RESUMEN
Multiple subsets of FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent dendritic cells (DCs) control T-cell tolerance and immunity. In mice, Batf3-dependent CD103(+) DCs efficiently enter lymph nodes and cross-present antigens, rendering this conserved DC subset a promising target for tolerance induction or vaccination. However, only limited numbers of CD103(+) DCs can be isolated with current methods. Established bone marrow culture protocols efficiently generate monocyte-derived DCs or produce a mixture of FLT3L-dependent DC subsets. We show that CD103(+) DC development requires prolonged culture time and continuous action of both FLT3L and granulocyte macrophage colony-stimulating factor (GM-CSF), explained by a dual effect of GM-CSF on DC precursors and differentiating CD103(+) DCs. Accordingly, we established a novel method to generate large numbers of CD103(+) DCs (iCD103-DCs) with limited presence of other DC subsets. iCD103-DCs develop in a Batf3- and Irf8-dependent fashion, express a CD8α/CD103 DC gene signature, cross-present cell-associated antigens, and respond to TLR3 stimulation. Thus, iCD103-DCs reflect key features of tissue CD103(+) DCs. Importantly, iCD103-DCs express high levels of CCR7 upon maturation and migrate to lymph nodes more efficiently than classical monocyte-derived DCs. Finally, iCD103-DCs induce T cell-mediated protective immunity in vivo. Our study provides insights into CD103(+) DC development and function.
Asunto(s)
Antígenos CD/inmunología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula/métodos , Células Dendríticas/citología , Células Dendríticas/inmunología , Cadenas alfa de Integrinas/inmunología , Proteínas Represoras/inmunología , Animales , Antígenos CD/análisis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/análisis , Diferenciación Celular , Células Cultivadas , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Inmunidad Celular , Cadenas alfa de Integrinas/análisis , Proteínas de la Membrana/inmunología , Ratones , Proteínas Represoras/análisis , Linfocitos T/inmunología , Receptor Toll-Like 3/inmunologíaRESUMEN
Plasmacytoid dendritic cells (pDCs) express the I-type lectin receptor Siglec-H and produce interferon α (IFNα), a critical anti-viral cytokine during the acute phase of murine cytomegalovirus (MCMV) infection. The ligands and biological functions of Siglec-H still remain incompletely defined in vivo. Thus, we generated a novel bacterial artificial chromosome (BAC)-transgenic "pDCre" mouse which expresses Cre recombinase under the control of the Siglec-H promoter. By crossing these mice with a Rosa26 reporter strain, a representative fraction of Siglec-H⺠pDCs is terminally labeled with red fluorescent protein (RFP). Interestingly, systemic MCMV infection of these mice causes the downregulation of Siglec-H surface expression. This decline occurs in a TLR9- and MyD88-dependent manner. To elucidate the functional role of Siglec-H during MCMV infection, we utilized a novel Siglec-H deficient mouse strain. In the absence of Siglec-H, the low infection rate of pDCs with MCMV remained unchanged, and pDC activation was still intact. Strikingly, Siglec-H deficiency induced a significant increase in serum IFNα levels following systemic MCMV infection. Although Siglec-H modulates anti-viral IFNα production, the control of viral replication was unchanged in vivo. The novel mouse models will be valuable to shed further light on pDC biology in future studies.
Asunto(s)
Células Dendríticas/inmunología , Infecciones por Herpesviridae/inmunología , Interferón-alfa/inmunología , Lectinas/inmunología , Modelos Inmunológicos , Muromegalovirus/fisiología , Células Plasmáticas/inmunología , Receptores de Superficie Celular/inmunología , Animales , Células Dendríticas/metabolismo , Células Dendríticas/patología , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/patología , Interferón-alfa/genética , Interferón-alfa/metabolismo , Lectinas/genética , Lectinas/metabolismo , Ratones , Ratones Noqueados , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
Self-reactive and polyreactive B cells generated during B cell development are silenced by either apoptosis, clonal deletion, receptor editing or anergy to avoid autoimmunity. The specific contribution of apoptosis to normal B cell development and self-tolerance is incompletely understood. Here, we quantify self-reactivity, polyreactivity and apoptosis during physiologic B lymphocyte development. Self-reactivity and polyreactivity are most abundant in early immature B cells and diminish significantly during maturation within the bone marrow. Minimal apoptosis still occurs at this site, however B cell receptors cloned from apoptotic B cells show comparable self-reactivity to that of viable cells. Apoptosis increases dramatically only following immature B cells leaving the bone marrow sinusoids, but above 90% of cloned apoptotic transitional B cells are not self-reactive/polyreactive. Our data suggests that an apoptosis-independent mechanism, such as receptor editing, removes most self-reactive B cells in the bone marrow. Mechanistically, lack of survival signaling rather than clonal deletion appears to be the underpinning cause of apoptosis in most transitional B cells in the periphery.
Asunto(s)
Apoptosis , Linfocitos B , Supresión Clonal , Ratones Endogámicos C57BL , Animales , Apoptosis/inmunología , Supresión Clonal/inmunología , Linfocitos B/inmunología , Ratones , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/genética , Diferenciación Celular/inmunología , Médula Ósea/inmunología , Femenino , Células Precursoras de Linfocitos B/inmunologíaRESUMEN
Autoreactive B cells generated during B cell development are inactivated by clonal deletion, receptor editing or anergy. Up to 97% of immature B cells appear to die before completing maturation, but the anatomic sites and reasons underlying this massive cell loss are not fully understood. Here, we directly quantitated apoptosis and clonal deletion during physiologic B lymphocyte development using Rosa26INDIA apoptosis indicator mice. Immature B cells displayed low levels of apoptosis in the bone marrow but started dying at high levels in the periphery upon release from bone marrow sinusoids into the blood circulation. Clonal deletion of self-reactive B cells was neither a major contributor to apoptosis in the bone marrow nor the periphery. Instead, most peripheral transitional 1 B cells did not encounter the signals required for positive selection into the mature B cell compartments. This study sheds new light on B cell development and suggests that receptor editing and/or anergy efficiently control most primary autoreactivity in mice.
RESUMEN
Foxp3(+) regulatory T cells (Tregs) balance the mammalian immune system by mechanisms that are yet to be elucidated in their entirety. Methods employed to quantify the regulatory activity of Tregs in vitro are an important tool in cellular immunology, but can be technically demanding and subjected to variation. In this manuscript, we describe in detail a robust Treg suppression assay based on the flow cytometric quantification of both CD4(+) and CD8(+) effector T cell functions. This method can provide valuable insights into the immunosuppressive activity of Foxp3(+) Tregs and is versatile with regard to genetic or pharmacologic manipulations. Additionally, novel regulatory immune cells can be characterized by using this assay.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Tolerancia Inmunológica/inmunología , Técnicas Inmunológicas/métodos , Linfocitos T Reguladores/inmunología , Animales , Separación Celular/métodos , Células Cultivadas , Femenino , Citometría de Flujo , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismoRESUMEN
Regulatory T cells (Tregs) play a non-redundant role in maintenance of immune homeostasis. This is achieved by suppressing both, priming of naïve cells and effector cell functions. Although Tregs have been implicated in modulating allergic immune responses, their influence on distinct phases of development of allergies remains unclear. In this study, by using bacterial artificial chromosome (BAC)-transgenic Foxp3-DTR (DEREG) mice we demonstrate that the absence of Foxp3(+) Tregs during the allergen challenge surprisingly does not exacerbate allergic airway inflammation in BALB/c mice. As genetic disposition due to strain specificity may contribute significantly to development of allergies, we performed similar experiment in C57BL/6 mice, which are less susceptible to allergy in the model of sensitization used in this study. We report that the genetic background does not influence the consequence of this depletion regimen. These results signify the temporal regulation exerted by Foxp3(+) Tregs in limiting allergic airway inflammation and may influence their application as potential therapeutics.