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Acute myeloid leukemia (AML) is initiated and sustained by a hierarchy of leukemia stem cells (LSCs), and elimination of this cell population is required for curative therapies. Here we show that transmembrane and immunoglobulin domain containing 2 (TMIGD2), a recently discovered co-stimulatory immune receptor, is aberrantly expressed by human AML cells, and can be used to identify and enrich functional LSCs. We demonstrate that TMIGD2 is required for the development and maintenance of AML and self-renewal of LSCs but is not essential for normal hematopoiesis. Mechanistically, TMIGD2 promotes proliferation, blocks myeloid differentiation and increases cell-cycle of AML cells via an ERK1/2-p90RSK-CREB signaling axis. Targeting TMIGD2 signaling with anti-TMIGD2 monoclonal antibodies attenuates LSC self-renewal and reduces leukemia burden in AML patient-derived xenograft models but has negligible effect on normal hematopoietic stem/progenitor cells. Thus, our studies reveal the function of TMIGD2 in LSCs and provide a promising therapeutic strategy for AML.
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Leucemia Mieloide Aguda , Células Madre Neoplásicas , Humanos , Células Madre Hematopoyéticas , Transducción de Señal , Hematopoyesis , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/tratamiento farmacológicoRESUMEN
How dedifferentiated stem-like tumor cells evade immunosurveillance remains poorly understood. We show that the lineage-plasticity regulator SOX9, which is upregulated in dedifferentiated tumor cells, limits the number of infiltrating T lymphocytes in premalignant lesions of mouse basal-like breast cancer. SOX9-mediated immunosuppression is required for the progression of in situ tumors to invasive carcinoma. SOX9 induces the expression of immune checkpoint B7x/B7-H4 through STAT3 activation and direct transcriptional regulation. B7x is upregulated in dedifferentiated tumor cells and protects them from immunosurveillance. B7x also protects mammary gland regeneration in immunocompetent mice. In advanced tumors, B7x targeting inhibits tumor growth and overcomes resistance to anti-PD-L1 immunotherapy. In human breast cancer, SOX9 and B7x expression are correlated and associated with reduced CD8+ T cell infiltration. This study, using mouse models, cell lines, and patient samples, identifies a dedifferentiation-associated immunosuppression mechanism and demonstrates the therapeutic potential of targeting the SOX9-B7x pathway in basal-like breast cancer.
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Neoplasias de la Mama , Animales , Femenino , Humanos , Ratones , Linfocitos T CD8-positivos , Terapia de Inmunosupresión , Factor de Transcripción SOX9 , Inhibidor 1 de la Activación de Células T con Dominio V-Set/metabolismoRESUMEN
Human killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail (KIR3DL3) is expressed on natural killer (NK) cells and is a newly identified inhibitory receptor for B7 family member HERV-H LTR-associating protein 2 (HHLA2). Here, we summarize the isolation and expansion of KIR3DL3+ human NK cells, and in vitro functional characterization of in-house anti-KIR3DL3 monoclonal antibody (mAb). We also describe a human NK cell-based xenogeneic lung tumor model for testing the therapeutic activity of KIR3DL3 blockade in vivo. For complete details on the use and execution of this protocol, please refer to Wei et al. (2021).
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Células Asesinas Naturales , Neoplasias Pulmonares , Humanos , Receptores KIR/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , InmunoglobulinasRESUMEN
Cancer immunotherapy targeting the TIGIT/PVR pathway is currently facing challenges. KIR2DL5, a member of the human killer cell, immunoglobulin-like receptor (KIR) family, has recently been identified as another binding partner for PVR. The biology and therapeutic potential of the KIR2DL5/PVR pathway are largely unknown. Here we report that KIR2DL5 was predominantly expressed on human NK cells with mature phenotype and cytolytic function and that it bound to PVR without competition with the other 3 known PVR receptors. The interaction between KIR2DL5 on NK cells and PVR on target cells induced inhibitory synapse formation, whereas new monoclonal antibodies blocking the KIR2DL5-PVR interaction robustly augmented the NK cytotoxicity against PVR+ human tumors. Mechanistically, both intracellular ITIM and ITSM of KIR2DL5 underwent tyrosine phosphorylation after engagement, which was essential for KIR2DL5-mediated NK suppression by recruiting SHP-1 and/or SHP-2. Subsequently, ITIM/SHP-1/SHP-2 and ITSM/SHP-1 downregulated the downstream Vav1/ERK1/2/p90RSK/NF-κB signaling. KIR2DL5+ immune cells infiltrated in various types of PVR+ human cancers. Markedly, the KIR2DL5 blockade reduced tumor growth and improved overall survival across multiple NK cell-based humanized tumor models. Thus, our results revealed functional mechanisms of KIR2DL5-mediated NK cell immune evasion, demonstrated blockade of the KIR2DL5/PVR axis as a therapy for human cancers, and provided an underlying mechanism for the clinical failure of anti-TIGIT therapies.
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Células Asesinas Naturales , Neoplasias , Humanos , Transducción de Señal , Fosforilación , Neoplasias/terapia , Neoplasias/metabolismoRESUMEN
Somatic activating mutations in the epidermal growth factor receptor (EGFR) are one of the most common oncogenic drivers in cancers such as non-small-cell lung cancer (NSCLC), metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer. Molecular-targeted agents against EGFR signaling pathways have shown robust clinical efficacy, but patients inevitably experience acquired resistance. Although immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 have exhibited durable anti-tumor responses in a subset of patients across multiple cancer types, their efficacy is limited in cancers harboring activating gene alterations of EGFR. Increasing studies have demonstrated that upregulation of new B7/CD28 family members such as B7-H3, B7x and HHLA2, is associated with EGFR signaling and may contribute to resistance to EGFR-targeted therapies by creating an immunosuppressive tumor microenvironment (TME). In this review, we discuss the regulatory effect of EGFR signaling on the PD-1/PD-L1 pathway and new B7/CD28 family member pathways. Understanding these interactions may inform combination therapeutic strategies and potentially overcome the current challenge of resistance to EGFR-targeted therapies. We also summarize clinical data of anti-PD-1/PD-L1 therapies in EGFR-mutated cancers, as well as ongoing clinical trials of combination of EGFR-targeted therapies and anti-PD-1/PD-L1 immunotherapies.
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BACKGROUND: Adoptive immunotherapy using CD19-targeted Chimeric antigen receptor T cells (CAR-T) has revolutionized the treatment of relapsed/refractory diffuse large B-cell lymphoma (DLBCL). Data is limited on the propensity of infections and lymphohematopoietic reconstitution after Day 30 (D30) following CAR-T cell therapy. In this study, we evaluated the prevalence and nature of infectious complications in an expanded cohort of DLBCL patients treated with CD19 CAR-T therapy and its association with the dynamics of leukocyte subpopulation reconstitution post-CAR-T cell therapy. METHODS: We conducted a retrospective study including 19 patients who received axicabtagene ciloleucel and investigated associations between cytopenia and infectious complications after D30. RESULTS: Nineteen patients were included, consisting of 42% Hispanic, 32% Caucasian, 21% African-American, and 5% Asian subjects. Post-D30 of CAR-T infusion, 47% patients (n=9) developed an infection and 53% (n=10) remained infection-free. The most common infection type observed was viral (7 patients) followed by bacterial (5 patients) and fungal (3 patients). Of 25 total infectious events, 56% were grade 1 or 2 and 44% were grade 3 with 10 being viral in etiology. To determine the kinetics of lymphohematopoietic reconstitution and its association with infection risk, we evaluated the relationship between cytopenias and rates of infection after D30. Notably, compared to non-infection group, infection group had a higher median absolute lymphocyte count (ALC) (1,000/µL vs. 600/µL, P<0.05), a lower median absolute neutrophil count (ANC)/ALC ratio (1.6 vs. 3.1, P<0.05) and a lower median AMC/ALC at D30 (0.37 vs. 1.67, P<0.05). In addition, we observed that only 22% of patients had recovered ANC >1,500/µL in the infection group as opposed to 70% in the non-infection group at D90 (P<0.05). Fifty-eight percent of the patients (11/19) with relapsed refractory DLBCL achieved a complete response with a median follow-up of 233 days (7.7 months). CONCLUSIONS: Although CAR-T cell therapy is highly effective, infectious complications remain an important cause of morbidity and mortality. Low ANC/ALC and AMC/ALC ratios at D30 are potential novel predictors of infection and can be considered in future prophylactic strategies.
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The B7 family ligand HERV-H LTR-associating protein 2 (HHLA2) is an attractive target for cancer immunotherapy because of its coinhibitory function, overexpression in human cancers, and association with poor prognoses. However, the knowledge of the HHLA2 pathway is incomplete. HHLA2 has an established positive receptor transmembrane and immunoglobulin (Ig) domain containing 2 (TMIGD2) but a poorly characterized negative receptor human killer cell Ig-like receptor, three Ig domains, and long cytoplasmic tail (KIR3DL3). Here, KIR3DL3 and TMIGD2 simultaneously bound to different sites of HHLA2. KIR3DL3 was mainly expressed on CD56dim NK and terminally differentiated effector memory CD8+ T (CD8+ TEMRA) cells. KIR3DL3+ CD8+ TEMRA acquired an NK-like phenotype and function. HHLA2 engagement recruited KIR3DL3 to the immunological synapse and coinhibited CD8+ T and NK cell function and killing, inducing immune-evasive HHLA2+ tumors. KIR3DL3 recruited SHP-1 and SHP-2 to attenuate Vav1, ERK1/2, AKT, and NF-κB signaling. HHLA2+ tumors from human kidney, lung, gallbladder, and stomach were infiltrated by KIR3DL3+ immune cells. KIR3DL3 blockade inhibited tumor growth in multiple humanized mouse models. Thus, our findings elucidated the molecular and cellular basis for the inhibitory function of KIR3DL3, demonstrating that the KIR3DL3-HHLA2 pathway is a potential immunotherapeutic target for cancer.
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Inmunoglobulinas/inmunología , Neoplasias/inmunología , Receptores KIR/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Linfocitos T CD8-positivos/inmunología , Supervivencia Celular , Células Cultivadas , Humanos , Tolerancia Inmunológica , Inmunoglobulinas/genética , Células Asesinas Naturales/inmunología , Ratones Endogámicos BALB C , Neoplasias/tratamiento farmacológico , Receptores KIR/antagonistas & inhibidoresRESUMEN
Immune checkpoint inhibitors are a powerful new tool in the treatment of cancer, with prolonged responses in multiple diseases, including hematologic malignancies, such as Hodgkin lymphoma. However, in a recent report, we demonstrated that the PD-1 inhibitor nivolumab led to rapid progression in patients with adult T-cell leukemia/lymphoma (ATLL) (NCT02631746). We obtained primary cells from these patients to determine the cause of this hyperprogression. Analyses of clonality, somatic mutations, and gene expression in the malignant cells confirmed the report of rapid clonal expansion after PD-1 blockade in these patients, revealed a previously unappreciated origin of these malignant cells, identified a novel connection between ATLL cells and tumor-resident regulatory T cells (Tregs), and exposed a tumor-suppressive role for PD-1 in ATLL. Identifying the mechanisms driving this alarming outcome in nivolumab-treated ATLL may be broadly informative for the growing problem of rapid progression with immune checkpoint therapies.
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Antineoplásicos Inmunológicos/uso terapéutico , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Nivolumab/uso terapéutico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T Reguladores/patología , Adulto , Animales , Progresión de la Enfermedad , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/patología , Ratones , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Células Tumorales CultivadasRESUMEN
Highly suppressive tumor microenvironments often result in T cell dysfunction and inability to control tumors, but factors regulating this process remain elusive. A new study by Ma et al. (2019) reports that tumor cholesterol devitalizes T cells by modulating endoplasmic reticulum stress pathways, revealing a new mechanism underlying T cell exhaustion.
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Neoplasias , Microambiente Tumoral , Linfocitos T CD8-positivos , Colesterol , Estrés del Retículo Endoplásmico , HumanosRESUMEN
PURPOSE: To evaluate therapeutic activity of PAK inhibition in ATLL and to characterize the role of PAK isoforms in cell proliferation, survival, and adhesion of ATLL cells in preclinical models. EXPERIMENTAL DESIGN: Frequency and prognostic impact of PAK2 amplification were evaluated in an ATLL cohort of 370 cases. Novel long-term cultures and in vivo xenograft models were developed using primary ATLL cells from North American patients. Two PAK inhibitors were used to block PAK kinase activity pharmacologically. siRNA-based gene silencing approach was used to genetically knockdown (KD) PAK1 and PAK2 in ATLL cell lines. RESULTS: PAK1/2/4 are the three most abundantly expressed PAK family members in ATLL. PAK2 amplifications are seen in 24% of ATLLs and are associated with worse prognosis in a large patient cohort. The pan-PAK inhibitor PF-3758309 (PF) has strong in vitro and in vivo activity in a variety of ATLL preclinical models. These activities of PF are likely attributed to its ability to target several PAK isoforms simultaneously because genetic silencing of either PAK1 or PAK2 produced more modest effects. PAK2 plays a major role in CADM1-mediated stromal interaction, which is an important step in systemic dissemination of the disease. This finding is consistent with the observation that PAK2 amplification is more frequent in aggressive ATLLs and correlates with inferior outcome. CONCLUSIONS: PAK2, a gene frequently amplified in ATLL, facilitates CADM1-mediated stromal interaction and promotes survival of ATLL cells. Taken together, PAK inhibition may hold significant promise as a targeted therapy for aggressive ATLLs.
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Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirroles/farmacología , Quinasas p21 Activadas/antagonistas & inhibidores , Adulto , Animales , Adhesión Celular/efectos de los fármacos , Molécula 1 de Adhesión Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Amplificación de Genes , Humanos , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/metabolismo , Leucemia-Linfoma de Células T del Adulto/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Cultivo Primario de Células , ARN Interferente Pequeño/genética , Tasa de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas p21 Activadas/genéticaRESUMEN
BACKGROUND: B7x (B7-H4/B7S1/VTCN1), an inhibitory immune checkpoint molecule is a potential therapeutic target owing to its immunosuppressive effect and well-known expression in cancers. Immune checkpoints in canine bladder cancer are largely undefined. Here, we report the first evaluation on expression of B7x in spontaneous canine invasive bladder cancer, a novel model system for the study of invasive human urothelial carcinoma. OBJECTIVE: This work aims to study expression of immune checkpoint B7x in spontaneous canine invasive bladder cancer. METHODS: RNA-seq analysis was performed to determine B7x expression in tumor versus normal bladder. Gene ontology (GO) study was used to explore the biological role of B7x. B7x protein expression was evaluated by immunohistochemistry (IHC). TCGA and GTEx were used to examine B7x expression in 599 human bladder urothelial carcinoma (BLCA). RESULTS: RNA-seq analysis indicated 5.72 and 7.04 fold up regulation of B7x in tumors, using DESeq2 and edge R respectively (pâ<â0.00008). B7x was closely associated with immune processes in GO analysis. IHC results revealed 60% of cases as B7x positive. B7x intensity was scored as negative in 40% (nâ=â20/50), low in 24% (nâ=â12/50), medium in 14% (nâ=â7/50) and high in 22% (nâ=â11/50) of cases. In human BLCA, B7x expression was significantly associated with worse overall survival (pâ=â0.02). CONCLUSIONS: Our results suggest that B7x is over expressed in canine bladder cancer. Thus canine model can be vital in advancing the translational research on B7x, a new potential therapeutic target in human bladder cancer.
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Purpose: Immunotherapy targeting the PD-1/PD-L1 pathway has changed the treatment landscape of non-small cell lung carcinoma (NSCLC). We demonstrated that HHLA2, a newly identified immune inhibitory molecule, was widely expressed in NSCLC. We now compared the expression and function of PD-L1 with alternative immune checkpoints, B7x and HHLA2.Experimental Design: Expression was examined in tissue microarrays consisting of 392 resected NSCLC tumors. Effects of PD-L1, B7x, and HHLA2 on human T-cell proliferation and cytokine production were investigated.Results: PD-L1 expression was identified in 25% and 31% of tumors in the discovery and validation cohorts and was associated with higher stage and lymph node involvement. The multivariate analysis showed that stage, TIL status, and lymph node involvement were independently associated with PD-L1 expression. B7x was expressed in 69% and 68%, whereas HHLA2 was positive in 61% and 64% of tumors in the two sets. The coexpression of PD-L1 with B7x or HHLA2 was infrequent, 6% and 3%. The majority (78%) of PD-L1-negative cases expressed B7x, HHLA2, or both. The triple-positive group had more TIL infiltration than the triple-negative group. B7x-Ig and HHLA2-Ig inhibited TCR-mediated proliferation of CD4 and CD8 T cells more robustly than PD-L1-Ig. All three significantly suppressed cytokine productions by T cells.Conclusions: The majority of PD-L1-negative lung cancers express alternative immune checkpoints. The roles of the B7x and HHLA2 pathway in mediating immune evasion in PD-L1-negative tumors deserve to be explored to provide the rationale for an effective immunotherapy strategy in these tumors. Clin Cancer Res; 24(8); 1954-64. ©2018 AACR.
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Antígenos B7/genética , Antígeno B7-H1/genética , Biomarcadores de Tumor , Inmunoglobulinas/genética , Inmunomodulación/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Anciano , Anciano de 80 o más Años , Antígenos B7/metabolismo , Antígeno B7-H1/metabolismo , Proliferación Celular , Citocinas/metabolismo , Femenino , Humanos , Inmunoglobulinas/metabolismo , Inmunohistoquímica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mutación , Metástasis de la Neoplasia , Estadificación de Neoplasias , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Elucidation of mechanisms underlying the establishment, maintenance of and reactivation from HIV-1 latency is essential for the development of therapeutic strategies aimed at eliminating HIV-1 reservoirs. Microbial translocation, as a consequence of HIV-1-induced deterioration of host immune system, is known to result in a systemic immune activation and transient outbursts of HIV-1 viremia in chronic HIV-1 infection. How these microbes cause the robust HIV-1 reactivation remains elusive. Dendritic cells (DCs) have previously been shown to reactivate HIV-1 from latency; however, the precise role of DCs in reactivating HIV-1 from latently infected T-cell remains obscure. In this study, by using HIV-1 latently infected Jurkat T cells, we demonstrated that AIDS-associated pathogens as represented by Mycobacterium bovis (M. bovis) Bacillus Calmette-Guérin (BCG) and bacterial component lipopolysaccharide (LPS) were unable to directly reactivate HIV-1 from Jurkat T cells; instead, they mature DCs to secrete TNF-α to accomplish this goal. Moreover, we found that HIV-1 latently infected Jurkat T cells could also mature DCs and enhance their TNF-α production during co-culture in a CD40-CD40L-signaling-dependent manner. This in turn led to viral reactivation from Jurkat T cells. Our results reveal how DCs help AIDS-associated pathogens to trigger HIV-1 reactivation from latency.
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Síndrome de Inmunodeficiencia Adquirida/metabolismo , Células Dendríticas/inmunología , VIH-1/fisiología , Células Jurkat/virología , Factor de Necrosis Tumoral alfa/farmacología , Latencia del Virus , Síndrome de Inmunodeficiencia Adquirida/genética , Síndrome de Inmunodeficiencia Adquirida/virología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Técnicas de Cocultivo , Células Dendríticas/citología , VIH-1/genética , Humanos , Células Jurkat/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Activación ViralRESUMEN
HIV-1 latency is characterized by reversible silencing of viral transcription driven by the long terminal repeat (LTR) promoter of HIV-1. Cellular and viral factors regulating LTR activity contribute to HIV-1 latency, and certain repressive cellular factors modulate viral transcription silencing. Nef-associated factor 1 (Naf1) is a host nucleocytoplasmic shuttling protein that regulates multiple cellular signaling pathways and HIV-1 production. We recently reported that nuclear Naf1 promoted nuclear export of unspliced HIV-1 gag mRNA, leading to increased Gag production. Here we demonstrate new functions of Naf1 in regulating HIV-1 persistence. We found that Naf1 contributes to the maintenance of HIV-1 latency by inhibiting LTR-driven HIV-1 gene transcription in a nuclear factor kappa B-dependent manner. Interestingly, Naf1 knockdown significantly enhanced viral reactivation in both latently HIV-1-infected Jurkat T cells and primary central memory CD4+ T cells. Furthermore, Naf1 knockdown in resting CD4+ T cells from HIV-1-infected individuals treated with antiretroviral therapy significantly increased viral reactivation upon T-cell activation, suggesting an important role of Naf1 in modulating HIV-1 latency in vivo Our findings provide new insights for a better understanding of HIV-1 latency and suggest that inhibition of Naf1 activity to activate latently HIV-1-infected cells may be a potential therapeutic strategy. IMPORTANCE: HIV-1 latency is characterized mainly by a reversible silencing of LTR promoter-driven transcription of an integrated provirus. Cellular and viral proteins regulating LTR activity contribute to the modulation of HIV-1 latency. In this study, we found that the host protein Naf1 inhibited HIV-1 LTR-driven transcription of HIV genes and contributed to the maintenance of HIV-1 latency. Our findings provide new insights into the effects of host modulation on HIV-1 latency, which may lead to a potential therapeutic strategy for HIV persistence by targeting the Naf1 protein.
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Linfocitos T CD4-Positivos/metabolismo , Regulación Viral de la Expresión Génica , Infecciones por VIH/genética , VIH-1/genética , Ribonucleoproteínas/genética , Latencia del Virus/genética , Linfocitos T CD4-Positivos/virología , Núcleo Celular/metabolismo , Núcleo Celular/virología , Silenciador del Gen , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH , VIH-1/crecimiento & desarrollo , VIH-1/metabolismo , Interacciones Huésped-Patógeno , Humanos , Células Jurkat , FN-kappa B/genética , FN-kappa B/metabolismo , Cultivo Primario de Células , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Ribonucleoproteínas/antagonistas & inhibidores , Ribonucleoproteínas/metabolismo , Transducción de Señal , Transcripción Genética , Activación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
HIV-1 depends on host-cell-encoded factors to complete its life cycle. A comprehensive understanding of how HIV-1 manipulates host machineries during viral infection can facilitate the identification of host targets for antiviral drugs or gene therapy. The cellular protein Naf1 (HIV-1 Nef-associated factor 1) is a CRM1-dependent nucleo-cytoplasmic shuttling protein, and has been identified to regulate multiple receptor-mediated signal pathways in inflammation. The cytoplasm-located Naf1 can inhibit NF-κB activation through binding to A20, and the loss of Naf1 controlled NF-κB activation is associated with multiple autoimmune diseases. However, the effect of Naf1 on HIV-1 mRNA expression has not been characterized. In this study we found that the nucleus-located Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA. We demonstrated that the association between Naf1 and CRM1 was required for this function as the inhibition or knockdown of CRM1 expression significantly impaired Naf1-promoted HIV-1 production. The mutation of Naf1 nuclear export signals (NESs) that account for CRM1 recruitment for nuclear export decreased Naf1 function. Additionally, the mutation of the nuclear localization signal (NLS) of Naf1 diminished its ability to promote HIV-1 production, demonstrating that the shuttling property of Naf1 is required for this function. Our results reveal a novel role of Naf1 in enhancing HIV-1 production, and provide a potential therapeutic target for controlling HIV-1 infection.
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Proteínas Portadoras/metabolismo , Carioferinas/metabolismo , Precursores del ARN/metabolismo , Transporte de ARN , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/química , Proteínas Portadoras/genética , Supervivencia Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , VIH-1/fisiología , Humanos , Inmunoprecipitación , Carioferinas/antagonistas & inhibidores , Carioferinas/genética , Microscopía Confocal , Señales de Exportación Nuclear , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Mutación Puntual , Interferencia de ARN , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Proteína Exportina 1RESUMEN
P-selectin glycoprotein ligand-1 (PSGL-1) has been proved to serve as the functional receptor for enterovirus 71 (EV71). We found the abundant expression of PSGL-1 on monocyte-derived dendritic cells (MDDCs). However, we have previously demonstrated that MDDCs did not support efficient replication of EV71. Dendritic cells (DCs) have been described to be subverted by various viruses including EV71 for viral dissemination, we thus explore the potential contribution of PSGL-1 on DC-mediated EV71 transmission. We found that the cell-surface-expressing PSGL-1 on MDDCs mediated EV71 binding, and intriguingly, these loaded-viruses on MDDCs could be transferred to encountered target cells; Prior-treatment with PSGL-1 antibodies or interference with PSGL-1 expression diminished MDDC-mediated EV71 transfer and rescued virus-induced cell death. Our data uncover a novel role of PSGL-1 in DC-mediated EV71 spread, and provide an insight into blocking primary EV71 infection.
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Anticuerpos Monoclonales/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/virología , Enterovirus Humano A/efectos de los fármacos , Glicoproteínas de Membrana/inmunología , Anticuerpos Monoclonales/inmunología , Muerte Celular/inmunología , Línea Celular , Células Dendríticas/inmunología , Enterovirus Humano A/metabolismo , Citometría de Flujo , Interacciones Huésped-Patógeno , Humanos , Células Jurkat , Glicoproteínas de Membrana/metabolismo , ARN Interferente Pequeño , Receptores Virales/metabolismo , Acoplamiento Viral/efectos de los fármacosRESUMEN
BACKGROUND: Enterovirus 71 (EV71) is the main causative agent of hand, foot and mouth disease that occurs in young children. Neither antiviral agents nor vaccines are available for efficiently combating viral infection. Study of EV71-host interplay is important for understanding viral infection and developing strategies for prevention and therapy. Here the interactions of EV71 with human dendritic cells were analyzed. METHODS: EV71 capture, endocytosis, infection, and degradation in monocyte-derived dendritic cells (MDDCs) were detected by Flow cytometry or real-time (RT-) PCR, and MDDCs-mediated EV71 trans-infection of RD cells was determined via coculture system. Cell morphology or viability was monitored with microscopy or flow cytometry. SiRNA interference was used to knock down gene expression. RESULTS: MDDCs can bind EV71, but these loaded-EV71 particles in MDDCs underwent a rapid degradation in the absence of efficient replication; once the captured EV71 encountered susceptible cells, MDDCs efficiently transferred surface-bound viruses to target cells. The molecule of DC-SIGN (DC-specific intercellular adhesion molecule-3 grabbing nonintegrin) mediated viral binding and transfer, because interference of DC-SIGN expression with specific siRNAs reduced EV71 binding and impaired MDDC-mediated viral trans-infection, and exogenous expression of DC-SIGN molecule on Raji cell initiated viral binding and subsequent transmission. CONCLUSION: MDDCs could bind efficiently EV71 viruses through viral binding to DC-SIGN molecule, and these captured-viruses could be transferred to susceptible cells for robust infection. The novel finding of DC-mediated EV71 dissemination might facilitate elucidation of EV71 primary infection and benefit searching for new clues for preventing viruses from initial infection.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/virología , Enterovirus Humano A/inmunología , Enterovirus Humano A/fisiología , Interacciones Huésped-Patógeno , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/metabolismo , Acoplamiento Viral , Moléculas de Adhesión Celular/genética , Células Cultivadas , Técnicas de Cocultivo , Humanos , Lectinas Tipo C/genética , Receptores de Superficie Celular/genéticaRESUMEN
Altered cytokine profiles and imbalanced frequencies of CD4(+) T helper cell subsets are thought to be linked with HIV-1/AIDS pathogenesis, but the causes need to be further clarified. Histamine, a biogenic amine with many functions, shows enhancement in HIV-1 infected individuals, which are considered to link with disease progression, but is poorly understood. This study investigated histamine-assisted HIV-1 modulation of dendritic cell (DC) functions. Histamine and HIV-1 showed a synergistic role in induction of interleukin-10; histamine inhibited HIV-1-induced IL-12 production from MDDCs (monocyte-derived DCs); notably, histamine augmented HIV-1-induced MDDC functional polarization and skewed naïve T cell differentiation toward regulatory T cells (Tregs). The results indicate the novel role of histamine in HIV-1-induced DC functional regulation, which promoted Treg cell differentiation and up-regulated immunosuppressive factors. These findings help to bridge the correlation between elevated histamine and increased Treg cell frequency in HIV-1 infected individuals, and add to our understanding of HIV-1-induced immunosuppression.