The ETS1 Transcription Factor Is Implicated in Human and Murine Intermediate NK Cell Development Stages

Abstract Introduction: While key regulators of early NK cell development and differentiation have been identified, few studies have looked at transcription factor (TF) dynamics and regulatory interactions during subsequent stages of NK cell maturation. Epigenetic landscapes are highly dynamic during cellular differentiation, with TFs playing an important role in the establishment and activation of specific DNA elements, such as enhancers, and subsequent programming of gene expression. ETS1 is a TF that is expressed in adult immune tissues and is critical for the development of lymphoid cells. A role for ETS1 has been described in early NK cell development by activating core transcriptional regulators such as T-BET and ID2. However, despite its continual expression in subsequent stages of NK maturation, the role of ETS1 in NK maturation is not well characterized. Methods and Results: We used FACS to isolate purified human NK cells at various maturation stages as established previously (Freud et. al. Cell Reports, 2016, 16:379-91), ranging from intermediate precursors (Stage 3) through to fully developed and mature peripheral NK cells (Stage 6). Epigenetic programming of cells during lineage maturation allows us to identify critical TFs that are active at each stage of development. We employed Illumina EPIC/850K methylation arrays and RNA sequencing to interrogate epigenetic changes at regulatory elements and TF dynamics at multiple stages along the NK developmental axis. Analysis of TF recognition motifs within hypomethylated regions revealed strong enrichment of specific motif sequences implicating T-box (T-BET and Eomes), along with RUNX and ETS TF families in specific programming of epigenetic patterns during NK development. In studying the expression of TFs that potentially bind these motifs, ETS1 exhibited the highest and most consistent expression throughout NK development. Interestingly, despite consistently high expression, ETS motifs were continually programmed throughout NK maturation, including a significant degree of modification between tonsillar Stages 4A to 4B, where NK cells acquire the ability to produce IFN-γ and significantly gain cytotoxic capability and functional maturity. Among the genes that are upregulated at this stage is the NK-cell-specific gene, NKp46. The progressive hypomethylation of regulatory regions enriched in ETS motifs led us to believe that ETS1 has a continuous role in full NK cell maturation. To test our hypothesis, we developed a novel genetically engineered mouse line with a NK cell intermediate stage-specific conditional deletion of Ets1 mediated by NKp46-driven Cre expression, NKp46-Cre-Ets1fl/fl (NKp46-Ets1fl/fl). This allowed us to study the role of ETS1 in the transition between immature and mature NK cell stages in vivo. Using a comprehensive NK cell development panel for multi-color flow cytometry, we found a drastic reduction of total NK cells in NKp46-Ets1fl/fl mice (n=7) compared to the Ets1fl/fl (n=7) and the NKp46-Cre (n=7) controls in bone marrow (3.2x104 ± 5.9x103, 2.9x105 ± 5.7x104, 2.6x105 ± 8.0x104 total NK cells respectively; p= 0.0007), spleen (3.1x104 ± 7.2x103, 1.2x106 ± 2.4x105, 1.5x106 ± 7.7x105 total NK cells respectively; p= 0.0091) and blood (21 ± 6, 385 ± 35, 185 ± 35 NK cells/uL whole blood respectively; p= 0.0001). Supporting our hypothesis, we indeed observed that while CD11b-/CD27+/- immature NK cell populations in our model are unaltered, the loss of ETS1 is associated with a decrease in CD11b+/CD27+/- mature NK cell populations. Conclusions: Our findings demonstrate that in addition to its role in early NK establishment, persistent ETS1 expression is important in intermediate differentiation stages in both human and murine NK cell development. This constitutes a significant step forward in understanding the role of ETS1 as a master transcriptional regulator in the entire NK cell developmental axis. Current studies are ongoing to dissect the mechanism by which ETS1 affects NK cell development and function in the NKp46-Ets1fl/fl mice. (*EH and JW are recipients of Pelotonia Graduate and Undergraduate student fellowships respectively and contributed equally to this work. This work was partly supported by OCRA, NIH R01 CA159296, NIH R01 CA208353, P01CA95426, R35 CA197734 and OSUCCC Leukemia Tissue Bank and Genetically Engineered Mouse Modeling Core supported by P30CA016058) Disclosures No relevant conflicts of interest to declare.

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T-BET and EOMES Accelerate and Enhance Functional Differentiation of Human Natural Killer Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.732511 ◽

2021 ◽

Vol 12 ◽

Author(s):

Laura Kiekens ◽

Wouter Van Loocke ◽

Sylvie Taveirne ◽

Sigrid Wahlen ◽

Eva Persyn ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Biology ◽

Nk Cell ◽

Current Knowledge ◽

Functional Differentiation ◽

Cell Maturation ◽

Hematopoietic Stem ◽

And Function

T-bet and Eomes are transcription factors that are known to be important in maturation and function of murine natural killer (NK) cells. Reduced T-BET and EOMES expression results in dysfunctional NK cells and failure to control tumor growth. In contrast to mice, the current knowledge on the role of T-BET and EOMES in human NK cells is rudimentary. Here, we ectopically expressed either T-BET or EOMES in human hematopoietic progenitor cells. Combined transcriptome, chromatin accessibility and protein expression analyses revealed that T-BET or EOMES epigenetically represses hematopoietic stem cell quiescence and non-NK lineage differentiation genes, while activating an NK cell-specific transcriptome and thereby drastically accelerating NK cell differentiation. In this model, the effects of T-BET and EOMES are largely overlapping, yet EOMES shows a superior role in early NK cell maturation and induces faster NK receptor and enhanced CD16 expression. T-BET particularly controls transcription of terminal maturation markers and epigenetically controls strong induction of KIR expression. Finally, NK cells generated upon T-BET or EOMES overexpression display improved functionality, including increased IFN-γ production and killing, and especially EOMES overexpression NK cells have enhanced antibody-dependent cellular cytotoxicity. Our findings reveal novel insights on the regulatory role of T-BET and EOMES in human NK cell maturation and function, which is essential to further understand human NK cell biology and to optimize adoptive NK cell therapies.

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An improved method to produce clinical scale natural killer cells from human pluripotent stem cells

10.1101/614792 ◽

2019 ◽

Author(s):

Huang Zhu ◽

Dan S. Kaufman

Keyword(s):

Stem Cells ◽

Nk Cells ◽

Natural Killer ◽

Pluripotent Stem Cells ◽

Nk Cell ◽

Embryonic Stem ◽

Human Pluripotent Stem Cells ◽

Genetically Engineered ◽

Cell Populations ◽

Clinical Scale

AbstractHuman natural killer (NK) cell-based adoptive anti-cancer immunotherapy has gained intense interest with many clinical trials actively recruiting patients to treat a variety of both hematological malignancies and solid tumors. Most of these trials use primary NK cells isolated either from peripheral blood (PB-NK cells) or umbilical cord blood (UCB-NK cells), though these sources require NK cell collection for each patient leading to donor variability and heterogeneity in the NK cell populations. In contrast, NK cells derived human embryonic stem cells (hESC-NK cells) or induced pluripotent stem cells (hiPSC-NK cells) provide more homogeneous cell populations that can be grown at clinical scale, and genetically engineered if desired. These characteristics make hESC/iPSC-derived NK cells an ideal cell population for developing standardized, “off-the-shelf” immunotherapy products. Additionally, production of NK cells from undifferentiated human pluripotent stem cells enables studies to better define pathways that regulate human NK cell development and function. Our group previously established a stromal-free, two-stage culture system to derive NK cells from hESC/hiPSC in vitro followed by clinical-scale expansion of these cells using interleukin-21 expressing artificial antigen-presenting cells. However, prior to differentiation, this method requires single cell adaption of hESCs/hiPSCs which takes months. Recently we optimized this method by adapting the mouse embryonic fibroblast-dependent hESC/hiPSC to feeder-free culture conditions. These feeder-free hESC/hiPSCs are directly used to generate hemato-endothelial precursor cells. This new method produces mature, functional NK cells with higher efficiency to enable rapid production of an essentially unlimited number of homogenous NK cells that can be used for standardized, targeted immunotherapy for the treatment of refractory cancers and infectious diseases.

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The p110δ of PI3K plays a critical role in NK cell terminal maturation and cytokine/chemokine generation

Journal of Experimental Medicine ◽

10.1084/jem.20072327 ◽

2008 ◽

Vol 205 (10) ◽

pp. 2419-2435 ◽

Cited By ~ 69

Author(s):

Hailong Guo ◽

Asanga Samarakoon ◽

Bart Vanhaesebroeck ◽

Subramaniam Malarkannan

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Critical Role ◽

Cell Receptor ◽

Cell Development ◽

Effector Functions ◽

Antiviral Responses

Phosphatidylinositol 3-kinases (PI3Ks) play a critical role in regulating B cell receptor– and T cell receptor–mediated signaling. However, their role in natural killer (NK) cell development and functions is not well understood. Using mice expressing p110δD910A, a catalytically inactive p110δ, we show that these mice had reduced NK cellularity, defective Ly49C and Ly49I NK subset maturation, and decreased CD27High NK numbers. p110δ inactivation marginally impaired NK-mediated cytotoxicity against tumor cells in vitro and in vivo. However, NKG2D, Ly49D, and NK1.1 receptor–mediated cytokine and chemokine generation by NK cells was severely affected in these mice. Further, p110δD910A/D910A NK cell–mediated antiviral responses through natural cytotoxicity receptor 1 were reduced. Analysis of signaling events demonstrates that p110δD910A/D910A NK cells had a reduced c-Jun N-terminal kinase 1/2 phosphorylation in response to NKG2D-mediated activation. These results reveal a previously unrecognized role of PI3K-p110δ in NK cell development and effector functions.

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Differential requirements for the Ets transcription factor Elf-1 in the development of NKT cells and NK cells

Blood ◽

10.1182/blood-2010-09-309468 ◽

2011 ◽

Vol 117 (6) ◽

pp. 1880-1887 ◽

Cited By ~ 26

Author(s):

Hak-Jong Choi ◽

Yanbiao Geng ◽

Hoonsik Cho ◽

Sha Li ◽

Pramod Kumar Giri ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Nk Cells ◽

Nk Cell ◽

Nkt Cells ◽

Cell Development ◽

Intrinsic Defect ◽

Nkt Cell ◽

Ets Family ◽

And Function

Abstract E26 Transformation specific (Ets) family transcription factors control the expression of a large number of genes regulating hematopoietic cell development and function. Two such transcription factors, Ets-1 and myeloid Elf-1–like factor (MEF), have been shown to play critical roles in both natural killer (NK)– and NKT-cell development, but not in the development of conventional T cells. In this study, we address the role of E74-like factor 1 (Elf-1), another Ets family transcription factor that is closely related to MEF but divergent from Ets-1, in NK- and NKT-cell development using Elf-1–deficient (Elf-1−/−) mice. Whereas the proportion of NK cells in Elf-1−/− mice was normal, the proportion of NKT cells was significantly reduced in the thymus and periphery of Elf-1−/− mice compared with wild-type (WT) mice. Although Ets-1–deficient mice lack NKT cells altogether, Elf-1−/− mice exhibited only a partial block in NKT-cell development caused by a cell-intrinsic defect in the selection, survival, and maturation of NKT cells. In addition, residual NKT cells found in Elf-1−/− mice produced less cytokine upon antigen stimulation compared with WT NKT cells. Our data demonstrate that Elf-1 plays an important and nonredundant role in the development and function of NKT cells, but is not involved in NK-cell development.

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Transcription Factors Associated With IL-15 Cytokine Signaling During NK Cell Development

Frontiers in Immunology ◽

10.3389/fimmu.2021.610789 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xiang Wang ◽

Xiang-Yu Zhao

Keyword(s):

Nk Cells ◽

Developmental Stages ◽

Nk Cell ◽

Current Knowledge ◽

Cell Development ◽

Cytokine Signaling ◽

Hematopoietic Stem ◽

Downstream Signaling ◽

Downstream Target

Natural killer (NK) cells are lymphocytes primarily involved in innate immunity and possess important functional properties in anti-viral and anti-tumor responses; thus, these cells have broad potential for clinical utilization. NK cells originate from hematopoietic stem cells (HSCs) through the following two independent and continuous processes: early commitment from HSCs to IL-15-responsive NK cell progenitors (NKPs) and subsequent differentiation into mature NK cells in response to IL-15. IL-15 is the most important cytokine for NK cell development, is produced by both hematopoietic and nonhematopoietic cells, and functions through a distinct delivery process termed transpresentation. Upon being transpresented to NK cells, IL-15 contributes to NK cell development via the activation of several downstream signaling pathways, including the Ras–MEK–MAPK, JAK–STAT5, and PI3K–ATK–mTOR pathways. Nonetheless, the exact role of IL-15 in NK cell development has not been discussed in a consecutive and comprehensive manner. Here, we review current knowledge about the indispensable role of IL-15 in NK cell development and address which cells produce IL-15 to support NK cell development and when IL-15 exerts its function during multiple developmental stages. Specifically, we highlight how IL-15 supports NK cell development by elucidating the distinct transpresentation of IL-15 to NK cells and revealing the downstream target of IL-15 signaling during NK cell development.

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Heterogeneity Among Ly-49C Natural Killer (NK) Cells: Characterization of Highly Related Receptors with Differing Functions and Expression Patterns

Journal of Experimental Medicine ◽

10.1084/jem.184.6.2085 ◽

1996 ◽

Vol 184 (6) ◽

pp. 2085-2090 ◽

Cited By ~ 77

Author(s):

Jack Brennan ◽

Suzanne Lemieux ◽

J. Douglas Freeman ◽

Dixie L. Mager ◽

Fumio Takei

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Expression Patterns ◽

Flow Cytometric Analysis ◽

Cdna Clones ◽

The Novel ◽

Color Flow ◽

Flow Cytometric

Ly-49C is a member of the polymorphic family of murine NK cell inhibitory receptors. The 5E6 antibody that defines a subset of NK cells responsible for the rejection of parental H-2d bone marrow by F1 mice has been shown previously to react with Ly-49C. Here, the 5E6 antibody was found to detect two Ly-49C-related molecules in B6 mice. Two cDNA clones were isolated from B6 NK cells, one identical to previously reported Ly-49CB6 and the other a novel cDNA. The deduced amino acid sequence of the latter differs from that of Ly-49CBALB at only 4 residues, whereas the previously reported Ly-49CB6 differs at 22 residues. Flow cytometric analyses of COS cells transfected with the two cDNAs showed that the 5E6 antibody binds to both Ly-49 molecules, while another anti-Ly-49C antibody, 4LO3311, binds to the newly described Ly-49C but not the previously reported Ly-49CB6. Two-color flow cytometric analysis detected 5E6+4LO3311− as well as 5E6+4LO3311+ subsets of NK cells from B6, but not BALB/c, mice. The level of Ly-49C expression on B6 NK cells detected by the 4LO3311 antibody was substantially lower than that on BALB/c NK cells. Binding specificity of the novel Ly-49CB6 was indistinguishable from that of Ly-49CBALB, whereas no binding was detectable with previously reported Ly-49CB6. These results demonstrate that the newly described Ly-49CB6, not the previously reported Ly-49CB6, is the probable B6 allelic form of Ly49C. The previously reported Ly-49CB6 must be encoded by a separate gene and should be renamed Ly-49I. The implication of these results with respect to the role of Ly-49C in hybrid resistance is discussed.

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The Dual Role of Innate Lymphoid and Natural Killer Cells in Cancer. From Phenotype to Single-Cell Transcriptomics, Functions and Clinical Uses

Cancers ◽

10.3390/cancers13205042 ◽

2021 ◽

Vol 13 (20) ◽

pp. 5042

Author(s):

Stefania Roma ◽

Laura Carpen ◽

Alessandro Raveane ◽

Francesco Bertolini

Keyword(s):

Nk Cells ◽

Single Cell ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Cell Activity ◽

Lymphoid Cells ◽

Cell Populations ◽

Transcriptional Level

The role of innate lymphoid cells (ILCs), including natural killer (NK) cells, is pivotal in inflammatory modulation and cancer. Natural killer cell activity and count have been demonstrated to be regulated by the expression of activating and inhibitory receptors together with and as a consequence of different stimuli. The great majority of NK cell populations have an anti-tumor activity due to their cytotoxicity, and for this reason have been used for cellular therapies in cancer patients. On the other hand, the recently classified helper ILCs are fundamentally involved in inflammation and they can be either helpful or harmful in cancer development and progression. Tissue niche seems to play an important role in modulating ILC function and conversion, as observed at the transcriptional level. In the past, these cell populations have been classified by the presence of specific cellular receptor markers; more recently, due to the advent of single-cell RNA sequencing (scRNA-seq), it has been possible to also explore them at the transcriptomic level. In this article we review studies on ILC (and NK cell) classification, function and their involvement in cancer. We also summarize the potential application of NK cells in cancer therapy and give an overview of the most recent studies involving ILCs and NKs at scRNA-seq, focusing on cancer. Finally, we provide a resource for those who wish to start single-cell transcriptomic analysis on the context of these innate lymphoid cell populations.

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Full Activation of Kinase Protein Kinase B by Phosphoinositide-Dependent Protein Kinase-1 and Mammalian Target of Rapamycin Complex 2 Is Required for Early Natural Killer Cell Development and Survival

Frontiers in Immunology ◽

10.3389/fimmu.2020.617404 ◽

2021 ◽

Vol 11 ◽

Author(s):

Junming He ◽

Jun Zhao ◽

Yuhe Quan ◽

Xinlei Hou ◽

Meixiang Yang ◽

...

Keyword(s):

Protein Kinase ◽

Nk Cells ◽

Nk Cell ◽

Protein Kinase B ◽

Killer Cell ◽

Cell Development ◽

Developmental Defect ◽

Dependent Protein Kinase ◽

Dependent Protein

The role of PI3K-mTOR pathway in regulating NK cell development has been widely reported. However, it remains unclear whether NK cell development depends on the protein kinase B (PKB), which links PI3K and mTOR, perhaps due to the potential redundancy of PKB. PKB has two phosphorylation sites, threonine 308 (T308) and serine 473 (S473), which can be phosphorylated by phosphoinositide-dependent protein kinase-1 (PDK1) and mTORC2, respectively. In this study, we established a mouse model in which PKB was inactivated through the deletion of PDK1 and Rictor, a key component of mTORC2, respectively. We found that the single deletion of PDK1 or Rictor could lead to a significant defect in NK cell development, while combined deletion of PDK1 and Rictor severely hindered NK cell development at the early stage. Notably, ectopic expression of myristoylated PKB significantly rescued this defect. In terms of mechanism, in PDK1/Rictor-deficient NK cells, E4BP4, a transcription factor for NK cell development, was less expressed, and the exogenous supply of E4BP4 could alleviate the developmental defect of NK cell in these mice. Besides, overexpression of Bcl-2 also helped the survival of PDK1/Rictor-deficient NK cells, suggesting an anti-apoptotic role of PKB in NK cells. In summary, complete phosphorylation of PKB at T308 and S473 by PDK1 and mTORC2 is necessary for optimal NK cell development, and PKB regulates NK cell development by promoting E4BP4 expression and preventing cell apoptosis.

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The Role of microRNAs in NK Cell Development and Function

Cells ◽

10.3390/cells10082020 ◽

2021 ◽

Vol 10 (8) ◽

pp. 2020

Author(s):

Arash Nanbakhsh ◽

Subramaniam Malarkannan

Keyword(s):

Nk Cells ◽

Cellular Therapy ◽

Nk Cell ◽

Cell Development ◽

Clinical Use ◽

Effector Functions ◽

Genetic Manipulations ◽

Cytotoxic Proteins ◽

And Function

The clinical use of natural killer (NK) cells is at the forefront of cellular therapy. NK cells possess exceptional antitumor cytotoxic potentials and can generate significant levels of proinflammatory cytokines. Multiple genetic manipulations are being tested to augment the anti-tumor functions of NK cells. One such method involves identifying and altering microRNAs (miRNAs) that play essential roles in the development and effector functions of NK cells. Unique miRNAs can bind and inactivate mRNAs that code for cytotoxic proteins. MicroRNAs, such as the members of the Mirc11 cistron, downmodulate ubiquitin ligases that are central to the activation of the obligatory transcription factors responsible for the production of inflammatory cytokines. These studies reveal potential opportunities to post-translationally enhance the effector functions of human NK cells while reducing unwanted outcomes. Here, we summarize the recent advances made on miRNAs in murine and human NK cells and their relevance to NK cell development and functions.

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The transcription factor c-Myc enhances KIR gene transcription through direct binding to an upstream distal promoter element

Blood ◽

10.1182/blood-2008-07-166389 ◽

2009 ◽

Vol 113 (14) ◽

pp. 3245-3253 ◽

Cited By ~ 32

Author(s):

Frank Cichocki ◽

Rebecca J. Hanson ◽

Todd Lenvik ◽

Michelle Pitt ◽

Valarie McCullar ◽

...

Keyword(s):

Transcription Factor ◽

Nk Cells ◽

Nk Cell ◽

Cell Development ◽

Target Cells ◽

Promoter Element ◽

Kir Genes ◽

Factor C ◽

Distal Promoter ◽

Kir Gene

Abstract The killer cell immunoglobulin-like receptor (KIR) repertoire of natural killer (NK) cells determines their ability to detect infected or transformed target cells. Although epigenetic mechanisms play a role in KIR gene expression, work in the mouse suggests that other regulatory elements may be involved at specific stages of NK-cell development. Here we report the effects of the transcription factor c-Myc on KIR expression. c-Myc directly binds to, and promotes transcription from, a distal element identified upstream of most KIR genes. Binding of endogenous c-Myc to the distal promoter element is significantly enhanced upon interleukin-15 (IL-15) stimulation in peripheral blood NK cells and correlates with an increase in KIR transcription. In addition, the overexpression of c-Myc during NK-cell development promotes transcription from the distal promoter element and contributes to the overall transcription of multiple KIR genes. Our data demonstrate the significance of the 5′ promoter element upstream of the conventional KIR promoter region and support a model whereby IL-15 stimulates c-Myc binding at the distal KIR promoter during NK-cell development to promote KIR transcription. This finding provides a direct link between NK-cell activation signals and KIR expression required for acquisition of effector function during NK-cell education.

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