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1.
J Immunol ; 206(10): 2265-2270, 2021 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-33931486

RESUMEN

NK cells are cytotoxic lymphocytes displaying strong antimetastatic activity. Mouse models and in vitro studies suggest a prominent role of the mechanistic target of rapamycin (mTOR) kinase in the control of NK cell homeostasis and antitumor functions. However, mTOR inhibitors are used as chemotherapies in several cancer settings. The impact of such treatments on patients' NK cells is unknown. We thus performed immunophenotyping of circulating NK cells from metastatic breast cancer patients treated with the mTOR inhibitor everolimus over a three-month period. Everolimus treatment resulted in inhibition of mTORC1 activity in peripheral NK cells, whereas mTORC2 activity was preserved. NK cell homeostasis was profoundly altered with a contraction of the NK cell pool and an overall decrease in their maturation. Phenotype and function of the remaining NK cell population was less affected. This is, to our knowledge, the first in vivo characterization of the role of mTOR in human NK cells.


Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Everolimus/administración & dosificación , Células Asesinas Naturales/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Transducción de Señal/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/patología , Femenino , Estudios de Seguimiento , Francia/epidemiología , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Persona de Mediana Edad , Metástasis de la Neoplasia , Estudios Prospectivos , Serina-Treonina Quinasas TOR/metabolismo , Resultado del Tratamiento
2.
J Immunol ; 200(11): 3769-3776, 2018 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-29695416

RESUMEN

Hemophagocytic lymphohistiocytosis (HLH) is a severe inflammatory condition that occurs in patients with genetic defects of cytotoxicity (familial HLH [FHL]) or secondary to other immunological disorders such as juvenile idiopathic arthritis. HLH is characterized by elevated levels of serum IL-18 and other cytokines. Moreover, a novel clinical entity has been recently identified in which constitutive NLRC4 inflammasome activation leads to severe HLH. Altogether, these clinical observations suggest that inflammasome activation is a central event in the development of all HLH forms and that inflammasome blockade could alleviate inflammation in FHL patients. To formally address this question, we invalidated genes encoding for Caspase-1 or the inflammasome adapter ASC in perforin-deficient mice that were subsequently infected with lymphocytic or mouse choriomeningitis virus as models of FHL. These deletions nearly abrogated IL-18 production occurring during HLH in all models. However, they did not reduce serum IFN-γ levels at the peak of the inflammatory reaction nor did they modulate inflammatory parameters at mid and late stages or fatal outcome. These data show that inflammasome blockade is not sufficient to prevent cytokine storm and lethality in mouse models of FHL and suggest that different pathophysiological mechanisms underlie HLH in genetic defects of cytotoxicity and genetic forms of inflammasome activation.


Asunto(s)
Inflamasomas/genética , Inflamación/genética , Linfohistiocitosis Hemofagocítica/genética , Eliminación de Secuencia/genética , Animales , Caspasa 1/genética , Línea Celular , Chlorocebus aethiops , Modelos Animales de Enfermedad , Humanos , Interferón gamma/genética , Interleucina-18/genética , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Perforina/genética , Células Vero
3.
Cytokine ; 113: 446-452, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30413290

RESUMEN

Type I interferonopathies are characterized by an increase of circulating type I interferon (IFN) concentration. Type I interferonopathies refer to rare Mendelian genetic disorders such as Aicardi-Goutières Syndrome (AGS) as well as more frequent and polygenic auto-immune diseases like systemic lupus erythematosus (SLE). Yet, detection of type I IFN in these patients remains challenging as its amount is usually very low in patients' sera. Thus, the detection of interferon-stimulating genes has been proposed as an alternative for the detection of this cytokine but sensitivy, specificity and predictive values of the assay have not been reported so far. In this study, we propose two different methods based on Nanostring or RT-qPCR to measure in the clinical routine the IFN response, defined as a set of transcripts that are systemically induced by IFNs. The IFN signature is composed of 6 IFN stimulated genes (ISGs) and has a strong predictive value for the diagnosis of type I interferonopathies. The use of this simple test might represent a gold standard for the evaluation of various autoimmune diseases. Moreover, this test could also be used to monitor patients treated with drugs targeting type I IFN pathway. When comparing both methods - Nanostring and qPCR - in terms of analytical performance, they provided similar results but Nanostring was quicker, easier to multiplex, and almost fully-automated, which represent a more reliable assay for the daily clinical practice.


Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso , Regulación de la Expresión Génica , Interferón Tipo I/metabolismo , Lupus Eritematoso Sistémico , Malformaciones del Sistema Nervioso , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Enfermedades Autoinmunes del Sistema Nervioso/diagnóstico , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Femenino , Humanos , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/metabolismo , Masculino , Malformaciones del Sistema Nervioso/diagnóstico , Malformaciones del Sistema Nervioso/metabolismo
4.
Clin Immunol ; 177: 12-17, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-26794911

RESUMEN

Obesity is associated with increased cancer rates and higher susceptibility to infections. The adipose tissue of obese individuals is inflammatory and may negatively impact on innate and adaptive immunity in a systemic way. Here, we explored the phenotype and function of peripheral Natural Killer (NK) cells of patients in correlation with their body mass index (BMI). We found that high BMI was associated with an increased activation status of peripheral NK cells, as measured by surface levels of CD69 and levels of granzyme-B. However, these activated NK cells had an impaired capacity to degranulate or to produce cytokines/chemokines when exposed to tumor cell lines deficient in MHC-I expression or coated with antibodies. This suggests that chronic stimulation of NK cells during obesity may lead to their incapacity to respond normally and eliminate target cells, which could contribute to the greater susceptibility of obese individuals to develop cancers or infectious diseases.


Asunto(s)
Células Asesinas Naturales/inmunología , Obesidad/inmunología , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Adulto Joven
6.
J Immunother Cancer ; 11(11)2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37940345

RESUMEN

BACKGROUND: Multidrug resistance-1 (MDR1) transporter limits the intracellular accumulation of chemotherapies (paclitaxel, anthracyclines) used in breast cancer (BC) treatment. In addition to tumor cells, MDR1 is expressed on immune cell subsets in which it confers chemoresistance. Among human T cells, MDR1 is expressed by most CD8+ T cells, and a subset of CD4+ T helper (Th) cells. Here we explored the expression, function and regulation of MDR1 on CD4+ T cells and investigated the role of this population in response to neoadjuvant chemotherapy (NAC) in BC. METHODS: Phenotypic and functional characteristics of MDR1+ CD4 Th cells were assessed on blood from healthy donors and patients with BC by flow cytometry. These features were extended to CD4+ Th cells from untreated breast tumor by flow cytometry and RNA-sequencing (RNA-seq). We performed in vitro polarization assays to decipher MDR1 regulation on CD4 Th cells. We evaluated in vitro the impact of chemotherapy agents on MDR1+ CD4+ Th cells. We analyzed the impact of NAC treatment on MDR1+ CD4+ Th cells from blood and tumors and their association with treatment efficacy in two independent BC cohorts and in a public RNA-seq data set of BC tumor biopsies before and after NAC. Finally, we performed single cell (sc) RNAseq of blood CD4+ memory T cells from NAC-treated patients and combined them with an scRNAseq public data set. RESULTS: MDR1+ CD4 Th cells were strongly enriched in Th1.17 polyfunctional cells but also in Th17 cells, both in blood and untreated breast tumor tissues. Mechanistically, Tumor growth factor (TGF)-ß1 was required for MDR1 induction during in vitro Th17 or Th1.17 polarization. MDR1 expression conferred a selective advantage to Th1.17 and Th17 cells following paclitaxel treatment in vitro and in vivo in NAC-treated patients. scRNAseq demonstrated MDR1 association with tumor Th1.17 and Th with features of cytotoxic cells. Enrichment in MDR1+ CD4+ Th1.17 and Th17 cells, in blood and tumors positively correlated with pathological response. Absence of early modulation of Th1.17 and Th17 in NAC-resistant patients, argue for its use as a biomarker for chemotherapy regimen adjustment. CONCLUSION: MDR1 favored the enrichment of Th1.17 and Th17 in blood and tumor after NAC that correlated to clinical response.


Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Linfocitos T CD8-positivos , Terapia Neoadyuvante , Linfocitos T CD4-Positivos , Células Th17 , Paclitaxel/farmacología , Paclitaxel/uso terapéutico
7.
Cancers (Basel) ; 13(8)2021 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-33924428

RESUMEN

Regulatory T cells (Tregs) are present in a large majority of solid tumors and are mainly associated with a poor prognosis, as their major function is to inhibit the antitumor immune response contributing to immunosuppression. In this review, we will investigate the mechanisms involved in the recruitment, amplification and stability of Tregs in the tumor microenvironment (TME). We will also review the strategies currently developed to inhibit Tregs' deleterious impact in the TME by either inhibiting their recruitment, blocking their expansion, favoring their plastic transformation into other CD4+ T-cell subsets, blocking their suppressive function or depleting them specifically in the TME to avoid severe deleterious effects associated with Treg neutralization/depletion in the periphery and normal tissues.

8.
Elife ; 102021 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-33507150

RESUMEN

Antiviral effectors such as natural killer (NK) cells have impaired functions in chronic hepatitis B (CHB) patients. The molecular mechanism responsible for this dysfunction remains poorly characterised. We show that decreased cytokine production capacity of peripheral NK cells from CHB patients was associated with reduced expression of NKp30 and CD16, and defective mTOR pathway activity. Transcriptome analysis of patients NK cells revealed an enrichment for transcripts expressed in exhausted T cells suggesting that NK cell dysfunction and T cell exhaustion employ common mechanisms. In particular, the transcription factor TOX and several of its targets were over-expressed in NK cells of CHB patients. This signature was predicted to be dependent on the calcium-associated transcription factor NFAT. Stimulation of the calcium-dependent pathway recapitulated features of NK cells from CHB patients. Thus, deregulated calcium signalling could be a central event in both T cell exhaustion and NK cell dysfunction occurring during chronic infections.


Asunto(s)
Factores de Restricción Antivirales/inmunología , Hepatitis B Crónica/inmunología , Células Asesinas Naturales/inmunología , Infección Persistente/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven
9.
SLAS Discov ; 24(1): 25-37, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30184441

RESUMEN

Natural killer (NK) cells are essential players of the innate immune response that secrete cytolytic factors and cytokines such as IFN-γ when contacting virus-infected or tumor cells. They represent prime targets in immunotherapy as defects in NK cell functions are hallmarks of many pathological conditions, such as cancer and chronic infections. The functional screening of chemical libraries or biologics would greatly help identify new modulators of NK cell activity, but commonly used methods such as flow cytometry are not easily scalable to high-throughput settings. Here we describe an efficient assay to measure the natural cytotoxicity of primary NK cells where the bioluminescent enzyme NanoLuc is constitutively expressed in the cytoplasm of target cells and is released in co-culture supernatants when lysis occurs. We fully characterized this assay using either purified NK cells or total peripheral blood mononuclear cells (PBMCs), including some patient samples, as effector cells. A pilot screen was also performed on a library of 782 metabolites, xenobiotics, and common drugs, which identified dextrometorphan and diphenhydramine as novel NK cell inhibitors. Finally, this assay was further improved by developing a dual-reporter cell line to simultaneously measure NK cell cytotoxicity and IFN-γ secretion in a single well, extending the potential of this system.


Asunto(s)
Células Asesinas Naturales/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Línea Celular , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos/métodos , Citometría de Flujo/métodos , Células HEK293 , Humanos , Interferón gamma/metabolismo , Células K562 , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Luciferasas/metabolismo , Proyectos Piloto
10.
Front Immunol ; 9: 704, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29706958

RESUMEN

Multiple myeloma (MM) is a proliferation of tumoral plasma B cells that is still incurable. Natural killer (NK) cells can recognize and kill MM cells in vitro and can limit MM growth in vivo. Previous reports have shown that NK cell function is impaired during MM progression and suggested that treatment with immunomodulatory drugs (IMIDs) such as lenalidomide (LEN) could enhance it. However, the effects of IMIDs on NK cells have been tested mostly in vitro or in preclinical models and supporting evidence of their effect in vivo in patients is lacking. Here, we monitored NK cell activity in blood samples from 10 MM patients starting after frontline induction chemotherapy (CTX) consisting either of association of bortezomib-lenalidomide-dexamethasone (Velcade Revlimid Dexamethasone) or autologous stem-cell transplantation (SCT). We also monitored NK cell activity longitudinally each month during 1 year, after maintenance therapy with LEN. Following frontline chemotherapy, peripheral NK cells displayed a very immature phenotype and retained poor reactivity toward target cells ex vivo. Upon maintenance treatment with LEN, we observed a progressive normalization of NK cell maturation, likely caused by discontinuation of chemotherapy. However, LEN treatment neither activated NK cells nor improved their capacity to degranulate or to secrete IFN-γ or MIP1-ß following stimulation with MHC-I-deficient or antibody-coated target cells. Upon LEN discontinuation, there was no reduction of NK cell effector function either. These results caution against the use of LEN as single therapy to improve NK cell activity in patients with cancer and call for more preclinical assessments of the potential of IMIDs in NK cell activation.


Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Citotoxicidad Inmunológica/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Lenalidomida/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/inmunología , Anciano , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores , Femenino , Humanos , Inmunofenotipificación , Células Asesinas Naturales/metabolismo , Lenalidomida/administración & dosificación , Lenalidomida/efectos adversos , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/metabolismo , Fenotipo , Resultado del Tratamiento
11.
Cancers (Basel) ; 9(10)2017 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-28956813

RESUMEN

The control of cellular metabolism is now recognized as key to regulate functional properties of immune effectors such as T or Natural Killer (NK) cells. During persistent infections or in the tumor microenvironment, multiple metabolic changes have been highlighted in T cells that contribute to their dysfunctional state or exhaustion. NK cells may also undergo major phenotypic and functional modifications when infiltrating tumors that could be linked to metabolic alterations. The mammalian target of rapamycin (mTOR) kinase is a central regulator of cellular metabolism. mTOR integrates various extrinsic growth or immune signals and modulates metabolic pathways to fulfill cellular bioenergetics needs. mTOR also regulates transcription and translation thereby adapting cellular pathways to the growth or activation signals that are received. Here, we review the role and regulation of mTOR in NK cells, with a special focus on cytokines that target mTOR such as IL-15 and TGF-ß. We also discuss how NK cell metabolic activity could be enhanced or modulated to improve their effector anti-tumor functions in clinical settings.

12.
Elife ; 62017 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-28875936

RESUMEN

NK cell education is the process through which chronic engagement of inhibitory NK cell receptors by self MHC-I molecules preserves cellular responsiveness. The molecular mechanisms responsible for NK cell education remain unclear. Here, we show that mouse NK cell education is associated with a higher basal activity of the mTOR/Akt pathway, commensurate to the number of educating receptors. This higher activity was dependent on the SHP-1 phosphatase and essential for the improved responsiveness of reactive NK cells. Upon stimulation, the mTOR/Akt pathway amplified signaling through activating NK cell receptors by enhancing calcium flux and LFA-1 integrin activation. Pharmacological inhibition of mTOR resulted in a proportional decrease in NK cell reactivity. Reciprocally, acute cytokine stimulation restored reactivity of hyporesponsive NK cells through mTOR activation. These results demonstrate that mTOR acts as a molecular rheostat of NK cell reactivity controlled by educating receptors and uncover how cytokine stimulation overcomes NK cell education.


Asunto(s)
Células Asesinas Naturales/inmunología , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Citocinas/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Ratones Endogámicos C57BL , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo
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