmunología , Citometría de Flujo , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Presentación de Antígeno , Receptores de Antígenos de Linfocitos B/genética , Análisis de Expresión Génica de una Sola Célula , Carga Tumoral , Interferón Tipo IRESUMEN
Regulatory B cells (Bregs) ameliorate autoimmune disease and prevent allograft rejection. Conversely, they hinder effective clearance of pathogens and malignancies. Breg activity is mainly attributed to IL-10 expression, but also utilizes additional regulatory mechanisms such as TGF-ß, FasL, IL-35, and TIGIT. Although Bregs are present in various subsets defined by phenotypic markers (including canonical B cell subsets), our understanding of Bregs has been limited by the lack of a broadly inclusive and specific phenotypic or transcriptional marker. TIM-1, a broad marker for Bregs first identified in transplant models, plays a major role in Breg maintenance and induction. Here, we expand on the role of TIM-1+ Bregs in immune tolerance and propose TIM-1 as a unifying marker for Bregs that utilize various inhibitory mechanisms in addition to IL-10. Further, this review provides an in-depth assessment of our understanding of Bregs in transplantation as elucidated in murine models and clinical studies. These studies highlight the major contribution of Bregs in preventing allograft rejection, and their ability to serve as highly predictive biomarkers for clinical transplant outcomes.
Asunto(s)
Enfermedades Autoinmunes , Linfocitos B Reguladores , Animales , Tolerancia Inmunológica , Ratones , Transducción de Señal , Tolerancia al TrasplanteRESUMEN
IL-10+ regulatory B cells (Bregs) inhibit immune responses in various settings. While Bregs appear to inhibit inflammatory cytokine expression by CD4+ T cells and innate immune cells, their reported impact on CD8+ T cells is contradictory. Moreover, it remains unclear which effects of Bregs are direct versus indirect. Finally, the subanatomical localization of Breg suppressive function and the nature of their intercellular interactions remain unknown. Using novel tamoxifen-inducible B cell-specific IL-10 knockout mice, we found that Bregs inhibit CD8+ T cell proliferation and inhibit inflammatory cytokine expression by both CD4+ and CD8+ T cells. Sort-purified Bregs from IL-10-reporter mice were adoptively transferred into wild-type hosts and examined by live-cell imaging. Bregs localized to the T:B border, specifically entered the T cell zone, and made more frequent and longer contacts with both CD4+ and CD8+ T cells than did non-Bregs. These Breg:T cell interactions were antigen-specific and reduced subsequent T:DC contacts. Thus, Bregs inhibit T cells through direct cognate interactions that subsequently reduce DC:T cell interactions.
Asunto(s)
Linfocitos B Reguladores/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Comunicación Celular , Células Dendríticas/inmunología , Interleucina-10/fisiología , Linfocitos T Reguladores/inmunología , Animales , Linfocitos B Reguladores/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Células Dendríticas/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Interleucina-10/metabolismo , Linfocitos T Reguladores/metabolismoRESUMEN
B cells give rise to polarized subsets, including B effector 1 (Be1) cells and regulatory B cells, which can promote or inhibit immune responses through expression of IFN-γ and IL-10, respectively. Such subsets likely explain why B cell depletion can either ameliorate or exacerbate inflammatory diseases; however, these cells remain poorly understood because of the absence of specific markers. Although T cell Ig and mucin domain-containing molecule (TIM)-1 broadly identifies IL-10+ regulatory B cells, no similar markers for Be1 cells have been described. We now show that TIM-4 is expressed by a subset of B cells distinct from those expressing TIM-1. Although TIM-1+ B cells are enriched for IL-10, TIM-4+ B cells are enriched for IFN-γ. TIM-1+ B cells enhanced the growth of B16-F10 melanoma. In contrast, TIM-4+ B cells decreased B16-F10 metastasis and s.c. tumor growth, and this was IFN-γ dependent. TIM-1+ B cells prolonged islet allograft survival in B-deficient mice, whereas TIM-4+ B cells accelerated rejection in an IFN-γ-dependent manner. Moreover, TIM-4+ B cells promoted proinflammatory Th differentiation in vivo, increasing IFN-γ while decreasing IL-4, IL-10, and Foxp3 expression by CD4+ T cells-effects that are opposite from those of TIM-1+ B cells. Importantly, a monoclonal anti-TIM-4 Ab promoted allograft tolerance, and this was dependent on B cell expression of TIM-4. Anti-TIM-4 downregulated T-bet and IFN-γ expression by TIM-4+ B cells and indirectly increased IL-10 expression by TIM-1+ B cells. Thus, TIM-4+ B cells are enriched for IFN-γ-producing proinflammatory Be1 cells that enhance immune responsiveness and can be specifically targeted with anti-TIM-4.
Asunto(s)
Aloinjertos/inmunología , Subgrupos de Linfocitos B/inmunología , Rechazo de Injerto , Interferón gamma/biosíntesis , Melanoma Experimental/inmunología , Proteínas de la Membrana/inmunología , Tolerancia al Trasplante , Animales , Anticuerpos Monoclonales/administración & dosificación , Diferenciación Celular , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-4/inmunología , Activación de Linfocitos , Melanoma Experimental/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Metástasis de la Neoplasia , Células TH1/fisiologíaRESUMEN
PURPOSE OF REVIEW: Regulatory B cells (Bregs) are potent inhibitors of the immune system with the capacity to suppress autoimmune and alloimmune responses. Murine transplant models showing that Bregs can promote allograft tolerance are now supported by clinical data showing that patients who develop operational tolerance have higher frequency of Bregs. Breg function has been widely studied resulting in improved understanding of their biology and effector mechanisms. However, our overall understanding of Bregs remains poor due the lack of specific marker, limited knowledge of how and where they act in vivo, and whether different Breg subpopulations exhibit different functions. RECENT FINDINGS: In this review we detail murine and human phenotypic markers used to identify Bregs, their induction, maintenance, and mechanisms of immune suppression. We highlight recent advances in the field including their use as biomarkers to predict allograft rejection, in-vitro expansion of Bregs, and the effects of commonly used immunosuppressive drugs on their induction and frequency. SUMMARY: Clinical data continue to emerge in support of Bregs playing an important role in preventing transplant rejection. Hence, it is necessary for the transplant field to better comprehend the mechanisms of Breg induction and approaches to preserve or even enhance their activity to improve long-term transplant outcomes.
Asunto(s)
Linfocitos B Reguladores/inmunología , Tolerancia al Trasplante/genética , Trasplante/métodos , HumanosRESUMEN
B cells can be polarized to express various cytokines. The roles of IFNγ and IL-10, expressed respectively by B effector 1 (Be1) and Bregs, have been established in pathogen clearance, tumor growth, autoimmunity and allograft rejection. However, the in vivo role of B cell IL-4, produced by Be2 cells, remains to be established. We developed B-IL-4/13 iKO mice carrying a tamoxifen-inducible B cell-specific deletion of IL-4 and IL-13. After alloimmunization, B-IL-4/13 iKO mice exhibited decreased IL-4+ Th2 cells and IL-10+ Bregs without impact on Th1, Tregs, or CD8 T cell responses. B-IL-4/13 iKO mice rejected islet allografts more rapidly, even when treated with tolerogenic anti-TIM-1 mAb. In ovalbumin-induced allergic airway disease (AAD), B-IL-4/13 iKO mice had reduced inflammatory cells in BAL, and preserved lung histology with markedly decreased infiltration by IL-4+ and IL-5+ CD4+ T cells. Hence, B cell IL-4 is a major driver of Th2 responses in vivo which promotes allograft survival, and conversely, worsens AAD.
Asunto(s)
Linfocitos B Reguladores , Hipersensibilidad , Aloinjertos , Animales , Rechazo de Injerto , Interleucina-10 , Interleucina-4/genética , Ratones , Ratones Endogámicos C57BLRESUMEN
Regulatory mechanisms pertaining to the self-renewal of stem cells remain incompletely understood. Here, we show that functional interactions between small GTPase Rap1 and the adhesion molecule E-cadherin uniquely regulate the self-renewal of human embryonic stem cells (hESCs). Inhibition of Rap1 suppresses colony formation and self-renewal of hESCs, whereas overexpression of Rap1 augments hESC clonogenicity. Rap1 does not directly influence the expression of the pluripotency genes Oct4 and Nanog. Instead, it affects the endocytic recycling pathway involved in the formation and maintenance of E-cadherin-mediated cell-cell cohesion, which is essential for the colony formation and self-renewal of hESCs. Conversely, distinct from epithelial cells, disruption of E-cadherin mediated cell-cell adhesions induces lysosome delivery and degradation of Rap1. This in turn leads to a further downregulation of E-cadherin function and a subsequent reduction in hESC clonogenic capacity. These findings provide the first demonstration that the interplay between Rap1 and E-cadherin along the endocytic recycling pathway serves as a timely and efficient mechanism to regulate hESC self-renewal. Given the availability of specific activators for Rap1, this work provides a new perspective to enable better maintenance of human pluripotent stem cells.
Asunto(s)
Cadherinas/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Benzamidas/farmacología , Western Blotting , Cadherinas/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Humanos , Microscopía Fluorescente , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Complejo Shelterina , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteínas de Unión a Telómeros/genéticaRESUMEN
In addition to their role in humoral immunity, B cells can exhibit regulatory activity. Such B cells have been termed regulatory B cells (Bregs). Bregs have been shown to inhibit inflammatory immune responses in a variety of autoimmune, alloimmune, and infectious settings. Breg activity is frequently IL-10-dependent, although a number of other mechanisms have been identified. However, our understanding of Bregs has been hampered by their rarity, lack of a specific phenotypic marker, and poor insight into their induction and maintenance. A variety of B-cell subsets enriched for IL-10+ Bregs have been identified in multiple murine disease models that can adoptively transfer Breg activity. However, most of these B-cell subsets actually contain only a minority of all IL-10+ B cells. In contrast, TIM-1 identifies over 70% of IL-10-producing B cells, irrespective of other markers. Thus, TIM-1 can be considered a broad marker for IL-10-expressing Bregs. Moreover, TIM-1 signaling plays a direct role in both the maintenance and induction of Bregs under physiological conditions, in response to both TIM-1 ligation and to apoptotic cells. TIM-1 expression has also been reported on IL-10+ human B cells. Together, these findings suggest that TIM-1 may represent a novel therapeutic target for modulating the immune response and provide insight into the signals involved in the generation and induction of Bregs. Here, we provide the methods to analyze and purify the murine TIM-1+ B-cell subset for further in vitro and in vivo experiments. We also provide methods for in vitro analysis and in vivo tracking of Bregs using IL-10-reporter mice.
Asunto(s)
Citometría de Flujo/métodos , Receptor Celular 1 del Virus de la Hepatitis A/metabolismo , Interleucina-10/aislamiento & purificación , Animales , Linfocitos B/inmunología , Linfocitos B Reguladores/inmunología , Interleucina-10/metabolismo , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
Prior experience of pathogen-associated stimuli reduces morbidity and mortality to newly encountered infections through innate immune training, which can be enhanced by childhood vaccination. Fibroblastic reticular cells (FRCs) are stromal cells in lymphoid organs that support lymphocyte localization and survival and modulate adaptive immune responses. IL-17 signaling is important for FRC metabolism and proliferation during inflammatory responses. Here, we show that FRC-intrinsic IL-17 signaling was required for protective antibody-mediated immunity to the gut bacterial pathogen Citrobacter rodentium. We asked whether prior activation of FRC through nonspecific inflammatory "training" of the gut would alter subsequent immune response to C. rodentium. Inflammatory training increased the number of activated FRC in mesenteric LN (MLN) and enhanced the antibody response to C. rodentium in an IL-17dependent manner. FRC demonstrated cardinal features of innate immune training, including increased epigenetic markers of activation and increased metabolic response to infection. Enhanced responses were still evident 6 weeks after training. The kinetics of bacterial infection were not changed by inflammatory training, but colon inflammation was paradoxically reduced. Mechanistically, IL-10 production by activated B cells was required for colon protective effects of inflammatory training. Enhancing tissue protective B cell responses thus led to increased production of antibody and IL-10, allowing clearance of infection with reduced tissue inflammation. These data identify a new mode of immune training through FRC to modulate future adaptive responses and better preserve host health.
Asunto(s)
Linfocitos B/inmunología , Fibroblastos/inmunología , Inmunidad Mucosa/inmunología , Interleucina-10/biosíntesis , Interleucina-17/inmunología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Early immunological biomarkers that predict rejection and chronic allograft loss are needed to inform preemptive therapy and improve long-term outcomes. Here, we prospectively examined the ratio of interleukin-10 (IL-10) to tumor necrosis factor-α (TNFα) produced by transitional-1 B cells (T1B) 3 months after transplantation as a predictive biomarker for clinical and subclinical renal allograft rejection and subsequent clinical course. In both Training (n = 162) and Internal Validation (n = 82) Sets, the T1B IL-10/TNFα ratio 3 months after transplantation predicted both clinical and subclinical rejection anytime in the first year. The biomarker also predicted subsequent late rejection with a lead time averaging 8 months. Among biomarker high-risk patients, 60% had early rejection, of which 48% recurred later in the first posttransplant year. Among high-risk patients without early rejection, 74% developed rejection later in the first year. In contrast, only 5% of low-risk patients had early and 5% late rejection. The biomarker also predicted rejection in an External Validation Set (n = 95) and in key patient subgroups, confirming generalizability. Biomarker high-risk patients exhibited progressively worse renal function and decreased 5-year graft survival compared to low-risk patients. Treatment of B cells with anti-TNFα in vitro augmented the IL-10/TNFα ratio, restored regulatory activity, and inhibited plasmablast differentiation. To conclude, the T1B IL-10/TNFα ratio was validated as a strong predictive biomarker of renal allograft outcomes and provides a rationale for preemptive therapeutic intervention with TNF blockade.
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Rechazo de Injerto , Trasplante de Riñón , Aloinjertos , Citocinas , Humanos , Riñón/fisiología , Células Precursoras de Linfocitos BRESUMEN
Clonality is, at present, the only means by which the self-renewal potential of a given stem cell can be determined. To assess the clonality of human embryonic stem cells (hESC), a protocol involving seeding wells at low cell densities is commonly used to surmount poor cloning efficiencies. However, factors influencing the accuracy of such an assay have not been fully elucidated. Using clonogenic assays together with time-lapse microscopy, numerical analyses, and regulated gene expression strategies, we found that individual and collective cell movements are inherent properties of hESCs and that they markedly impact the accuracy of clonogenic assays. Analyses of cell motility using mean-square displacement and paired migration correlation indicated that cell movements become more straight-line or ballistic and less random-walk as separation distance decreases. Such motility-induced reaggregation (rather than a true clone) occurs approximately 70% of the time if the distance between two hESCs is <6.4 mum, and is not observed if the distance is >150 mum. Furthermore, newly formed small hESC colonies have a predisposition toward the formation of larger colonies through asymmetric colony expansion and movement, which would not accurately reflect self-renewal and proliferative activity of a true hESC clone. Notably, inhibition of Rho-associated kinase markedly upregulated hESC migration and reaggregation, producing considerable numbers of false-positive colonies. Conversely, E-cadherin upregulation significantly augmented hESC clonogenicity via improved survival of single hESCs without influencing cell motility. This work reveals that individual cell movement, asymmetric colony expansion, Rho-associated kinase, and E-cadherin all work together to influence hESC clonogenicity, and provides additional guidance for improvement of clonogenic assays in the analysis of hESC self-renewal.
Asunto(s)
Cadherinas/metabolismo , Movimiento Celular , Proliferación Celular , Células Madre Embrionarias/fisiología , Quinasas Asociadas a rho/metabolismo , Algoritmos , Cadherinas/genética , Técnicas de Cultivo de Célula/métodos , Línea Celular , Supervivencia Celular/fisiología , Simulación por Computador , Células Madre Embrionarias/citología , Células Madre Embrionarias/enzimología , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Modelos Biológicos , Factores de Tiempo , Grabación en Video , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genéticaRESUMEN
Human erythropoietin (EPO) is a pleiotropic cytokine with remarkable tissue-protective activities in addition to its well-established role in red blood cell production. Unfortunately, conventional mammalian cell cultures are unlikely to meet the anticipated market demands for recombinant EPO because of limited capacity and high production costs. Plant expression systems may address these limitations to enable practical, cost-effective delivery of EPO in tissue injury prevention therapeutics. In this study, we produced human EPO in tobacco and demonstrated that plant-derived EPO had tissue-protective activity. Our results indicated that targeting to the endoplasmic reticulum (ER) provided the highest accumulation levels of EPO, with a yield approaching 0.05% of total soluble protein in tobacco leaves. The codon optimization of the human EPO gene for plant expression had no clear advantage; furthermore, the human EPO signal peptide performed better than a tobacco signal peptide. In addition, we found that glycosylation was essential for the stability of plant recombinant EPO, whereas the presence of an elastin-like polypeptide fusion had a limited positive impact on the level of EPO accumulation. Confocal microscopy showed that apoplast and ER-targeted EPO were correctly localized, and N-glycan analysis demonstrated that complex plant glycans existed on apoplast-targeted EPO, but not on ER-targeted EPO. Importantly, plant-derived EPO had enhanced receptor-binding affinity and was able to protect kidney epithelial cells from cytokine-induced death in vitro. These findings demonstrate that tobacco plants may be an attractive alternative for the production of large amounts of biologically active EPO.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Eritropoyetina/farmacología , Inflamación/tratamiento farmacológico , Nicotiana/genética , Células Cultivadas , Retículo Endoplásmico/metabolismo , Eritropoyetina/genética , Glicosilación , Humanos , Riñón/citología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Señales de Clasificación de Proteína , Proteínas Recombinantes , Nicotiana/metabolismoRESUMEN
B cells shape the alloimmune response through polarized subsets. These cells inhibit or promote immune responses by expressing suppressive or proinflammatory cytokines. Their summed activity dictates the influence of B cells on the alloimmune response. We review the evidence for regulatory B cells and effector B cells in mice and humans, discuss current limitations in their phenotypic identification, and discuss regulatory B cells as a signature for clinical renal allograft tolerance and predictive markers for allograft outcomes. We discuss the effects of therapeutic agents on regulatory B cells and potential approaches to augment their numbers as a therapeutic tool.
Asunto(s)
Subgrupos de Linfocitos B/fisiología , Linfocitos B Reguladores/fisiología , Tolerancia al Trasplante , Animales , Subgrupos de Linfocitos B/metabolismo , Linfocitos B Reguladores/metabolismo , Biomarcadores/metabolismo , Humanos , Ratones , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
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
Human embryonic stem cells (hESCs) offer great hope in regenerative medicine. Their ability to give rise to almost any type of cell present in the adult body makes them an invaluable tool in finding cures for a variety of diseases. While considerable protocols have been devised to efficiently differentiate hESCs into various cells types including cells of hematopoietic origin, this protocol will focus on the derivation of dendritic cells (DC), a potent antigen-presenting cell. DCs are a highly important arm of the immune system, as they represent one of the few cells that bridge the innate and adaptive systems, leading to effective pathogen clearance. The study of DCs has led to potential applications in diverse fields, such as vaccine development, tumor immunology, and transplantation. In this protocol, we describe two different methods of differentiating hESCs into DCs. The first method uses OP9 bone marrow stromal supporting cells as a coculture system, while the second method utilizes the formation of embryoid body (EB, cellular aggregate) as an approach. To assure the successful outcome and subsequent assessment of the differentiated DCs, supporting protocols have been included in this chapter.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Dendríticas/citología , Células Madre Embrionarias/citología , Adulto , Animales , Antígenos CD/inmunología , Células de la Médula Ósea/citología , Línea Celular , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/inmunología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/inmunología , Células Nutrientes/citología , Fibroblastos/citología , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Interleucina-4/farmacología , Ratones , Esferoides Celulares/citología , Células del Estroma/citologíaRESUMEN
Embryonic stem cells (ESCs) possess immune privileged properties and have the capacity to modulate immune activation. However, the mechanisms by which ESCs inhibit immune activation remain mostly unknown. We have previously shown that ESC-derived factors block dendritic cell maturation, thereby indirectly affecting T cell activation. Here, we show that ESC-derived factors also directly affect T cell activation. We provide the first demonstration that ESC-derived factors significantly down-regulated the expressions of IL-2 and IFN-γ, while markedly up-regulating the expression of IL-10, TGF-ß, and Treg transcription factor Foxp3 in CD4+ CD25+ T cells. Furthermore, ESC-derived factors robustly suppressed T cell proliferation in response to the protein kinase C-θ (PKC-θ) activator phorbol 12-myristate 13-acetate (PMA). Western blot analysis indicated that ESC-derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. The impact of ESC-derived factors on PKC-θ activation appeared to be specific since other upstream T cell signaling components were not affected. In conclusion, ESCs appear to directly impact T cell activation and polarization by negatively regulating the PKC-θ pathway.
Asunto(s)
Células Madre Embrionarias/metabolismo , Isoenzimas/metabolismo , Activación de Linfocitos/inmunología , Proteína Quinasa C/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T/inmunología , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Ciclosporina/farmacología , Citocinas/biosíntesis , Activación Enzimática/efectos de los fármacos , Humanos , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Proteína Quinasa C-theta , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismoRESUMEN
Embryonic stem cells (ESC) possess inherent properties of immune privilege with the capacity to evade allogeneic immune responses. Moreover, ESCs have been shown to prevent immune activation in response to third party antigen presenting cells in vitro and have the capacity to promote allograft survival in vivo. However, clinical use of human ESCs to treat immunological disorders may risk teratoma or ectopic tissue formation. Here, we show that cellular extracts from both human and mouse ESCs retain the immune modulatory properties of intact cells. ESC-extracts that contained 12-24 µg of total protein effectively prevented T cell proliferation in allogeneic mixed lymphocyte reactions (MLR), whereas control fibroblast extracts did not affect proliferation. Cellular mechanisms underlying hESC extract-mediated immune modulation involve the maturation of monocyte derived dendritic cells (mDC). hESC extract-treated mDCs had reduced surface expression of co-stimulatory and maturation markers CD80, HLA-DR and CD83 and secreted lower levels of IL12p40. Accordingly, hESC extract-treated DCs were found to be poor stimulators of purified allogeneic T cells compared to those DCs treated with vehicle or fibroblast extracts. Our results demonstrate that ESC extracts retain the immune modulatory properties of ESCs and for the first time demonstrates that ESC derived factors can inhibit human mDC maturation and function.
Asunto(s)
Diferenciación Celular/fisiología , Células Dendríticas/citología , Células Madre Embrionarias/citología , Monocitos/citología , Animales , Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Células Madre Embrionarias/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Antígenos HLA-DR/metabolismo , Humanos , Inmunoglobulinas/metabolismo , Interleucina-10/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Fagocitosis/efectos de los fármacos , Fagocitosis/fisiología , Reacción en Cadena de la Polimerasa , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Antígeno CD83RESUMEN
Indoleamine 2,3-dioxygenase (IDO) catabolizes tryptophan to N-formyl kynurenine and has a proapoptotic role in renal tubular epithelial cells (TEC) in response to IFN-gamma and TNF-alpha in vitro. TEC produce abundant amounts of IDO in vitro in response to inflammation but a pathological role for IDO in renal injury remains unknown. We investigated the role of IDO in a mouse model of renal ischemia-reperfusion injury (IRI). IRI was induced by clamping the renal pedicle of C57BL/6 mice for 45 min at 32 degrees C. Here, we demonstrate upregulation of IDO in renal tissue at 2 h after reperfusion which reached maximal levels at 24 h. Inhibition of IDO following IRI prevented the increase in serum creatinine observed in vehicle-treated mice (86.4 +/- 25 micromol/l, n = 11) compared with mice treated with 1-methyl-D-tryptophan, a specific inhibitor of IDO (33.7 +/- 8.7 micromol/l, n = 10, P = 0.031). The role of IDO in renal IRI was further supported by results in IDO-KO mice which maintained normal serum creatinine levels (32.5 +/- 2.0 micromol/l, n = 6) following IRI compared with wild-type mice (123 +/- 30 micromol/l, n = 9, P = 0.008). Our data suggest that attenuation of IDO expression within the kidney may represent a novel strategy to reduce renal injury as a result of ischemia reperfusion.
Asunto(s)
Indolamina-Pirrol 2,3,-Dioxigenasa/biosíntesis , Enfermedades Renales/etiología , Daño por Reperfusión/etiología , Animales , Células Epiteliales/enzimología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indolamina-Pirrol 2,3,-Dioxigenasa/deficiencia , Riñón/irrigación sanguínea , Riñón/patología , Túbulos Renales/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión/patología , Triptófano/análogos & derivados , Triptófano/farmacología , Regulación hacia ArribaRESUMEN
Exposure of renal tubular epithelial cells (TEC) to IFN-gamma/TNF-alpha leads to Fas/FasL-mediated self-injury, which contributes to allograft rejection. Indoleamine 2,3-dioxygenase (IDO) converts tryptophan to N-formyl-kynurenine and contributes to immune privilege in tissues by increasing Fas-mediated T cell apoptosis. However, renal expression of IDO and its role in promoting Fas-mediated TEC death have not been examined. IDO expression was analyzed by RT-PCR and Western blot. Apoptosis was measured by fluorescence-activated cell sorting analysis and terminal deoxytransferase-mediated dUTP nick end labeling. We demonstrated that functional IDO is expressed in TEC and is increased by IFN-gamma/TNF-alpha exposure. Increased IDO activity promoted TEC apoptosis, whereas inhibition of IDO by its specific inhibitor 1-methyl-d-tryptophan attenuated IFN-gamma/TNF-alpha-mediated TEC apoptosis and augmented TEC survival. Transgenic expression of IDO resulted in increased TEC apoptosis in the absence of proinflammatory cytokine exposure, supporting a central role for IDO in TEC injury. Inhibition of IDO-mediated TEC death by a caspase-8-specific inhibitor (Z-IETD-FMK), as well as the absence of an IDO effect in Fas-deficient and FasL-deficient TEC, supports a Fas/FasL-dependent, caspase-8-mediated mechanism for IDO-enhanced TEC death. These data suggest that renal IDO expression may be deleterious during renal inflammation, because it enhances TEC self-injury through Fas/FasL interactions. Thus attenuation of IDO may represent a novel strategy to promote kidney function following ischemia and renal allograft rejection.