RESUMEN
Regulatory T cells (Tregs) are crucial for suppressing autoimmunity and inflammation mediated by conventional T cells. To be useful, some Tregs should have overlapping specificity with relevant self-reactive or pathogen-specific clones. Whether matching recognition between Tregs and non-Tregs might arise through stochastic or deterministic mechanisms has not been addressed. We tested the hypothesis that some Tregs that arise in the thymus or that are induced during Ag-driven expansion of conventional CD4+ T cells might be clonally related to non-Tregs by virtue of asymmetric Foxp3 induction during cell division. We isolated mouse CD4+ thymocytes dividing in vivo, wherein sibling cells exhibited discordant expression of Foxp3 and CD25. Under in vitro conditions that stimulate induced Tregs from conventional mouse CD4+ T cells, we found a requirement for cell cycle progression to achieve Foxp3 induction. Moreover, a substantial fraction of sibling cell pairs arising from induced Treg stimulation also contained discordant expression of Foxp3. Division-linked yet asymmetric induction of Treg fate offers potential mechanisms to anticipate peripheral self-reactivity during thymic selection as well as produce precise, de novo counterregulation during CD4+ T cell-mediated immune responses.
RESUMEN
Unequal transmission of nutritive signaling during cell division establishes fate disparity between sibling lymphocytes, but how asymmetric signaling becomes organized is not understood. We show that receptor-associated class I phosphatidylinositol 3-kinase (PI3K) signaling activity, indexed by phosphatidylinositol (3,4,5)-trisphosphate (PIP3) staining, is spatially restricted to the microtubule-organizing center and subsequently to one pole of the mitotic spindle in activated T and B lymphocytes. Asymmetric PI3K activity co-localizes with polarization of antigen receptor components implicated in class I PI3K signaling and with facultative glucose transporters whose trafficking is PI3K dependent and whose abundance marks cells destined for differentiation. Perturbation of class I PI3K activity disrupts asymmetry of upstream antigen receptors and downstream glucose transporter traffic. The roles of PI3K signaling in nutrient utilization, proliferation, and gene expression may have converged with the conserved role of PI3K signaling in cellular symmetry breaking to form a logic for regenerative lymphocyte divisions.
Asunto(s)
Linfocitos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Diferenciación Celular , Humanos , Transducción de SeñalRESUMEN
Anabolic metabolism in lymphocytes promotes plasmablast and cytotoxic T cell differentiation at the expense of self-renewal. Heightened expression and function of the transcription factor IFN regulatory factor 4 (IRF4) accompany enhanced anabolic induction and full commitment to functional differentiation in B cells and CD8+ T cells. In this study, we used a genetic approach to determine whether IRF4 plays an analogous role in Th1 cell induction. Our findings indicate that IRF4 promotes determined Th1 cell differentiation in tandem with anabolic metabolism of CD4+ T cells. IRF4-deficient CD4+ T cells stimulated in vitro exhibit impaired induction of Th1 gene expression and defective silencing of T cell factor 1 expression. IRF4-deficient CD4+ T cells also undergo a shift toward catabolic metabolism, with reduced mammalian target of rapamycin activation, cell size, and nutrient uptake, as well as increased mitochondrial clearance. These findings suggest that the ability to remodel metabolic states can be an essential gateway for altering cell fate.
RESUMEN
Upon infection, an activated CD4+ T cell produces terminally differentiated effector cells and renews itself for continued defense. In this study, we show that differentiation and self-renewal arise as opposing outcomes of sibling CD4+ T cells. After influenza challenge, antigen-specific cells underwent several divisions in draining lymph nodes (LN; DLNs) while maintaining expression of TCF1. After four or five divisions, some cells silenced, whereas some cells maintained TCF1 expression. TCF1-silenced cells were T helper 1-like effectors and concentrated in the lungs. Cells from earliest divisions were memory-like and concentrated in nondraining LN. TCF1-expressing cells from later divisions in the DLN could self-renew, clonally yielding a TCF1-silenced daughter cell as well as a sibling cell maintaining TCF1 expression. Some TCF1-expressing cells in DLNs acquired an alternative, follicular helper-like fate. Modeled differentiation experiments in vitro suggested that unequal PI3K/mechanistic target of rapamycin signaling drives intraclonal cell fate heterogeneity. Asymmetric division enables self-renewal to be coupled to production of differentiated CD4+ effector T cells during clonal selection.
Asunto(s)
División Celular Asimétrica/fisiología , Linfocitos T CD4-Positivos/inmunología , Animales , División Celular , Células Cultivadas , Factor Nuclear 1-alfa del Hepatocito/análisis , Factor Nuclear 1-alfa del Hepatocito/genética , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/fisiología , Serina-Treonina Quinasas TOR/fisiologíaRESUMEN
Regeneration requires related cells to diverge in fate. We show that activated lymphocytes yield sibling cells with unequal elimination of aged mitochondria. Disparate mitochondrial clearance impacts cell fate and reflects larger constellations of opposing metabolic states. Differentiation driven by an anabolic constellation of PI3K/mTOR activation, aerobic glycolysis, inhibited autophagy, mitochondrial stasis, and ROS production is balanced with self-renewal maintained by a catabolic constellation of AMPK activation, mitochondrial elimination, oxidative metabolism, and maintenance of FoxO1 activity. Perturbations up and down the metabolic pathways shift the balance of nutritive constellations and cell fate owing to self-reinforcement and reciprocal inhibition between anabolism and catabolism. Cell fate and metabolic state are linked by transcriptional regulators, such as IRF4 and FoxO1, with dual roles in lineage and metabolic choice. Instructing some cells to utilize nutrients for anabolism and differentiation while other cells catabolically self-digest and self-renew may enable growth and repair in metazoa.
Asunto(s)
Proteína Forkhead Box O1/genética , Factores Reguladores del Interferón/genética , Activación de Linfocitos/genética , Linfocitos/metabolismo , Mitocondrias/metabolismo , Animales , Autofagia/genética , Diferenciación Celular/genética , Proteína Forkhead Box O1/metabolismo , Glucólisis , Hematopoyesis/genética , Factores Reguladores del Interferón/metabolismo , Metabolismo/genética , Ratones , Mitocondrias/genética , Fosfatidilinositol 3-Quinasas/genética , Especies Reactivas de Oxígeno/metabolismo , Regeneración/genética , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Selected CD8+ T cells must divide, produce differentiated effector cells, and self-renew, often repeatedly. We now show that silencing expression of the transcription factor TCF1 marks loss of self-renewal by determined effector cells and that this requires cell division. In acute infections, the first three CD8+ T cell divisions produce daughter cells with unequal proliferative signaling but uniform maintenance of TCF1 expression. The more quiescent initial daughter cells resemble canonical central memory cells. The more proliferative, effector-prone cells from initial divisions can subsequently undergo division-dependent production of a TCF1-negative effector daughter cell along with a self-renewing TCF1-positive daughter cell, the latter also contributing to the memory cell pool upon resolution of infection. Self-renewal in the face of effector cell determination may promote clonal amplification and memory cell formation in acute infections, sustain effector regeneration during persistent subclinical infections, and be rate limiting, but remediable, in chronic active infections and cancer.
Asunto(s)
Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Autorrenovación de las Células , Animales , Ciclo Celular , Diferenciación Celular , División Celular , Proliferación Celular , Células Clonales , Silenciador del Gen , Ratones Endogámicos C57BL , Factor 1 de Transcripción de Linfocitos T/metabolismoRESUMEN
Metazoan sibling cells often diverge in activity and identity, suggesting links between growth signals and cell fate. We show that unequal transduction of nutrient-sensitive PI3K/AKT/mTOR signaling during cell division bifurcates transcriptional networks and fates of kindred cells. A sibling B lymphocyte with stronger signaling, indexed by FoxO1 inactivation and IRF4 induction, undergoes PI3K-driven Pax5 repression and plasma cell determination, while its sibling with weaker PI3K activity renews a memory or germinal center B cell fate. PI3K-driven effector T cell determination silences TCF1 in one sibling cell, while its PI3K-attenuated sibling self-renews in tandem. Prior to bifurcations achieving irreversible plasma or effector cell fate determination, asymmetric signaling during initial divisions specifies a more proliferative, differentiation-prone lymphocyte in tandem with a more quiescent memory cell sibling. By triggering cell division but transmitting unequal intensity between sibling cells, nutrient-sensitive signaling may be a frequent arbiter of cell fate bifurcations during development and repair.
Asunto(s)
Linfocitos T CD8-positivos/citología , Diferenciación Celular/fisiología , Células Madre Hematopoyéticas/citología , Fosfatidilinositol 3-Quinasas/metabolismo , Células Plasmáticas/citología , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/metabolismo , Linaje de la Célula , Citometría de Flujo , Técnicas de Sustitución del Gen , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Células Plasmáticas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Humanized mice represent an important model to study the development and function of the human immune system. While it is known that mouse thymic stromal cells can support human T-cell development, the extent of interspecies cross-talk and the degree to which these systems recapitulate normal human T-cell development remain unclear. To address these questions, we compared conventional and non-conventional T-cell development in a neonatal chimera humanized mouse model with that seen in human fetal and neonatal thymus samples, and also examined the impact of a human HLA-A2 transgene expressed by the mouse stroma. Given that dynamic migration and cell-cell interactions are essential for T-cell differentiation, we also studied the intrathymic migration pattern of human thymocytes developing in a murine thymic environment. We found that both conventional T-cell development and intra-thymic migration patterns in humanized mice closely resemble human thymopoiesis. Additionally, we show that developing human thymocytes engage in short, serial interactions with other human hematopoietic-derived cells. However, non-conventional T-cell differentiation in humanized mice differed from both fetal and neonatal human thymopoiesis, including a marked deficiency of Foxp3(+) T-cell development. These data suggest that although the murine thymic microenvironment can support a number of aspects of human T-cell development, important differences remain, and additional human-specific factors may be required.
Asunto(s)
Diferenciación Celular , Movimiento Celular , Linfocitos T/citología , Linfocitos T/fisiología , Animales , Biomarcadores , Comunicación Celular , Microambiente Celular , Expresión Génica , Genes Reporteros , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Antígeno HLA-A2/metabolismo , Humanos , Sistema Inmunológico/citología , Sistema Inmunológico/fisiología , Linfopoyesis , Ratones , Ratones Transgénicos , Modelos Animales , Organogénesis , Fenotipo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/fisiología , Timocitos/citología , Timocitos/fisiología , Timo/citología , Timo/embriología , Timo/fisiologíaRESUMEN
The ordered migration of thymocytes from the cortex to the medulla is critical for the appropriate selection of the mature T cell repertoire. Most studies of thymocyte migration rely on mouse models, but we know relatively little about how human thymocytes find their appropriate anatomical niches within the thymus. Moreover, the signals that retain CD4+CD8+ double-positive (DP) thymocytes in the cortex and prevent them from entering the medulla prior to positive selection have not been identified in mice or humans. Here, we examined the intrathymic migration of human thymocytes in both mouse and human thymic stroma and found that human thymocyte subsets localized appropriately to the cortex on mouse thymic stroma and that MHC-dependent interactions between human thymocytes and mouse stroma could maintain the activation and motility of DP cells. We also showed that CXCR4 was required to retain human DP thymocytes in the cortex, whereas CCR7 promoted migration of mature human thymocytes to the medulla. Thus, 2 opposing chemokine gradients control the migration of thymocytes from the cortex to the medulla. These findings point to significant interspecies conservation in thymocyte-stroma interactions and provide the first evidence that chemokines not only attract mature thymocytes to the medulla, but also play an active role in retaining DP thymocytes in the cortex prior to positive selection.
Asunto(s)
Quimiotaxis de Leucocito , Receptores CCR7/metabolismo , Receptores CXCR4/metabolismo , Timocitos/citología , Timo/fisiología , Animales , Comunicación Celular , Diferenciación Celular , Citometría de Flujo , Humanos , Ratones , Microscopía Fluorescente , Subgrupos de Linfocitos T/citología , Timo/embriologíaRESUMEN
Monkeypox virus (MPXV) is an orthopoxvirus closely related to variola virus, the causative agent of smallpox. Human MPXV infection results in a disease that is similar to smallpox and can also be fatal. Two clades of MPXV have been identified, with viruses of the central African clade displaying more pathogenic properties than those within the west African clade. The monkeypox inhibitor of complement enzymes (MOPICE), which is not expressed by viruses of the west African clade, has been hypothesized to be a main virulence factor responsible for increased pathogenic properties of central African strains of MPXV. To gain a better understanding of the role of MOPICE during MPXV-mediated disease, we compared the host adaptive immune response and disease severity following intrabronchial infection with MPXV-Zaire (n = 4), or a recombinant MPXV-Zaire (n = 4) lacking expression of MOPICE in rhesus macaques (RM). Data presented here demonstrate that infection of RM with MPXV leads to significant viral replication in the peripheral blood and lungs and results in the induction of a robust and sustained adaptive immune response against the virus. More importantly, we show that the loss of MOPICE expression results in enhanced viral replication in vivo, as well as a dampened adaptive immune response against MPXV. Taken together, these findings suggest that MOPICE modulates the anti-MPXV immune response and that this protein is not the sole virulence factor of the central African clade of MPXV.
Asunto(s)
Monkeypox virus/inmunología , Monkeypox virus/patogenicidad , Mpox/inmunología , Mpox/patología , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismo , Inmunidad Adaptativa , Animales , Linfocitos B/inmunología , Sangre/virología , ADN Viral/química , ADN Viral/genética , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Pulmón/virología , Macaca mulatta , Masculino , Datos de Secuencia Molecular , Mpox/virología , Enfermedades de los Primates/inmunología , Enfermedades de los Primates/patología , Enfermedades de los Primates/virología , Análisis de Secuencia de ADN , Piel/patología , Linfocitos T/inmunología , Proteínas Virales/genética , Factores de Virulencia/genéticaRESUMEN
Rhesus macaque rhadinovirus (RRV) is closely related to Kaposi sarcoma-associated herpesvirus (KSHV) and is associated with the development of B-cell hyperplasia and persistent lymphadenopathy resembling multicentric Castleman disease in rhesus macaques (RMs) coinfected with simian immunodeficiency virus (SIV). Here we investigated whether RMs experimentally infected with SIV and RRV can develop other disease manifestations observed in HIV- and KSHV-infected patients. As reported earlier, inoculation of SIV-infected RMs with RRV results in persistent RRV infection, whereas immunocompetent animals infected with RRV exhibit viremia 2 weeks after infection, followed by a period of no virus detection until they are subsequently made immunodeficient by SIV infection. A subset of animals developed abnormal cellular proliferations characterized as extranodal lymphoma and a proliferative mesenchymal lesion. In situ hybridization and immunohistochemistry analysis indicate RRV is present in both malignancies, and DNA microarray analysis detected viral interleukin-6 (vIL-6) and viral FLICE-like inhibitory protein (vFLIP) transcripts. Reverse-transcriptase polymerase chain reaction analysis confirmed vIL-6 and vFLIP expression, and that of RRV open reading frames 72 and 73, homologs of KSHV open reading frames shown to be expressed in primary effusion lymphoma. These data support the utility of the RRV-/SIV-infected RM as an excellent animal model to investigate KSHV-like pathogenesis.
Asunto(s)
Modelos Animales de Enfermedad , Infecciones por VIH/virología , VIH , Infecciones por Herpesviridae/metabolismo , Herpesvirus Humano 8/metabolismo , Linfoma no Hodgkin/metabolismo , Rhadinovirus/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/metabolismo , Virus de la Inmunodeficiencia de los Simios , Infecciones Tumorales por Virus/metabolismo , Animales , Enfermedad de Castleman/metabolismo , Enfermedad de Castleman/virología , Regulación Leucémica de la Expresión Génica , Regulación Viral de la Expresión Génica , Infecciones por VIH/metabolismo , Infecciones por Herpesviridae/virología , Humanos , Linfoma no Hodgkin/virología , Macaca mulatta , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Infecciones Tumorales por Virus/virología , Proteínas Virales/biosíntesisRESUMEN
Previously, we showed that the Vpu protein from subtype C human immunodeficiency virus type 1 (HIV-1) was efficiently targeted to the cell surface, suggesting that this protein has biological properties that differ from the well-studied subtype B Vpu protein. In this study, we have further analyzed the biological properties of the subtype C Vpu protein. Flow cytometric analysis revealed that the subtype B Vpu (strain HXB2) was more efficient at down-regulating CD4 surface expression than the Vpu proteins from four subtype C clinical isolates. We constructed a simian-human immunodeficiency virus virus, designated as SHIV(SCVpu), in which the subtype B vpu gene from the pathogenic SHIV(KU-1bMC33) was substituted with the vpu from a clinical isolate of subtype C HIV-1 (strain C.96.BW16B01). Cell culture studies revealed that SHIV(SCVpu) replicated with slightly reduced kinetics when compared with the parental SHIV(KU-1bMC33) and that the viral Env and Gag precursor proteins were synthesized and processed similarly compared to the parental SHIV(KU-1bMC33). To determine if substitution of the subtype C Vpu protein affected the pathogenesis of the virus, three pig-tailed macaques were inoculated with SHIV(SCVpu) and circulating CD4+ T-cell levels and viral loads were monitored for up to 44 weeks. Our results show that SHIV(SCVpu) caused a more gradual decline in the rate of CD4+ T cells in pig-tailed macaques compared to those inoculated with parental subtype B SHIV(KU-1bMC33). These results show for the first time that different Vpu proteins of HIV-1 can influence the rate at which CD4+ T-cell loss occurs in the SHIV/pig-tailed macaque model.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , VIH-1/patogenicidad , Proteínas del Virus de la Inmunodeficiencia Humana/inmunología , Virus de la Inmunodeficiencia de los Simios , Proteínas Reguladoras y Accesorias Virales/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Linfocitos T CD4-Positivos/ultraestructura , Línea Celular , Modelos Animales de Enfermedad , Citometría de Flujo , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , VIH-1/inmunología , Células HeLa , Proteínas del Virus de la Inmunodeficiencia Humana/química , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Recuento de Linfocitos , Macaca nemestrina , Datos de Secuencia Molecular , Plásmidos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/ultraestructura , Linfocitos T/virología , Factores de Tiempo , Transfección , Carga Viral , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
Rhesus rhadinovirus (RRV) is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) and causes KSHV-like diseases in immunocompromised rhesus macaques (RM) that resemble KSHV-associated diseases including multicentric Castleman's disease and non-Hodgkin's lymphoma. RRV retains a majority of open reading frames (ORFs) postulated to be involved in the pathogenesis of KSHV and is the closest available animal model to KSHV infection in humans. Here we describe the generation of a recombinant clone of RRV strain 17577 (RRV(17577)) utilizing bacterial artificial chromosome (BAC) technology. Characterization of the RRV BAC demonstrated that it is a pathogenic molecular clone of RRV(17577), producing virus that behaves like wild-type RRV both in vitro and in vivo. Specifically, BAC-derived RRV displays wild-type growth properties in vitro and readily infects simian immunodeficiency virus-infected RM, inducing B cell hyperplasia, persistent lymphadenopathy, and persistent infection in these animals. This RRV BAC will allow for rapid genetic manipulation of the RRV genome, facilitating the creation of recombinant versions of RRV that harbor specific alterations and/or deletions of viral ORFs. This system will provide insights into the roles of specific RRV genes in various aspects of the viral life cycle and the RRV-associated pathogenesis in vivo in an RM model of infection. Furthermore, the generation of chimeric versions of RRV containing KSHV genes will allow analysis of the function and contributions of KSHV genes to viral pathogenesis by using a relevant primate model system.