RESUMEN
Immunotherapy is a promising treatment for triple-negative breast cancer (TNBC), but patients relapse, highlighting the need to understand the mechanisms of resistance. We discovered that in primary breast cancer, tumor cells that resist T cell attack are quiescent. Quiescent cancer cells (QCCs) form clusters with reduced immune infiltration. They also display superior tumorigenic capacity and higher expression of chemotherapy resistance and stemness genes. We adapted single-cell RNA-sequencing with precise spatial resolution to profile infiltrating cells inside and outside the QCC niche. This transcriptomic analysis revealed hypoxia-induced programs and identified more exhausted T cells, tumor-protective fibroblasts, and dysfunctional dendritic cells inside clusters of QCCs. This uncovered differential phenotypes in infiltrating cells based on their intra-tumor location. Thus, QCCs constitute immunotherapy-resistant reservoirs by orchestrating a local hypoxic immune-suppressive milieu that blocks T cell function. Eliminating QCCs holds the promise to counteract immunotherapy resistance and prevent disease recurrence in TNBC.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Humanos , Inmunosupresores/uso terapéutico , Inmunoterapia , Recurrencia Local de Neoplasia , Linfocitos T/patología , Neoplasias de la Mama Triple Negativas/patología , Microambiente TumoralRESUMEN
Alzheimer's disease (AD) is the most common form of incurable dementia and represents a critical public health issue as the world's population ages. Although microglial dysregulation is a cardinal feature of AD, the extensive heterogeneity of these immunological cells in the brain has impeded our understanding of their contribution to this disease. Here, we identify a pathogenic microglial subset which expresses the CD11c surface marker as the sole producer of Osteopontin (OPN) in the 5XFAD mouse model of AD. OPN production divides Disease-Associated Microglia (DAM) into two functionally distinct subsets, i.e., a protective CD11c+OPN- subset that robustly ingests amyloid ß (Aß) in a noninflammatory fashion and a pathogenic CD11c+OPN+ subset that produces proinflammatory cytokines and fails to ingest significant amounts of Aß. Genetic ablation of OPN or administration of monoclonal anti-OPN antibody to 5XFAD mice reduces proinflammatory microglia, plaque formation, and numbers of dystrophic neurites and results in improved cognitive function. Analysis of brain tissue from AD patients indicates that levels of OPN-producing CD11c+ microglia correlate strongly with the degree of cognitive deficit and AD neuropathology. These findings define an OPN-dependent pathway to disease driven by a distinct microglial subset, and identify OPN as a novel therapeutic target for potentially effective immunotherapy to treat AD.
Asunto(s)
Enfermedad de Alzheimer , Ratones , Animales , Enfermedad de Alzheimer/metabolismo , Microglía/metabolismo , Péptidos beta-Amiloides/metabolismo , Osteopontina/metabolismo , Ratones Transgénicos , Modelos Animales de Enfermedad , Placa Amiloide/metabolismoRESUMEN
Expression of Itgax (encoding the CD11c surface protein) and Spp1 (encoding osteopontin; OPN) has been associated with activated microglia that can develop in healthy brains and some neuroinflammatory disorders. However, whether CD11c and OPN expression is a consequence of microglial activation or represents a portion of the genetic program expressed by a stable microglial subset is unknown. Here, we show that OPN production in the brain is confined to a small CD11c+ microglial subset that differentiates from CD11c- precursors in perinatal life after uptake of apoptotic neurons. Our analysis suggests that coexpression of OPN and CD11c marks a microglial subset that is expressed at birth and persists into late adult life, independent of environmental activation stimuli. Analysis of the contribution of OPN to the intrinsic functions of this CD11c+ microglial subset indicates that OPN is required for subset stability and the execution of phagocytic and proinflammatory responses, in part through OPN-dependent engagement of the αVß3-integrin receptor. Definition of OPN-producing CD11c+ microglia as a functional microglial subset provides insight into microglial differentiation in health and disease.
Asunto(s)
Antígenos CD11/metabolismo , Microglía/fisiología , Osteopontina/metabolismo , Animales , Encéfalo/metabolismo , Antígenos CD11/genética , Femenino , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Inflamación/metabolismo , Activación de Macrófagos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neurogénesis , Osteopontina/genética , Fagocitos/metabolismo , Transcriptoma/genéticaRESUMEN
Regulatory T cells (Treg) can impede antitumor immunity and currently represent a major obstacle to effective cancer immunotherapy. Targeting tumor-infiltrating regulatory Treg while sparing systemic Treg represents an optimal approach to this problem. Here, we provide evidence that the interleukin 23 receptor (IL23R) expressed by tumor-infiltrating Treg promotes suppressive activity. Disruption of the IL23R results in increased responsiveness of destabilized Treg to the IL12 cytokine, the production of γ-interferon, and the recruitment of CD8 T cells that inhibit tumor growth. Since the Treg destabilization pathway that is initiated by IL23R blockade is distinct and independent from the destabilization pathway coupled to glucocorticoid-induced TNFR-related protein (GITR) activation, we examined the impact of the coordinate induction of the two destabilization pathways on antitumor immune responses. Combined GITR and IL23R antibody treatment of mice inoculated with MC38 tumors resulted in robust and synergistic antitumor responses. These findings indicate that the delineation of independent Treg destabilization pathways may allow improved approaches to the development of combination immunotherapy for cancers.
Asunto(s)
Neoplasias , Linfocitos T Reguladores , Anticuerpos Bloqueadores , Humanos , Factores Inmunológicos/metabolismo , Inmunoterapia , Interleucina-23/metabolismo , Neoplasias/metabolismoRESUMEN
In the past decade, the study of NK cells was transformed by the discovery of three ways these "innate" immune cells display adaptive immune behavior, including the ability to form long-lasting, Ag-specific memories of a wide variety of immunogens. In this review, we examine these types of NK cell memory, highlighting their unique features and underlying similarities. We explore those similarities in depth, focusing on the role that Ly49 receptors play in various types of NK cell memory. From this Ly49 dependency, we will build a model by which we understand the three types of NK cell memory as aspects of what is ultimately the same adaptive immune process, rather than separate facets of NK cell biology. We hope that a defined model for NK cell memory will empower collaboration between researchers of these three fields to further our understanding of this surprising and clinically promising immune response.
Asunto(s)
Células Asesinas Naturales/inmunología , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Animales , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunidad Innata , Memoria InmunológicaRESUMEN
Influenza A virus (IAV) increases the presentation of class I human leukocyte antigen (HLA) proteins that limit antiviral responses mediated by natural killer (NK) cells, but molecular mechanisms for these processes have not yet been fully elucidated. We observed that infection with A/Fort Monmouth/1/1947(H1N1) IAV significantly increased the presentation of HLA-B, -C, and -E on lung epithelial cells. Virus entry was not sufficient to induce HLA upregulation because UV-inactivated virus had no effect. Aberrant internally deleted viral RNAs (vRNAs) known as mini viral RNAs (mvRNAs) and defective interfering RNAs (DI RNAs) expressed from an IAV minireplicon were sufficient for inducing HLA upregulation. These defective RNAs bind to retinoic acid-inducible gene I (RIG-I) and initiate mitochondrial antiviral signaling (MAVS) protein-dependent antiviral interferon (IFN) responses. Indeed, MAVS was required for HLA upregulation in response to IAV infection or ectopic mvRNA/DI RNA expression. The effect was partially due to paracrine signaling, as we observed that IAV infection or mvRNA/DI RNA-expression stimulated production of IFN-ß and IFN-λ1 and conditioned media from these cells elicited a modest increase in HLA surface levels in naive epithelial cells. HLA upregulation in response to aberrant viral RNAs could be prevented by the Janus kinase (JAK) inhibitor ruxolitinib. While HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein; we determined that NS1 limits cell-intrinsic and paracrine mechanisms of HLA upregulation. Taken together, our findings indicate that aberrant IAV RNAs stimulate HLA presentation, which may aid viral evasion of innate immunity.IMPORTANCE Human leukocyte antigens (HLAs) are cell surface proteins that regulate innate and adaptive immune responses to viral infection by engaging with receptors on immune cells. Many viruses have evolved ways to evade host immune responses by modulating HLA expression and/or processing. Here, we provide evidence that aberrant RNA products of influenza virus genome replication can trigger retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS)-dependent remodeling of the cell surface, increasing surface presentation of HLA proteins known to inhibit the activation of an immune cell known as a natural killer (NK) cell. While this HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein, which limits RIG-I activation and interferon production by the infected cell.
Asunto(s)
Genes MHC Clase I/genética , Antígenos HLA/metabolismo , Subtipo H1N1 del Virus de la Influenza A/genética , Células A549 , Proteínas Adaptadoras Transductoras de Señales/genética , Proteína 58 DEAD Box/genética , Bases de Datos Genéticas , Células Epiteliales/virología , Interacciones Huésped-Patógeno/genética , Humanos , Inmunidad Innata , Virus de la Influenza A/genética , Gripe Humana/genética , Células Asesinas Naturales/metabolismo , Pulmón/virología , ARN Viral/genética , Transducción de Señal , Activación Transcripcional , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/genéticaRESUMEN
Adaptive natural killer (NK) cell memory represents a new frontier in immunology. Work over the last decade has discovered and confirmed the existence of NK cells with antigen-specific memories, which had previously been considered a unique property of T and B cells. These findings have shown that antigen-specific NK cells gain their specificity without the use of RAG proteins, representing a novel mechanism for generating antigen specificity, but the details of this mechanism have remained a mystery. We have discovered that members of the Ly49 family of surface receptors are critically involved in both the sensitization and the challenge phases of an NK cell memory response, as is antigen presentation from their binding partner, the class I MHC. Moreover, we demonstrate that the Ly49-interacting component of a presented antigen dictates the specificity of the NK cell memory response, implicating Ly49 receptors themselves in antigen-specific recognition. Finally, we demonstrate that adaptive NK cell memories can protect against an otherwise lethal melanoma without T cell or B cell support. These findings offer insight into the mechanism behind NK cell antigen specificity and demonstrate the clinical potential of this adaptive immune cell.
Asunto(s)
Dermatitis por Contacto/prevención & control , Memoria Inmunológica , Células Asesinas Naturales/inmunología , Melanoma Experimental/terapia , Subfamilia A de Receptores Similares a Lectina de Células NK/genética , Péptidos/inmunología , Inmunidad Adaptativa/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Vacunas contra el Cáncer/administración & dosificación , Dermatitis por Contacto/genética , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/patología , Dinitrofluorobenceno/administración & dosificación , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Masculino , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Subfamilia A de Receptores Similares a Lectina de Células NK/inmunología , Oxazoles/administración & dosificación , Péptidos/administración & dosificación , Péptidos/síntesis química , VacunaciónRESUMEN
Monocytes differentiate into macrophages, which deactivate invading pathogens. Macrophages can be resistant to cell death mechanisms in some situations, and the mechanisms involved are not clear. Here, using mouse immune cells, we investigated whether the differentiation of macrophages affects their susceptibility to cell death by the ripoptosome/necrosome pathways. We show that treatment of macrophages with a mimetic of second mitochondrial activator of caspases (SMAC) resulted in ripoptosome-driven cell death that specifically depended on tumor necrosis factor α (TNFα) expression and the receptor-interacting serine/threonine protein kinase 1 (RipK1)-RipK3-caspase-8 interaction in activated and cycling macrophages. Differentiation of macrophages increased the expression of pro-inflammatory cytokines but reduced RipK1-dependent cell death and the RipK3-caspase-8 interaction. The expression of the anti-apoptotic mediators, X-linked inhibitor of apoptosis protein (XIAP) and caspase-like apoptosis regulatory protein (cFLIPL), also increased in differentiated macrophages, which inhibited caspase activation. The resistance to cell death was abrogated in XIAP-deficient macrophages. However, even in the presence of increased XIAP expression, inhibition of the mitogen-activated protein kinase (MAPK) p38 and MAPK-activated protein kinase 2 (MK2) made differentiated macrophages susceptible to cell death. These results suggest that the p38/MK2 pathway overrides apoptosis inhibition by XIAP and that acquisition of resistance to cell death by increased expression of XIAP and cFLIPL may allow inflammatory macrophages to participate in pathogen control for a longer duration.
Asunto(s)
Inflamación/inmunología , Proteínas Inhibidoras de la Apoptosis/inmunología , Macrófagos/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Animales , Apoptosis , Diferenciación Celular , Células Cultivadas , Macrófagos/citología , Ratones Endogámicos C57BLAsunto(s)
Agricultura/legislación & jurisprudencia , Productos Agrícolas/genética , Unión Europea , Edición Génica/legislación & jurisprudencia , Plantas Modificadas Genéticamente/genética , Agricultura/economía , Animales , Bélgica , Brasil , Cruzamiento/legislación & jurisprudencia , Sistemas CRISPR-Cas/genética , Productos Agrícolas/economía , Estudios de Evaluación como Asunto , Francia , HumanosRESUMEN
The immune response to influenza virus infection comprises both innate and adaptive defenses. NK cells play an early role in the destruction of tumors and virally-infected cells. NK cells express a variety of inhibitory receptors, including those of the Ly49 family, which are functional homologs of human killer-cell immunoglobulin-like receptors (KIR). Like human KIR, Ly49 receptors inhibit NK cell-mediated lysis by binding to major histocompatibility complex class I (MHC-I) molecules that are expressed on normal cells. During NK cell maturation, the interaction of NK cell inhibitory Ly49 receptors with their MHC-I ligands results in two types of NK cells: licensed ("functional"), or unlicensed ("hypofunctional"). Despite being completely dysfunctional with regard to rejecting MHC-I-deficient cells, unlicensed NK cells represent up to half of the mature NK cell pool in rodents and humans, suggesting an alternative role for these cells in host defense. Here, we demonstrate that after influenza infection, MHC-I expression on lung epithelial cells is upregulated, and mice bearing unlicensed NK cells (Ly49-deficient NKCKD and MHC-I-deficient B2m-/- mice) survive the infection better than WT mice. Importantly, transgenic expression of an inhibitory self-MHC-I-specific Ly49 receptor in NKCKD mice restores WT influenza susceptibility, confirming a direct role for Ly49. Conversely, F(ab')2-mediated blockade of self-MHC-I-specific Ly49 inhibitory receptors protects WT mice from influenza virus infection. Mechanistically, perforin-deficient NKCKD mice succumb to influenza infection rapidly, indicating that direct cytotoxicity is necessary for unlicensed NK cell-mediated protection. Our findings demonstrate that Ly49:MHC-I interactions play a critical role in influenza virus pathogenesis. We suggest a similar role may be conserved in human KIR, and their blockade may be protective in humans.
Asunto(s)
Antígenos Ly/metabolismo , Evasión Inmune , Virus de la Influenza A/inmunología , Células Asesinas Naturales/inmunología , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Receptores KIR/metabolismo , Mucosa Respiratoria/inmunología , Animales , Antígenos Ly/genética , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Cruzamientos Genéticos , Inmunidad Innata , Virus de la Influenza A/fisiología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Células Asesinas Naturales/virología , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Ratones Noqueados , Ratones Transgénicos , Subfamilia A de Receptores Similares a Lectina de Células NK/agonistas , Subfamilia A de Receptores Similares a Lectina de Células NK/antagonistas & inhibidores , Subfamilia A de Receptores Similares a Lectina de Células NK/genética , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptores KIR/agonistas , Receptores KIR/antagonistas & inhibidores , Receptores KIR/genética , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Mucosa Respiratoria/virología , Organismos Libres de Patógenos Específicos , Análisis de Supervivencia , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1005446.].
RESUMEN
NK cells play a major role in immune defense against human and murine CMV (MCMV) infection. Although the MCMV genome encodes for MHC class I-homologous decoy ligands for inhibitory NK cell receptors to evade detection, some mouse strains have evolved activating receptors, such as Ly49H, to recognize these ligands and initiate an immune response. In this study, we demonstrate that approximately half of the Ly49H-expressing (Ly49H(+)) NK cells in the spleen and liver of C57BL/6 mice also express the inhibitory NKR-P1B receptor. During MCMV infection, the NKR-P1B(-)Ly49H(+) NK cell subset proliferates to constitute the bulk of the NK cell population. This NK cell subset also confers better protection against MCMV infection compared with the NKR-P1B(+)Ly49H(+) subset. The two populations are composed of cells that differ in their surface expression of receptors such as Ly49C/I and NKG2A/C/E, as well as developmental markers, CD27 and CD11b, and the high-affinity IL-2R (CD25) following infection. Although the NKR-P1B(+) NK cells can produce effector molecules such as IFNs and granzymes, their proliferation is inhibited during infection. A similar phenotype in MCMV-infected Clr-b-deficient mice, which lack the ligand for NKR-P1B, suggests the involvement of ligands other than the host Clr-b. Most interestingly, genetic deficiency of the NKR-P1B, but not Clr-b, results in accelerated virus clearance and recovery from MCMV infection. This study is particularly significant because the mouse NKR-P1B:Clr-b receptor:ligand system represents the closest homolog of the human NKR-P1A:LLT1 system and may have a direct relevance to human CMV infection.
Asunto(s)
Infecciones por Herpesviridae/inmunología , Células Asesinas Naturales/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Animales , Ligandos , Ratones , Ratones Endogámicos C57BL , Muromegalovirus/inmunología , Muromegalovirus/fisiología , Subfamilia B de Receptores Similares a Lectina de Células NK/deficiencia , Subfamilia B de Receptores Similares a Lectina de Células NK/genéticaRESUMEN
Beyond its role in genomic organization and compaction, the nucleosome is believed to participate in the regulation of gene transcription. Here, we report a computational method to evaluate the nucleosome sensitivity for a transcription factor over a given stretch of the genome. Sensitive factors are predicted to be those with binding sites preferentially contained within nucleosome boundaries and lacking 10 bp periodicity. Based on these criteria, the Acute Myeloid Leukemia-1a (AML-1a) transcription factor, a regulator of immune gene expression, was identified as potentially sensitive to nucleosomal regulation within the mouse Ly49 gene family. This result was confirmed in RMA, a cell line with natural expression of Ly49, using MNase-Seq to generate a nucleosome map of chromosome 6, where the Ly49 gene family is located. Analysis of this map revealed a specific depletion of nucleosomes at AML-1a binding sites in the expressed Ly49A when compared to the other, silent Ly49 genes. Our data suggest that nucleosome-based regulation contributes to the expression of Ly49 genes, and we propose that this method of predicting nucleosome sensitivity could aid in dissecting the regulatory role of nucleosomes in general.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subfamilia A de Receptores Similares a Lectina de Células NK/genética , Nucleosomas/genética , Nucleosomas/metabolismo , Animales , Sitios de Unión/genética , Línea Celular , Mapeo Cromosómico , Biología Computacional , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Regulación de la Expresión Génica , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ratones , Familia de Multigenes , Nucleosomas/inmunología , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
MHC-I-specific receptors play a vital role in NK cell-mediated "missing-self" recognition, which contributes to NK cell activation. In contrast, MHC-independent NK recognition mechanisms are less well characterized. In this study, we investigated the role of NKR-P1B:Clr-b (Klrb1:Clec2d) interactions in determining the outcome of murine hematopoietic cell transplantation in vivo. Using a competitive transplant assay, we show that Clr-b(-/-) bone marrow (BM) cells were selectively rejected by wild-type B6 recipients, to a similar extent as H-2D(b-/-) MHC-I-deficient BM cells. Selective rejection of Clr-b(-/-) BM cells was mitigated by NK depletion of recipient mice. Competitive rejection of Clr-b(-/-) BM cells also occurred in allogeneic transplant recipients, where it was reversed by selective depletion of NKR-P1B(hi) NK cells, leaving the remaining NKR-P1B(lo) NK subset and MHC-I-dependent missing-self recognition intact. Moreover, competitive rejection of Clr-b(-/-) hematopoietic cells was abrogated in Nkrp1b-deficient recipients, which lack the receptor for Clr-b. Of interest, similar to MHC-I-deficient NK cells, Clr-b(-/-) NK cells were hyporesponsive to both NK1.1 (NKR-P1C)-stimulated and IL-12/18 cytokine-primed IFN-γ production. These findings support a unique and nonredundant role for NKR-P1B:Clr-b interactions in missing-self recognition of normal hematopoietic cells and suggest that optimal BM transplant success relies on MHC-independent tolerance mechanisms. These findings provide a model for human NKR-P1A:LLT1 (KLRB1:CLEC2D) interactions in human hematopoietic cell transplants.
Asunto(s)
Trasplante de Médula Ósea/métodos , Células Asesinas Naturales/inmunología , Lectinas Tipo C/inmunología , Proteínas de la Membrana/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Citometría de Flujo , Expresión Génica/inmunología , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Antígeno de Histocompatibilidad H-2D/genética , Antígeno de Histocompatibilidad H-2D/inmunología , Antígeno de Histocompatibilidad H-2D/metabolismo , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Células Asesinas Naturales/metabolismo , Lectinas Tipo C/deficiencia , Lectinas Tipo C/genética , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Subfamilia B de Receptores Similares a Lectina de Células NK/deficiencia , Subfamilia B de Receptores Similares a Lectina de Células NK/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HomólogoRESUMEN
Murine Ly49 receptors, which are expressed mainly on NK and NKT cells, interact with MHC class I (MHC-I) molecules with varying specificity. Differing reports of Ly49/MHC binding affinities may be affected by multiple factors, including cis versus trans competition and species origin of the MHC-I L chain (ß2-microglobulin). To determine the contribution of each of these factors, Ly49G, Ly49I, Ly49O, Ly49V, and Ly49Q receptors from the 129 mouse strain were expressed individually on human 293T cells or the mouse cell lines MHC-I-deficient C1498, H-2(b)-expressing MC57G, and H-2(k)-expressing L929. The capacity to bind to H-2D(b)- and H-2K(b)-soluble MHC-I tetramers containing either human or murine ß2-microglobulin L chains was tested for all five Ly49 receptors in all four cell lines. We found that most of these five inhibitory Ly49 receptors show binding for one or both self-MHC-I molecules in soluble tetramer binding assays when three conditions are fulfilled: 1) lack of competing cis interactions, 2) tetramer L chain is of mouse origin, and 3) Ly49 is expressed in mouse and not human cell lines. Furthermore, Ly49Q, the single known MHC-I receptor on plasmacytoid dendritic cells, was shown to bind H-2D(b) in addition to H-2K(b) when the above conditions were met, suggesting that Ly49Q functions as a pan-MHC-Ia receptor on plasmacytoid dendritic cells. In this study, we have optimized the parameters for soluble tetramer binding analyses to enhance future Ly49 ligand identification and to better evaluate specific contributions by different Ly49/MHC-I pairs to NK cell education and function.
Asunto(s)
Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/inmunología , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Células T Asesinas Naturales/inmunología , Animales , Diferenciación Celular , Separación Celular , Pruebas Inmunológicas de Citotoxicidad , Citometría de Flujo/métodos , Células HEK293 , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Ligandos , Ratones , Ratones Noqueados , Subfamilia A de Receptores Similares a Lectina de Células NK/genética , Subfamilia A de Receptores Similares a Lectina de Células NK/inmunología , Unión Proteica , Ingeniería de Proteínas , Especificidad de la EspecieRESUMEN
Although most CD8+ T cells are equipped to kill infected or transformed cells, a subset may regulate immune responses and preserve self-tolerance. Here, we describe a CD8 lineage that is instructed to differentiate into CD8 T regulatory cells (Tregs) by a surprisingly restricted set of T cell receptors (TCRs) that recognize MHC-E (mouse Qa-1) and several dominant self-peptides. Recognition and elimination of pathogenic target cells that express these Qa-1-self-peptide complexes selectively inhibits pathogenic antibody responses without generalized immune suppression. Immunization with synthetic agonist peptides that mobilize CD8 Tregs in vivo efficiently inhibit antigraft antibody responses and markedly prolong heart and kidney organ graft survival. Definition of TCR-dependent differentiation and target recognition by this lineage of CD8 Tregs may open the way to new therapeutic approaches to inhibit pathogenic antibody responses.
Asunto(s)
Linfocitos T CD8-positivos , Linfocitos T Reguladores , Ratones , Animales , Receptores de Antígenos de Linfocitos T , Péptidos , Tolerancia Inmunológica , Antígenos de Histocompatibilidad Clase IRESUMEN
Alveolar macrophages (AM) hold lung homeostasis intact. In addition to the defense against inhaled pathogens and deleterious inflammation, AM also maintain pulmonary surfactant homeostasis, a vital lung function that prevents pulmonary alveolar proteinosis. Signals transmitted between AM and pneumocytes of the pulmonary niche coordinate these specialized functions. However, the mechanisms that guide the metabolic homeostasis of AM remain largely elusive. We show that the NK cell-associated receptor, NKR-P1B, is expressed by AM and is essential for metabolic programming. Nkrp1b-/- mice are vulnerable to pneumococcal infection due to an age-dependent collapse in the number of AM and the formation of lipid-laden AM. The AM of Nkrp1b-/- mice show increased uptake but defective metabolism of surfactant lipids. We identify a physical relay between AM and alveolar type-II pneumocytes that is dependent on pneumocyte Clr-g expression. These findings implicate the NKR-P1B:Clr-g signaling axis in AM-pneumocyte communication as being important for maintaining metabolism in AM.
Asunto(s)
Proteinosis Alveolar Pulmonar , Surfactantes Pulmonares , Ratones , Animales , Macrófagos Alveolares/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Proteinosis Alveolar Pulmonar/metabolismo , Surfactantes Pulmonares/metabolismo , Muerte CelularRESUMEN
BACKGROUND: Much of the reluctance about using cytology specimens rather than histology specimens to assess programmed death ligand 1 (PD-L1) expression for guiding the use of immune modulating drugs in the management of non-small cell lung cancer (NSCLC) is based on the belief that the alcohol-based fixatives favored by cytopathologists might reduce the antigenicity of PD-L1 and lead to artifactually low expression levels and false-negative reporting. Therefore, this study was performed to determine whether there is any difference in PD-L1 expression between endobronchial ultrasound (EBUS)-guided aspirates of NSCLC fixed in alcohol-based fixatives and those fixed in neutral buffered formalin (NBF), the standard laboratory fixative for histology specimens. METHODS: The expression of PD-L1 was compared in 50 paired EBUS aspirates of NSCLC taken from the same lymph node during the same procedure. One aspirate of each pair was fixed in an alcohol-based fixative, and the other was fixed in NBF. RESULTS: In none of the 50 pairs was there any significant difference, qualitative or quantitative, in the strength, pattern, or extent of PD-L1 expression. In the great majority, the expression was identical, regardless of fixation. CONCLUSIONS: There is no evidence from this study showing that the use of alcohol-based fixatives has any effect on the expression of PD-L1 or its interpretation. Notwithstanding the general challenges in accurately assessing such expression in cytology specimens, pathologists should feel able to interpret them with confidence, and clinicians should feel able to rely on the results.