RESUMEN
BACKGROUND AND AIMS: The anti-MAdCAM-1 antibody ontamalimab demonstrated efficacy in a phase II trial in ulcerative colitis and results of early terminated phase III trials are pending, but its precise mechanisms of action are still unclear. Thus, we explored the mechanisms of action of ontamalimab and compared it to the anti-α4ß7 antibody vedolizumab. METHODS: We studied MAdCAM-1 expression with RNA sequencing and immunohistochemistry. The mechanisms of action of ontamalimab were assessed with fluorescence microscopy, dynamic adhesion and rolling assays. We performed in vivo cell trafficking studies in mice and compared ontamalimab and vedolizumab surrogate [-s] antibodies in experimental models of colitis and wound healing. We analysed immune cell infiltration under anti-MAdCAM-1 and anti-α4ß7 treatment by single-cell transcriptomics and studied compensatory trafficking pathways. RESULTS: MAdCAM-1 expression was increased in active inflammatory bowel disease. Binding of ontamalimab to MAdCAM-1 induced the internalization of the complex. Functionally, ontamalimab blocked T cell adhesion similar to vedolizumab, but also inhibited L-selectin-dependent rolling of innate and adaptive immune cells. Despite conserved mechanisms in mice, the impact of ontamalimab-s and vedolizumab-s on experimental colitis and wound healing was similar. Single-cell RNA sequencing demonstrated enrichment of ontamalimab-s-treated lamina propria cells in specific clusters, and in vitro experiments indicated that redundant adhesion pathways are active in these cells. CONCLUSIONS: Ontamalimab has unique and broader mechanisms of action compared to vedolizumab. However, this seems to be compensated for by redundant cell trafficking circuits and leads to similar preclinical efficacy of anti-α4ß7 and anti-MAdCAM-1 treatment. These results will be important for the interpretation of pending phase III data.
Asunto(s)
Colitis Ulcerosa , Enfermedades Inflamatorias del Intestino , Animales , Ratones , Fármacos Gastrointestinales/farmacología , Fármacos Gastrointestinales/uso terapéutico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Colitis Ulcerosa/tratamiento farmacológico , Inflamación/tratamiento farmacológico , IntegrinasRESUMEN
Due to their ability to knock down the expression of any gene, siRNAs have been heralded as ideal candidates for treating a wide variety of diseases, including those involving "undruggable" targets. However, the therapeutic potential of siRNAs remains severely limited by a lack of effective delivery vehicles. Recently, lipid nanoparticles (LNPs) containing ionizable cationic lipids have been developed for hepatic siRNA delivery. However, their suitability for delivery to other cell types has not been determined. We have modified LNPs for preferential targeting to dendritic cells (DCs), central regulators of immune responses. To achieve directed delivery, we coated LNPs with a single-chain antibody (scFv; DEC-LNPs), specific to murine DEC205, which is highly expressed on distinct DC subsets. Here we show that injection of siRNAs encapsulated in DEC-LNPs are preferentially delivered to DEC205(+) DCs. Gene knockdown following uptake of DEC-LNPs containing siRNAs specific for the costimulatory molecules CD40, CD80, and CD86 dramatically decreases gene expression levels. We demonstrate the functionality of this knockdown with a mixed lymphocyte response (MLR). Overall, we report that injection of LNPs modified to restrict their uptake to a distinct cell population can confer profound gene knockdown, sufficient to inhibit powerful immune responses like the MLR.
Asunto(s)
Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígenos CD40/metabolismo , Células Dendríticas/inmunología , Lípidos/química , ARN Interferente Pequeño/administración & dosificación , Animales , Regulación de la Expresión Génica , Inyecciones , Hígado/metabolismo , Ratones , Terapia Molecular Dirigida , Nanopartículas/administración & dosificación , Nanopartículas/químicaRESUMEN
Necrotic cell death triggers a range of biological responses including a strong adaptive immune response, yet we know little about the cellular pathways that control necrotic cell death. Inhibitor studies suggest that proteases, and in particular cathepsins, drive necrotic cell death. The cathepsin B-selective inhibitor CA-074-Me blocks all forms of programmed necrosis by an unknown mechanism. We found that cathepsin B deficiency does not prevent induction of pyroptosis and lysosome-mediated necrosis suggesting that CA-074-Me blocks necrotic cell death by targeting cathepsins other than cathepsin B. A single cathepsin, cathepsin C, drives necrotic cell death mediated by the lysosome-destabilizing agent Leu-Leu-OMe (LLOMe). Here we present evidence that cathepsin C-deficiency and CA-074-Me block LLOMe killing in a distinct and cell type-specific fashion. Cathepsin C-deficiency and CA-074-Me block LLOMe killing of all myeloid cells, except for neutrophils. Cathepsin C-deficiency, but not CA-074-Me, blocks LLOMe killing of neutrophils suggesting that CA-074-Me does not target cathepsin C directly, consistent with inhibitor studies using recombinant cathepsin C. Unlike other cathepsins, cathepsin C lacks endoproteolytic activity, and requires activation by other lysosomal proteases, such as cathepsin D. Consistent with this theory, we found that lysosomotropic agents and cathepsin D downregulation by siRNA block LLOMe-mediated necrosis. Our findings indicate that a proteolytic cascade, involving cathepsins C and D, controls LLOMe-mediated necrosis. In contrast, cathepsins C and D were not required for pyroptotic cell death suggesting that distinct cathepsins control pyroptosis and lysosome-mediated necrosis.
Asunto(s)
Catepsina C/fisiología , Catepsina D/fisiología , Lisosomas/enzimología , Animales , Apoptosis , Catepsina B/antagonistas & inhibidores , Catepsina B/fisiología , Dipéptidos/farmacología , Lisosomas/efectos de los fármacos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , NecrosisRESUMEN
BACKGROUND: The evolutionarily conserved DNA mismatch repair (MMR) system corrects base-substitution and insertion-deletion mutations generated during erroneous replication. The mutation or inactivation of many MMR factors strongly predisposes to cancer, where the resulting tumors often display resistance to standard chemotherapeutics. A new direction to develop targeted therapies is the harnessing of synthetic genetic interactions, where the simultaneous loss of two otherwise non-essential factors leads to reduced cell fitness or death. High-throughput screening in human cells to directly identify such interactors for disease-relevant genes is now widespread, but often requires extensive case-by-case optimization. Here we asked if conserved genetic interactors (CGIs) with MMR genes from two evolutionary distant yeast species (Saccharomyces cerevisiae and Schizosaccharomyzes pombe) can predict orthologous genetic relationships in higher eukaryotes. METHODS: High-throughput screening was used to identify genetic interaction profiles for the MutSα and MutSß heterodimer subunits (msh2Δ, msh3Δ, msh6Δ) of fission yeast. Selected negative interactors with MutSß (msh2Δ/msh3Δ) were directly analyzed in budding yeast, and the CGI with SUMO-protease Ulp2 further examined after RNA interference/drug treatment in MSH2-deficient and -proficient human cells. RESULTS: This study identified distinct genetic profiles for MutSα and MutSß, and supports a role for the latter in recombinatorial DNA repair. Approximately 28% of orthologous genetic interactions with msh2Δ/msh3Δ are conserved in both yeasts, a degree consistent with global trends across these species. Further, the CGI between budding/fission yeast msh2 and SUMO-protease Ulp2 is maintained in human cells (MSH2/SENP6), and enhanced by Olaparib, a PARP inhibitor that induces the accumulation of single-strand DNA breaks. This identifies SENP6 as a promising new target for the treatment of MMR-deficient cancers. CONCLUSION: Our findings demonstrate the utility of employing evolutionary distance in tractable lower eukaryotes to predict orthologous genetic relationships in higher eukaryotes. Moreover, we provide novel insights into the genome maintenance functions of a critical DNA repair complex and propose a promising targeted treatment for MMR deficient tumors.
RESUMEN
Cells of the innate immune system are essential for host defenses against primary microbial pathogen infections, yet their involvement in effective memory responses of vaccinated individuals has been poorly investigated. Here we show that memory T cells instruct innate cells to become potent effector cells in a systemic and a mucosal model of infection. Memory T cells controlled phagocyte, dendritic cell, and NK or NK T cell mobilization and induction of a strong program of differentiation, which included their expression of effector cytokines and microbicidal pathways, all of which were delayed in nonvaccinated hosts. Disruption of IFN-γ signaling in Ly6C+ monocytes, dendritic cells, and macrophages impaired these processes and the control of pathogen growth. These results reveal how memory T cells, through rapid secretion of IFN-γ, orchestrate extensive modifications of host innate immune responses that are essential for effective protection of vaccinated hosts.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Interferón gamma/inmunología , Activación de Linfocitos/inmunología , Traslado Adoptivo , Animales , Antígenos Ly , Diferenciación Celular/inmunología , Citocinas/inmunología , Células Dendríticas/inmunología , Femenino , Herpesvirus Humano 2/inmunología , Inmunidad Innata , Inmunización , Células Asesinas Naturales/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/inmunología , Células T Asesinas Naturales/inmunología , Fagocitosis/inmunología , Receptores de Interferón/genética , Receptor de Interferón gammaRESUMEN
Effective therapeutic vaccines often require activation of T cell-mediated immunity. Robust T cell activation, including CD8 T cell responses, can be achieved using antibodies or antibody fragments to direct antigens of interest to professional antigen presenting cells. This approach represents an important advance in enhancing vaccine efficacy. Nucleic acid aptamers present a promising alternative to protein-based targeting approaches. We have selected aptamers that specifically bind the murine receptor, DEC205, a C-type lectin expressed predominantly on the surface of CD8α(+) dendritic cells (DCs) that has been shown to be efficient at facilitating antigen crosspresentation and subsequent CD8(+) T cell activation. Using a minimized aptamer conjugated to the model antigen ovalbumin (OVA), DEC205-targeted antigen crosspresentation was verified in vitro and in vivo by proliferation and cytokine production by primary murine CD8(+) T cells expressing a T cell receptor specific for the major histocompatibility complex (MHC) I-restricted OVA257-264 peptide SIINFEKL. Compared with a nonspecific ribonucleic acid (RNA) of similar length, DEC205 aptamer-OVA-mediated antigen delivery stimulated strong proliferation and production of interferon (IFN)-γ and interleukin (IL)-2. The immune responses elicited by aptamer-OVA conjugates were sufficient to inhibit the growth of established OVA-expressing B16 tumor cells. Our results demonstrate a new application of aptamer technology for the development of effective T cell-mediated vaccines.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos/inmunología , Aptámeros de Nucleótidos/administración & dosificación , Aptámeros de Nucleótidos/genética , Animales , Antígenos/administración & dosificación , Linfocitos T CD8-positivos/inmunología , Células CHO , Cricetinae , Cricetulus , Células Dendríticas/metabolismo , Inmunidad Celular , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
We have developed a selection scheme to generate nucleic acid sequences that recognize and directly internalize into mammalian cells without the aid of conventional delivery methods. To demonstrate the generality of the technology, two independent selections with different starting pools were performed against distinct target cells. Each selection yielded a single highly functional sequence, both of which folded into a common core structure. This internalization signal can be adapted for use as a general purpose reagent for transfection into a wide variety of cell types including primary cells.
Asunto(s)
ARN/química , Transfección , Animales , Secuencia de Bases , Línea Celular , Endocitosis , Femenino , Citometría de Flujo , Biblioteca de Genes , Técnicas de Transferencia de Gen , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Membrana Mucosa/metabolismo , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Vagina/metabolismoRESUMEN
RNA interference (RNAi) describes a highly conserved pathway, present in eukaryotic cells, for regulating gene expression. Small stretches of double-stranded RNA, termed small interfering RNAs (siRNAs), utilize this pathway to bind homologous mRNA, resulting in site-specific mRNA cleavage and subsequent protein degradation. The ubiquitous presence of the RNAi machinery, combined with its specificity and efficacy, makes it an attractive mechanism for reducing aberrant gene expression in therapeutic settings. However, a major obstacle to utilizing RNAi in the clinic is siRNA delivery. Administered siRNAs must make contact with the appropriate cell types and, following internalization, gain access to the cytosol where the RNAi machinery resides. This must be achieved so that silencing is maximized, whilst minimizing any undesirable off-target effects. Recently, the utility of siRNAs as a microbicide, usually applied to the genital mucosa for preventing transmission of sexually transmitted diseases including HIV-1 and HSV-2, has been investigated. In this review we will describe these studies and discuss potential strategies for improving gene silencing.
Asunto(s)
Técnicas de Transferencia de Gen , Genitales/metabolismo , Membrana Mucosa/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Antiinfecciosos/farmacología , Genitales/efectos de los fármacos , Humanos , Membrana Mucosa/efectos de los fármacosRESUMEN
Sexually transmitted infections (STIs) are a major cause of morbidity and mortality worldwide. Although a vaccine is available for HPV, no effective vaccines exist for the HIV-1 and HSV-2 viral pathogens, and there are no cures for these infections. Furthermore, recent setbacks in clinical trials, such as the failure of the STEP trial to prevent HIV-1 infection, have emphasized the need to develop alternative approaches to interrupt the transmission of these pathogens. One alternative strategy is represented by the use of topically applied microbicides, and such agents are being developed against various viruses. RNAi-based microbicides have recently been demonstrated to prevent HSV-2 transmission, and may be useful for targeting multiple STIs. In this review, microbicides that are under development for the prevention of STIs are described, with a focus on topically applied microbicidal siRNAs.
Asunto(s)
Antiinfecciosos , ARN Interferente Pequeño , Enfermedades de Transmisión Sexual/tratamiento farmacológico , Antiinfecciosos/química , Antiinfecciosos/uso terapéutico , Transmisión de Enfermedad Infecciosa/prevención & control , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/prevención & control , Infecciones por VIH/transmisión , Herpes Genital/tratamiento farmacológico , Herpes Genital/prevención & control , Herpes Genital/transmisión , Humanos , Infecciones por Papillomavirus/tratamiento farmacológico , Infecciones por Papillomavirus/prevención & control , Infecciones por Papillomavirus/transmisión , Interferencia de ARN , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Enfermedades de Transmisión Sexual/mortalidad , Enfermedades de Transmisión Sexual/prevención & controlRESUMEN
A vaginal microbicide should prevent pathogen transmission without disrupting tissue barriers to infection. Ideally, it would not need to be applied immediately before sexual intercourse, when compliance is a problem. Intravaginal administration of small interfering RNA (siRNA) lipoplexes targeting Herpes Simplex Virus Type 2 (HSV-2) genes protects mice from HSV-2. However, protection is short-lived, and the transfection lipid on its own unacceptably enhances transmission. Here, we show that cholesterol-conjugated (chol)-siRNAs without lipid silence gene expression in the vagina without causing inflammation or inducing interferons. A viral siRNA prevents transmission within a day of challenge, whereas an siRNA targeting the HSV-2 receptor nectin-1 protects for a week, but protection is delayed for a few days until the receptor is downmodulated. Combining siRNAs targeting a viral and host gene protects mice from HSV-2 for a week, irrespective of the time of challenge. Therefore, intravaginal siRNAs could provide sustained protection against viral transmission.
Asunto(s)
Antivirales/administración & dosificación , Transmisión de Enfermedad Infecciosa/prevención & control , Herpes Genital/prevención & control , Herpes Genital/transmisión , Herpesvirus Humano 2/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Administración Intravaginal , Animales , Antivirales/química , Moléculas de Adhesión Celular/antagonistas & inhibidores , Femenino , Silenciador del Gen , Ratones , Ratones Endogámicos BALB C , Nectinas , ARN Interferente Pequeño/química , Análisis de Supervivencia , Vagina/patología , Vagina/virologíaRESUMEN
Herpes simplex virus 2 (HSV-2) infection causes significant morbidity and is an important cofactor for the transmission of HIV infection. A microbicide to prevent sexual transmission of HSV-2 would contribute substantially to controlling the spread of HIV and other infections. Because RNA interference (RNAi) provides effective antiviral defence in plants and other organisms, several studies have focused on harnessing RNAi to inhibit viral infection. Here we show that vaginal instillation of small interfering RNAs (siRNAs) targeting HSV-2 protects mice from lethal infection. siRNAs mixed with lipid are efficiently taken up by epithelial and lamina propria cells and silence gene expression in the mouse vagina and ectocervix for at least nine days. Intravaginal application of siRNAs targeting the HSV-2 UL27 and UL29 genes (which encode an envelope glycoprotein and a DNA binding protein, respectively) was well tolerated, did not induce interferon-responsive genes or cause inflammation, and protected mice when administered before and/or after lethal HSV-2 challenge. These results suggest that siRNAs are attractive candidates for the active component of a microbicide designed to prevent viral infection or transmission.
Asunto(s)
Herpes Genital/prevención & control , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/patogenicidad , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Administración Intravaginal , Animales , Línea Celular , Cuello del Útero/virología , Femenino , Silenciador del Gen , Genes Esenciales/genética , Genes Virales/genética , Herpes Genital/complicaciones , Herpesvirus Humano 2/fisiología , Inflamación , Interferones/fisiología , Liposomas/administración & dosificación , Ratones , Ratones Endogámicos BALB C , ARN Interferente Pequeño/administración & dosificación , Factores de Tiempo , Vagina/virología , Proteínas Virales/genética , Replicación ViralRESUMEN
The intracellular bacterium Listeria monocytogenes infects dendritic cells (DC) and other APCs and induces potent cell-mediated protective immunity. However, heat-killed bacteria fail to do so. This study explored whether DC differentially respond to live and killed Listeria and how this affects T cell activation. To control for bacterial number, a replication-deficient strain, Lmdd, defective in D-alanine biosynthesis, was used. We found that DC internalize both live and heat-killed Lmdd and similarly up-regulate the expression of costimulatory molecules, a necessary step for T cell activation. However, only live Lmdd-infected DC stimulate T cells to express the early activation marker CD69 and enhance T cell activation upon TCR engagement. Infection with live, but not heat-killed, Lmdd induces myeloid DC to secrete copious amounts of IFN-beta, which requires bacterial cytosolic invasion. Exposure to high concentrations of IFN-beta sensitizes naive T cells for Ag-dependent activation.
Asunto(s)
Células Dendríticas/inmunología , Interferón beta/biosíntesis , Listeria monocytogenes/inmunología , Listeria monocytogenes/patogenicidad , Células Mieloides/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Complejo CD3/inmunología , Calor , Técnicas In Vitro , Interferón beta/genética , Lectinas Tipo C , Listeria monocytogenes/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.
Asunto(s)
Sistemas de Liberación de Medicamentos , ARN Interferente Pequeño/administración & dosificación , Receptores de Superficie Celular/fisiología , Animales , Anticuerpos/química , Células COS , VIH-1 , Humanos , Interferones/metabolismo , Melanoma Experimental/terapia , Ratones , Receptores del VIH/fisiología , Proteínas Recombinantes de FusiónRESUMEN
Soluble heat shock fusion proteins (Hsfp) stimulate mice to produce CD8+ CTL, indicating that these proteins are cross-presented by dendritic cells (DC) to naive CD8 T cells. We report that cross-presentation of these proteins depends upon their binding to DC receptors, likely belonging to the scavenger receptor superfamily. Hsfp entered DC by receptor-mediated endocytosis that was either inhibitable by cytochalasin D or not inhibitable, depending upon aggregation state and time. Most endocytosed Hsfp was transported to lysosomes, but not the small cross-presented fraction that exited early from the endocytic pathway and required access to proteasomes and TAP. Naive CD8 T cell (2C and OT-I) responses to DC incubated with Hsfp at 1 microM were matched by incubating DC with cognate octapeptides at 1-10 pM, indicating that display of very few class I MHC-peptide complexes per DC can be sufficient for cross-presentation. With an Hsfp (heat shock protein-OVA) having peptide sequences for both CD4+ (OT-II) and CD8+ (OT-I) cells, the CD4 cells responded far more vigorously than the CD8 cells and many more class II MHC-peptide than class I MHC-peptide complexes were displayed.
Asunto(s)
Proteínas Bacterianas/inmunología , Chaperoninas/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/fisiología , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células Cultivadas , Chaperonina 60 , Chaperoninas/genética , Chaperoninas/metabolismo , Reactividad Cruzada/genética , Células Dendríticas/enzimología , Endocitosis/inmunología , Endosomas/inmunología , Endosomas/metabolismo , Lisosomas/inmunología , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas de la Nucleocápside , Nucleoproteínas/genética , Nucleoproteínas/inmunología , Nucleoproteínas/metabolismo , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Fagocitosis/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/inmunología , Transporte de Proteínas/inmunología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al ARN/metabolismo , Receptores Inmunológicos/metabolismo , Receptores Depuradores , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/inmunología , Solubilidad , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/inmunología , Proteínas del Núcleo Viral/metabolismoRESUMEN
We describe a role for myeloid differentiation factor 88 (MyD88) in the induction of functional CTLs in vivo, in response to exogenously administered Ag, using a heat shock fusion protein, hsp65-P1, as a model Ag. CD8 T cells transferred into MyD88-deficient animals produce normal numbers of CD8 effector cells that have normal activation marker profiles after immunization with hsp65-P1. However, these CD8 T cells produced significantly less IFN-gamma and showed reduced killing activity. This reduction in activation of functional CTLs appears to be unrelated to Toll-like receptor 4 function, because in vitro hsp65-P1-experienced Toll-like receptor 4-deficient dendritic cells (DCs), but not MyD88-deficient DCs, activated CD8 T cells to a similar extent to wild-type DCs. We identify a cross-presentation defect in MyD88-deficient DCs that, when treated with hsp65-P1 fusion protein, results in surface display of fewer SIYRYYGL/class I MHC complexes. Thus, MyD88 plays a role in the developmental maturation of DCs that allows them to prime CD8 T cells through cross-presentation.
Asunto(s)
Presentación de Antígeno , Antígenos de Diferenciación/fisiología , Citotoxicidad Inmunológica , Inmunización , Receptores Inmunológicos/fisiología , Linfocitos T Citotóxicos/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Presentación de Antígeno/genética , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/metabolismo , Antígenos de Diferenciación/genética , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Chaperonina 60 , Chaperoninas/administración & dosificación , Chaperoninas/genética , Chaperoninas/inmunología , Chaperoninas/metabolismo , Citotoxicidad Inmunológica/genética , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunización/métodos , Inyecciones Subcutáneas , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mycobacterium tuberculosis/inmunología , Factor 88 de Diferenciación Mieloide , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/trasplanteRESUMEN
Recombinant heat shock fusion proteins (Hsfp) injected into mice without added adjuvants can stimulate production of CD8 cytolytic T cells. Because initiation of productive immune responses generally requires dendritic cell (DC) activation, the question arises as to whether the Hsfp can activate DC independently of contaminating LPS. Using microarray analyses of DC from LPS-insensitive mice having a point mutation in Toll-like receptor 4 (Tlr4) (C3H/HeJ), or lacking Tlr4 (B10/ScNCr), we show here that unlike a LPS standard, Hsfp activated DC from HeJ mice almost as well as DC from wild-type mice. Consistent with the microarray analysis, the Hsfp's ability to activate DC was not eliminated by polymyxin B but was destroyed by proteinase K. The Hsfp did not, however, stimulate DC from mice lacking Tlr4. In vivo the CD8 T cell response to the Hsfp in mice lacking Tlr4 was impaired: the responding CD8 cells initially proliferated vigorously but their development into cytolytic effector cells was diminished. Overall, the results indicate that this Hsfp can activate DC independently of LPS but still requires Tlr4 for an optimal CD8 T cell response.
Asunto(s)
Proteínas Bacterianas/fisiología , Chaperoninas/fisiología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Proteínas Recombinantes de Fusión/fisiología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Animales , Proteínas Bacterianas/administración & dosificación , Diferenciación Celular/inmunología , Chaperonina 60 , Chaperoninas/administración & dosificación , Citotoxicidad Inmunológica/genética , Células Dendríticas/efectos de los fármacos , Relación Dosis-Respuesta Inmunológica , Endopeptidasa K/farmacología , Perfilación de la Expresión Génica , Lipopolisacáridos/administración & dosificación , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimixina B/farmacología , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Linfocitos T Citotóxicos/citología , Receptor Toll-Like 4 , Receptores Toll-Like , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
We identify and consider some characteristics of a peptide antagonist for the Ag-specific receptor on 2C cells (the 2C TCR). The peptide, GNYSFYAL (called GNY), binds to H-2K(b), and a very high-resolution crystal structure of the GNY-K(b) complex at 1.35 A is described. Although the GNY peptide does not bind to L(d), the potency of GNY-K(b) as an antagonist is evident from its ability to specifically inhibit 2C TCR-mediated reactions to an allogenic agonist complex (QLSPFPFDL-L(d)), as well as to a syngeneic agonist complex (SIYRYYGL-K(b)). The crystal structure and the activities of alanine-substituted peptide variants point to the properties of the peptide P4 side chain and the conformation of the Tyr-P6 side chain as the structural determinants of GNYSFYAL antagonist activity.
Asunto(s)
Isoantígenos/fisiología , Oligopéptidos/química , Oligopéptidos/fisiología , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/fisiología , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/inmunología , Animales , Arginina/metabolismo , Línea Celular , Línea Celular Tumoral , Células Clonales , Cristalografía por Rayos X , Pruebas Inmunológicas de Citotoxicidad , Femenino , Antígenos H-2/metabolismo , Antígeno de Histocompatibilidad H-2D , Lisina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oligopéptidos/metabolismo , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos T/agonistas , Serina/metabolismo , Relación Estructura-ActividadRESUMEN
Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.
Asunto(s)
Lentivirus/genética , Lentivirus/fisiología , Interferencia de ARN/fisiología , Células Dendríticas , Genes Reporteros , Células HeLa , Humanos , Técnicas In VitroRESUMEN
Naive T cells do not proliferate in normal individuals in the absence of antigen stimulation, but they proliferate spontaneously when T cells are severely depleted. We show here that coculture of syngeneic dendritic cells (DC) with naive T cells expressing a single T cell receptor also results in T cell proliferation in the absence of foreign antigen. As in lymphopenic mice, where T cell proliferation depends upon DC, this response in the coculture system requires interaction of the T cells' T cell receptor with self-peptide-MHCs on DC. This in vitro proliferation also requires soluble factors, including IL-15 secreted by DC, and can be inhibited potently by cell-cell contact with CD4+CD25+ regulatory T cells. The coculture system described may illuminate mechanisms that maintain stable numbers of T cells in normal individuals.
Asunto(s)
Células Dendríticas/metabolismo , Linfocitos T/citología , Animales , Linfocitos T CD4-Positivos/metabolismo , División Celular , Técnicas de Cocultivo , Citocinas/metabolismo , Femenino , Proteínas de Homeodominio/genética , Homeostasis , Interleucina-15/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Interleucina-2 , SolubilidadRESUMEN
Many murine T cell clones grow continuously in culture despite weekly ligation of their TCR by antigen. To learn how the cultured cells avoid or minimize antigen-induced cell death (AICD), we compared Fas and tumor necrosis factor (TNF) receptors (TNFR) on several long-term cultured CD8(+) T cell clones with those on naive and activated naive cells expressing the same TCR (2C). In contrast to the naive cells, Fas was absent on the cultured clones and the TNFR-II receptor, present initially at high levels on the cultured cells, was rapidly down-modulated in response to TCR ligation and had virtually disappeared by 2 h, when only approximately 10% of the cloned cells had been induced to express TNF-alpha. The extent of AICD of the cultured clones in response to cognate peptide-MHC on the presenting cells used for routine stimulation of the cultures was also considerably less than the massive cell death of the clones following exposure to anti-CD3 antibody plate-bound at high density.