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Pulmonary ischemia-reperfusion injury (IRI) is a major contributor to poor lung transplant outcomes. We recently demonstrated a central role of airway-centered NK cells in mediating IRI; however, there are no existing effective therapies for directly targeting NK cells in humans. We hypothesized that a depleting anti-CD94 monoclonal antibody (mAb) would provide therapeutic benefit in mouse and human models of IRI based on high levels of KLRD1 (CD94) transcripts in bronchoalveolar lavage samples from lung transplant patients. We found that CD94 is highly expressed on mouse and human NK cells, with increased expression during IRI. Anti-mouse and anti-human mAbs against CD94 showed effective NK cell depletion in mouse and human models and blunted lung damage and airway epithelial killing. In two different allogeneic orthotopic lung transplant mouse models, anti-CD94 treatment during induction reduced early lung injury and chronic inflammation relative to control therapies. Anti-CD94 did not increase donor antigen-presenting cells that could alter long-term graft acceptance. Lung transplant induction regimens incorporating anti-CD94 treatment may safely improve early clinical outcomes.
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BACKGROUND: Cryopreserved peripheral blood mononuclear cells (PBMCs) are frequently collected and provide disease- and treatment-relevant data in clinical studies. Here, we developed combined protein (40 antibodies) and transcript single-cell (sc)RNA sequencing (scRNA-seq) in PBMCs. RESULTS: Among 31 participants in the Women's Interagency HIV Study (WIHS), we sequenced 41,611 cells. Using Boolean gating followed by Seurat UMAPs (tool for visualizing high-dimensional data) and Louvain clustering, we identified 50 subsets among CD4+ T, CD8+ T, B, NK cells, and monocytes. This resolution was superior to flow cytometry, mass cytometry, or scRNA-seq without antibodies. Combined protein and transcript scRNA-seq allowed for the assessment of disease-related changes in transcriptomes and cell type proportions. As a proof-of-concept, we showed such differences between healthy and matched individuals living with HIV with and without cardiovascular disease. CONCLUSIONS: In conclusion, combined protein and transcript scRNA sequencing is a suitable and powerful method for clinical investigations using PBMCs.
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Infecciones por VIH , Leucocitos Mononucleares , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica/métodos , Infecciones por VIH/genética , Humanos , Leucocitos Mononucleares/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , TranscriptomaRESUMEN
Interleukin-1ß (IL-1ß) is abundant in the tumor microenvironment, where this cytokine can promote tumor growth, but also antitumor activities. We studied IL-1ß during early tumor progression using a model of orthotopically introduced 4T1 breast cancer cells. Whereas there is tumor progression and spontaneous metastasis in wild-type (WT) mice, in IL-1ß-deficient mice, tumors begin to grow but subsequently regress. This change is due to recruitment and differentiation of inflammatory monocytes in the tumor microenvironment. In WT mice, macrophages heavily infiltrate tumors, but in IL-1ß-deficient mice, low levels of the chemokine CCL2 hamper recruitment of monocytes and, together with low levels of colony-stimulating factor-1 (CSF-1), inhibit their differentiation into macrophages. The low levels of macrophages in IL-1ß-deficient mice result in a relatively high percentage of CD11b+ dendritic cells (DCs) in the tumors. In WT mice, IL-10 secretion from macrophages is dominant and induces immunosuppression and tumor progression; in contrast, in IL-1ß-deficient mice, IL-12 secretion by CD11b+ DCs prevails and supports antitumor immunity. The antitumor immunity in IL-1ß-deficient mice includes activated CD8+ lymphocytes expressing IFN-γ, TNF-α, and granzyme B; these cells infiltrate tumors and induce regression. WT mice with 4T1 tumors were treated with either anti-IL-1ß or anti-PD-1 Abs, each of which resulted in partial growth inhibition. However, treating mice first with anti-IL-1ß Abs followed by anti-PD-1 Abs completely abrogated tumor progression. These data define microenvironmental IL-1ß as a master cytokine in tumor progression. In addition to reducing tumor progression, blocking IL-1ß facilitates checkpoint inhibition.
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Anticuerpos Monoclonales/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Interleucina-1beta/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Antígeno CD11b/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Factores Estimulantes de Colonias/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Femenino , Granzimas/farmacología , Humanos , Terapia de Inmunosupresión/métodos , Inflamación/metabolismo , Interferón gamma/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Microambiente Tumoral/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Despite the decades-old knowledge that males and people with diabetes mellitus (DM) are at increased risk for coronary artery disease (CAD), the reasons for this association are only partially understood. Among the immune cells involved, recent evidence supports a critical role of T cells as drivers and modifiers of CAD. CD4+ T cells are commonly found in atherosclerotic plaques. We aimed to understand the relationship of CAD with sex and DM by single-cell RNA (scRNA-Seq) and antibody sequencing (CITE-Seq) of CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) of 61 men and women who underwent cardiac catheterization were interrogated by scRNA-Seq combined with 49 surface markers (CITE-Seq). CAD severity was quantified using Gensini scores, with scores above 30 considered CAD+ and below 6 considered CAD-. Four pairs of groups were matched for clinical and demographic parameters. To test how sex and DM changed cell proportions and gene expression, we compared matched groups of men and women, as well as diabetic and non-diabetic subjects. We analyzed 41,782 single CD4+ T cell transcriptomes for sex differences in 16 women and 45 men with and without coronary artery disease and with and without DM. We identified 16 clusters in CD4+ T cells. The proportion of cells in CD4+ effector memory cluster 8 (CD4T8, CCR2+ Em) was significantly decreased in CAD+, especially among DM+ participants. This same cluster, CD4T8, was significantly decreased in female participants, along with two other CD4+ T cell clusters. In CD4+ T cells, 31 genes showed significant and coordinated upregulation in both CAD and DM. The DM gene signature was partially additive to the CAD gene signature. We conclude that (1) CAD and DM are clearly reflected in PBMC transcriptomes, and (2) significant differences exist between women and men and (3) between subjects with DM and non-DM.
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Enfermedad de la Arteria Coronaria , Diabetes Mellitus , Linfocitos T CD4-Positivos , Angiografía Coronaria , Enfermedad de la Arteria Coronaria/genética , Diabetes Mellitus/genética , Femenino , Humanos , Leucocitos Mononucleares , Masculino , Caracteres Sexuales , Análisis de la Célula IndividualRESUMEN
The natural cytotoxicity receptors (NCRs; NKp30, NKp44, and NKp46) were first defined as activating receptors on human NK cells that are important in recognition of and response to tumors. A flurry of recent research, however, has revealed that differential splicing can occur during transcription of each of the NCR genes, resulting in some transcripts that encode receptor isoforms with inhibitory functions. These alternative transcripts can arise in certain tissue microenvironments and appear to be induced by cytokines. Evidence indicates that some of the inhibitory NCRs are triggered by specific ligands, such as the interaction of the inhibitory isoform of NKp44 with PCNA on the surface of tumor cells. Here, we review the different NCR splice variants, cytokines that modulate their expression, their functional impacts on innate immune cells, and their differential expression in the contexts of cancer, pregnancy, and infections. The recent discovery of these inhibitory NCR isoforms has revealed novel innate immune checkpoints, many of which still lack defined ligands and clear mechanisms driving their expression. These NCR checkpoint pathways offer exciting potential therapeutic targets to manipulate innate immune functions under defined pathological conditions, such as cancer, pregnancy disorders, and pathogen exposure.
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Empalme Alternativo , Citotoxicidad Inmunológica/genética , Inmunidad Innata/genética , Receptores Inmunológicos/genética , Animales , Microambiente Celular/genética , Microambiente Celular/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunidad Innata/efectos de los fármacos , Receptores Inmunológicos/metabolismoRESUMEN
Central nervous system acute lymphoblastic leukemia (CNS-ALL) is a major clinical problem. Prophylactic therapy is neurotoxic, and a third of the relapses involve the CNS. Increased expression of interleukin 15 (IL-15) in leukemic blasts is associated with increased risk for CNS-ALL. Using in vivo models for CNS leukemia caused by mouse T-ALL and human xenografts of ALL cells, we demonstrate that expression of IL-15 in leukemic cells is associated with the activation of natural killer (NK) cells. This activation limits the outgrowth of leukemic cells in the periphery, but less in the CNS because NK cells are excluded from the CNS. Depletion of NK cells in NOD/SCID mice enabled combined systemic and CNS leukemia of human pre-B-ALL. The killing of human leukemia lymphoblasts by NK cells depended on the expression of the NKG2D receptor. Analysis of bone marrow (BM) diagnostic samples derived from children with subsequent CNS-ALL revealed a significantly high expression of the NKG2D and NKp44 receptors. We suggest that the CNS may be an immunologic sanctuary protected from NK-cell activity. CNS prophylactic therapy may thus be needed with emerging NK cell-based therapies against hematopoietic malignancies.
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Neoplasias del Sistema Nervioso Central/inmunología , Células Asesinas Naturales/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Animales , Animales Recién Nacidos , Células Cultivadas , Neoplasias del Sistema Nervioso Central/metabolismo , Neoplasias del Sistema Nervioso Central/mortalidad , Neoplasias del Sistema Nervioso Central/patología , Humanos , Interleucina-15/metabolismo , Células Jurkat , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologíaRESUMEN
NKp44 (NCR2) is a distinct member of natural cytotoxicity receptors (NCRs) family that can induce cytokine production and cytolytic activity in human NK cells. Heparan sulfate proteoglycans (HSPGs) are differentially expressed in various normal and cancerous tissues. HSPGs were reported to serve as ligands/co-ligands for NKp44 and other NCRs. However, HSPG expression is not restricted to either group and can be found also in NK cells. Our current study reveals that NKp44 function can be modulated through interactions with HSPGs on NK cells themselves in -cis rather than on target cells in -trans. The intimate interaction of NKp44 and the NK cell-associated HSPG syndecan-4 (SDC4) in -cis can directly regulate membrane distribution of NKp44 and constitutively dampens the triggering of the receptor. We further demonstrate, that the disruption of NKp44 and SDC4 interaction releases the receptor to engage with its ligands in -trans and therefore enhances NKp44 activation potential and NK cell functional response.
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Proteoglicanos de Heparán Sulfato/metabolismo , Células Asesinas Naturales/inmunología , Receptor 2 Gatillante de la Citotoxidad Natural/metabolismo , Sindecano-4/metabolismo , Sitios de Unión/genética , Sitios de Unión/inmunología , Línea Celular Tumoral , Citocinas/biosíntesis , Humanos , Neoplasias/inmunología , Unión Proteica/inmunología , Receptores Inmunológicos/inmunologíaRESUMEN
NK cells express activating receptors that signal through ITAM-bearing adapter proteins. The phosphorylation of each ITAM creates binding sites for SYK and ZAP70 protein tyrosine kinases to propagate downstream signaling including the induction of Ca 2 + influx. While all immature and mature human NK cells co-express SYK and ZAP70, clonally driven memory or adaptive NK cells can methylate SYK genes and signaling is mediated exclusively using ZAP70. Here, we examined the role of SYK and ZAP70 in a clonal human NK cell line KHYG1 by CRISPR-based deletion using a combination of experiments and mechanistic computational modeling. Elimination of SYK resulted in more robust Ca + + influx after cross-linking of the CD16 and NKp30 receptors and enhanced phosphorylation of downstream proteins, whereas ZAP70 deletion diminished these responses. By contrast, ZAP70 depletion increased proliferation of the NK cells. As immature T cells express both SYK and ZAP70 but mature T cells often express only ZAP70, we transduced the human Jurkat cell line with SYK and found that expression of SYK increased proliferation but diminished TCR-induced Ca 2 + flux and activation. We performed transcriptional analysis of the matched sets of variant Jurkat and KHYG1 cells and observed profound alterations caused by SYK expression. As depletion of SYK in NK cells increased their activation, primary human NK cells were transduced with a CD19-targeting CAR and were CRISPR edited to ablate SYK or ZAP70. Deletion of SYK resulted in more robust cytotoxic activity and cytokine production, providing a new therapeutic strategy of NK cell engineering for cancer immunotherapy.
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Primary graft dysfunction (PGD) limits clinical benefit after lung transplantation, a life-prolonging therapy for patients with end-stage disease. PGD is the clinical syndrome resulting from pulmonary ischemia-reperfusion injury (IRI), driven by innate immune inflammation. We recently demonstrated a key role for NK cells in the airways of mouse models and human tissue samples of IRI. Here, we used 2 mouse models paired with human lung transplant samples to investigate the mechanisms whereby NK cells migrate to the airways to mediate lung injury. We demonstrate that chemokine receptor ligand transcripts and proteins are increased in mouse and human disease. CCR5 ligand transcripts were correlated with NK cell gene signatures independently of NK cell CCR5 ligand secretion. NK cells expressing CCR5 were increased in the lung and airways during IRI and had increased markers of tissue residency and maturation. Allosteric CCR5 drug blockade reduced the migration of NK cells to the site of injury. CCR5 blockade also blunted quantitative measures of experimental IRI. Additionally, in human lung transplant bronchoalveolar lavage samples, we found that CCR5 ligand was associated with increased patient morbidity and that the CCR5 receptor was increased in expression on human NK cells following PGD. These data support a potential mechanism for NK cell migration during lung injury and identify a plausible preventative treatment for PGD.
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Lesión Pulmonar , Daño por Reperfusión , Animales , Humanos , Ratones , Células Asesinas Naturales , Ligandos , Pulmón/metabolismo , Lesión Pulmonar/metabolismo , Receptores CCR5/genética , Daño por Reperfusión/metabolismoRESUMEN
IL-12 is an essential cytokine involved in the generation of memory or memory-like NK cells. Mouse cytomegalovirus infection triggers NK receptor-induced, ligand-specific IL-12-dependent NK cell expansion, yet specific IL-12 stimulation ex vivo leading to NK cell proliferation and expansion is not established. Here, we show that IL-12 alone can sustain human primary NK cell survival without providing IL-2 or IL-15 but was insufficient to promote human NK cell proliferation. IL-12 signaling analysis revealed STAT5 phosphorylation and weak mTOR activation, which was enhanced by activating NK receptor upregulation and crosslinking leading to STAT5-dependent, rapamycin-sensitive, or TGFß-sensitive NK cell IL-12-dependent expansion, independently of IL-12 receptor upregulation. Prolonged IL-2 culture did not impair IL-12-dependent ligand-specific NK cell expansion. These findings demonstrate that activating NK receptor stimulation promotes differential IL-12 signaling, leading to human NK cell expansion, and suggest adopting strategies to provide IL-12 signaling in vivo for ligand-specific IL-2-primed NK cell-based therapies.
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Interleucina-12 , Factor de Transcripción STAT5 , Proliferación Celular , Humanos , Interleucina-2/farmacología , Ligandos , Receptores de Células Asesinas NaturalesRESUMEN
Human adaptive-like natural killer (NK) cells express low levels of FcεRIγ (FcRγ-/low) and are reported to accumulate during COVID-19 infection; however, the mechanism underlying and regulating FcRγ expression in NK cells has yet to be fully defined. We observed lower FcRγ protein expression in NK cell subsets from lung transplant patients during rapamycin treatment, suggesting a link with reduced mTOR activity. Further, FcRγ-/low NK cell subsets from healthy donors displayed reduced mTOR activity. We discovered that FcRγ upregulation is dependent on cell proliferation progression mediated by IL-2, IL-15, or IL-12, is sensitive to mTOR suppression, and is inhibited by TGFß or IFNα. Accordingly, the accumulation of adaptive-like FcRγ-/low NK cells in COVID-19 patients corresponded to increased TGFß and IFNα levels and disease severity. Our results show that an adaptive-like NK cell phenotype is induced by diminished cell proliferation and has an early prognostic value for increased TGFß and IFNα levels in COVID-19 infection associated with disease severity.
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COVID-19 , Proliferación Celular , Humanos , Células Asesinas Naturales , Fenotipo , Serina-Treonina Quinasas TOR , Factor de Crecimiento Transformador betaRESUMEN
Clinical outcomes after lung transplantation, a life-saving therapy for patients with end-stage lung diseases, are limited by primary graft dysfunction (PGD). PGD is an early form of acute lung injury with no specific pharmacologic therapies. Here, we present a large multicenter study of plasma and bronchoalveolar lavage (BAL) samples collected on the first posttransplant day, a critical time for investigations of immune pathways related to PGD. We demonstrated that ligands for NKG2D receptors were increased in the BAL from participants who developed severe PGD and were associated with increased time to extubation, prolonged intensive care unit length of stay, and poor peak lung function. Neutrophil extracellular traps (NETs) were increased in PGD and correlated with BAL TNF-α and IFN-γ cytokines. Mechanistically, we found that airway epithelial cell NKG2D ligands were increased following hypoxic challenge. NK cell killing of hypoxic airway epithelial cells was abrogated with NKG2D receptor blockade, and TNF-α and IFN-γ provoked neutrophils to release NETs in culture. These data support an aberrant NK cell/neutrophil axis in human PGD pathogenesis. Early measurement of stress ligands and blockade of the NKG2D receptor hold promise for risk stratification and management of PGD.
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Trasplante de Pulmón , Disfunción Primaria del Injerto , Humanos , Subfamilia K de Receptores Similares a Lectina de Células NK , Disfunción Primaria del Injerto/etiología , Factor de Necrosis Tumoral alfa , Trasplante de Pulmón/efectos adversos , Pulmón/metabolismoRESUMEN
mAb-based blocking of the immune checkpoints involving the CTLA4-B7 and PD1-PDL1 inhibitory axes enhance T-cell-based adaptive immune responses in patients with cancer. We show here that antitumor responses by natural killer (NK) cells can be enhanced by a checkpoint-blocking mAb, 14-25-9, which we developed against proliferating cell nuclear antigen (PCNA). PCNA is expressed on the surface of cancer cells and acts as an inhibitory ligand for the NK-cell receptor, NKp44-isoform1. We tested for cytoplasmic- and membrane-associated PCNA by FACS- and ImageStream-based staining of cell lines and IHC of human cancer formalin fixed, paraffin embedded tissues. The mAb, 14-25-9, inhibited binding of chimeric NKp44 receptor to PCNA and mostly stained the cytoplasm and membrane of tumor cells, whereas commercial antibody (clone PC10) stained nuclear PCNA. NK functions were measured using ELISA-based IFNγ secretion assays and FACS-based killing assays. The NK92-NKp44-1 cell line and primary human NK cells showed increased IFNγ release upon coincubation with mAb 14-25-9 and various solid tumor cell lines and leukemias. Treatment with 14-25-9 also increased NK cytotoxic activity. In vivo efficacy was evaluated on patient-derived xenografts (PDX)-bearing NSG mice. In PDX-bearing mice, intravenous administration of mAb 14-25-9 increased degranulation (CD107a expression) of intratumorally injected patient autologous or allogeneic NK cells, as well as inhibited tumor growth when treated long term. Our study describes a mAb against the NKp44-PCNA innate immune checkpoint that can enhance NK-cell antitumor activity both in vitro and in vivo.
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Anticuerpos Monoclonales/farmacología , Citotoxicidad Inmunológica/inmunología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Células Asesinas Naturales/efectos de los fármacos , Receptor 2 Gatillante de la Citotoxidad Natural/antagonistas & inhibidores , Antígeno Nuclear de Célula en Proliferación/química , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Animales , Apoptosis , Proliferación Celular , Citotoxicidad Inmunológica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Receptor 2 Gatillante de la Citotoxidad Natural/inmunología , Antígeno Nuclear de Célula en Proliferación/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Ebola virus (EBOV) uses evasion mechanisms that directly interfere with host T-cell antiviral responses. By steric shielding of human leukocyte antigen class-1, the Ebola glycoprotein (GP) blocks interaction with T-cell receptors (TCRs), thus rendering T cells unable to attack virus-infected cells. It is likely that this mechanism could promote increased natural killer (NK) cell activity against GP-expressing cells by preventing the engagement of NK inhibitory receptors; however, we found that primary human NK cells were less reactive to GP-expressing HEK293T cells. This was manifested as reduced cytokine secretion, a reduction in NK degranulation, and decreased lysis of GP-expressing target cells. We also demonstrated reduced recognition of GP-expressing cells by recombinant NKG2D and NKp30 receptors. In accordance, we showed a reduced monoclonal antibody-based staining of NKG2D and NKp30 ligands on GP-expressing target cells. Trypsin digestion of the membrane-associated GP led to a recovery of the recognition of membrane-associated NKG2D and NKp30 ligands. We further showed that membrane-associated GP did not shield recognition by KIR2DL receptors; in accordance, GP expression by target cells significantly perturbed signal transduction through activating, but not through inhibitory, receptors. Our results suggest a novel evasion mechanism employed by the EBOV to specifically avoid the NK cell immune response.
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Proliferating cell nuclear antigen (PCNA) is considered as a hub protein and is a key regulator of DNA replication, repair, cell cycle control, and apoptosis. PCNA is overexpressed in many cancer types, and PCNA overexpression is correlated with cancer virulence. Membrane-associated PCNA is a ligand for the NKp44 (NCR2) innate immune receptor. The purpose of this study was to characterize the PCNA-binding site within NKp44. We have identified NKp44-derived linear peptide (pep8), which can specifically interact with PCNA and partly block the NKp44-PCNA interaction. We then tested whether NKp44-derived pep8 (NKp44-pep8) fused to cell-penetrating peptides (CPPs) can be employed for targeting the intracellular PCNA for the purpose of anticancer therapy. Treatment of tumor cells with NKp44-pep8, fused to R11-NLS cell-penetrating peptide (R11-NLS-pep8), reduced cell viability and promoted cell death, in various murine and human cancer cell lines. Administration of R11-NLS-pep8 to tumor-bearing mice suppressed tumor growth in the 4T1 breast cancer and the B16 melanoma in vivo models. We therefore identified the NKp44 binding site to PCNA and further developed an NKp44-peptide-based agent that can inhibit tumor growth through interfering with the function of intracellular PCNA in the tumor cell.
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Péptidos de Penetración Celular/metabolismo , Receptor 2 Gatillante de la Citotoxidad Natural/metabolismo , Neoplasias/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Dominios y Motivos de Interacción de Proteínas , Animales , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Péptidos de Penetración Celular/química , Femenino , Humanos , Inmunofenotipificación , Masculino , Ratones , Receptor 2 Gatillante de la Citotoxidad Natural/química , Antígeno Nuclear de Célula en Proliferación/química , Unión Proteica , Proteínas Recombinantes de Fusión , Resonancia por Plasmón de SuperficieRESUMEN
The natural cytotoxicity receptor (NCR) family is constituted by NKp46, NKp44, and NKp30 in humans, which are expressed mainly on natural killer (NK) cells and are encoded by the ncr1, ncr2, and ncr3 genes, respectively. NCRs have classically been defined as activating receptors that trigger cytotoxicity and cytokine responses by NK cells upon engaging with ligands on tumor cells. Several new findings, however, have challenged this model and identified alternative mechanisms regulating the function of NCRs. Recent reports indicate that ligand matters, since the interaction of NKp44 with distinct ligands on target cells can either activate or inhibit NK cells. Also, the NCRs have been found to interact with distinct specificities to various heparan sulfate glycosaminoglycans, which are complex polysaccharides found in extracellular matrix or on cell surface heparan sulfate proteoglycans (HSPGs). The NCRs can engage with HSPGs in trans as a co-ligand on the target cells or in cis on the NK cell surface to regulate receptor-ligand interactions and NK cell activation. A number of splice variants of ncr2 and ncr3 have also been identified, and a predominant expression of certain variants results in inhibitory signaling through NKp44 and NKp30. Several recent studies have found that the selective expression of some of these inhibitory splice variants can significantly influence outcome in the contexts of cancer, infection, and pregnancy. These findings establish that NCR functions are more diverse than originally thought, and better understanding of their splice variant expression profiles and ligand interactions are needed to establish their functional regulation in the context of human health.
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The natural killer (NK) cell activating receptor NKp46/NCR1 plays a critical role in elimination of virus-infected and tumor cells. The NCR1 gene can be transcribed into five different splice variants, but the functional importance and physiological distribution of NKp46 isoforms are not yet fully understood. Here, we shed light on differential expression of NKp46 splice variants in viral respiratory tract infections and their functional difference at the cellular level. NKp46 was the most predominantly expressed natural cytotoxicity receptor in the nasal lavage of patients infected with four respiratory viruses: respiratory syncytia virus, adenovirus, human metapneumovirus, or influenza A. Expression of NKp30 was far lower and NKp44 was absent in all patients. Domain 1-negative NKp46 splice variants (i.e., NKp46 isoform d) were the predominantly expressed isoform in nasal lavage following viral infections. Using our unique anti-NKp46 mAb, D2-9A5, which recognizes the D2 extracellular domain, and a commercial anti-NKp46 mAb, 9E2, which recognizes D1 domain, allowed us to identify a small subset of NKp46 D1-negative splice variant-expressing cells within cultured human primary NK cells. This NKp46 D1-negative subset also showed higher degranulation efficiency in term of CD107a surface expression. NK-92 cell lines expressing NKp46 D1-negative and NKp46 D1-positive splice variants also showed functional differences when interacting with targets. A NKp46 D1-negative isoform-expressing NK-92 cell line showed enhanced degranulation activity. To our knowledge, we provide the first evidence showing the physiological distribution and functional importance of human NKp46 splice variants under pathological conditions.
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Adipose tissue inflammation and dysfunction are considered central in the pathogenesis of obesity-related dysmetabolism, but their role in the rapid metabolic recovery upon obesity reversal is less well defined. We hypothesized that changes in adipose tissue endocrine and paracrine mechanisms may support the rapid improvement of obesity-induced impairment in cellular lipid handling. C57Bl-6J mice were fed ad libitum either normal chow (NC) or high-fat diet (HFF) for 10 weeks. A dietary obesity reversal group was fed HFF for 8 weeks and then switched to NC for 2 weeks (HFFâNC). Whole-body glucose homeostasis rapidly nearly normalized in the HFFâNC mice (fasting glucose and insulin fully normalized, glucose and insulin tolerance tests reversed 82% to the NC group levels). During 2 weeks of the dietary reversal, the liver was significantly cleared from ectopic fat, and functionally, glucose production from pyruvate, alanine or fructose was normalized. In contrast, adipose tissue inflammation (macrophage infiltration and polarization) largely remained as in HFF, though obesity-induced adipose tissue macrophage lipid accumulation decreased by ~50%, and adipose tissue MAP kinase hyperactivation was reversed. Ex vivo, mild changes in adipose tissue adipocytokine secretion profile were noted. These corresponded to partial or full reversal of the excess cellular lipid droplet accumulation induced by HFF adipose tissue conditioned media in hepatoma or macrophage cells, respectively. We propose that early after initiating reversal of nutritional obesity, rapid metabolic normalization largely precedes resolution of adipose tissue inflammation. Nevertheless, we demonstrate a hitherto unrecognized contribution of adipose tissue to the rapid improvement in lipid handling by the liver and by macrophages.
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Tejido Adiposo/fisiología , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Macrófagos/fisiología , Obesidad/metabolismo , Animales , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Endogámicos C57BL , Pérdida de PesoRESUMEN
NKp44 and NKp30 splice variant profiles have been shown to promote diverse cellular functions. Moreover, microenvironment factors such as TGF-ß, IL-15 and IL-18 are able to influence both NKp44 and NKp30 splice variant profiles, leading to cytokine-associated profiles. Placenta and cancerous tissues have many similarities; both are immunologically privileged sites and both share immune tolerance mechanisms to support tissue development. Therefore, we studied the profiles of NKp44 and NKp30 splice variants in these states by comparing (i) decidua from pregnancy disorder and healthy gestation and (ii) matched normal and cancer tissue. Decidua samples had high incidence of both NKp44 and NKp30. In cancerous state it was different; while NKp30 expression was evident in most cancerous and matched normal tissues, NKp44 incidence was lower and was mostly associated with the cancerous tissues. A NKp44-1dominant inhibitory profile predominated in healthy pregnancy gestation. Interestingly, the NKp44-2/3 activation profile becomes the leading profile in spontaneous abortions, whereas balanced NKp44 profiles were observed in preeclampsia. In contrast, a clear preference for the NKp30a/b profile was evident in the 1st trimester decidua, yet no significant differences were observed for NKp30 profiles between healthy gestation and spontaneous abortions/preeclampsia. Both cancerous and matched normal tissues manifested balanced NKp30c inhibitory and NKp30a/b activation profiles with a NKp44-1dominant profile. However, a shift in NKp30 profiles between matched normal and cancer tissue was observed in half of the cases. To summarize, NKp44 and NKp30 splice variants profiles are tissue/condition specific and demonstrate similarity between placenta and cancerous tissues.