RESUMEN
BACKGROUND: Ischemic stroke (IS) is a fearful disease that can cause a variety of immune events. Nevertheless, precise immune-related mechanisms have yet to be systematically elucidated. This study aimed to identify immune-related signatures using machine learning and to validate them with animal experiments and single cell analysis. METHODS: In this study, we screened 24 differentially expressed genes (DEGs) while identifying immune-related signatures that may play a key role in IS development through a comprehensive strategy between least absolute shrinkage and selection operation (LASSO) regression, support vector machine (SVM) and immune-related genes. In addition, we explored immune infiltration using the CIBERSORT algorithm. Finally, we performed validation in mouse brain tissue and single cell analysis. RESULTS: We identified 24 DEGs for follow-up analysis. ID3 and SLC22A4 were finally identified as the better immune-related signatures through a comprehensive strategy among DEGs, LASSO, SVM and immune-related genes. RT-qPCR, western blot, and immunofluorescence revealed a significant decrease in ID3 and a significant increase in SLC22A4 in the middle cerebral artery occlusion group. Single cell analysis revealed that ID3 was mainly concentrated in endothelial_2 cells and SLC22A4 in astrocytes in the MCAO group. A CIBERSORT finds significantly altered levels of immune infiltration in IS patients. CONCLUSIONS: This study focused on immune-related signatures after stroke and ID3 and SLC22A4 may be new therapeutic targets to promote functional recovery after stroke. Furthermore, the association of ID3 and SLC22A4 with immune cells may be a new direction for post-stroke immunotherapy.
Asunto(s)
Proteínas Inhibidoras de la Diferenciación , Accidente Cerebrovascular Isquémico , Proteínas de Transporte de Catión Orgánico , Accidente Cerebrovascular , Simportadores , Animales , Humanos , Ratones , Algoritmos , Astrocitos , Western Blotting , Proteínas Inhibidoras de la Diferenciación/inmunología , Proteínas Inhibidoras de la Diferenciación/metabolismo , Accidente Cerebrovascular Isquémico/genética , Proteínas de Neoplasias , Proteínas de Transporte de Catión Orgánico/inmunología , Proteínas de Transporte de Catión Orgánico/metabolismo , Accidente Cerebrovascular/inmunología , Accidente Cerebrovascular/metabolismo , Simportadores/inmunología , Simportadores/metabolismoRESUMEN
Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias Pancreáticas/terapia , Receptores Quiméricos de Antígenos/inmunología , Animales , Linfocitos T CD8-positivos/citología , Línea Celular Tumoral , Células HEK293 , Humanos , Proteínas Inhibidoras de la Diferenciación/inmunología , Masculino , Ratones , Ratones Noqueados , Ratones Desnudos , Ratones SCID , Proteínas de Neoplasias/inmunología , Factores de Transcripción SOXC/inmunologíaRESUMEN
Tissue-resident memory CD8+ T cells (Trm) provide host protection through continuous surveillance of non-lymphoid tissues. Using single-cell RNA-sequencing (scRNA-seq) and genetic reporter mice, we identified discrete lineages of intestinal antigen-specific CD8+ T cells, including a Blimp1hiId3lo tissue-resident effector cell population most prominent in the early phase of acute viral and bacterial infections and a molecularly distinct Blimp1loId3hi tissue-resident memory population that subsequently accumulated at later infection time points. These Trm populations exhibited distinct cytokine production, secondary memory potential, and transcriptional programs including differential roles for transcriptional regulators Blimp1, T-bet, Id2, and Id3 in supporting and maintaining intestinal Trm. Extending our analysis to malignant tissue, we also identified discrete populations of effector-like and memory-like CD8+ T cell populations with tissue-resident gene-expression signatures that shared features of terminally exhausted and progenitor-exhausted T cells, respectively. Our findings provide insight into the development and functional heterogeneity of Trm cells, which has implications for enhancing vaccination and immunotherapy approaches.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Neoplasias/terapia , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Células Cultivadas , Inmunoterapia/métodos , Proteína 2 Inhibidora de la Diferenciación/genética , Proteína 2 Inhibidora de la Diferenciación/inmunología , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/inmunología , Proteínas Inhibidoras de la Diferenciación/metabolismo , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neoplasias/inmunología , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/inmunología , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismoRESUMEN
Antigen-specific Th1 cells could be a passage to the infection sites during infection to execute effector functions, such as help CD8+ T cells to localize in these sites by secretion of anti-viral cytokines-IFN-γ or direct cytotoxicity of antigen-bearing cells. However, the molecular components that modulate Th1 cell differentiation and function in response to viral infection remain incompletely understood. Here, we reported that both inhibitor of DNA binding 3(Id3) protein and inhibitor of DNA binding 2(Id2) protein promoted Th1 cell differentiation. Depletion of Id3 or Id2 led to severe defect of Th1 cell differentiation during influenza virus infection. Whereas depletion of both Id3 and Id2 in CD4+ T cells restrained Th1 cell differentiation to a greater extent, indicating that Id3 and Id2 nonredundantly regulate Th1 cell differentiation. Moreover, deletion of E-proteins, the antagonists of Id proteins, greatly enhanced Th1 cell differentiation. Mechanistic study indicated that E-proteins suppressed Th1 cell differentiation by directly binding to the regulatory elements of Th1 cell master regulator T-bet and regulate T-bet expression. Thus, our findings identified Id-protein's importance for Th1 cells and clarified the nonredundant role of Id3 and Id2 in regulating Th1 cell differentiation, providing novel insight that Id3-Id2-E protein axis are essential for Th1 cell polarization.
Asunto(s)
Diferenciación Celular/inmunología , Proteína 2 Inhibidora de la Diferenciación/inmunología , Proteínas Inhibidoras de la Diferenciación/inmunología , Infecciones por Orthomyxoviridae/inmunología , Orthomyxoviridae/inmunología , Células TH1/inmunología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Regulación de la Expresión Génica/inmunología , Proteína 2 Inhibidora de la Diferenciación/genética , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/metabolismo , Ratones Noqueados , Ratones Transgénicos , Orthomyxoviridae/fisiología , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/virología , Elementos Reguladores de la Transcripción/genética , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/inmunología , Proteínas de Dominio T Box/metabolismo , Células TH1/metabolismo , Células TH1/virologíaRESUMEN
γδ NKT cells are neonatal-derived γδ T lymphocytes that are grouped together with invariant NKT cells based on their shared innate-like developmental program characterized by the transcription factor PLZF (promyelocytic leukemia zinc finger). Previous studies have demonstrated that the population size of γδ NKT cells is tightly controlled by Id3-mediated inhibition of E-protein activity in mice. However, how E proteins promote γδ NKT cell development and expansion remains to be determined. In this study, we report that the transcription factor Egr2, which also activates PLZF expression in invariant NKT cells, is essential for regulating γδ NKT cell expansion. We observed a higher expression of Egr family genes in γδ NKT cells compared with the conventional γδ T cell population. Loss of function of Id3 caused an expansion of γδ NKT cells, which is accompanied by further upregulation of Egr family genes as well as PLZF. Deletion of Egr2 in Id3-deficient γδ NKT cells prevented cell expansion and blocked PLZF upregulation. We further show that this Egr2-mediated γδ NKT cell expansion is dependent on c-Myc. c-Myc knockdown attenuated the proliferation of Id3-deficient γδ NKT cells, whereas c-Myc overexpression enhanced the proliferation of Id3/Egr2-double-deficient γδ NKT cells. Therefore, our data reveal a regulatory circuit involving Egr2-Id3-E2A, which normally restricts the population size of γδ NKT cells by adjusting Egr2 dosage and c-Myc expression.
Asunto(s)
Proliferación Celular/fisiología , Proteína 2 de la Respuesta de Crecimiento Precoz/inmunología , Regulación de la Expresión Génica/inmunología , Proteínas Inhibidoras de la Diferenciación/inmunología , Células T Asesinas Naturales/inmunología , Proteínas Proto-Oncogénicas c-myc/inmunología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Proteínas Inhibidoras de la Diferenciación/genética , Ratones , Ratones Noqueados , Células T Asesinas Naturales/citología , Proteína de la Leucemia Promielocítica con Dedos de Zinc/genética , Proteína de la Leucemia Promielocítica con Dedos de Zinc/inmunología , Proteínas Proto-Oncogénicas c-myc/genética , Receptores de Antígenos de Linfocitos T gamma-deltaRESUMEN
All innate lymphoid cells (ILCs) require the small helix-loop-helix transcription factor ID2, but the functions of ID2 are not well understood in these cells. We show that mature natural killer (NK) cells, the prototypic ILCs, developed in mice lacking ID2 but remained as precursor CD27+CD11b- cells that failed to differentiate into CD27-CD11b+ cytotoxic effectors. We show that ID2 limited chromatin accessibility at E protein binding sites near naïve T lymphocyte-associated genes including multiple chemokine receptors, cytokine receptors, and signaling molecules and altered the NK cell response to inflammatory cytokines. In the absence of ID2, CD27+CD11b- NK cells expressed ID3, a helix-loop-helix protein associated with naïve T cells, and they transitioned from a CD8 memory precursor-like to a naïve-like chromatin accessibility state. We demonstrate that ID3 was required for the development of ID2-deficient NK cells, indicating that completely unfettered E protein function is incompatible with NK cell development. These data solidify the roles of ID2 and ID3 as mediators of effector and naïve gene programs, respectively, and revealed a critical role for ID2 in promoting a chromatin state and transcriptional program in CD27+CD11b- NK cells that supports cytotoxic effector differentiation and cytokine responses.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Diferenciación Celular/inmunología , Proteína 2 Inhibidora de la Diferenciación/inmunología , Células Asesinas Naturales/inmunología , Linfocitos T/inmunología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/genética , Cromatina/genética , Cromatina/inmunología , Cromatina/metabolismo , Regulación de la Expresión Génica/inmunología , Proteína 2 Inhibidora de la Diferenciación/genética , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/inmunología , Proteínas Inhibidoras de la Diferenciación/metabolismo , Células Asesinas Naturales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T/metabolismoRESUMEN
Inhibitor of DNA binding (Id) proteins, including Id1-4, are transcriptional regulators involved in promoting cell proliferation and survival in various cell types. Although upregulation of Id proteins is associated with a broad spectrum of tumors, recent studies have identified that Id3 plays a tumor-suppressor role in the development of Burkitt's lymphoma in humans and hepatosplenic T cell lymphomas in mice. In this article, we report rapid lymphoma development in Id2/Id3 double-knockout mice that is caused by unchecked expansion of invariant NKT (iNKT) cells or a unique subset of innate-like CD1d-independent T cells. These populations began to expand in neonatal mice and, upon malignant transformation, resulted in mortality between 3 and 11 mo of age. The malignant cells also gave rise to lymphomas upon transfer to Rag-deficient and wild-type hosts, reaffirming their inherent tumorigenic potential. Microarray analysis revealed a significantly modified program in these neonatal iNKT cells that ultimately led to their malignant transformation. The lymphoma cells demonstrated chromosome instability along with upregulation of several signaling pathways, including the cytokine-cytokine receptor interaction pathway, which can promote their expansion and migration. Dysregulation of genes with reported driver mutations and the NF-κB pathway were found to be shared between Id2/Id3 double-knockout lymphomas and human NKT tumors. Our work identifies a distinct premalignant state and multiple tumorigenic pathways caused by loss of function of Id2 and Id3. Thus, conditional deletion of Id2 and Id3 in developing T cells establishes a unique animal model for iNKT and relevant innate-like lymphomas.
Asunto(s)
Proteína 2 Inhibidora de la Diferenciación/inmunología , Proteínas Inhibidoras de la Diferenciación/inmunología , Linfoma/inmunología , Células T Asesinas Naturales/inmunología , Subgrupos de Linfocitos T/inmunología , Traslado Adoptivo , Animales , Separación Celular , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Humanos , Linfoma/patología , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la PolimerasaRESUMEN
The generation of high-affinity antibodies requires germinal center (GC) development and differentiation of long-lived plasma cells in a multilayered process that is tightly controlled by the activity of multiple transcription factors. Here, we reveal a new layer of complexity by demonstrating that dynamic changes in Id3 and E-protein activity govern both GC and plasma cell differentiation. We show that down-regulation of Id3 in B cells is essential for releasing E2A and E2-2, which in a redundant manner are required for antigen-induced B cell differentiation. We demonstrate that this pathway controls the expression of multiple key factors, including Blimp1, Xbp1, and CXCR4, and is therefore critical for establishing the transcriptional network that controls GC B cell and plasma cell differentiation.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/inmunología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Diferenciación Celular/inmunología , Centro Germinal/inmunología , Proteínas Inhibidoras de la Diferenciación/inmunología , Células Plasmáticas/inmunología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Proteínas Inhibidoras de la Diferenciación/genética , Ratones , Ratones Noqueados , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Receptores CXCR4/genética , Receptores CXCR4/inmunología , Factor de Transcripción 4 , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/inmunologíaRESUMEN
The molecular mechanisms by which signaling via transforming growth factor-ß (TGF-ß) and interleukin 4 (IL-4) control the differentiation of CD4(+) IL-9-producing helper T cells (TH9 cells) remain incompletely understood. We found here that the DNA-binding inhibitor Id3 regulated TH9 differentiation, as deletion of Id3 increased IL-9 production from CD4(+) T cells. Mechanistically, TGF-ß1 and IL-4 downregulated Id3 expression, and this process required the kinase TAK1. A reduction in Id3 expression enhanced binding of the transcription factors E2A and GATA-3 to the Il9 promoter region, which promoted Il9 transcription. Notably, Id3-mediated control of TH9 differentiation regulated anti-tumor immunity in an experimental melanoma-bearing model in vivo and also in human CD4(+) T cells in vitro. Thus, our study reveals a previously unrecognized TAK1-Id3-E2A-GATA-3 pathway that regulates TH9 differentiation.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Proteínas Inhibidoras de la Diferenciación/inmunología , Interleucina-9/biosíntesis , Proteínas de Neoplasias/inmunología , Animales , Diferenciación Celular , Células Cultivadas , Citometría de Flujo , Humanos , Proteínas Inhibidoras de la Diferenciación/genética , Interleucina-9/inmunología , Ratones , Proteínas de Neoplasias/genética , Reacción en Cadena de la Polimerasa , Transducción de Señal/inmunologíaRESUMEN
Sustained Ag persistence in chronic infection results in a deregulated CD8(+) T cell response that is characterized by T cell exhaustion and cell death of Ag-specific CD8(+) T cells. Yet, the underlying transcriptional mechanisms regulating CD8(+) T cell exhaustion and cell death are poorly defined. Using the experimental mouse model of lymphocytic choriomeningitis virus infection, we demonstrate that the transcriptional regulator Id3 controls cell death of virus-specific CD8(+) T cells in chronic infection. By comparing acute and chronic infection, we showed that Id3 (-) virus-specific CD8(+) T cells were less abundant, whereas the absolute numbers of Id3 (+) virus-specific CD8(+) T cells were equal in chronic and acute infection. Phenotypically, Id3 (-) and Id3 (+) cells most prominently differed with regard to expression of the surface receptor 2B4; although Id3 (-) cells were 2B4(+), almost all Id3 (+) cells lacked expression of 2B4. Lineage-tracing experiments showed that cells initially expressing Id3 differentiated into Id3 (-)2B4(+) cells; in turn, these cells were terminally differentiated and highly susceptible to cell death under conditions of persisting Ag. Enforced Id3 expression specifically increased the persistence of 2B4(+) virus-specific CD8(+) T cells by decreasing susceptibility to Fas/Fas ligand-mediated cell death. Thus, our findings reveal that the transcriptional regulator Id3 promotes the survival of virus-specific CD8(+) T cells in chronic infection and suggest that targeting Id3 might be beneficial for preventing cell death of CD8(+) T cells in chronic infection or for promoting cell death of uncontrolled, hyperactive CD8(+) T cells to prevent immunopathology.
Asunto(s)
Antígenos CD/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas Inhibidoras de la Diferenciación/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Receptores Inmunológicos/inmunología , Traslado Adoptivo , Animales , Antígenos CD/metabolismo , Apoptosis/genética , Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Western Blotting , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular Tumoral , Enfermedad Crónica , Perros , Proteína Ligando Fas/inmunología , Proteína Ligando Fas/metabolismo , Citometría de Flujo , Expresión Génica/inmunología , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/metabolismo , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/metabolismo , Virus de la Coriomeningitis Linfocítica/fisiología , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones Transgénicos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Receptores Inmunológicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/inmunología , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Receptor fas/inmunología , Receptor fas/metabolismoRESUMEN
Macrophages are responsible for the control of inflammation and healing, and their malfunction results in cardiometabolic disorders. TGF-ß is a pleiotropic growth factor with dual (protective and detrimental) roles in atherogenesis. We have previously shown that in human macrophages, TGF-ß1 activates Smad2/3 signaling and induces a complex gene expression program. However, activated genes were not limited to known Smad2/3-dependent ones, which prompted us to study TGF-ß1-induced signaling in macrophages in detail. Analysis of Id3 regulatory sequences revealed a novel enhancer, located between +4517 and 4662 bp, but the luciferase reporter assay demonstrated that this enhancer is not Smad2/3 dependent. Because Id3 expression is regulated by Smad1/5 in endothelial cells, we analyzed activation of Smad1/5 in macrophages. We demonstrate here for the first time, to our knowledge, that TGF-ß1, but not BMPs, activates Smad1/5 in macrophages. We show that an ALK5/ALK1 heterodimer is responsible for the induction of Smad1/5 signaling by TGF-ß1 in mature human macrophages. Activation of Smad1/5 by TGF-ß1 induces not only Id3, but also HAMP and PLAUR, which contribute to atherosclerotic plaque vulnerability. We suggest that the balance between Smad1/5- and Smad2/3-dependent signaling defines the outcome of the effect of TGF-ß on atherosclerosis where Smad1/5 is responsible for proatherogenic effects, whereas Smad2/3 regulate atheroprotective effects of TGF-ß.
Asunto(s)
Macrófagos/inmunología , Placa Aterosclerótica/inmunología , Transducción de Señal/inmunología , Proteína Smad1/inmunología , Proteína Smad5/inmunología , Factor de Crecimiento Transformador beta1/inmunología , Receptores de Activinas Tipo II/inmunología , Proteínas Morfogenéticas Óseas/inmunología , Células Cultivadas , Hepcidinas/inmunología , Humanos , Proteínas Inhibidoras de la Diferenciación/inmunología , Macrófagos/patología , Proteínas de Neoplasias/inmunología , Placa Aterosclerótica/patología , Proteínas Serina-Treonina Quinasas/inmunología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/inmunología , Proteína Smad2/inmunología , Proteína smad3/inmunologíaRESUMEN
Regulatory T (Treg) cells suppress the development of inflammatory disease, but our knowledge of transcriptional regulators that control this function remains incomplete. Here we show that expression of Id2 and Id3 in Treg cells was required to suppress development of fatal inflammatory disease. We found that T cell antigen receptor (TCR)-driven signaling initially decreased the abundance of Id3, which led to the activation of a follicular regulatory T (TFR) cell-specific transcription signature. However, sustained lower abundance of Id2 and Id3 interfered with proper development of TFR cells. Depletion of Id2 and Id3 expression in Treg cells resulted in compromised maintenance and localization of the Treg cell population. Thus, Id2 and Id3 enforce TFR cell checkpoints and control the maintenance and homing of Treg cells.
Asunto(s)
Inflamación/inmunología , Proteína 2 Inhibidora de la Diferenciación/inmunología , Proteínas Inhibidoras de la Diferenciación/inmunología , Linfocitos T Reguladores/inmunología , Animales , Secuencia de Bases , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Proliferación Celular , Femenino , Factores de Transcripción Forkhead/biosíntesis , Regulación de la Expresión Génica/inmunología , Proteínas Fluorescentes Verdes/genética , Inflamación/genética , Proteína 2 Inhibidora de la Diferenciación/biosíntesis , Proteína 2 Inhibidora de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/biosíntesis , Proteínas Inhibidoras de la Diferenciación/genética , Interleucina-10/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/inmunología , Receptores CXCR5/biosíntesis , Análisis de Secuencia de ARNRESUMEN
The possibility to modulate ex vivo human NK cell differentiation towards specific phenotypes will contribute to a better understanding of NK cell differentiation and facilitate tailored production of NK cells for immunotherapy. In this study, we show that addition of a specific low dose of IL-12 to an ex vivo NK cell differentiation system from cord blood CD34(+) stem cells will result in significantly increased proportions of cells with expression of CD62L as well as KIRs and CD16 which are preferentially expressed on mature CD56(dim) peripheral blood NK cells. In addition, the cells displayed decreased expression of receptors such as CCR6 and CXCR3, which are typically expressed to a lower extent by CD56(dim) than CD56(bright) peripheral blood NK cells. The increased number of CD62L and KIR positive cells prevailed in a population of CD33(+)NKG2A(+) NK cells, supporting that maturation occurs via this subtype. Among a series of transcription factors tested we found Gata3 and TOX to be significantly downregulated, whereas ID3 was upregulated in the IL-12-modulated ex vivo NK cells, implicating these factors in the observed changes. Importantly, the cells differentiated in the presence of IL-12 showed enhanced cytokine production and cytolytic activity against MHC class I negative and positive targets. Moreover, in line with the enhanced CD16 expression, these cells exhibited improved antibody-dependent cellular cytotoxicity for B-cell leukemia target cells in the presence of the clinically applied antibody rituximab. Altogether, these data provide evidence that IL-12 directs human ex vivo NK cell differentiation towards more mature NK cells with improved properties for potential cancer therapies.
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Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Diferenciación Celular/inmunología , Interleucina-2/inmunología , Células Asesinas Naturales/inmunología , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antígenos CD34/inmunología , Antígenos CD34/metabolismo , Antineoplásicos/inmunología , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Células Cultivadas , Relación Dosis-Respuesta a Droga , Sangre Fetal/citología , Sangre Fetal/inmunología , Sangre Fetal/metabolismo , Citometría de Flujo , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/inmunología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/inmunología , Humanos , Inmunoterapia Adoptiva/métodos , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/inmunología , Interleucina-2/farmacología , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Selectina L/inmunología , Selectina L/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Receptores CCR6/inmunología , Receptores CCR6/metabolismo , Receptores CXCR3/inmunología , Receptores CXCR3/metabolismo , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Receptores KIR/inmunología , Receptores KIR/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , RituximabRESUMEN
Disease outcome is known to be influenced by defined subsets of invariant NKT (iNKT) cells residing in distinct locations within peripheral tissue. However, the factors governing the development of these unique iNKT sublineages during thymic development are unknown. In this study we explored the mechanism by which E protein transcription factors and their negative regulators, the Id proteins, control the development of iNKT sublineages after positive selection. We found that E proteins directly bound the promyelocytic leukemia zinc finger (PLZF) promoter and were required for expression of this lineage-defining transcription factor and for the maturation and expansion of thymic iNKT cells. Moreover, expression of the negative regulators of E proteins, Id2 and Id3, defined distinct iNKT cell sublineages. Id3 was expressed in PLZF(high) NKT2 cells and loss of Id3 allowed for increased thymic iNKT cell expansion and abundance of the PLZF(+) NKT2 sublineage. Id2 was expressed in T-BET(+) NKT1 cells, and both Id proteins were required for the formation of this sublineage. Thus, we provide insight into E and Id protein regulation of iNKT cell proliferation and differentiation to specific sublineages during development in the thymus.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/inmunología , Diferenciación Celular/fisiología , Proliferación Celular , Proteína 2 Inhibidora de la Diferenciación/inmunología , Proteínas Inhibidoras de la Diferenciación/inmunología , Células T Asesinas Naturales/inmunología , Timo/inmunología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/biosíntesis , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Proteína 2 Inhibidora de la Diferenciación/biosíntesis , Proteína 2 Inhibidora de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/biosíntesis , Proteínas Inhibidoras de la Diferenciación/genética , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/inmunología , Ratones , Ratones Transgénicos , Células T Asesinas Naturales/citología , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Timo/citología , Timo/metabolismoRESUMEN
Upon infection, CD8(+) T cells proliferate and differentiate into armed effector cells capable of eliminating the assaulting pathogen. Although the majority of the antigen-specific T cells will die as the immune response wanes, a few will survive indefinitely to establish the memory population and provide long-lived protection against reinfection. E protein transcription factors and their inhibitors, ID proteins, operate to balance expression of genes that control CD8(+) T cell differentiation through this process. Here, we discuss the role of ID2 and ID3 in promoting the generation and survival of effector and memory populations, particularly highlighting their reciprocal roles in shaping the CD8(+) T cell response unique to the inflammatory milieu. We further examine this coordinated control of gene expression in the context of additional transcription factors within the transcriptional network that programs CD8(+) effector and memory T cell differentiation.
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Memoria Inmunológica , Proteínas Inhibidoras de la Diferenciación/inmunología , Linfocitos T/inmunología , Animales , Diferenciación Celular , Supervivencia Celular , Regulación de la Expresión Génica , Humanos , Linfocitos T/citologíaRESUMEN
UNLABELLED: In aâ¯phase II clinical study, pretreated multiple myeloma patients with relapsing or stable disease received autologous anticancer vaccine containing dendritic cells loaded with Id-protein. Patients received aâ¯total of 6 vaccine doses intradermally in monthly intervals. No clinical responses were observed. During the follow-up with aâ¯median of 33.1 months (range: 11-43 months), the disease remained stable in 7/11 (64%) of patients. Immune responses measured by ELISpot were noted in 3/11 (27%) and DTH skin test for Id-protein was positive in 8/11 (73%) of patients; out of those, 1/11 (9%) and 5/11 (46%), respectively, had preexisting immune response to Id-protein before the vaccination began. Outcomes were compared to those of aâ¯control group of 13 patients. Aâ¯trend to lower cumulative incidence of progression in the vaccinated group was observed at 12 months from the first vaccination (p= 0.099). More patients from the control group compared to vaccinated patients required active anticancer therapy [4/11 (36%) vs. 8/13 (62%)]. Vaccines based on dendritic cells loaded with Id-protein are safe and induce specific immune response in multiple myeloma patients. Our results suggest that the vaccination could stabilize the disease in approximately two-thirds of patients. KEYWORDS: dendritic cells, immunotherapy, anticancer vaccines, Id-protein, multiple myeloma.
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Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/inmunología , Inmunoterapia , Proteínas Inhibidoras de la Diferenciación/inmunología , Proteínas Inhibidoras de la Diferenciación/metabolismo , Mieloma Múltiple/terapia , Adyuvantes Inmunológicos , Anciano , Estudios de Casos y Controles , Células Dendríticas/trasplante , Ensayo de Inmunoadsorción Enzimática , Femenino , Hemocianinas/inmunología , Humanos , Hipersensibilidad Tardía , Masculino , Persona de Mediana Edad , Mieloma Múltiple/inmunología , Estadificación de Neoplasias , Pronóstico , Tasa de Supervivencia , VacunaciónRESUMEN
The double-positive (DP) to single-positive (SP) transition during T cell development is initiated by downregulation of the E protein transcription factors HEB and E2A. Here, we have demonstrated that in addition to regulating the onset of this transition, HEB and E2A also play a separate role in CD4(+) lineage choice. Deletion of HEB and E2A in DP thymocytes specifically blocked the development of CD4(+) lineage T cells. Furthermore, deletion of the E protein inhibitors Id2 and Id3 allowed CD4(+) T cell development but blocked CD8(+) lineage development. Analysis of the CD4(+) lineage transcriptional regulators ThPOK and Gata3 placed HEB and E2A upstream of CD4(+) lineage specification. These studies identify an important role for E proteins in the activation of CD4(+) lineage differentiation as thymocytes undergo the DP to SP transition.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Linfocitos T CD4-Positivos/inmunología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Antígenos CD4/genética , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/inmunología , Proteína 2 Inhibidora de la Diferenciación/deficiencia , Proteína 2 Inhibidora de la Diferenciación/genética , Proteína 2 Inhibidora de la Diferenciación/inmunología , Proteínas Inhibidoras de la Diferenciación/deficiencia , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/inmunología , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores CCR7/metabolismo , Regulación hacia ArribaRESUMEN
Recent studies have identified a number of transcriptional regulators, including E proteins, EBF1, FOXO1, and PAX5, that act together to orchestrate the B-cell fate. However, it still remains unclear as to how they are linked at the earliest stages of B-cell development. Here, we show that lymphocyte development in HEB-ablated mice exhibits a partial developmental arrest, whereas B-cell development in E2A(+/-)HEB(-/-) mice is completely blocked at the LY6D(-) common lymphoid progenitor stage. We show that the transcription signatures of E2A- and HEB-ablated common lymphoid progenitors significantly overlap. Notably, we found that Foxo1 expression was substantially reduced in the LY6D(-) HEB- and E2A-deficient cells. Finally, we show that E2A binds to enhancer elements across the FOXO1 locus to activate Foxo1 expression, linking E2A and FOXO1 directly in a common pathway. In summary, the data indicate that the earliest event in B-cell specification involves the induction of FOXO1 expression and requires the combined activities of E2A and HEB.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Factores de Transcripción Forkhead/genética , Células Progenitoras Linfoides/inmunología , Animales , Antígenos Ly/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteína Forkhead Box O1 , Proteínas Ligadas a GPI/inmunología , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/genética , Hematopoyesis/inmunología , Proteína 2 Inhibidora de la Diferenciación/genética , Proteína 2 Inhibidora de la Diferenciación/inmunología , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/inmunología , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Id3-/- mice represent a model for T cell mediated primary Sjogren's syndrome (PSS). An intriguing feature of this disease model is the early appearance of impaired salivary function or exocrinopathy prior to lymphocytic infiltration of the salivary glands. This phenomenon prompted us to examine the role of cytokines produced by T cells in the systemic regulation of gland function. A comprehensive examination of serum cytokine profiles revealed elevated levels of IL-13 in Id3-/- mice. We found that the increase in serum IL-13 levels in Id3-/- mice was largely dependent on αß T cells. Removal of αß T cells in Id3-/- mice also eliminates disease symptoms, including lymphocytic infiltration in the gland tissues, and impaired saliva production. We further show that the number of mast cells in the salivary glands of Id3-/- mice is significantly increased, in a trend inversely related to the saliva production. This increase in the number of mast cells is also dependent on the presence of αß T cells. Treatment of young Id3-/- mice with anti-IL-13 antibodies over a two-month period resulted in a reduction of both serum IL-13 levels and the number of mast cells in the salivary gland tissues, as well as correspondingly improved saliva production. These findings indicate a potentially important role for IL-13 in gland regulation and disease pathology.
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Linfocitos T CD4-Positivos/inmunología , Proteínas Inhibidoras de la Diferenciación/inmunología , Interleucina-13/inmunología , Mastocitos/patología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Síndrome de Sjögren/inmunología , Animales , Separación Celular , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Proteínas Inhibidoras de la Diferenciación/deficiencia , Proteínas Inhibidoras de la Diferenciación/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Síndrome de Sjögren/genética , Síndrome de Sjögren/patologíaRESUMEN
The molecular mechanisms that direct transcription of the gene encoding the transcription factor Foxp3 in CD4(+) T cells remain ill-defined. We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells). We identify two transforming growth factor-ß1 (TGF-ß1)-dependent mechanisms that were vital for activation of Foxp3 transcription and were defective in Id3(-/-) CD4(+) T cells. Enhanced binding of the transcription factor E2A to the Foxp3 promoter promoted Foxp3 transcription. Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter. Furthermore, Id3(-/-) T cells showed greater differentiation into the T(H)17 subset of helper T cells in vitro and in a mouse asthma model. Therefore, a network of factors acts in a TGF-ß-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.