RESUMEN
Natural killer (NK) cells are innate lymphoid cells (ILCs) contributing to immune responses to microbes and tumors. Historically, their classification hinged on a limited array of surface protein markers. Here, we used single-cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to dissect the heterogeneity of NK cells. We identified three prominent NK cell subsets in healthy human blood: NK1, NK2 and NK3, further differentiated into six distinct subgroups. Our findings delineate the molecular characteristics, key transcription factors, biological functions, metabolic traits and cytokine responses of each subgroup. These data also suggest two separate ontogenetic origins for NK cells, leading to divergent transcriptional trajectories. Furthermore, we analyzed the distribution of NK cell subsets in the lung, tonsils and intraepithelial lymphocytes isolated from healthy individuals and in 22 tumor types. This standardized terminology aims at fostering clarity and consistency in future research, thereby improving cross-study comparisons.
Asunto(s)
Células Asesinas Naturales , Análisis de la Célula Individual , Humanos , Análisis de la Célula Individual/métodos , Células Asesinas Naturales/inmunología , Transcriptoma , Neoplasias/inmunología , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Tonsila Palatina/inmunología , Tonsila Palatina/citología , Perfilación de la Expresión Génica , Pulmón/inmunología , Citocinas/metabolismoRESUMEN
Inflammation can support or restrain cancer progression and the response to therapy. Here, we searched for primary regulators of cancer-inhibitory inflammation through deep profiling of inflammatory tumor microenvironments (TMEs) linked to immune-dependent control in mice. We found that early intratumoral accumulation of interferon gamma (IFN-γ)-producing natural killer (NK) cells induced a profound remodeling of the TME and unleashed cytotoxic T cell (CTL)-mediated tumor eradication. Mechanistically, tumor-derived prostaglandin E2 (PGE2) acted selectively on EP2 and EP4 receptors on NK cells, hampered the TME switch, and enabled immune evasion. Analysis of patient datasets across human cancers revealed distinct inflammatory TME phenotypes resembling those associated with cancer immune control versus escape in mice. This allowed us to generate a gene-expression signature that integrated opposing inflammatory factors and predicted patient survival and response to immune checkpoint blockade. Our findings identify features of the tumor inflammatory milieu associated with immune control of cancer and establish a strategy to predict immunotherapy outcomes.
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Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inflamación/inmunología , Neoplasias/inmunología , Escape del Tumor/inmunología , Animales , Dinoprostona/metabolismo , Humanos , Inmunoterapia , Inflamación/genética , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Ratones , Neoplasias/terapia , Fenotipo , Pronóstico , Prostaglandina-Endoperóxido Sintasas/genética , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
Natural killer (NK) cells are a vital part of the innate immune system capable of rapidly clearing mutated or infected cells from the body and promoting an immune response. Here, we find that NK cells activated by viral infection or tumor challenge increase uptake of fatty acids and their expression of carnitine palmitoyltransferase I (CPT1A), a critical enzyme for long-chain fatty acid oxidation. Using a mouse model with an NK cell-specific deletion of CPT1A, combined with stable 13C isotope tracing, we observe reduced mitochondrial function and fatty acid-derived aspartate production in CPT1A-deficient NK cells. Furthermore, CPT1A-deficient NK cells show reduced proliferation after viral infection and diminished protection against cancer due to impaired actin cytoskeleton rearrangement. Together, our findings highlight that fatty acid oxidation promotes NK cell metabolic resilience, processes that can be optimized in NK cell-based immunotherapies.
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Neoplasias , Virosis , Humanos , Metabolismo de los Lípidos , Células Asesinas Naturales , Ácidos GrasosRESUMEN
Tumor immune escape is a major factor contributing to cancer progression and unresponsiveness to cancer therapies. Tumors can produce prostaglandin E2 (PGE2 ), an inflammatory mediator that directly acts on Natural killer (NK) cells to inhibit antitumor immunity. However, precisely how PGE2 influences NK cell tumor-restraining functions remains unclear. Here, we report that following PGE2 treatment, human NK cells exhibited altered expression of specific activating receptors and a reduced ability to degranulate and kill cancer targets. Transcriptional analysis uncovered that PGE2 also differentially modulated the expression of chemokine receptors by NK cells, inhibiting CXCR3 but increasing CXCR4. Consistent with this, PGE2-treated NK cells exhibited decreased migration to CXCL10 but increased ability to migrate toward CXCL12. Using live cell imaging, we showed that in the presence of PGE2 , NK cells were slower and less likely to kill cancer target cells following conjugation. Imaging the sequential stages of NK cell killing revealed that PGE2 impaired NK cell polarization, but not the re-organization of synaptic actin or the release of perforin itself. Together, these findings demonstrate that PGE2 affects multiple but select NK cell functions. Understanding how cancer cells subvert NK cells is necessary to more effectively harness the cancer-inhibitory function of NK cells in treatments.
Asunto(s)
Dinoprostona , Células Asesinas Naturales , Humanos , Dinoprostona/metabolismo , Línea Celular Tumoral , InmunidadRESUMEN
The impact of radiotherapy on the interaction between immune cells and cancer cells is important not least because radiotherapy can be used alongside immunotherapy as a cancer treatment. Unexpectedly, we found that X-ray irradiation of cancer cells induced significant resistance to natural killer (NK) cell killing. This was true across a wide variety of cancer-cell types as well as for antibody-dependent cellular cytotoxicity. Resistance appeared 72 h postirradiation and persisted for 2 wk. Resistance could also occur independently of radiotherapy through pharmacologically induced cell-cycle arrest. Crucially, multiple steps in NK-cell engagement, synapse assembly, and activation were unaffected by target cell irradiation. Instead, radiotherapy caused profound resistance to perforin-induced calcium flux and lysis. Resistance also occurred to a structurally similar bacterial toxin, streptolysin O. Radiotherapy did not affect the binding of pore-forming proteins at the cell surface or membrane repair. Rather, irradiation instigated a defect in functional pore formation, consistent with phosphatidylserine-mediated perforin inhibition. In vivo, radiotherapy also led to a significant reduction in NK cell-mediated clearance of cancer cells. Radiotherapy-induced resistance to perforin also constrained chimeric antigen receptor T-cell cytotoxicity. Together, these data establish a treatment-induced resistance to lymphocyte cytotoxicity that is important to consider in the design of radiotherapy-immunotherapy protocols.
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Citotoxicidad Inmunológica , Neoplasias/metabolismo , Radioterapia , Citotoxicidad Celular Dependiente de Anticuerpos , Proteínas Bacterianas , Línea Celular Tumoral , Membrana Celular/metabolismo , Humanos , Inmunoterapia , Células Asesinas Naturales/inmunología , Perforina/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , EstreptolisinasRESUMEN
Natural killer (NK) cells can kill infected or transformed cells via a lytic immune synapse. Diseased cells may exhibit altered mechanical properties but how this impacts NK cell responsiveness is unknown. We report that human NK cells were stimulated more effectively to secrete granzymes A and B, FasL (also known as FasLG), granulysin and IFNγ, by stiff (142â kPa) compared to soft (1â kPa) planar substrates. To create surrogate spherical targets of defined stiffness, sodium alginate was used to synthesise soft (9â kPa), medium (34â kPa) or stiff (254â kPa) cell-sized beads, coated with antibodies against activating receptor NKp30 (also known as NCR3) and the integrin LFA-1 (also known as ITGAL). Against stiff beads, NK cells showed increased degranulation. Polarisation of the microtubule-organising centre and lytic granules were impaired against soft targets, which instead resulted in the formation of unstable kinapses. Thus, by varying target stiffness to characterise the mechanosensitivity of immune synapses, we identify soft targets as a blind spot in NK cell recognition. This article has an associated First Person interview with the co-first authors of the paper.
Asunto(s)
Células Asesinas Naturales , Centro Organizador de los Microtúbulos , Línea Celular , Citotoxicidad Inmunológica , Humanos , Antígeno-1 Asociado a Función de Linfocito , SinapsisRESUMEN
BACKGROUND: Mucopolysaccharidosis IIIC (MPSIIIC) is one of four Sanfilippo diseases sharing clinical symptoms of severe cognitive decline and shortened lifespan. The missing enzyme, heparan sulfate acetyl-CoA: α-glucosaminide-N-acetyltransferase (HGSNAT), is bound to the lysosomal membrane, therefore cannot cross the blood-brain barrier or diffuse between cells. We previously demonstrated disease correction in MPSIIIC mice using an Adeno-Associated Vector (AAV) delivering HGSNAT via intraparenchymal brain injections using an AAV2 derived AAV-truetype (AAV-TT) serotype with improved distribution over AAV9. METHODS: Here, intraparenchymal AAV was delivered in sheep using catheters or Hamilton syringes, placed using Brainlab cranial navigation for convection enhanced delivery, to reduce proximal vector expression and improve spread. RESULTS: Hamilton syringes gave improved AAV-GFP distribution, despite lower vector doses and titres. AAV-TT-GFP displayed moderately better transduction compared to AAV9-GFP but both serotypes almost exclusively transduced neurons. Functional HGSNAT enzyme was detected in 24-37% of a 140g gyrencephalic sheep brain using AAV9-HGSNAT with three injections in one hemisphere. CONCLUSIONS: Despite variabilities in volume and titre, catheter design may be critical for efficient brain delivery. These data help inform a clinical trial for MPSIIIC.
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Mucopolisacaridosis III , Animales , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Encéfalo , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos , Heparitina Sulfato/metabolismo , Mucopolisacaridosis/genética , Mucopolisacaridosis/terapia , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/metabolismo , Mucopolisacaridosis III/terapia , Ovinos , Terapia GenéticaRESUMEN
Natural killer (NK) cells form immune synapses to ascertain the state of health of cells they encounter. If a target cell triggers NK cell cytotoxicity, lytic granules containing proteins including perforin and granzyme B, are secreted into the synaptic cleft inducing target cell death. Secretion of these proteins also occurs from activated cytotoxic T lymphocytes (CTLs) where they have recently been reported to complex with thrombospondin-1 (TSP-1) in specialized structures termed supramolecular attack particles (SMAPs). Here, using an imaging method to define the position of each NK cell after removal, secretions from individual cells were assessed. NK cell synaptic secretion, triggered by ligation of NKp30 or NKG2D, included vesicles and SMAPs which contained TSP-1, perforin, and granzyme B. Individual NK cells secreted SMAPs, CD63+ vesicles, or both. A similar number of SMAPs were secreted per cell for both NK cells and CTLs, but NK cell SMAPs were larger. These data establish an unexpected diversity in NK cell synaptic secretions.
Asunto(s)
Células Asesinas Naturales , Sinapsis , Granzimas/metabolismo , Humanos , Células Asesinas Naturales/química , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Perforina/metabolismo , Sinapsis/química , Sinapsis/inmunología , Sinapsis/metabolismo , Linfocitos T Citotóxicos/química , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Trombospondina 1/metabolismoRESUMEN
Type 2 inflammation is a defining feature of infection with parasitic worms (helminths), as well as being responsible for widespread suffering in allergies. However, the precise mechanisms involved in T helper (Th) 2 polarization by dendritic cells (DCs) are currently unclear. We have identified a previously unrecognized role for type I IFN (IFN-I) in enabling this process. An IFN-I signature was evident in DCs responding to the helminth Schistosoma mansoni or the allergen house dust mite (HDM). Further, IFN-I signaling was required for optimal DC phenotypic activation in response to helminth antigen (Ag), and efficient migration to, and localization with, T cells in the draining lymph node (dLN). Importantly, DCs generated from Ifnar1-/- mice were incapable of initiating Th2 responses in vivo These data demonstrate for the first time that the influence of IFN-I is not limited to antiviral or bacterial settings but also has a central role to play in DC initiation of Th2 responses.
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Células Dendríticas/inmunología , Interferón Tipo I/metabolismo , Células Th2/inmunología , Alérgenos/inmunología , Animales , Ratones , Ratones Noqueados , Pyroglyphidae/inmunología , Receptor de Interferón alfa y beta/deficiencia , Schistosoma mansoni/inmunologíaRESUMEN
Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.
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Macrófagos , Microscopía , Membrana Celular , Pulmón , MicrotúbulosRESUMEN
Membrane nanotubes are transient long-distance connections between cells that can facilitate intercellular communication (for example, by trafficking vesicles or transmitting calcium-mediated signals), but they can also contribute to pathologies (for example, by directing the spread of viruses). Recent data have revealed considerable heterogeneity in their structures, processes of formation and functional properties, in part dependent on the cell types involved. Despite recent progress in this young research field, further research is sorely needed.
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Comunicación Celular/fisiología , Membrana Celular/fisiología , Uniones Intercelulares/fisiología , Animales , Células Cultivadas , HumanosRESUMEN
Cross-talk between NK cells and dendritic cells (DCs) is important in Th1 immune responses, including antitumor immunity and responses to infections. DCs also play a crucial role in polarizing Th2 immunity, but the impact of NK cell-DC interactions in this context remains unknown. In this study, we stimulated human monocyte-derived DCs in vitro with different pathogen-associated molecules: LPS or polyinosinic-polycytidylic acid, which polarize a Th1 response, or soluble egg Ag from the helminth worm Schistosoma mansoni, a potent Th2-inducing Ag. Th2-polarizing DCs were functionally distinguishable from Th1-polarizing DCs, and both showed distinct morphology and dynamics from immature DCs. We then assessed the outcome of autologous NK cells interacting with these differently stimulated DCs. Confocal microscopy showed polarization of the NK cell microtubule organizing center and accumulation of LFA-1 at contacts between NK cells and immature or Th2-polarizing DCs but not Th1-polarizing DCs, indicative of the assembly of an activating immune synapse. Autologous NK cells lysed immature DCs but not DCs treated with LPS or polyinosinic-polycytidylic acid as reported previously. In this study, we demonstrated that NK cells also degranulated in the presence of Th2-polarizing DCs. Moreover, time-lapse live-cell microscopy showed that DCs that had internalized fluorescently labeled soluble egg Ag were efficiently lysed. Ab blockade of NK cell-activating receptors NKp30 or DNAM-1 abrogated NK cell lysis of Th2-polarizing DCs. Thus, these data indicate a previously unrecognized role of NK cell cytotoxicity and NK cell-activating receptors NKp30 and DNAM-1 in restricting the pool of DCs involved in Th2 immune responses.
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Antígenos de Diferenciación de Linfocitos T/metabolismo , Células Dendríticas/inmunología , Células Asesinas Naturales/inmunología , Receptor 3 Gatillante de la Citotoxidad Natural/metabolismo , Schistosoma mansoni/inmunología , Células TH1/inmunología , Células Th2/inmunología , Animales , Antígenos Helmínticos/inmunología , Diferenciación Celular , Células Cultivadas , Citotoxicidad Inmunológica , Humanos , Lipopolisacáridos/inmunología , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Poli I-C/inmunología , Imagen de Lapso de TiempoRESUMEN
Recombinant adeno-associated viruses (AAVs) are popular in vivo gene transfer vehicles. However, vector doses needed to achieve therapeutic effect are high and some target tissues in the central nervous system remain difficult to transduce. Gene therapy trials using AAV for the treatment of neurological disorders have seldom led to demonstrated clinical efficacy. Important contributing factors are low transduction rates and inefficient distribution of the vector. To overcome these hurdles, a variety of capsid engineering methods have been utilized to generate capsids with improved transduction properties. Here we describe an alternative approach to capsid engineering, which draws on the natural evolution of the virus and aims to yield capsids that are better suited to infect human tissues. We generated an AAV capsid to include amino acids that are conserved among natural AAV2 isolates and tested its biodistribution properties in mice and rats. Intriguingly, this novel variant, AAV-TT, demonstrates strong neurotropism in rodents and displays significantly improved distribution throughout the central nervous system as compared to AAV2. Additionally, sub-retinal injections in mice revealed markedly enhanced transduction of photoreceptor cells when compared to AAV2. Importantly, AAV-TT exceeds the distribution abilities of benchmark neurotropic serotypes AAV9 and AAVrh10 in the central nervous system of mice, and is the only virus, when administered at low dose, that is able to correct the neurological phenotype in a mouse model of mucopolysaccharidosis IIIC, a transmembrane enzyme lysosomal storage disease, which requires delivery to every cell for biochemical correction. These data represent unprecedented correction of a lysosomal transmembrane enzyme deficiency in mice and suggest that AAV-TT-based gene therapies may be suitable for treatment of human neurological diseases such as mucopolysaccharidosis IIIC, which is characterized by global neuropathology.
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Cápside/fisiología , Terapia Genética/métodos , Ingeniería de Proteínas/métodos , Animales , Dependovirus/genética , Femenino , Vectores Genéticos , Masculino , Ratones , Ratones Endogámicos C57BL , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/terapia , Células Fotorreceptoras/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Retina/fisiología , Distribución Tisular , Transducción GenéticaRESUMEN
Aims: Investigators have proposed that cardiovascular magnetic resonance (CMR) should have restrictions similar to those of ionizing imaging techniques. We aimed to investigate the acute effect of 1.5 T CMR on leucocyte DNA integrity, cell counts, and function in vitro, and in a large cohort of patients in vivo. Methods and results: In vitro study: peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers, and histone H2AX phosphorylation (γ-H2AX) expression, leucocyte counts, and functional parameters were quantified using flow cytometry under the following conditions: (i) immediately following PBMC isolation, (ii) after standing on the benchside as a temperature and time control, (iii) after a standard CMR scan. In vivo study: blood samples were taken from 64 consecutive consenting patients immediately before and after a standard clinical scan. Samples were analysed for γ-H2AX expression and leucocyte counts. CMR was not associated with a significant change in γ-H2AX expression in vitro or in vivo, although there were significant inter-patient variations. In vitro cell integrity and function did not change with CMR. There was a significant reduction in circulating T cells in vivo following CMR. Conclusion: 1.5 T CMR was not associated with DNA damage in vitro or in vivo. Histone H2AX phosphorylation expression varied markedly between individuals; therefore, small studies using γ-H2AX as a marker of DNA damage should be interpreted with caution. Cardiovascular magnetic resonance was not associated with loss of leucocyte viability or function in vitro. Cardiovascular magnetic resonance was associated with a statistically significant reduction in viable leucocytes in vivo.
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Técnicas de Imagen Cardíaca/efectos adversos , Leucocitos Mononucleares/efectos de la radiación , Imagen por Resonancia Cinemagnética/efectos adversos , Adulto , Daño del ADN/efectos de la radiación , Femenino , Humanos , Leucocitos Mononucleares/química , Leucocitos Mononucleares/citología , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
BACKGROUND: Chediak-Higashi syndrome (CHS) is a rare disorder caused by biallelic mutations in the lysosomal trafficking regulator gene (LYST), resulting in formation of giant lysosomes or lysosome-related organelles in several cell types. The disease is characterized by immunodeficiency and a fatal hemophagocytic lymphohistiocytosis caused by impaired function of cytotoxic lymphocytes, including natural killer (NK) cells. OBJECTIVE: We sought to determine the underlying biochemical cause of the impaired cytotoxicity of NK cells in patients with CHS. METHODS: We generated a human cell model of CHS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. We used a combination of classical techniques to evaluate lysosomal function and cell activity in the model system and super-resolution microscopy to visualize F-actin and lytic granules in normal and LYST-deficient NK cells. RESULTS: Loss of LYST function in a human NK cell line, NK92mi, resulted in inhibition of NK cell cytotoxicity and reproduced other aspects of the CHS cellular phenotype, including the presence of significantly enlarged lytic granules with defective exocytosis and impaired integrity of endolysosomal compartments. The large granules had an acidic pH and normal activity of lysosomal enzymes and were positive for the proteins essential for lytic granule exocytosis. Visualization of the actin meshwork openings at the immunologic synapse revealed that the cortical actin acts as a barrier for secretion of such large granules at the cell-cell contact site. Decreasing the cortical actin density at the immunologic synapse or decreasing the lytic granule size restored the ability of LYST-deficient NK cells to degranulate and kill target cells. CONCLUSION: The cortical actin and granule size play significant roles in NK cell cytotoxic function. We present evidence that the periodicity of subsynaptic actin is an important factor limiting the release of large lytic granules from NK cells from patients with CHS and could be a novel target for pharmaceutical intervention.
Asunto(s)
Actinas/inmunología , Síndrome de Chediak-Higashi/inmunología , Gránulos Citoplasmáticos/inmunología , Células Asesinas Naturales/inmunología , Línea Celular , Citoesqueleto/inmunología , Humanos , Proteínas de Transporte Vesicular/genéticaRESUMEN
An emerging new paradigm is that immune cell activation is controlled by transient interactions between supramolecular assemblies of receptors and ligands. Current immunotherapy biologic pharmaceuticals that activate or desensitize NK cells are, however, individual molecules that do not replicate this nanoscale organization of proteins. Here, we use nanoscale graphene oxide (NGO) as a template to generate soluble nanoscale clusters of Natural Killer cell-activating antibodies. We control nanocluster size and molecular number to mimic reported values for cell surface proteins. These NGO-templated molecular nanoclusters, used to stimulate NK cells via the CD16 receptor, successfully induced cellular activation, indicated by degranulation of cytolytic granules and IFN-γ secretion. Importantly, activation significantly exceeded that induced by the same antibodies applied as a solution of individual molecules. These results demonstrate that future immunotherapies could be enhanced by assembling immunomodulatory drugs into nanoclusters and establish NGO-templating as a candidate technology.
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Anticuerpos Monoclonales/farmacología , Grafito/farmacología , Inmunoconjugados/farmacología , Células Asesinas Naturales/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Nanoconjugados , Anticuerpos Monoclonales/química , Degranulación de la Célula/efectos de los fármacos , Células Cultivadas , Grafito/química , Humanos , Inmunoconjugados/química , Células Asesinas Naturales/inmunología , Nanoconjugados/química , Óxidos/química , Óxidos/farmacología , Receptores de IgG/inmunologíaRESUMEN
Cells can extend the limits of their transcriptome by using proteins captured from other cells. Through an exchange of specific proteins, tools and information can be shared to establish integrated communities of cells that are better able to coordinate stages of an immune response. Transferred proteins can also contribute to pathology by allowing, for example, infection of cell types not otherwise infected. Here, I present the case for considering the intercellular transfer of cell-surface proteins between immune cells as commonplace and important.
Asunto(s)
Comunicación Celular/inmunología , Espacio Extracelular/inmunología , Inmunidad , Proteínas de la Membrana/metabolismo , Animales , Transporte Biológico Activo/genética , Transporte Biológico Activo/inmunología , Comunicación Celular/genética , Espacio Extracelular/genética , Humanos , Inmunidad/genética , Proteínas de la Membrana/genéticaRESUMEN
As multiple myeloma (MM) progresses, natural killer (NK)-cell responses decline against malignant plasma cells. The immunomodulatory drug lenalidomide is widely used for treatment of MM but its influence on NK-cell biology is unclear. Here, we report that lenalidomide lowers the threshold for NK-cell activation, causing a 66% decrease in the 50% effective concentration (EC50) for activation through CD16, and a 38% decrease in EC50 for NK group 2 member D (NKG2D)-mediated activation, allowing NK cells to respond to lower doses of ligand. In addition, lenalidomide augments NK-cell responses, causing a twofold increase in the proportion of primary NK cells producing interferon-γ (IFN-γ), and a 20-fold increase in the amount of IFN-γ produced per cell. Importantly, lenalidomide did not trigger IFN-γ production in unstimulated NK cells. Thus, lenalidomide enhances the NK-cell arm of the immune response, without activating NK cells inappropriately. Of particular clinical importance, lenalidomide also allowed NK cells to be activated by lower doses of rituximab, an anti-CD20 monoclonal antibody (mAb) widely used to treat B-cell malignancies. This supports combined use of lenalidomide and rituximab in a clinical setting. Finally, superresolution microscopy revealed that lenalidomide increased the periodicity of cortical actin at immune synapses, resulting in an increase in the area of the actin mesh predicted to be penetrable to vesicles containing IFN-γ. NK cells from MM patients also responded to lenalidomide in this way. This indicates that nanometer-scale rearrangements in cortical actin, a recently discovered step in immune synapse assembly, are a potential new target for therapeutic compounds.
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Actinas/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Talidomida/análogos & derivados , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Células Cultivadas , Proteínas Ligadas a GPI/metabolismo , Humanos , Sinapsis Inmunológicas/efectos de los fármacos , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Lenalidomida , Recuento de Linfocitos , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Receptores de IgG/metabolismo , Talidomida/farmacologíaRESUMEN
Natural killer (NK) cells discriminate between healthy and unhealthy target cells through a balance of activating and inhibitory signals at direct intercellular contacts called immune synapses. Rearrangements in the cellular cytoskeleton have long been known to be critical in assembly of immune synapses. Here, through bringing together the vast literature on this subject, the number of different ways in which the cytoskeleton is important becomes evident. The dynamics of filamentous actin are critical in (i) creating the nanometer-scale organization of NK cell receptors, (ii) establishing cellular polarity, (iii) coordinating immune receptor and integrin-mediated signaling, and (iv) directing secretion of lytic granules and cytokines. The microtubule network also is important in the delivery of lytic granules and vesicles containing cytokines to the immune synapse. Together, these data establish that the cytoskeleton acts as a central regulator of this complex and dynamic process - and an enormous amount of NK cell biology is controlled through the cytoskeleton.