RESUMO
According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34-CD117+ ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34-CD117+ ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.
Assuntos
Células Matadoras Naturais/imunologia , Linfócitos/imunologia , Tonsila Palatina/imunologia , Animais , Antígenos CD34/metabolismo , Antígeno CD56/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Imunidade Inata , Ativação Linfocitária , Camundongos , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
The current model of murine innate lymphoid cell (ILC) development holds that mouse ILCs are derived downstream of the common lymphoid progenitor through lineage-restricted progenitors. However, corresponding lineage-restricted progenitors in humans have yet to be discovered. Here we identified a progenitor population in human secondary lymphoid tissues (SLTs) that expressed the transcription factor RORγt and was unique in its ability to generate all known ILC subsets, including natural killer (NK) cells, but not other leukocyte populations. In contrast to murine fate-mapping data, which indicate that only ILC3s express Rorγt, these human progenitor cells as well as human peripheral blood NK cells and all mature ILC populations expressed RORγt. Thus, all human ILCs can be generated through an RORγt(+) developmental pathway from a common progenitor in SLTs. These findings help establish the developmental signals and pathways involved in human ILC development.
Assuntos
Células Matadoras Naturais/fisiologia , Linfonodos/imunologia , Subpopulações de Linfócitos/fisiologia , Células Progenitoras Linfoides/fisiologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Tonsila Palatina/imunologia , Adulto , Animais , Antígenos CD34/metabolismo , Diferenciação Celular , Linhagem Celular , Criança , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genéticaRESUMO
IL-15 is a proinflammatory cytokine that plays an essential role in the development and activation of natural killer (NK) cells. Adipose tissue acts as an endocrine organ that secretes cytokines and is an important reservoir for lymphocytes. We hypothesized that activation of the IL-15 signaling in adipose tissue will activate and expand the NK cell population and control tumor growth. We recently developed an adipocyte-targeting recombinant adeno-associated viral (rAAV) vector with minimal off-target transgene expression in the liver. Here, we used this rAAV system to deliver an IL-15/IL-15Rα complex to the abdominal fat by intraperitoneal (i.p.) injection. Adipose IL-15/IL-15Rα complex gene transfer led to the expansion of NK cells in the adipose tissue and spleen in normal mice without notable side effects. The i.p. injection of rAAV-IL-15/IL-15Rα complex significantly suppressed the growth of Lewis lung carcinoma implanted subcutaneously and exerted a significant survival advantage in a B16-F10 melanoma metastasis model. The antitumor effects were associated with the expansion of the NK cells in the blood, spleen, abdominal fat, and tumor, as well as the enhancement of NK cell maturity. Our proof-of-concept preclinical studies demonstrate the safety and efficacy of the adipocyte-specific IL-15/IL-15Rα complex vector as a novel cancer immune gene therapy.
Assuntos
Terapia Genética , Subunidade alfa de Receptor de Interleucina-15/genética , Interleucina-15/farmacologia , Neoplasias/terapia , Gordura Abdominal/efeitos dos fármacos , Gordura Abdominal/imunologia , Adipócitos/efeitos dos fármacos , Adipócitos/imunologia , Adipócitos/metabolismo , Animais , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/terapia , Proliferação de Células/genética , Dependovirus , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/farmacologia , Humanos , Interleucina-15/genética , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos , Metástase Neoplásica , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Transdução de Sinais/genéticaRESUMO
Human natural killer (NK) cells originate from CD34(+) hematopoietic progenitor cells, but the discrete stages of NK cell differentiation in vivo have not been elucidated. We identify and functionally characterize, from human lymph nodes and tonsils, four NK cell developmental intermediates spanning the continuum of differentiation from a CD34(+) NK cell progenitor to a functionally mature NK cell. Analyses of each intermediate stage for CD34, CD117, and CD94 cell surface expression, lineage differentiation potentials, capacity for cytokine production and natural cytotoxicity, and ETS-1, GATA-3, and T-BET expression provide evidence for a new model of human NK cell differentiation in secondary lymphoid tissues.
Assuntos
Diferenciação Celular/imunologia , Células Matadoras Naturais/citologia , Biomarcadores , Linhagem da Célula/imunologia , Células Cultivadas , Citocinas/biossíntese , Testes Imunológicos de Citotoxicidade , Perfilação da Expressão Gênica , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismoRESUMO
Human CD56(bright) natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIRs), high interferon-gamma (IFN-gamma) production, but little cytotoxicity. CD56(dim) NK cells have high KIR expression, produce little IFN-gamma, yet display high cytotoxicity. We hypothesized that, if human NK maturation progresses from a CD56(bright) to a CD56(dim) phenotype, an intermediary NK cell must exist, which demonstrates more functional overlap than these 2 subsets, and we used CD94 expression to test our hypothesis. CD94(high)CD56(dim) NK cells express CD62L, CD2, and KIR at levels between CD56(bright) and CD94(low)CD56(dim) NK cells. CD94(high)CD56(dim) NK cells produce less monokine-induced IFN-gamma than CD56(bright) NK cells but much more than CD94(low)CD56(dim) NK cells because of differential interleukin-12-mediated STAT4 phosphorylation. CD94(high)CD56(dim) NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56(bright) NK cells but lower than CD94(low)CD56(dim) NK cells. Collectively, our data suggest that the density of CD94 surface expression on CD56(dim) NK cells identifies a functional and likely developmental intermediary between CD56(bright) and CD94(low)CD56(dim) NK cells. This supports the notion that, in vivo, human CD56(bright) NK cells progress through a continuum of differentiation that ends with a CD94(low)CD56(dim) phenotype.
Assuntos
Antígeno CD56/imunologia , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Subfamília D de Receptores Semelhantes a Lectina de Células NK/imunologia , Células Cultivadas , Humanos , Interferon gama/imunologia , Interleucina-12/imunologia , Células Matadoras Naturais/citologia , Selectina L/imunologia , Subpopulações de Linfócitos/citologia , Fosforilação/imunologia , Fator de Transcrição STAT4/imunologiaRESUMO
IL-15 is required for NK cell development and homeostasis in vivo. Because IL-15 is presented in trans via its high-affinity IL-15Ralpha-chain to cells expressing the IL-15Rbetagamma complex, we postulated that certain IL-15-bearing cells must be required for NK cell homeostasis. Using IL-15(WT/WT) and IL-15(-/-) mice, bone marrow chimeras with normal cellularity, and a selective depletion of CD11c(hi) dendritic cells (DCs), we demonstrate that ablation of the resting CD11c(hi) DC population results in a highly significant decrease in the absolute number of mature NK cells. In contrast, administration of Flt3 ligand increases the CD11c(hi) DC population, which, when expressing IL-15, significantly expands mature NK cells via enhanced survival and proliferation. In summary, a CD11c(hi) DC population expressing IL-15 is required to maintain NK cell homeostasis under conditions of normal cellularity and also is required to mediate Flt3 ligand-induced NK cell expansion in vivo.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Homeostase/imunologia , Células Matadoras Naturais/citologia , Proteínas de Membrana/fisiologia , Animais , Antígeno CD11c/biossíntese , Diferenciação Celular/imunologia , Proliferação de Células , Sobrevivência Celular/imunologia , Feminino , Humanos , Interleucina-15/deficiência , Interleucina-15/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , Ligantes , Proteínas de Membrana/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Recombinantes/administração & dosagemRESUMO
Natural killer (NK) cells can have potent antileukemic activity following haplo-mismatched, T cell-depleted stem cell transplantations for the treatment of acute myeloid leukemia (AML), but they are not successful in eradicating de novo AML. Here, we have used a mouse model of de novo AML to elucidate the mechanisms by which AML evades NK cell surveillance. NK cells in leukemic mice displayed a marked reduction in the cytolytic granules perforin and granzyme B. Further, as AML progressed, we noted the selective loss of an immature subset of NK cells in leukemic mice and in AML patients. This absence was not due to elimination by cell death or selective reduction in proliferation, but rather to the result of a block in NK cell differentiation. Indeed, NK cells from leukemic mice and humans with AML showed lower levels of TBET and EOMES, transcription factors that are critical for terminal NK cell differentiation. Further, the microRNA miR-29b, a regulator of T-bet and EOMES, was elevated in leukemic NK cells. Finally, deletion of miR-29b in NK cells reversed the depletion of this NK cell subset in leukemic mice. These results indicate that leukemic evasion of NK cell surveillance occurs through miR-mediated dysregulation of lymphocyte development, representing an additional mechanism of immune escape in cancer.
Assuntos
Imunidade Inata , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/imunologia , MicroRNAs/imunologia , RNA Neoplásico/imunologia , Evasão Tumoral , Animais , Linhagem Celular Tumoral , Granzimas/genética , Granzimas/imunologia , Humanos , Células Matadoras Naturais/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Perforina/genética , Perforina/imunologia , RNA Neoplásico/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologiaRESUMO
Molecular characterization of human natural killer (NK) cells will require targeted gene delivery to inhibit and activate specific signaling pathways, yet to our knowledge, an effective means to deliver such products for long-term gene expression without disrupting normal cellular processes has not been described. In this study, we have developed a retroviral strategy to effectively express gene products in the NK cell, whereby its effector functions of cytotoxicity and cytokine production remain intact. Using an EBV/retroviral hybrid vector, we demonstrate infection of human peripheral blood NK cells with simultaneous expression of a marker for infection--the enhanced green fluorescent protein (EGFP)--along with various genes of interest. This technique results in successful infection of the CD56dim NK population that predominates among human peripheral blood NK and is the effector of antibody-dependent cellular cytotoxicity and natural killing. In addition, we demonstrate infection of the CD56bright NK subset as well as the NK-92 cell line. In summary, we have devised an efficient and reproducible methodology for the targeted delivery of gene products to human NK cells that should now provide opportunities to dissect the molecular processes critical to normal NK cell physiology.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/genética , Herpesvirus Humano 4/genética , Células Matadoras Naturais/metabolismo , Retroviridae/genética , Antígeno CD56/imunologia , Células Cultivadas , Expressão Gênica , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Interferon gama/análise , Interferon gama/metabolismo , Células Matadoras Naturais/imunologiaRESUMO
The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase-independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression--but not activity--of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/ß-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase-independent and PP2A-mediated inhibition of JAK2 and ß-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1-positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/ß-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 2/metabolismo , Animais , Apoptose , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ativadores de Enzimas/farmacologia , Cloridrato de Fingolimode , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/enzimologia , Humanos , Janus Quinase 2/metabolismo , Células K562 , Camundongos , Camundongos Transgênicos , Células-Tronco Neoplásicas/enzimologia , Propilenoglicóis/farmacologia , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismoRESUMO
Natural killer (NK) cells contribute to host immunity, including tumor surveillance, through the production of interferon gamma (IFN-gamma). Although there is some knowledge about molecular mechanisms that induce IFN-gamma in NK cells, considerably less is known about the mechanisms that reduce its expression. Here, we investigate the role of the Hlx transcription factor in IFN-gamma production by NK cells. Hlx expression is induced in monokine-activated NK cells, but with delayed kinetics compared to IFN-gamma. Ectopic Hlx expression decreases IFN-gamma synthesis in primary human NK cells and IFN-gamma promoter activity in an NK-like cell line. Hlx protein levels inversely correlate with those of STAT4, a requisite factor for optimal IFN-gamma transcription. Mechanistically, we provide evidence indicating that Hlx overexpression accelerates dephosphorylation and proteasome-dependent degradation of the active Y693-phosphorylated form of STAT4. Thus, Hlx expression in activated NK cells temporally controls and limits the monokine-induced production of IFN-gamma, in part through the targeted depletion of STAT4.
Assuntos
Regulação para Baixo , Proteínas de Homeodomínio/metabolismo , Interferon gama/biossíntese , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Monocinas/farmacologia , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Genes Homeobox/genética , Proteínas de Homeodomínio/genética , Humanos , Interferon gama/genética , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Transcrição STAT4/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
In humans, T cells differentiate in thymus and B cells develop in bone marrow (BM), but the natural killer (NK) precursor cell(s) and site(s) of NK development are unclear. The CD56bright NK subset predominates in lymph nodes (LN) and produces abundant cytokines compared to the cytolytic CD56dim NK cell that predominates in blood. Here, we identify a novel CD34dimCD45RA(+) hematopoietic precursor cell (HPC) that is integrin alpha4beta7bright. CD34dimCD45RA(+)beta7bright HPCs constitute <1% of BM CD34(+) HPCs and approximately 6% of blood CD34(+) HPCs, but >95% of LN CD34(+) HPCs. They reside in the parafollicular T cell regions of LN with CD56bright NK cells, and when stimulated by IL-15, IL-2, or activated LN T cells, they become CD56bright NK cells. The data identify a new NK precursor and support a model of human NK development in which BM-derived CD34dimCD45RA(+)beta7bright HPCs reside in LN where endogenous cytokines drive their differentiation to CD56bright NK cells in vivo.