RESUMEN
Innate lymphoid cells (ILC) are RAG-independent lymphocytes with important roles in innate immunity, and include group-1 (natural killer (NK) cell, ILC1), group-2 (ILC2), and group-3 (lymphoid tissue inducer (LTi), NCR(+) ILC3) subsets. Group-3 ILC express Rorγt, produce interleukin (IL)-22, and are critically important in the normal function of mucosal tissues. Here, we describe a novel model cell line for the study of ILC function and differentiation. The parental MNK cell line, derived from NKR-P1B(+) fetal thymocytes, shows a capacity to differentiate in γc cytokines. One IL-7-responsive subline, designated MNK-3, expresses Rorγt and produces high levels of IL-22 in response to IL-23 and IL-1ß stimulation. MNK-3 cells display surface markers and transcript expression characteristic of group-3 ILC, including IL-7Rα (CD127), c-kit (CD117), CCR6, Thy1 (CD90), RANK, RANKL, and lymphotoxin (LTα1ß2). Using an in vitro assay of LTi cell activity, MNK-3 cells induce ICAM-1 and VCAM-1 expression on stromal cells in a manner dependent upon LTα1ß2 expression. A second IL-2-responsive subline, MNK-1, expresses several NK cell receptors, perforin and granzymes, and shows some cytotoxic activity. Thus, MNK-1 cells serve as a model of ILC1/NK development and differentiation, whereas MNK-3 cells provide an attractive in vitro system to study the function of ILC3/LTi cells.
Asunto(s)
Diferenciación Celular/inmunología , Inmunidad Innata , Linfocitos/citología , Linfocitos/inmunología , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Linaje de la Célula , Análisis por Conglomerados , Citocinas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Inmunofenotipificación , Subgrupos Linfocitarios/citología , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Linfocitos/metabolismo , Ratones , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Fenotipo , Receptores de Células Asesinas Naturales/genética , Receptores de Células Asesinas Naturales/metabolismoRESUMEN
We report an in vitro stroma-dependent system for the clonal growth and differentiation of natural killer (NK) cells from lymphoid-restricted bone marrow progenitors or bone marrow NK1.1+ cells. Strikingly, the potential to initiate expression of specific Ly49 receptors becomes increasingly restricted as NK cells develop. Moreover, when NK cells express a Ly49 receptor specific for stromal cell class I MHC, they are less likely to initiate expression of another Ly49 receptor in the clonal culture system. The results indicate multiple roles for stromal cells in NK cell development, in supporting clonal growth, in initiation of Ly49 receptor expression, and in formation of the NK cell receptor repertoire.
Asunto(s)
Antígenos Ly/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Antígenos , Antígenos Ly/genética , Antígenos de Superficie , Diferenciación Celular , Células Cultivadas , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Lectinas Tipo C , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Subfamilia B de Receptores Similares a Lectina de Células NK , Proteínas , Proteínas Proto-Oncogénicas c-kit/análisis , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Receptores Similares a Lectina de Células NK , Factores de Tiempo , Microglobulina beta-2/genéticaRESUMEN
We recently described a population of fetal thymocytes with a CD117+NK1.1+CD90lowCD25- phenotype, which were shown to contain committed T cell and NK cell progenitors. However, the characterization of a single cell with a restricted T and NK cell precursor potential was lacking. Here, using an in vitro model for T and NK cell differentiation, we provide conclusive evidence demonstrating the existence of a clonal lineage-restricted T and NK cell progenitor. These results establish that fetal thymocytes with a CD117+NK1.1+CD90lowCD25- phenotype represent bipotent T and NK cell progenitors.
Asunto(s)
Células Asesinas Naturales/inmunología , Proteínas , Células Madre/inmunología , Subgrupos de Linfocitos T/inmunología , Timo/embriología , Timo/inmunología , Animales , Antígenos/biosíntesis , Antígenos Ly , Antígenos de Superficie , Linaje de la Célula/inmunología , Células Clonales , Ensayo de Unidades Formadoras de Colonias , Inmunofenotipificación , Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Lectinas Tipo C , Ratones , Ratones Endogámicos C57BL , Subfamilia B de Receptores Similares a Lectina de Células NK , Técnicas de Cultivo de Órganos , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-kit/biosíntesis , Células Madre/citología , Células Madre/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Timo/citologíaRESUMEN
To study molecular events involved in B lymphocyte development and V(D)J rearrangement, we have established an efficient system for the differentiation of embryonic stem (ES) cells into mature Ig-secreting B lymphocytes. Here, we show that B lineage cells generated in vitro from ES cells are functionally analogous to normal fetal liver-derived or bone marrow-derived B lineage cells at three important developmental stages: first, they respond to Flt-3 ligand during an early lymphopoietic progenitor stage; second, they become targets for Abelson murine leukemia virus (A-MuLV) infection at a pre-B cell stage; third, they secrete Ig upon stimulation with lipopolysaccharide at a mature mitogen-responsive stage. Moreover, the ES cell-derived A-MuLV-transformed pre-B (EAB) cells are phenotypically and functionally indistinguishable from standard A-MuLV-transformed pre-B cells derived from infection of mouse fetal liver or bone marrow. Notably, EAB cells possess functional V(D)J recombinase activity. In particular, the generation of A-MuLV transformants from ES cells will provide an advantageous system to investigate genetic modifications that will help to elucidate molecular mechanisms in V(D)J recombination and in A-MuLV-mediated transformation.
Asunto(s)
Linfocitos B/fisiología , Reordenamiento Génico de Linfocito B , Células Madre Hematopoyéticas/fisiología , Virus de la Leucemia Murina de Abelson , Animales , Diferenciación Celular/efectos de los fármacos , Técnicas de Cocultivo , Hematopoyesis , Células Madre Hematopoyéticas/efectos de los fármacos , Ligandos , Proteínas de la Membrana/farmacología , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
The mouse NK1.1 Ag originally defined as NK cell receptor (NKR)-P1C (CD161) mediates NK cell activation. Here, we show that another member of the mouse CD161 family, NKR-P1B, represents a novel NK1.1 Ag. In contrast to NKR-P1C, which functions as an activating receptor, NKR-P1B inhibits NK cell activation. Association of NKR-P1B with Src homology 2-containing protein tyrosine phosphatase-1 provides a molecular mechanism for this inhibition. The existence of these two NK1.1 Ags with opposite functions suggests a potential role for NKR-P1 molecules, such as those of the Ly-49 gene family, in regulating NK cell function.
Asunto(s)
Antígenos de Superficie/metabolismo , Antígenos/metabolismo , Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Lectinas Tipo C , Proteínas/metabolismo , Receptores Inmunológicos/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos/genética , Antígenos Ly , Antígenos de Superficie/genética , Células Sanguíneas/inmunología , Sangre Fetal/inmunología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Modelos Inmunológicos , Datos de Secuencia Molecular , Familia de Multigenes , Subfamilia B de Receptores Similares a Lectina de Células NK , Fosforilación , Unión Proteica , Proteína Fosfatasa 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas/genética , Receptores Inmunológicos/genética , Proteínas Tirosina Fosfatasas con Dominio SH2 , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Dominios Homologos srcRESUMEN
We recently identified a stage in fetal ontogeny (NK1.1+/CD117+) that defines committed progenitors for T and NK lymphocytes. These cells are found in the fetal thymus as early as day 13 of gestation, but are absent in the fetal liver. Nonetheless, multipotent precursors derived from both the fetal thymus and fetal liver are capable of rapidly differentiating to the NK1.1+ stage upon transfer into fetal thymic organ culture (FTOC). This suggests that expression of NK1.1 marks a thymus-induced lineage commitment event. We now report that a subset of the most immature fetal thymocytes (NK1.1-/CD117+) is capable of up-regulating NK1.1 expression spontaneously upon short-term in vitro culture. Interestingly, fetal liver-derived CD117+ precursors remain NK1.1- upon similar culture. Spontaneous up-regulation of NK1.1 surface expression is minimally affected by transcriptional blockade, mitogen-induced activation, or exposure of these cells to exogenous cytokines or stromal cells. These data suggest that induction of NK1.1 expression on cultured thymocytes may be predetermined by exposure to the thymic microenvironment in vivo. Importantly, multipotent CD117+ thymocytes subdivided on the basis of NK1.1 expression after short-term in vitro culture show distinct precursor potential in lymphocyte lineage reconstitution assays. This demonstrates that even the earliest precursor thymocyte population, although phenotypically homogeneous, contains a functionally heterogeneous subset of lineage-committed progenitors. These findings characterize a thymus-induced pathway in the control of lymphocyte lineage commitment to the T and NK cell fates.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Células Asesinas Naturales/metabolismo , Biosíntesis de Proteínas , Subgrupos de Linfocitos T/metabolismo , Timo/citología , Animales , Antígenos/genética , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos Ly , Antígenos de Superficie , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Células Madre Hematopoyéticas/citología , Células Asesinas Naturales/citología , Lectinas Tipo C , Hígado/citología , Hígado/embriología , Ratones , Ratones Endogámicos C57BL , Subfamilia B de Receptores Similares a Lectina de Células NK , Técnicas de Cultivo de Órganos , Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/metabolismo , Subgrupos de Linfocitos T/citología , Timo/embriología , Transcripción GenéticaRESUMEN
T cells and natural killer (NK) cells are presumed to share a common intrathymic precursor. The development of conventional alpha beta T lymphocytes begins within the early fetal thymus, after the colonization of multipotent CD117+ precursors. Irrevocable commitment to the T lineage is marked by thymus-induced expression of CD25. However, the contribution of the fetal thymus to NK lineage commitment and differentiation remains largely unappreciated. Recently, we demonstrated that the development of functional mouse NK cells occurs first in the fetal thymus. Moreover, the appearance of mature fetal thymic NK cells (NK1.1+/CD117-) is preceded by a thymus-induced developmental stage (NK1.1+/CD117+) that marks lineage commitment of multipotent hematopoietic precursors to the T and NK-cell fates. Commitment to the T/NK bipotent stage is induced by fetal thymic stroma, but is not thymus dependent. Recent data indicate that CD90+/CD117lo fetal blood prothymocytes exhibit NK lineage potential and are phenotypically and functionally identical to fetal thymic NK1.1+/CD117+ progenitors. This finding also indicates that full commitment of circulating precursors to the T-cell lineage occurs after thymus colonization. In this review, we discuss recent insights into the cellular and molecular events involved in fetal mouse T and NK lineage commitment and differentiation to unipotent progenitors.
Asunto(s)
Células Asesinas Naturales/citología , Linfocitos T/citología , Animales , Diferenciación Celular , Linaje de la Célula , Células Madre Hematopoyéticas , Humanos , Linfocitos , Ratones , Timo/citología , Timo/embriologíaRESUMEN
We recently identified a fetal thymic developmental stage (NK1.1+/CD117(lo)) that characterizes committed T/NK progenitors. We now report the existence of phenotypically and functionally identical T/NK progenitors in mouse fetal blood and spleen but not in fetal liver. These precursors are indistinguishable from previously characterized fetal blood "prothymocytes" (CD90+/CD117(lo)), with the exception that they express NK1.1, lack markers associated with T lineage commitment, maintain a germline TCRbeta locus, and can give rise to both T and NK cells. Moreover, NK1.1+/CD90+/CD117(lo) fetal blood precursors are present in athymic nude mice. These results suggest that the T/NK lineage commitment pathway is thymus-independent. In contrast, full commitment to the alphabeta T lineage does not precede thymus colonization.
Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Timo/fisiología , Animales , Linfocitos B/citología , Células de la Médula Ósea/citología , Diferenciación Celular , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Técnicas de Cocultivo , Femenino , Sangre Fetal/citología , Expresión Génica , Células Asesinas Naturales/citología , Células Asesinas Naturales/fisiología , Ratones , Ratones Desnudos , Técnicas de Cultivo de Órganos , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Bazo/citología , Bazo/fisiología , Células del Estroma/citología , Timo/citologíaRESUMEN
Natural killer (NK) cells mediate MHC-unrestricted cytolysis of virus-infected cells and tumor cells. In the adult mouse, NK cells are bone marrow-derived lymphocytes that mature predominantly in extrathymic locations but have also been suggested to share a common intrathymic progenitor with T lymphocytes. However, mature NK cells are thought to be absent in mouse fetal ontogeny. We report the existence of thymocytes with a mature NK cell phenotype (NK1.1+/CD117-) as early as day 13 of gestation, approximately 3 days before the appearance of CD4+/CD8+ cells in T lymphocyte development. These mature fetal thymic NK cells express genes associated with NK cell effector function and, when freshly isolated, display MHC-unrestricted cytolytic activity in vitro. Moreover, the capacity of fetal thymic NK cells for sustained growth both in vitro and in vivo, in addition to their close phenotypic resemblance to early precursor thymocytes, confounds previous assessments of NK lineage precursor function. Thus, mature NK cells may have been inadvertently included in previous attempts to identify multipotent and bipotent precursor thymocytes. These results provide the first evidence of functional NK lymphocytes in mouse fetal ontogeny and demonstrate that NK cell maturation precedes alpha beta T cell development in the fetal thymus.
Asunto(s)
Desarrollo Embrionario y Fetal/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Traslado Adoptivo , Animales , Diferenciación Celular/inmunología , División Celular/inmunología , Separación Celular , Células Cultivadas , Citotoxicidad Inmunológica/genética , Regulación del Desarrollo de la Expresión Génica/inmunología , Inmunofenotipificación , Células Asesinas Naturales/metabolismo , Complejo Mayor de Histocompatibilidad/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trasplante de Células Madre , Células Madre/citología , Células Madre/inmunología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
C4Dlow cells are a population of lymphoid lineage-restricted progenitor cells representing the earliest precursors present in the adult thymus. Paradoxically, thymic progenitors with a similar phenotype in fetal mice and adult RAG-2-deficient (RAG-2-/-) mice lack this characteristic low-level expression of CD4. We now show that radiation-induced differentiation of CD4+ CD8+ double positive thymocytes in RAG-2-/- mice results in the appearance of low levels of CD4 on thymocytes that are phenotypically identical to C4Dlow progenitor cells present in the normal adult thymus. This suggests that CD4 surface expression can be passively transferred from double positive cells to early progenitor thymocytes. Analysis of mixed bone marrow chimeras, reconstituted with hematopoietic stem cells from both CD4-/- (CD45.2) and CD4wt (CD45.1) congenic mice, revealed a CD4low phenotype on cells derived from CD4-/- bone marrow cells. Furthermore, these CD4-/- -derived "C4Dlow" progenitors were capable of reconstituting lymphocyte-depleted fetal thymi, with all thymocytes displaying a CD4-/- phenotype. This directly demonstrates that genetically CD4-deficient thymic progenitor cells can passively acquire a C4Dlow phenotype. Moreover, CD4 expression on C4Dlow progenitor thymocytes is sensitive to mild acid treatment, indicating that CD4 may not exist as an integral cell surface molecule on this thymocyte population. Our findings demonstrate that low-level CD4 surface expression can be passively acquired by intrathymic progenitor cells from the surrounding thymic microenvironment, suggesting that other cell surface molecules expressed at low levels may also result from an acquired phenotype.
Asunto(s)
Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/clasificación , Células Madre/clasificación , Células Madre/inmunología , Timo/citología , Timo/inmunología , Animales , Médula Ósea/inmunología , Antígenos CD4/biosíntesis , Antígenos CD4/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Quimera por Radiación/inmunología , Células Madre/metabolismo , Timo/metabolismoRESUMEN
Bipotent progenitors for T and natural killer (NK) lymphocytes are thought to exist among early precursor thymocytes. The identification and functional properties of such a progenitor population remain undefined. We report the identification of a novel developmental stage during fetal thymic ontogeny that delineates a population of T/NK-committed progenitors (NK1. 1(+)/CD117(+)/CD44(+)/CD25(-)). Thymocytes at this stage in development are phenotypically and functionally distinguishable from the pool of multipotent lymphoid-restricted (B, T, and NK) precursor thymocytes. Exposure of multipotent precursor thymocytes or fetal liver- derived hematopoietic progenitors to thymic stroma induces differentiation to the bipotent developmental stage. Continued exposure to a thymic microenvironment results in predominant commitment to the T cell lineage, whereas coculture with a bone marrow-derived stromal cell line results in the generation of mature NK cells. Thus, the restriction point to T and NK lymphocyte destinies from a multipotent progenitor stage is marked by a thymus-induced differentiation step.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Linfocitos T/fisiología , Animales , Diferenciación Celular , Linaje de la Célula , Femenino , Feto/inmunología , Células Asesinas Naturales/fisiología , Ratones , Ratones Endogámicos C57BL , Embarazo , Proteínas Proto-Oncogénicas c-kit/análisisRESUMEN
Swine offer an excellent, but previously unused, model for brain interstitial ionizing and non-ionizing radiation research. Significant advantages include size, cost, maneuverability, availability and conditioning. The methodology and some experimental results from studies of the effects of interstitial microwave hyperthermia and iridium-192 irradiation are presented.