RESUMEN
The classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127- and CD127+ early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127- and CD127+ ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127- ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127+ ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis.
Asunto(s)
Linfocitos B/metabolismo , Células Asesinas Naturales/metabolismo , Células Progenitoras Linfoides/metabolismo , Linfopoyesis/genética , Linfocitos T/metabolismo , Adolescente , Adulto , Animales , Linfocitos B/citología , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Cultivadas , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad alfa del Receptor de Interleucina-7/genética , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Células Asesinas Naturales/citología , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/trasplante , Masculino , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Persona de Mediana Edad , Trasplante de Células Madre , Linfocitos T/citología , Trasplante Heterólogo , Adulto JovenRESUMEN
In this study, we identify transmembrane protein 131-like (TMEM131L) as a novel regulator of thymocyte proliferation and demonstrate that it corresponds to a not as yet reported inhibitor of Wnt signaling. Short hairpin RNA-mediated silencing of TMEM131L in human CD34(+) hematopoietic progenitors, which were then grafted in NOD-SCID/IL-2rγ(null) mice, resulted in both thymocyte hyperproliferation and multiple pre- and post-ß-selection intrathymic developmental defects. Consistent with deregulated Wnt signaling, TMEM131L-deficient thymocytes expressed Wnt target genes at abnormally high levels, and they displayed both constitutive phosphorylation of Wnt coreceptor LRP6 and ß-catenin intranuclear accumulation. Using T cell factor reporter assays, we found that membrane-associated TMEM131L inhibited canonical Wnt/ß-catenin signaling at the LRP6 coreceptor level. Whereas membrane-associated TMEM131L did not affect LRP6 expression under basal conditions, it triggered lysosome-dependent degradation of its active phosphorylated form following Wnt activation. Genetic mapping showed that phosphorylated LRP6 degradation did not depend on TMEM131L cytoplasmic part but rather on a conserved extracellular domain proximal to the membrane. Collectively, these data indicate that, during thymopoiesis, stage-specific surface translocation of TMEM131L may regulate immature single-positive thymocyte proliferation arrest by acting through mixed Wnt-dependent and -independent mechanisms.
Asunto(s)
Proliferación Celular , Proteínas de la Membrana/metabolismo , Timocitos/citología , Vía de Señalización Wnt/fisiología , Animales , Citometría de Flujo , Células HEK293 , Humanos , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timocitos/inmunologíaRESUMEN
To model the developmental pattern of human prothymocytes and thymopoiesis, we used NOD-scid/γc(-/-) mice grafted with human umbilical cord blood CD34(+) hematopoietic progenitor cells (HPCs). Human prothymocytes developed in the murine bone marrow (BM) from multipotent CD34(++)CD38(lo)lineage(-) HPCs to CD34(++)CD7(+)CD2(-) pro-T1 cells that progressed in a Notch-dependent manner to CD34(+)CD7(++)CD2(+) pro-T2 cells, which migrated to the thymus. BM prothymocyte numbers peaked 1 mo after graft, dropped at mo 2, and persisted at low levels thereafter, with only a few CD34(+)CD7(lo) prothymocytes with limited T potential being detected by mo 5. As a consequence, thymopoiesis in this xenogeneic setting began by weeks 4-6, peaked at mo 3, and decreased thenceforth. Analyzing mice grafted at 2, 4 or 8, mo of age showed that in an "older" BM, prothymocyte differentiation was perturbed and resulted in CD34(+)CD7(lo) prothymocytes with limited T potential. Whereas the early drop in BM thymopoietic activity was related to a Notch-independent loss of T potential by CD34(++)CD38(lo)lineage(-) HPCs, the later age-dependent production decline of prothymocytes was linked to a more complex mix of cell-intrinsic and microenvironmental defects. Accordingly, and contrasting with what was observed with umbilical cord blood HPCs, CD34(+) HPCs from human adult BM displayed only marginal thymopoietic activity when grafted into young 2-mo-old NOD-scid/γc(-/-) mice. These data demonstrate that the developmental pattern of BM prothymocytes during human late fetal and early postnatal life can be reproduced in humanized mice, and they suggest that onset of human thymus involution relates to decreased colonization by prothymocytes.
Asunto(s)
Diferenciación Celular/inmunología , Células Progenitoras Linfoides/citología , Linfopoyesis/fisiología , Linfocitos T/citología , Timo/citología , Animales , Células de la Médula Ósea/citología , Linaje de la Célula/inmunología , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HeterólogoRESUMEN
The mechanisms regulating the emergence of BM prothymocytes remain poorly characterized. Genome-wide transcriptome analyses looking for genes expressed in human prothymocytes led to the identification of AF1q/MLLT11 as a candidate gene conceivably involved in this process. Analysis of AF1q protein subcellular localization and intracellular trafficking showed that despite pronounced karyophily, it was subjected to constitutive nuclear export followed by ubiquitin-mediated degradation in the centrosomal area. Using in vitro assays based on either forced expression or shRNA-mediated silencing of AF1q, we provide evidence that the protein promotes T- over B-cell differentiation in multipotent hematopoietic progenitors. At the molecular level, AF1q confers to multipotent progenitors an increased susceptibility to Delta-like/Notch-mediated signaling. Consistent with these findings, enforced AF1q expression in humanized mice fosters the emergence of BM CD34(+)CD7(+) prothymocytes, enhances subsequent thymus colonization, and accelerates intrathymic T-cell development. In contrast, AF1q silencing provokes a global shift of BM lymphopoiesis toward the B-cell lineage, hinders prothymocyte development, inhibits thymus colonization, and leads to intrathymic accumulation of B cells. Our results indicate that AF1q cooperates with the Notch signaling pathway to foster the emergence of BM prothymocytes and drive subsequent intrathymic specification toward the T-cell lineage.
Asunto(s)
Células Madre Hematopoyéticas/citología , Linfopoyesis , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Notch/metabolismo , Linfocitos T/citología , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Células Cultivadas , Silenciador del Gen , Células HeLa , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones SCID , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas/genética , Alineación de Secuencia , Transducción de Señal , Linfocitos T/metabolismoRESUMEN
Early interactions between lung dendritic cells (LDCs) and Mycobacterium tuberculosis, the etiological agent of tuberculosis, are thought to be critical for mounting a protective anti-mycobacterial immune response and for determining the outcome of infection. However, these interactions are poorly understood, at least at the molecular level. Here we show that M. tuberculosis enters human monocyte-derived DCs after binding to the recently identified lectin DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). By contrast, complement receptor (CR)3 and mannose receptor (MR), which are the main M. tuberculosis receptors on macrophages (Mphis), appeared to play a minor role, if any, in mycobacterial binding to DCs. The mycobacteria-specific lipoglycan lipoarabinomannan (LAM) was identified as a key ligand of DC-SIGN. Freshly isolated human LDCs were found to express DC-SIGN, and M. tuberculosis-derived material was detected in CD14(-)HLA-DR(+)DC-SIGN(+) cells in lymph nodes (LNs) from patients with tuberculosis. Thus, as for human immunodeficiency virus (HIV), which is captured by the same receptor, DC-SIGN-mediated entry of M. tuberculosis in DCs in vivo is likely to influence bacterial persistence and host immunity.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa , Mycobacterium tuberculosis/fisiología , Receptores de Superficie Celular/metabolismo , Antígenos CD , Células Dendríticas/citología , Células Dendríticas/inmunología , Citometría de Flujo , Células HeLa , Humanos , Inmunoglobulinas/metabolismo , Lipopolisacáridos/metabolismo , Pulmón/citología , Pulmón/inmunología , Pulmón/patología , Antígeno de Macrófago-1/metabolismo , Receptor de Manosa , Glicoproteínas de Membrana/metabolismo , Monocitos/citología , Mycobacterium tuberculosis/inmunología , Factores de Tiempo , Tuberculosis/inmunología , Tuberculosis/metabolismo , Tuberculosis/microbiología , Antígeno CD83RESUMEN
Polymorphonuclear neutrophils (PMNs) are rapidly recruited to tissues upon injury or infection. There, they can encounter local and/or recruited immature dendritic cells (iDCs), a colocalization that could promote at least transient interactions and mutually influence the two leukocyte populations. Using human live blood PMNs and monocyte-derived iDCs, we examined if these leukocytes actually interacted and whether this influenced DC function. Indeed, coculture with live but not apoptotic PMNs led to up-regulation of membrane CD40, CD86, and human leukocyte antigen (HLA)-DR on DCs. Whereas CD40 up-regulation was dependent on soluble factors released by PMNs, as determined in cultures conducted in different chambers, cell contact was necessary for CD86 and HLA-DR up-regulation, a process that was inhibited by anti-CD18 antibodies, indicating that CD18 ligation was required. We also found that via a cell contact-dependent mechanism, DCs acquired Candida albicans-derived antigens from live as well as from apoptotic PMNs and could thus elicit antigen-specific T lymphocyte responses. Altogether, our data demonstrate the occurrence of cross-talk between human PMNs and DCs and provide new insights into the immune processes occurring upstream of the interactions between DCs and T lymphocytes.
Asunto(s)
Presentación de Antígeno/inmunología , Comunicación Celular/inmunología , Células Dendríticas/inmunología , Activación de Linfocitos/inmunología , Neutrófilos/inmunología , Transducción de Señal/inmunología , Antígenos de Superficie/inmunología , Antígeno B7-2/inmunología , Antígenos CD18/inmunología , Antígenos CD40/inmunología , Candida albicans/inmunología , Candidiasis/inmunología , Células Cultivadas , Técnicas de Cocultivo , Antígenos HLA-DR/inmunología , Humanos , Inmunidad Celular/inmunología , Inmunofenotipificación , Ligadura , Fagocitosis/inmunología , Linfocitos T/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
That monocytes can differentiate into macrophages or dendritic cells (DCs) makes them an essential link between innate and adaptive immunity. However, little is known about how interactions with pathogens or T cells influence monocyte engagement toward DCs. We approached this point in cultures where granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 induced monocytes to differentiate into immature DCs. Activating monocytes with soluble CD40 ligand (CD40L) led to accelerated differentiation toward mature CD83(+) DCs with up-regulated human leukocyte antigen-DR, costimulatory molecules and CD116 (GM-CSF receptor), and down-regulation of molecules involved in antigen capture. Monocytes primed by phagocytosis of antibody-opsonized, killed Escherichia coli differentiated into DCs with an immature phenotype, whereas Zymosan priming yielded active DCs with an intermediate phenotype. Accordingly, DCs obtained from cultures with CD40L or after Zymosan priming had a decreased capacity to endocytose dextran, but only DCs cultured with CD40L had increased capacity to stimulate allogeneic T cells. DCs obtained after E. coli or Zymosan priming of monocytes produced high levels of proinflammatory tumor necrosis factor alpha and IL-6 as well as of regulatory IL-10, but they produced IL-12p70 only after secondary CD40 ligation. Thus, CD40 ligation on monocytes accelerates the maturation of DCs in the presence of GM-CSF/IL-4, whereas phagocytosis of different microorganisms does not alter and even facilitates their potential to differentiate into immature or active DCs, the maturation of which can be completed upon CD40 ligation. In vivo, such differences may correspond to DCs with different trafficking and T helper cell-stimulating capacities that could differently affect induction of adaptive immune responses to infections.
Asunto(s)
Ligando de CD40/farmacología , Células Dendríticas/citología , Monocitos/citología , Fagocitosis/fisiología , Antígenos CD/análisis , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Citocinas/metabolismo , Células Dendríticas/metabolismo , Humanos , Inmunofenotipificación , Prueba de Cultivo Mixto de Linfocitos , Monocitos/efectos de los fármacos , Monocitos/inmunología , Unión Proteica , Receptores de Quimiocina/análisisRESUMEN
Development of specifically labeled chemokines that retain their biological properties should be useful for analyzing their mechanisms of action both under physiological and pathological conditions. Here, we report the chemical synthesis and characterization of RANTES (regulated upon activation normal T cell expressed and secreted) derivatives that were biotinylated at residues 1, 25, 33, 45, or 67. Gel filtration and ultracentrifugation experiments showed that biotinylation at position 45 or 67 decreased the aggregation tendency of the chemokine to a dimeric state. Competition experiments, using a stably transfected CHO-K1 cell line overexpressing human CCR5, a RANTES receptor, indicated that derivatives biotinylated at positions 1, 25, and 67 bound to CCR5 with the same affinity as native RANTES. Flow cytometry analysis showed that RANTES biotinylated at residue 67 (B67-RANTES) bound more efficiently to primary macrophages than the other derivatives. Such binding was dependent on cell surface glycosaminoglycans (GAGs) since it was reduced when macrophages or HeLa cells expressing or not CCR5 were first treated with GAG-specific enzymes. In addition, B67-RANTES modulated CCR5 expression on lymphocytes and elicited chemotaxis of monocytes in the same manner as unmodified RANTES. Thus, B67-RANTES acts as a CCR5 agonist and may be useful to study the role of RANTES in pathologies such as, for example, human immunodeficiency virus (HIV) infection and inflammatory disorders.
Asunto(s)
Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/farmacología , Animales , Unión Competitiva , Biotinilación , Células CHO , Células Cultivadas , Quimiocina CCL5/química , Quimiocina CCL5/fisiología , Quimiotaxis , Cricetinae , Glicosaminoglicanos/metabolismo , Células HeLa , Humanos , Macrófagos/metabolismo , Monocitos/fisiología , Receptores CCR5/metabolismo , Linfocitos T/metabolismoRESUMEN
Dendritic cells (DC) are essential antigen-presenting cells that initiate and regulate adaptive immune responses. There are distinct DC populations of diverse origins, which develop from hematopoietic progenitors already committed to the lymphoid or the myeloid lineages and, in the latter case, even from terminally differentiated macrophages. One may assume that DC of lymphoid origin are dedicated to the adaptive immune system, along which they have phylogenetically co-evolved, whereas myeloid DC would be more involved as an interface between the innate and adaptive immune systems. However, any DC can ultimately present antigens in either an immunogenic or tolerogenic manner according to whether they are more or less or not at all activated towards maturation, depending on the condition under which they encountered antigen. Hence, DC either induce the appropriate immune response to pathogens or prevent autoimmune reactivity. Thus, besides default programming, which should be necessary to face the challenges of their usual setting, each type of DC can also display functions that are similar, in an instructive mode, to elicit immune responses deemed necessary for unexpected stimuli. In such a system, DC provide enough flexibility and sufficient redundancy to ensure that an essential function of the immune system, i.e., passing information from its innate to adaptive arms and affecting the latter's responses, occurs under optimal conditions. Working on the basis of such a unified theory of DC diversity should be useful for learning to adequately manipulate the immune system for the development of cellular immunotherapy.
Asunto(s)
Células Dendríticas/fisiología , Animales , Diferenciación Celular , Células Dendríticas/clasificación , Células Dendríticas/citología , HumanosRESUMEN
Activating cells of the immune system may stimulate human immunodeficiency virus type 1 (HIV-1) replication and contribute to select pathogenic variants in vivo. Here, we examined the possible effect of a major pathway of immune activation, CD40 interaction with its ligand (CD40L), on the susceptibility of monocyte-derived macrophages (MDMs) to various HIV-1 strains. Stimulation of MDMs with CD40L led to reduced replication of R5 HIV-1(Ba-L), whereas this strongly enhanced the replication of X4 HIV-1(Lai) as well as of X4 primary isolates, and this was associated with strong cytopathic effects. The replication of X4 strains was inhibited by stromal cell-derived factor 1, an indication of the restricted usage of CXCR4 as virus coreceptor in this case. CD40L induced the activation of mitogen-activated protein kinases ERK1/ERK2 and stimulated MDMs to secrete RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, interleukin 6 (IL-6), IL-1beta, and tumor necrosis factor alpha. From this data, it may be hypothesized that activated macrophages represent a favorable environment for the replication of classically T lymphocyte-tropic X4 variants and, thus, may contribute significantly to the selection of such variants at late stages of clinical HIV-1 infection.
Asunto(s)
Antígenos CD40/inmunología , VIH-1/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Ligando de CD40/farmacología , Células Cultivadas , Citocinas/biosíntesis , Activación Enzimática , VIH-1/efectos de los fármacos , VIH-1/aislamiento & purificación , VIH-1/fisiología , Humanos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/virología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fenotipo , Replicación ViralRESUMEN
Monocyte-derived dendritic cells (DCs) were used as an in vitro model of myeloid DCs in order to determine a minimum marker pattern with which to characterize and distinguish different stages of DC activation and maturation. Phenotypic changes induced on immature DCs by two prototypic stimuli, poly I:C and CD40 ligation, were first examined. Both elicited HLA-DR, CD40, CD86 and CXCR4 upregulation, and CCR5 downregulation, but only CD40 ligand-stimulated DCs became CD83(+)\CCR7(+), whereas poly I:C-stimulated DCs expressed lower CD83 levels and were mostly CCR7(--). CD40 ligation and poly I:C elicited increased production of inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha, of IL-10 and the CCL5 chemokine, but profiles differed as to higher IL-10, IL-12 and CCL22 (a CCR4 ligand important for T cell recruitment) levels for the former, and of CCL4 and CCL5 for the latter. Thus, a limited set of phenotypic markers, cytokine and chemokine production assays, may be used to distinguish the three stages in the life of DCs: immaturity, activation and full maturation. The ability of purified protein derivative-loaded DCs to stimulate autologous T cells to produce IL-2, IL-4 and interferon-gamma indeed depended on their activation stage and endocytic activity, which decreased upon maturation. We then examined whether ligation of CD4, CCR5 and\or CXCR4, the receptor and coreceptors of human immunodeficiency virus envelope gp120, respectively, affected DC activation or maturation, neither a monoclonal antibody to the gp120-binding site on CD4 nor CCL5 nor CXCL12, the natural ligands of CCR5 and CXCR4, respectively, nor gp120 altered the DC activation and maturation processes.
Asunto(s)
Antígenos CD40/inmunología , Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH/inmunología , Monocitos/inmunología , ARN Bicatenario/farmacología , Células Cultivadas , Quimiocinas CC/inmunología , Citocinas/biosíntesis , Endocitosis/inmunología , Humanos , Monocitos/citología , ARN Bicatenario/inmunología , Receptores de Quimiocina/inmunologíaRESUMEN
Neutrophils are increasingly thought to modulate dendritic cell (DC) functions. We investigated the role of the neutrophil-DC partnership in the response to Mycobacterium bovis BCG-the vaccine used against tuberculosis. We compared neutrophil-DC crosstalk in humans and mice, searching for functional differences. In both species, neutrophils captured fluorescent BCG-enhanced green fluorescent protein (EGFP) and were more phagocytic than DC. Non-apoptotic BCG-infected neutrophils clustered with immature DC, establishing intimate contacts with dendrites, at which fluorescent intact bacilli were observed. Physical interactions between neutrophils and DC were required for DC activation. Human BCG-infected DC produced interleukin (IL)-10, an inhibitory cytokine, whereas DC exposed to BCG-infected neutrophils produced low to undetectable amounts of the cytokine. Mouse BCG-infected neutrophils induced sustained IL-2 production by DC. Human DC exposed to BCG-infected neutrophils stimulated recall T cell reactivity from vaccinated donors. Mouse DC infected with recombinant ovalbumin (OVA)-producing BCG (rBCG(ova)) elicited proliferation of TCR-OVA-transgenic CD4 and CD8 T cells. Moreover, exposing DC to rBCG(ova)-infected neutrophils enhanced OVA presentation. Thus, in mice and humans, neutrophils help DC to cross-present BCG antigens to T cells. Our results suggest that this "ménage à trois" involving neutrophils, DC and T cells plays a major role in the immune response to BCG.
Asunto(s)
Comunicación Celular/inmunología , Células Dendríticas/microbiología , Mycobacterium bovis/inmunología , Neutrófilos/microbiología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Células Cultivadas , Técnicas de Cocultivo , Citocinas/biosíntesis , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Hibridomas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neutrófilos/inmunología , Neutrófilos/metabolismoRESUMEN
Infiltration of human immunodeficiency virus type 1 (HIV-1)-infected and uninfected monocytes/macrophages in organs and tissues is a general phenomenon observed in progression of acquired immunodeficiency syndrome (AIDS). HIV-1 protein Nef is considered as a progression factor in AIDS, and is released from HIV-1-infected cells. Here, we show that extracellular Nef increases migration of monocytes. This effect is (i) concentration-dependent, (ii) reaches the order of magnitude of that induced by formyl-methyonyl-leucyl-proline (fMLP) or CC chemokine ligand 2 (CCL2)/monocyte chemotactic protein (MCP)-1, (iii) inhibited by anti-Nef monoclonal antibodies as well as by heating, and (iv) depends on a concentration gradient of Nef. Further, Nef does not elicit monocytic THP-1 cells to express chemokines such as CCL2, macrophage inhibitory protein-1alpha (CCL3) and macrophage inhibitory protein-1beta (CCL4). These data suggest that extracellular Nef may contribute to disease progression as well as HIV-1 spreading through affecting migration of monocytes.
Asunto(s)
Movimiento Celular , Productos del Gen nef/metabolismo , VIH-1/metabolismo , Monocitos/fisiología , Células Cultivadas , Quimiocinas/metabolismo , Progresión de la Enfermedad , Infecciones por VIH/fisiopatología , Humanos , Monocitos/citología , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Here, we identify fetal bone marrow (BM)-derived CD34hiCD45RAhiCD7+ hematopoietic progenitors as thymus-colonizing cells. This population, virtually absent from the fetal liver (FL), emerges in the BM by development weeks 8-9, where it accumulates throughout the second trimester, to finally decline around birth. Based on phenotypic, molecular, and functional criteria, we demonstrate that CD34hiCD45RAhiCD7+ cells represent the direct precursors of the most immature CD34hiCD1a- fetal thymocytes that follow a similar dynamics pattern during fetal and early postnatal development. Histological analysis of fetal thymuses further reveals that early immigrants predominantly localize in the perivascular areas of the cortex, where they form a lymphostromal complex with thymic epithelial cells (TECs) driving their rapid specification toward the T lineage. Finally, using an ex vivo xenogeneic thymus-colonization assay, we show that BM-derived CD34hiCD45RAhiCD7+ progenitors are selectively recruited into the thymus parenchyma in the absence of exogenous cytokines, where they adopt a definitive T cell fate.
Asunto(s)
Linfocitos B/inmunología , Médula Ósea/embriología , Movimiento Celular , Células Madre Hematopoyéticas/fisiología , Timo/embriología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos B/fisiología , Médula Ósea/metabolismo , Diferenciación Celular , Humanos , Ratones , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Fenotipo , Timo/citología , Timo/inmunologíaRESUMEN
We analyzed the role of human immunodeficiency virus (HIV)-1 matrix protein (MA) during the virus replication afferent phase. Single-round infection of H9 T lymphocytes showed that the combined mutation of MA Lys residues 26-27 in MA reported nuclear localization signal (NLS)-1 impaired infectivity, abrogated 2-LTR-circle formation and significantly reduced integration. However, the mutation did not affect viral DNA docking to chromatin in either interphasic or mitotic cells, indicating that MA N-terminal basic domain should not represent a major determinant of HIV-1 nuclear import in T lymphocytes. These data point to a previously unreported role of MA in the late, post-chromatin-binding, afferent phase of HIV-1 replication cycle.
Asunto(s)
Productos del Gen gag/fisiología , Antígenos VIH/fisiología , VIH-1/fisiología , Proteínas Virales/fisiología , Ciclo Celular , Línea Celular , Cromatina/metabolismo , ADN Viral/metabolismo , Productos del Gen gag/genética , Antígenos VIH/genética , VIH-1/metabolismo , Humanos , Mutación , Linfocitos T/virología , Proteínas Virales/genética , Integración Viral , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
A novel membrane protein has been identified in the course of screening for differentially expressed cDNAs in human embryonic hematopoietic sites. This 37- to 38-kDa molecule, designated KLIP-1 (killer lineage protein), consisting of 350 amino acids and containing five transmembrane domains, is encoded by the 5093-bp KLIP-1 gene, composed of nine exons and located on chromosome 6 (6p21.1-6p21.2). We found the KLIP-1 protein to be expressed by nucleated hematopoietic cells, from early embryonic hematopoietic stem cells through mature adult blood lymphoid lineages, either as membrane or as cytoplasmic molecules. In day-30/32 human embryo sections, KLIP-1 protein expression is restricted to circulating hematopoietic cells at hematopoiesis sites. Membrane KLIP-1 is expressed by fetal and adult GP-A(+) erythroblasts, the fetal liver CD34(+) subset, fetal spleen, and adult bone marrow CD56(+) NK and CD19(+) B cells. Among mature blood cells, surface KLIP-1 expression is restricted to CD56(+) NK cells, indicating KLIP-1 to be a novel marker of this population. Altogether, these results indicate that membrane export of KLIP-1 antigen is developmentally and ontogenetically regulated. The high degree of conservation of the KLIP-1 protein sequence among mammals strongly suggests that it plays an important role during hematopoiesis and may exercise similar functions in human and mouse blood cells. The KLIP-1 molecule may therefore constitute a powerful tool for improving knowledge of both human hematopoiesis and NK cell ontogeny and immune functions.
Asunto(s)
Linaje de la Célula , Embrión de Mamíferos/citología , Hematopoyesis , Células Madre Hematopoyéticas/citología , Proteínas de la Membrana/fisiología , Adulto , Secuencia de Aminoácidos , Animales , Antígenos CD34 , Biomarcadores , Mapeo Cromosómico , Embrión de Mamíferos/química , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Distribución TisularRESUMEN
We examined the influence of mitosis on the kinetics of human immunodeficiency virus type 1 integration in T cells. Single-round infection of cells arrested in G1b or allowed to synchronously proceed through division showed that mitosis delays virus integration until 18-24 h postinfection, whereas integration reaches maximum levels by 15 h in G1b-arrested cells. Subcellular fractionation of metaphase-arrested cells indicated that, while nuclear envelope disassembly facilitates docking of viral DNA to chromatin, chromosome condensation directly antagonizes and therefore delays integration. As a result of the balance between the two effects, virus integration efficiency is eventually up to threefold greater in dividing cells. At the single-cell level, using a green fluorescent protein-expressing reporter virus, we found that passage through mitosis leads to prominent asymmetric segregation of the viral genome in daughter cells without interfering with provirus expression.
Asunto(s)
Ciclo Celular , Regulación Viral de la Expresión Génica , VIH-1/patogenicidad , Linfocitos T/virología , Integración Viral , Animales , Cromatina/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Infecciones por VIH/virología , VIH-1/genética , Humanos , Mitosis , Membrana Nuclear/metabolismoRESUMEN
Dendritic cells (DCs) are likely to play a key role in immunity against Mycobacterium tuberculosis, but the fate of the bacterium in these cells is still unknown. Here we report that, unlike macrophages (Mphis), human monocyte-derived DCs are not permissive for the growth of virulent M. tuberculosis H37Rv. Mycobacterial vacuoles are neither acidic nor fused with host cell lysosomes in DCs, in a mode similar to that seen in mycobacterial infection of Mphis. However, uptake of the fluid phase marker dextran, and of transferrin, as well as accumulation of the recycling endosome-specific small GTPase Rab11 onto the mycobacterial phagosome, are almost abolished in infected DCs, but not in Mphis. Moreover, communication between mycobacterial phagosomes and the host-cell biosynthetic pathway is impaired, given that <10% of M. tuberculosis vacuoles in DCs stained for the endoplasmic reticulum-specific proteins Grp78/BiP and calnexin. This correlates with the absence of the fusion factor N-ethylmaleimide-sensitive factor onto the vacuolar membrane in this cell type. Trafficking between the vacuoles and the host cell recycling and biosynthetic pathways is strikingly reduced in DCs, which is likely to impair access of intracellular mycobacteria to essential nutrients and may thus explain the absence of mycobacterial growth in this cell type. This unique location of M. tuberculosis in DCs is compatible with their T lymphocyte-stimulating functions, because M. tuberculosis-infected DCs have the ability to specifically induce cytokine production by autologous T lymphocytes from presensitized individuals. DCs have evolved unique subcellular trafficking mechanisms to achieve their Ag-presenting functions when infected by intracellular mycobacteria.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/microbiología , Líquido Intracelular/inmunología , Líquido Intracelular/microbiología , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/inmunología , Presentación de Antígeno , Diferenciación Celular/inmunología , División Celular/inmunología , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Chaperón BiP del Retículo Endoplásmico , Humanos , Inmunidad Innata , Líquido Intracelular/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Fagosomas/inmunología , Fagosomas/microbiología , Biosíntesis de Proteínas , Especificidad de la Especie , Tuberculina/inmunología , Tuberculina/metabolismo , Vacuolas/inmunología , Vacuolas/microbiologíaRESUMEN
We report that human T cells persistently infected with primate foamy virus type 1 (PFV-1) display an increased capacity to bind human immunodeficiency virus type 1 (HIV-1), resulting in increased cell permissiveness to HIV-1 infection and enhanced cell-to-cell virus transmission. This phenomenon is independent of HIV-1 receptor, CD4, and it is not related to PFV-1 Bet protein expression. Increased virus attachment is specifically inhibited by heparin, indicating that it should be mediated by interactions with heparan sulfate glycosaminoglycans expressed on the target cells. Given that both viruses infect similar animal species, the issue of whether coinfection with primate foamy viruses interferes with the natural course of lentivirus infections in nonhuman primates should be considered.