RESUMEN
Natural killer (NK) cells are cytotoxic type 1 innate lymphoid cells (ILCs) that defend against viruses and mediate anti-tumor responses, yet mechanisms controlling their development and function remain incompletely understood. We hypothesized that the abundantly expressed microRNA-142 (miR-142) is a critical regulator of type 1 ILC biology. Interleukin-15 (IL-15) signaling induced miR-142 expression, whereas global and ILC-specific miR-142-deficient mice exhibited a cell-intrinsic loss of NK cells. Death of NK cells resulted from diminished IL-15 receptor signaling within miR-142-deficient mice, likely via reduced suppressor of cytokine signaling-1 (Socs1) regulation by miR-142-5p. ILCs persisting in Mir142-/- mice demonstrated increased expression of the miR-142-3p target αV integrin, which supported their survival. Global miR-142-deficient mice exhibited an expansion of ILC1-like cells concurrent with increased transforming growth factor-ß (TGF-ß) signaling. Further, miR-142-deficient mice had reduced NK-cell-dependent function and increased susceptibility to murine cytomegalovirus (MCMV) infection. Thus, miR-142 critically integrates environmental cues for proper type 1 ILC homeostasis and defense against viral infection.
Asunto(s)
Homeostasis/inmunología , Inmunidad Innata/inmunología , Linfocitos/inmunología , MicroARNs/inmunología , Animales , Línea Celular , Femenino , Células HEK293 , Humanos , Células Asesinas Naturales/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Muromegalovirus/inmunología , Células 3T3 NIH , Receptores de Interleucina-15/inmunología , Transducción de Señal/inmunología , Proteínas Supresoras de la Señalización de Citocinas/inmunología , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
PURPOSE: Anti-CD20 monoclonal antibodies (mAb) are an important immunotherapy for B-cell lymphoma, and provide evidence that the immune system may be harnessed as an effective lymphoma treatment approach. ALT-803 is a superagonist IL-15 mutant and IL-15Rα-Fc fusion complex that activates the IL-15 receptor constitutively expressed on natural killer (NK) cells. We hypothesized that ALT-803 would enhance anti-CD20 mAb-directed NK-cell responses and antibody-dependent cellular cytotoxicity (ADCC). EXPERIMENTAL DESIGN: We tested this hypothesis by adding ALT-803 immunostimulation to anti-CD20 mAb triggering of NK cells in vitro and in vivo. Cell lines and primary human lymphoma cells were utilized as targets for primary human NK cells. Two complementary in vivo mouse models were used, which included human NK-cell xenografts in NOD/SCID-γc (-/-) mice. RESULTS: We demonstrate that short-term ALT-803 stimulation significantly increased degranulation, IFNγ production, and ADCC by human NK cells against B-cell lymphoma cell lines or primary follicular lymphoma cells. ALT-803 augmented cytotoxicity and the expression of granzyme B and perforin, providing one potential mechanism for this enhanced functionality. Moreover, in two distinct in vivo B-cell lymphoma models, the addition of ALT-803 to anti-CD20 mAb therapy resulted in significantly reduced tumor cell burden and increased survival. Long-term ALT-803 stimulation of human NK cells induced proliferation and NK-cell subset changes with preserved ADCC. CONCLUSIONS: ALT-803 represents a novel immunostimulatory drug that enhances NK-cell antilymphoma responses in vitro and in vivo, thereby supporting the clinical investigation of ALT-803 plus anti-CD20 mAbs in patients with indolent B-cell lymphoma.
Asunto(s)
Antineoplásicos/farmacología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Linfoma de Células B/inmunología , Linfoma de Células B/metabolismo , Proteínas/farmacología , Receptores de IgG/metabolismo , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Línea Celular Tumoral , Citotoxicidad Inmunológica/efectos de los fármacos , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Granzimas/genética , Granzimas/metabolismo , Humanos , Interferón gamma/biosíntesis , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/patología , Ratones , Ratones Noqueados , Perforina/genética , Perforina/metabolismo , Proteínas Recombinantes de Fusión , Rituximab/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Natural killer (NK) cells are lymphocytes that play an important role in the immune response to infection and malignancy. Recent studies in mice have shown that stimulation of NK cells with cytokines or in the context of a viral infection results in memory-like properties. We hypothesized that human NK cells exhibit such memory-like properties with an enhanced recall response after cytokine preactivation. In the present study, we show that human NK cells preactivated briefly with cytokine combinations including IL-12, IL-15, and IL-18 followed by a 7- to 21-day rest have enhanced IFN-γ production after restimulation with IL-12 + IL-15, IL-12 + IL-18, or K562 leukemia cells. This memory-like phenotype was retained in proliferating NK cells. In CD56(dim) NK cells, the memory-like IFN-γ response was correlated with the expression of CD94, NKG2A, NKG2C, and CD69 and a lack of CD57 and KIR. Therefore, human NK cells have functional memory-like properties after cytokine activation, which provides a novel rationale for integrating preactivation with combinations of IL-12, IL-15, and IL-18 into NK cell immunotherapy strategies.
Asunto(s)
Citocinas/inmunología , Interferón gamma/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígeno CD56/inmunología , Antígeno CD56/metabolismo , Proliferación Celular/efectos de los fármacos , Citocinas/farmacología , Citometría de Flujo , Humanos , Memoria Inmunológica/inmunología , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-12/inmunología , Interleucina-12/farmacología , Interleucina-15/inmunología , Interleucina-15/farmacología , Interleucina-18/inmunología , Interleucina-18/farmacología , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Activación de Linfocitos/efectos de los fármacos , Subfamília C de Receptores Similares a Lectina de Células NK/inmunología , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismo , Subfamília D de Receptores Similares a Lectina de las Células NK/inmunología , Subfamília D de Receptores Similares a Lectina de las Células NK/metabolismo , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/inmunología , Receptores de Interleucina-12/metabolismo , Receptores de Interleucina-18/genética , Receptores de Interleucina-18/inmunología , Receptores de Interleucina-18/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small RNA molecules that regulate a wide variety of cellular processes, typically by specific complementary targeting of the 3'UTR of mRNAs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. In this study, we report a mouse model of lymphocyte-restricted Dicer1 disruption to evaluate the role of Dicer1-dependent miRNAs in the development and function of NK cells. As expected, Dicer1-deficient NK cells had decreased total miRNA content. Furthermore, miRNA-deficient NK cells exhibited reduced survival and impaired maturation defined by cell surface phenotypic markers. However, Dicer1-deficient NK cells exhibited enhanced degranulation and IFN-γ production in vitro in response to cytokines, tumor target cells, and activating NK cell receptor ligation. Moreover, a similar phenotype of increased IFN-γ was evident during acute MCMV infection in vivo. miRs-15a/15b/16 were identified as abundant miRNAs in NK cells that directly target the murine IFN-γ 3'UTR, thereby providing a potential mechanism for enhanced IFN-γ production. These data suggest that the function of miRNAs in NK cell biology is complex, with an important role in NK cell development, survival, or homeostasis, while tempering peripheral NK cell activation. Further study of individual miRNAs in an NK cell specific fashion will provide insight into these complex miRNA regulatory effects in NK cell biology.
Asunto(s)
Células Asesinas Naturales/inmunología , MicroARNs/fisiología , Regiones no Traducidas 3' , Animales , Degranulación de la Célula , Supervivencia Celular , Citocinas/metabolismo , Citotoxicidad Inmunológica , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/fisiología , Infecciones por Herpesviridae/inmunología , Inmunidad Innata , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-15/farmacología , Tejido Linfoide/inmunología , Tejido Linfoide/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/biosíntesis , Muromegalovirus , Especificidad de Órganos , Ribonucleasa III/deficiencia , Ribonucleasa III/genética , Ribonucleasa III/fisiología , Organismos Libres de Patógenos EspecíficosRESUMEN
Relapsed or refractory (rel/ref) classical Hodgkin lymphoma (cHL) remains a clinical challenge, with limited effective treatment options available after stem cell transplantation. In a multicenter phase 2 study, the efficacy of lenalidomide in rel/ref cHL patients was evaluated at a dose of 25 mg/d on days 1-21 of a 28-day cycle. Patients remained on lenalidomide until disease progression or an unacceptable adverse event (AE) occurred. Thirty-eight cHL patients were enrolled with a median of 4 (range, 2-9) prior therapies; 87% had undergone prior stem cell transplantation and 55% of patients did not respond to their last prior therapy. Of 36 evaluable patients, responses were 1 complete remission (CR), 6 partial remissions (PRs), and 5 patients with stable disease (SD) for ≥ 6 months resulting in an International Working Committee (IWC) objective overall response rate (ORR) of 19% and a cytostatic ORR of 33%. Decreased chemokine (CCL17 and CCL22) plasma levels at 2 weeks were associated with a subsequent response. The treatment was well tolerated, and the most common grade 3/4 AEs were neutropenia (47%), anemia (29%), and thrombocytopenia (18%). Four patients discontinued lenalidomide because of rash, elevated transaminases/bilirubin, and cytopenias. We provide preliminary evidence of lenalidomide's activity in patients with rel/ref cHL, and therefore exploration of lenalidomide in combination with other active agents is warranted. This trial is registered at www.ClinicalTrials.gov as NCT00540007.
Asunto(s)
Antineoplásicos/uso terapéutico , Enfermedad de Hodgkin/tratamiento farmacológico , Talidomida/análogos & derivados , Adulto , Antineoplásicos/efectos adversos , Línea Celular Tumoral , Quimiocina CCL17/sangre , Quimiocina CCL22/sangre , Supervivencia sin Enfermedad , Femenino , Enfermedad de Hodgkin/sangre , Enfermedad de Hodgkin/terapia , Humanos , Estimación de Kaplan-Meier , Lenalidomida , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Recurrencia , Inducción de Remisión , Trasplante de Células Madre , Talidomida/efectos adversos , Talidomida/uso terapéuticoRESUMEN
Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g., granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates for mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes have been established, little is known about miRNAs in NK cells. Here, we used two next-generation sequencing (NGS) platforms to define the miRNA transcriptomes of resting and cytokine-activated primary murine NK cells, with confirmation by quantitative real-time PCR (qRT-PCR) and microarrays. We delineate a bioinformatics analysis pipeline that identified 302 known and 21 novel mature miRNAs from sequences obtained from NK cell small RNA libraries. These miRNAs are expressed over a broad range and exhibit isomiR complexity, and a subset is differentially expressed following cytokine activation. Using these miRNA NGS data, miR-223 was identified as a mature miRNA present in resting NK cells with decreased expression following cytokine activation. Furthermore, we demonstrate that miR-223 specifically targets the 3' untranslated region of murine GzmB in vitro, indicating that this miRNA may contribute to control of GzmB translation in resting NK cells. Thus, the sequenced NK cell miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Células Asesinas Naturales/metabolismo , MicroARNs/genética , Animales , Secuencia de Bases , Células Cultivadas , Biología Computacional/instrumentación , Biología Computacional/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Granzimas/genética , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Interleucina-15/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , MicroARNs/aislamiento & purificación , MicroARNs/metabolismo , MicroARNs/fisiología , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia de ARN/instrumentación , Análisis de Secuencia de ARN/métodos , Homología de Secuencia de Ácido NucleicoRESUMEN
Natural killer (NK) cells were identified by their ability to kill target cells without previous sensitization. However, without an antecedent "arming" event, NK cells can recognize, but are not equipped to kill, target cells. How NK cells become armed in vivo in healthy hosts is unclear. Because latent herpesviruses are highly prevalent and alter multiple aspects of host immunity, we hypothesized that latent herpesvirus infection would arm NK cells. Here we show that NK cells from mice latently infected with Murid herpesvirus 4 (MuHV-4) were armed as evidenced by increased granzyme B protein expression, cytotoxicity, and interferon-gamma production. NK-cell arming occurred rapidly in the latently infected host and did not require acute viral infection. Furthermore, NK cells armed by latent infection protected the host against a lethal lymphoma challenge. Thus, the immune environment created by latent herpesvirus infection provides a mechanism whereby host NK-cell function is enhanced in vivo.