RESUMEN
HIV-1 infects immature dendritic cells (iDCs), but infection is inefficient compared with activated CD4+ T cells and only involves a small subset of iDCs. We analyzed whether this could be attributed to specific cellular restrictions during the viral life cycle. To study env-independent restriction to HIV-1 infection, we used a single-round infection assay with HIV-1 pseudotyped with vesicular stomatitis virus G protein (HIV-VSVG). Small interfering RNA-mediated depletion of APOBEC3G/3F (A3G/3F), but not TRIM5alpha, enhanced HIV-1 infection of iDCs, indicating that A3G/3F controls the sensitivity of iDCs to HIV-1 infection. Furthermore, sequences of HIV reverse transcripts revealed G-to-A hypermutation of HIV genomes during iDC infection, demonstrating A3G/3F cytidine deaminase activity in iDCs. When we separated the fraction of iDCs that was susceptible to HIV, we found the cells to be deficient in A3G messenger RNA and protein. We also noted that during DC maturation, which further reduces susceptibility to infection, A3G levels increased. These findings highlight a role for A3G/3F in explaining the resistance of most DCs to HIV-1 infection, as well as the susceptibility of a fraction of iDCs. An increase in the A3G/3F-mediated intrinsic resistance of iDCs could result in a block of HIV infection at its mucosal point of entry.
Asunto(s)
Citosina Desaminasa/fisiología , Células Dendríticas/virología , VIH-1/crecimiento & desarrollo , Monocitos/citología , Nucleósido Desaminasas/fisiología , Proteínas Represoras/fisiología , Desaminasa APOBEC-3G , Factores de Restricción Antivirales , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Citidina Desaminasa , Citosina Desaminasa/genética , Citosina Desaminasa/metabolismo , ADN Viral/genética , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Citometría de Flujo , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , VIH-1/genética , Células HeLa , Humanos , Células Jurkat , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Nucleósido Desaminasas/genética , Nucleósido Desaminasas/metabolismo , Mutación Puntual , ARN Interferente Pequeño/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Homología de Secuencia de Ácido Nucleico , Transfección , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas , Proteínas del Envoltorio Viral/genética , Replicación Viral/genéticaRESUMEN
Current human immunodeficiency virus (HIV) vaccine approaches emphasize prime boost strategies comprising multiple doses of DNA vaccine and recombinant viral vectors. We are developing a protein-based approach that directly harnesses principles for generating T cell immunity. Vaccine is delivered to maturing dendritic cells in lymphoid tissue by engineering protein antigen into an antibody to DEC-205, a receptor for antigen presentation. Here we characterize the CD4+ T cell immune response to HIV gag and compare efficacy with other vaccine strategies in a single dose. DEC-205-targeted HIV gag p24 or p41 induces stronger CD4+ T cell immunity relative to high doses of gag protein, HIV gag plasmid DNA, or recombinant adenovirus-gag. High frequencies of interferon (IFN)-gamma- and interleukin 2-producing CD4+ T cells are elicited, including double cytokine-producing cells. In addition, the response is broad because the primed mice respond to an array of peptides in different major histocompatibility complex haplotypes. Long-lived T cell memory is observed. After subcutaneous vaccination, CD4+ and IFN-gamma-dependent protection develops to a challenge with recombinant vaccinia-gag virus at a mucosal surface, the airway. We suggest that a DEC-targeted vaccine, in part because of an unusually strong and protective CD4+ T cell response, will improve vaccine efficacy as a stand-alone approach or with other modalities.
Asunto(s)
Vacunas contra el SIDA/inmunología , Antígenos CD/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Productos del Gen gag/inmunología , VIH-1/inmunología , Lectinas Tipo C/inmunología , Receptores de Superficie Celular/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Adenoviridae , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/inmunología , Antígenos CD/genética , Relación Dosis-Respuesta Inmunológica , Productos del Gen gag/administración & dosificación , Productos del Gen gag/genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/inmunología , VIH-1/genética , Haplotipos/genética , Haplotipos/inmunología , Humanos , Inmunidad Mucosa/efectos de los fármacos , Inmunidad Mucosa/inmunología , Memoria Inmunológica/efectos de los fármacos , Memoria Inmunológica/inmunología , Inyecciones Subcutáneas , Lectinas Tipo C/deficiencia , Lectinas Tipo C/genética , Complejo Mayor de Histocompatibilidad/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Noqueados , Antígenos de Histocompatibilidad Menor , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Virus VacciniaRESUMEN
Dengue virus is a single-stranded, enveloped RNA virus that productively infects human dendritic cells (DCs) primarily at the immature stage of their differentiation. We now find that all four serotypes of dengue use DC-SIGN (CD209), a C-type lectin, to infect dendritic cells. THP-1 cells become susceptible to dengue infection after transfection of DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN), or its homologue L-SIGN, whereas the infection of dendritic cells is blocked by anti-DC-SIGN antibodies and not by antibodies to other molecules on these cells. Viruses produced by dendritic cells are infectious for DC-SIGN- and L-SIGN-bearing THP-1 cells and other permissive cell lines. Therefore, DC-SIGN may be considered as a new target for designing therapies that block dengue infection.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Células Dendríticas/virología , Virus del Dengue/fisiología , Lectinas Tipo C/fisiología , Receptores de Superficie Celular/fisiología , Anticuerpos Monoclonales/inmunología , Moléculas de Adhesión Celular/inmunología , Células Cultivadas , Dengue/clasificación , Virus del Dengue/clasificación , Citometría de Flujo , Humanos , Lectinas Tipo C/inmunología , Receptores de Superficie Celular/inmunología , Receptores Virales/fisiología , SerotipificaciónRESUMEN
The distribution of dendritic cells (DCs) and macrophages in the human spleen has received less attention than that of lymphocytes. Here we have addressed this problem with the human DEC-205/CD205 marker ('DEC'), which is an endocytic receptor on DCs that mediates efficient presentation of antigens. DEC was abundant on dendritic profiles in the white pulp but absent from the red pulp, the latter defined with antibodies to two antigens, mannose receptor/CD206 on sinusoidal lining cells, and macrosialin/CD68 on macrophages. Double staining with anti-DEC and anti-CD3 showed the expected concentration of DEC+ cells in the relatively small T-cell areas of the human spleen. DEC+ cells were also found in other regions of the white pulp. In all regions, the DEC+ cells were positive for major histocompatibility complex (MHC) class II and the CD11c integrin but largely immature, with low expression of B7-2/CD86 costimulator and DC-lysosome-associated membrane protein (LAMP)/CD208. When we concentrated on the perifollicular region between the red pulp and the marginal zone, we found macrophages that stained with antibodies to sialoadhesin/CD169 and DC-specific ICAM-3 grabbing non-integrin (SIGN)/CD209, and just inside these cells were DEC+ profiles. The DEC+ DCs were intertwined with cells that stained for the vascular addressin mucosal addressin cell adhesion molecule (MAdCAM). Therefore, anti-DEC-205/CD205 antibodies are useful for identifying DCs in human splenic white pulp and its border region with the red pulp.
Asunto(s)
Antígenos CD/análisis , Células Dendríticas/inmunología , Lectinas Tipo C/análisis , Receptores de Superficie Celular/análisis , Bazo/inmunología , Antígenos de Diferenciación Mielomonocítica/análisis , Complejo CD3/análisis , Moléculas de Adhesión Celular , Diferenciación Celular/inmunología , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Inmunoglobulinas/metabolismo , Macrófagos/inmunología , Antígenos de Histocompatibilidad Menor , Mucoproteínas/metabolismo , Linfocitos T/inmunologíaRESUMEN
SAMMA, a mandelic acid condensation polymer, exhibits a broad antimicrobial activity against several sexually transmitted pathogens including human immunodeficiency virus (HIV). Here we demonstrated that SAMMA suppressed HIV transmission by dendritic cells (DCs), one of the first target cells for primary infection. The greatest inhibitory effect was achieved when SAMMA was present during the co-culture with target cells. The inhibitory effect of SAMMA on DC-mediated HIV transmission was not due to cytotoxicity. Analysis of the level of DC-associated HIV p24 antigen revealed that SAMMA prevented HIV internalization by DCs when the virus was pre-incubated with the compound. In contrast, pre-incubation of DCs with SAMMA followed by wash-off did not affect the amount of cell-associated HIV p24 antigen. In addition, SAMMA blocked HIV glycoprotein-mediated cell-cell fusion. This study suggests that SAMMA prevents HIV infection through multiple mechanisms.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Infecciones por VIH/transmisión , Ácidos Mandélicos/farmacología , Polímeros/farmacología , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/virología , Glicoproteínas/metabolismo , Infecciones por VIH/virología , Humanos , Proteínas Virales/metabolismo , Internalización del Virus/efectos de los fármacosRESUMEN
DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin) is a myeloid pathogen-attachment factor C-type lectin which recognizes mannose- and fucose-containing oligosaccharide ligands on clinically relevant pathogens. Intracellular signaling initiated upon ligand engagement of DC-SIGN interferes with TLR-initiated signals, and modulates the T cell activating and polarizing ability of antigen-presenting cells. The C-terminal carbohydrate-recognition domain (CRD) of DC-SIGN is preceded by a neck domain composed of eight 23-residue repeats which mediate molecule multimerization, and whose polymorphism correlates with altered susceptibility to SARS and HIV infection. Naturally occurring isoforms and chimaeric molecules, in combination with established recognition properties, were used to define seven structural and functional epitopes on DC-SIGN. Three epitopes mapped to the CRD, one of which is multimerization-dependent and only exposed on DC-SIGN monomers. Epitopes within the neck domain were conformation-independent and unaltered upon molecule multimerization, but were differentially affected by neck domain truncations. Although neck-specific antibodies exhibited lower function-blocking ability, they were more efficient at inducing molecule internalization. Moreover, crosslinking of the different epitopes resulted in distinct levels of microclustering on the cell surface. The identification of independent epitopes on the DC-SIGN molecule might facilitate the design of reagents that modulate the T cell activating and polarizing ability of DC-SIGN-expressing cells without preventing its antigen- and pathogen-recognition capacities.
Asunto(s)
Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/inmunología , Mapeo Epitopo , Lectinas Tipo C/química , Lectinas Tipo C/inmunología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/inmunología , Anticuerpos Bloqueadores/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Línea Celular , Células Dendríticas/citología , Células Dendríticas/inmunología , Endocitosis/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Monocitos/citología , Estructura Terciaria de ProteínaRESUMEN
The infection of cultured monocyte-derived dendritic cells (DCs) with HIV-1 involves CD4 and CCR5 receptors, while transmission to T cells is enhanced at least in part by the lectin DC-SIGN/CD209. In the present study, we studied BDCA-1+ myeloid DCs isolated directly from human blood. These cells express CD4 and low levels of CCR5 and CXCR4 coreceptors, but not DC-SIGN. The myeloid DCs replicate two R5 viruses, BaL and YU2, and transfer infection to activated T cells. The virus productively infects a small fraction of the blood DCs that fail to mature in culture, as indicated by the maturation markers CD83 and DC-LAMP/CD208, and the expression of high CD86 and MHC class II, in contrast to many noninfected DCs. A greater proportion of BDCA-1+ DCs are infected when the virus is pseudotyped with the vesicular stomatitis envelope VSV-G (5-15%), as compared with the R5 virus (0.3-3.5%), indicating that HIV-1 coreceptors may limit the susceptibility of DCs to become infected, or the endocytic route of viral entry used by HIV/vesicular stomatitis virus enhances infectivity. When infected and noninfected cells are purified by cell sorting, the former uniformly express HIV p24 gag and are virtually inactive as stimulators of the allogeneic MLR, in contrast to potent stimulation by noninfected DCs from the same cultures. These results point to two roles for a small fraction of blood DCs in HIV-1 pathogenesis: to support productive infection and to evade the direct induction of T cell-mediated immunity.
Asunto(s)
Células Dendríticas/virología , VIH-1/patogenicidad , Antígenos de Superficie/metabolismo , Diferenciación Celular , Células Dendríticas/clasificación , Células Dendríticas/citología , Células Dendríticas/inmunología , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , VIH-1/fisiología , Humanos , Técnicas In Vitro , Prueba de Cultivo Mixto de Linfocitos , Linfocitos T/virología , Replicación ViralRESUMEN
The C-type lectin dendritic cell-specific ICAM 3-grabbing nonintegrin (DC-SIGN)/CD209 efficiently binds several pathogens, including HIV-1. DC-SIGN is expressed on monocyte-derived DCs in culture, and importantly, it is able to sequester HIV-1 within cells and facilitate transmission of virus to CD4+ T cells. To investigate DC-SIGN function, we have generated new mAbs. We report in this study that these and prior anti-DC-SIGN mAbs primarily label macrophages in the medullary sinuses of noninflamed human lymph node. In contrast, expression is not detected on most DCs in the T cell area, except for occasional cells. We also noted that IL-4 alone can induce expression of DC-SIGN in CD14+ monocytes and circulating blood DCs. However, blockade of DC-SIGN with Abs and DC-SIGN small interfering RNA did not result in a major reduction in the capacity of these DCs to transfer HIV to T cells, confirming significant DC-SIGN-independent mechanisms. The blocking approaches did reduce HIV-1 transmission by DC-SIGN-transfected cells by >90%. DC-SIGN blockade also did not reduce the ability of DCs to stimulate T cell proliferation in the MLR. These results indicate that DC-SIGN has the potential to contribute to macrophage function in normal human lymph node, and that DCs do not require DC-SIGN to transmit HIV or to initiate T cell responses.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/inmunología , Lectinas Tipo C/metabolismo , Ganglios Linfáticos/metabolismo , Macrófagos/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Anticuerpos Monoclonales , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Línea Celular , Células Clonales , Células Dendríticas/virología , Perros , VIH-1/inmunología , Humanos , Lectinas Tipo C/antagonistas & inhibidores , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Ganglios Linfáticos/citología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , ARN Interferente Pequeño/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Linfocitos T/virología , TransfecciónRESUMEN
Dendritic cells (DCs) undergo maturation during virus infection and thereby become potent stimulators of cell-mediated immunity. HIV-1 replicates in immature DCs, but we now find that infection is not accompanied by many components of maturation in either infected cells or uninfected bystanders. The infected cultures do not develop potent stimulating activity for the mixed leukocyte reaction (MLR), and the DCs producing HIV-1 gag p24 do not express CD83 and DC-lysosome-associated membrane protein maturation markers. If different maturation stimuli are applied to DCs infected with HIV-1, the infected cells selectively fail to mature. When DCs from HIV-1-infected patients are infected and cultured with autologous T cells, IL-10 was produced in 6 of 10 patients. These DC-T cell cocultures could suppress another immune response, the MLR. The regulation was partially IL-10-dependent and correlated in extent with the level of IL-10 produced. Suppressor cells only developed from infected patients, rather than healthy controls, and the DCs had to be exposed to live virus rather than HIV-1 gag peptides or protein. These results indicate that HIV-1-infected DCs have two previously unrecognized means to evade immune responses: maturation can be blocked reducing the efficacy of antigen presentation from infected cells, and T cell-dependent suppression can be induced.
Asunto(s)
Células Dendríticas/virología , VIH-1/metabolismo , Interleucina-10/biosíntesis , Linfocitos T/fisiología , Técnicas de Cocultivo , Células Dendríticas/fisiología , Células HeLa , HumanosRESUMEN
DC-SIGN (CD209) is a C-type lectin expressed by several groups of dendritic cells (DC), including those derived from blood monocytes and DC found beneath genital epithelium. DC-SIGN binds the envelope glycoprotein of HIV-1 and facilitates transmission of infectious virus to permissive CD4(+) T cells. We have compared the capacity of DC-SIGN in different cell types to bind, retain and transmit infectious HIV-1 to T cells. The analyzed cells included monocyte-derived DC, and three different DC-SIGN-expressing transfectants termed THP, 293 and HOS. Our results show that DC-SIGN transfectants were able to bind HIV-1 virions comparably to DC. However, only the THP monocytic cell line shared with DC the capacity to retain for several days virus that was infectious for T cells. In both THP-DC-SIGN transfectants and DC, but not in 293 cells, HIV-1 was localized to intracellular compartments that did not double label for endosomal and lysosomal markers or for DC-SIGN itself. Virus remained detectable in these compartments for at least 2 days. Anti-DC-SIGN antibodies blocked the binding and transmission of HIV-1 in DC-SIGN transfectants, as monitored by PCR for HIV LTR/gag and p24 ELISA. However anti-DC-SIGN antibodies did not block virus binding and transmission to T cells as well in DC as in THP-DC-SIGN transfectants. Thus, the function of DC-SIGN in HIV-1 transmission depends on its cellular context, since only DC and the THP monocyte cell line, but not 293 and HOS, are able to use DC-SIGN to retain HIV-1 in a highly infectious state for several days.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/metabolismo , Antígenos CD4/inmunología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Quimiocina CCL5/inmunología , Células Dendríticas/metabolismo , Infecciones por VIH/transmisión , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Linfocitos T/metabolismoRESUMEN
DC-SIGN and DC-SIGNR are two closely related membrane-associated C-type lectins that bind human immunodeficiency virus (HIV) envelope glycoprotein with high affinity. Binding of HIV to cells expressing DC-SIGN or DC-SIGNR can enhance the efficiency of infection of cells coexpressing the specific HIV receptors. DC-SIGN is expressed on some dendritic cells, while DC-SIGNR is localized to certain endothelial cell populations, including hepatic sinusoidal endothelial cells. We found that soluble versions of the hepatitis C virus (HCV) E2 glycoprotein and retrovirus pseudotypes expressing chimeric forms of both HCV E1 and E2 glycoproteins bound efficiently to DC-SIGN and DC-SIGNR expressed on cell lines and primary human endothelial cells but not to other C-type lectins tested. Soluble E2 bound to immature and mature human monocyte-derived dendritic cells (MDDCs). Binding of E2 to immature MDDCs was dependent on DC-SIGN interactions, while binding to mature MDDCs was partly independent of DC-SIGN, suggesting that other cell surface molecules may mediate HCV glycoprotein interactions. HCV interactions with DC-SIGN and DC-SIGNR may contribute to the establishment or persistence of infection both by the capture and delivery of virus to the liver and by modulating dendritic cell function.