ILC1s control leukemia stem cell fate and limit development of AML.

Type I innate lymphoid cells (ILC1s) are critical regulators of inflammation and immunity in mammalian tissues. However, their function in cancer is mostly undefined. Here, we show that a high density of ILC1s induces leukemia stem cell (LSC) apoptosis in mice. At a lower density, ILC1s prevent LSCs from differentiating into leukemia progenitors and promote their differentiation into non-leukemic cells, thus blocking the production of terminal myeloid blasts. All of these effects, which require ILC1s to produce interferon-γ after cell-cell contact with LSCs, converge to suppress leukemogenesis in vivo. Conversely, the antileukemia potential of ILC1s wanes when JAK-STAT or PI3K-AKT signaling is inhibited. The relevant antileukemic properties of ILC1s are also functional in healthy individuals and impaired in individuals with acute myeloid leukemia (AML). Collectively, these findings identify ILC1s as anticancer immune cells that might be suitable for AML immunotherapy and provide a potential strategy to treat AML and prevent relapse of the disease.

The IL-15-AKT-XBP1s signaling pathway contributes to effector functions and survival in human NK cells.

Interleukin 15 (IL-15) is one of the most important cytokines that regulate the biology of natural killer (NK) cells1. Here we identified a signaling pathway-involving the serine-threonine kinase AKT and the transcription factor XBP1s, which regulates unfolded protein response genes2,3-that was activated in response to IL-15 in human NK cells. IL-15 induced the phosphorylation of AKT, which led to the deubiquitination, increased stability and nuclear accumulation of XBP1s protein. XBP1s bound to and recruited the transcription factor T-BET to the gene encoding granzyme B, leading to increased transcription. XBP1s positively regulated the cytolytic activity of NK cells against leukemia cells and was also required for IL-15-mediated NK cell survival through an anti-apoptotic mechanism. Thus, the newly identified IL-15-AKT-XBP1s signaling pathway contributes to enhanced effector functions and survival of human NK cells.

CD56 Expression Marks Human Group 2 Innate Lymphoid Cell Divergence from a Shared NK Cell and Group 3 Innate Lymphoid Cell Developmental Pathway.

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.

The Broad Spectrum of Human Natural Killer Cell Diversity.

Natural killer (NK) cells provide protection against infectious pathogens and cancer. For decades it has been appreciated that two major NK cell subsets (CD56bright and CD56dim) exist in humans and have distinct anatomical localization patterns, phenotypes, and functions in immunity. In light of this traditional NK cell dichotomy, it is now clear that the spectrum of human NK cell diversity is much broader than originally appreciated as a result of variegated surface receptor, intracellular signaling molecule, and transcription factor expression; tissue-specific imprinting; and foreign antigen exposure. The recent discoveries of tissue-resident NK cell developmental intermediates, non-NK innate lymphoid cells, and the capacity for NK cells to adapt and differentiate into long-lived memory cells has added further complexity to this field. Here we review our current understanding of the breadth and generation of human NK cell diversity.

A Progenitor Cell Expressing Transcription Factor RORγt Generates All Human Innate Lymphoid Cell Subsets.

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.

Adipocyte CD1d Gene Transfer Induces T Cell Expansion and Adipocyte Inflammation in CD1d Knockout Mice.

CD1d, a lipid Ag-presenting molecule for invariant NKT (iNKT) cells, is abundantly expressed on adipocytes and regulates adipose homeostasis through iNKT cells. CD1d gene expression was restored in visceral adipose tissue adipocytes of CD1d knockout (KO) mice to investigate the interactions between adipocytes and immune cells within adipose tissue. We developed an adipocyte-specific targeting recombinant adeno-associated viral vector, with minimal off-target transgene expression in the liver, to rescue CD1d gene expression in visceral adipose tissue adipocytes of CD1d KO mice, followed by assessment of immune cell alternations in adipose tissue and elucidation of the underlying mechanisms of alteration. We report that adeno-associated virus-mediated gene transfer of CD1d to adipocytes in CD1d KO mice fails to rescue iNKT cells but leads to massive and selective expansion of T cells within adipose tissue, particularly CD8+ T effector cells, that is associated with adipocyte NLRP3 inflammasome activation, dysregulation of adipocyte functional genes, and upregulation of apoptotic pathway proteins. An NLRP3 inhibitor has no effect on T cell phenotypes whereas depletion of CD8+ T cells significantly attenuates inflammasome activation and abolishes the dysregulation of adipocyte functional genes induced by adipocyte CD1d. In contrast, adipocyte overexpression of CD1d fails to induce T cell activation in wild-type mice or in invariant TCR α-chain Jα18 KO mice that have a normal lymphocyte repertoire except for iNKT cells. Our studies uncover an adipocyte CD1d → CD8+ T cell → adipocyte inflammasome cascade, in which CD8+ T cells function as a key mediator of adipocyte inflammation likely induced by an allogeneic response against the CD1d molecule.

Differential Integrin Adhesome Expression Defines Human NK Cell Residency and Developmental Stage.

NK cells are innate immune cells that reside within tissue and circulate in peripheral blood. They interact with a variety of microenvironments, yet how NK cells engage with these varied microenvironments is not well documented. The adhesome represents a molecular network of defined and predicted integrin-mediated signaling interactions. In this study, we define the integrin adhesome expression profile of NK cells from human tonsil, peripheral blood, and those derived from human hematopoietic precursors through stromal cell coculture systems. We report that the site of cell isolation and NK cell developmental stage dictate differences in expression of adhesome associated genes and proteins. Furthermore, we define differences in cortical actin content associated with differential expression of actin regulating proteins, suggesting that differences in adhesome expression are associated with differences in cortical actin homeostasis. These data provide understanding of the diversity of human NK cell populations and how they engage with their microenvironment.

Acute Myeloid Leukemia Alters Group 1 Innate Lymphoid Cell Differentiation from a Common Precursor.

NK cells are known to be developmentally blocked and functionally inhibited in patients with acute myeloid leukemia (AML), resulting in poor clinical outcomes. In this study, we demonstrate that whereas NK cells are inhibited, closely related type 1 innate lymphoid cells (ILC1s) are enriched in the bone marrow of leukemic mice and in patients with AML. Because NK cells and ILC1s share a common precursor (ILCP), we asked if AML acts on the ILCP to alter developmental potential. A combination of ex vivo and in vivo studies revealed that AML skewing of the ILCP toward ILC1s and away from NK cells represented a major mechanism of ILC1 generation. This process was driven by AML-mediated activation of the aryl hydrocarbon receptor (AHR), a key transcription factor in ILCs, as inhibition of AHR led to decreased numbers of ILC1s and increased NK cells in the presence of AML. These results demonstrate a mechanism of ILC developmental skewing in AML and support further preclinical study of AHR inhibition in restoring normal NK cell development and function in the setting of AML.

Activation of the Intracellular Pattern Recognition Receptor NOD2 Promotes Acute Myeloid Leukemia (AML) Cell Apoptosis and Provides a Survival Advantage in an Animal Model of AML.

TLRs, a family of membrane-bound pattern recognition receptors found on innate immune cells, have been well studied in the context of cancer therapy. Activation of these receptors has been shown to induce inflammatory anticancer events, including differentiation and apoptosis, across a wide variety of malignancies. In contrast, intracellular pattern recognition receptors such as NOD-like receptors have been minimally studied. NOD2 is a member of the NOD-like receptor family that initiates inflammatory signaling in response to the bacterial motif muramyl dipeptide. In this study, we examined the influence of NOD2 in human acute myeloid leukemia (AML) cells, demonstrating that IFN-γ treatment upregulated the expression of NOD2 signaling pathway members SLC15A3 and SLC15A4, downstream signaling kinase RIPK2, and the NOD2 receptor itself. This priming allowed for effective induction of caspase-1-dependent cell death upon treatment with muramyl tripeptide phosphatidylethanolamine (MTP-PE), a synthetic ligand for NOD2. Furthermore, the combination of MTP-PE and IFN-γ on AML blasts generated an inflammatory cytokine profile and activated NK cells. In a murine model of AML, dual treatment with MTP-PE and IFN-γ led to a significant increase in mature CD27- CD11b+ NK cells as well as a significant reduction in disease burden and extended survival. These results suggest that NOD2 activation, primed by IFN-γ, may provide a novel therapeutic option for AML.

Notch Regulates Innate Lymphoid Cell Plasticity during Human NK Cell Development.

Human NK cells develop in tonsils through discrete NK cell developmental intermediates (NKDIs), yet the mechanistic regulation of this process is unclear. We demonstrate that Notch activation in human tonsil-derived stage 3 (CD34-CD117+CD94-NKp80-) and 4A (CD34-CD117+/-CD94+NKp80-) NKDIs promoted non-NK innate lymphoid cell differentiation at the expense of NK cell differentiation. In contrast, stage 4B (CD34-CD117+/-CD94+NKp80+) NKDIs were NK cell lineage committed despite Notch activation. Interestingly, whereas NK cell functional maturation from stage 3 and 4A NKDIs was independent of Notch activation, the latter was required for high NKp80 expression and a stage 4B-like phenotype by the NKDI-derived NK cells. The Notch-dependent effects required simultaneous engagement with OP9 stromal cells and were also stage-specific, with NOTCH1 and NOTCH2 receptors regulating stage 3 NKDIs and NOTCH1 primarily regulating stage 4A NKDIs. These data establish stage-specific and stromal-dependent roles for Notch in regulating human NK cell developmental plasticity and maturation.

Human AML activates the aryl hydrocarbon receptor pathway to impair NK cell development and function.

Acute myeloid leukemia (AML) can evade the mouse and human innate immune system by suppressing natural killer (NK) cell development and NK cell function. This is driven in part by the overexpression of microRNA (miR)-29b in the NK cells of AML patients, but how this occurs is unknown. In the current study, we demonstrate that the transcription factor aryl hydrocarbon receptor (AHR) directly regulates miR-29b expression. We show that human AML blasts activate the AHR pathway and induce miR-29b expression in NK cells, thereby impairing NK cell maturation and NK cell function, which can be reversed by treating NK cells with an AHR antagonist. Finally, we show that inhibition of constitutive AHR activation in AML blasts lowers their threshold for apoptosis and decreases their resistance to NK cell cytotoxicity. Together, these results identify the AHR pathway as a molecular mechanism by which AML impairs NK cell development and function. The results lay the groundwork in establishing AHR antagonists as potential therapeutic agents for clinical development in the treatment of AML.

The Raf Kinase Inhibitor Sorafenib Inhibits JAK-STAT Signal Transduction in Human Immune Cells.

Sorafenib is an oral multikinase inhibitor that was originally developed as a Raf kinase inhibitor. We hypothesized that sorafenib would also have inhibitory effects on cytokine signaling pathways in immune cells. PBMCs from normal donors were treated with varying concentrations of sorafenib and stimulated with IFN-α or IL-2. Phosphorylation of STAT1 and STAT5 was measured by flow cytometry and confirmed by immunoblot analysis. Changes in IFN-α- and IL-2-stimulated gene expression were measured by quantitative PCR, and changes in cytokine production were evaluated by ELISA. Cryopreserved PBMCs were obtained from cancer patients before and after receiving 400 mg sorafenib twice daily. Patient PBMCs were thawed, stimulated with IL-2 or IFN-α, and evaluated for phosphorylation of STAT1 and STAT5. Pretreatment of PBMCs with 10 µM sorafenib decreased STAT1 and STAT5 phosphorylation after treatment with IFN-α or IL-2. This inhibitory effect was observed in PBMCs from healthy donors over a range of concentrations of sorafenib (5-20 µM), IL-2 (2-24 nM), and IFN-α (10(1)-10(6) U/ml). This effect was observed in immune cell subsets, including T cells, B cells, NK cells, regulatory T cells, and myeloid-derived suppressor cells. Pretreatment with sorafenib also inhibited PBMC expression of IFN-α- and IL-2-regulated genes and inhibited NK cell production of IFN-γ, RANTES, MIP1-α, and MIG in response to IFN-α stimulation. PBMCs from patients receiving sorafenib therapy showed decreased responsiveness to IL-2 and IFN-α treatment. Sorafenib is a Raf kinase inhibitor that could have off-target effects on cytokine-induced signal transduction in immune effector cells.

PTEN is a negative regulator of NK cell cytolytic function.

Human NK cells are characterized by their ability to initiate an immediate and direct cytolytic response to virally infected or malignantly transformed cells. Within human peripheral blood, the more mature CD56(dim) NK cell efficiently kills malignant targets at rest, whereas the less mature CD56(bright) NK cells cannot. In this study, we show that resting CD56(bright) NK cells express significantly more phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein when compared with CD56(dim) NK cells. Consistent with this, forced overexpression of PTEN in NK cells resulted in decreased cytolytic activity, and loss of PTEN in CD56(bright) NK cells resulted in elevated cytolytic activity. Comparable studies in mice showed PTEN overexpression did not alter NK cell development or NK cell-activating and inhibitory receptor expression yet, as in humans, did decrease expression of downstream NK activation targets MAPK and AKT during early cytolysis of tumor target cells. Confocal microscopy revealed that PTEN overexpression disrupts the NK cell's ability to organize immunological synapse components including decreases in actin accumulation, polarization of the microtubule organizing center, and the convergence of cytolytic granules. In summary, our data suggest that PTEN normally works to limit the NK cell's PI3K/AKT and MAPK pathway activation and the consequent mobilization of cytolytic mediators toward the target cell and suggest that PTEN is among the active regulatory components prior to human NK cells transitioning from the noncytolytic CD56(bright) NK cell to the cytolytic CD56(dim) NK cells.

Overexpression of miR-155 causes expansion, arrest in terminal differentiation and functional activation of mouse natural killer cells.

It is known that microRNAs (miRs) are involved in lymphocyte development, homeostasis, activation, and occasionally malignant transformation. In this study, a miR-155 transgene (tg) was driven to be overexpressed off of the lck promoter in order to assess its effects on natural killer (NK) cell biology in vivo. miR-155 tg mice have an increase in NK-cell number with an excess of the CD11b(low)CD27(high) NK subset, indicative of a halt in terminal NK-cell differentiation that proved to be intrinsic to the cell itself. The increase in NK cells results, in part, from improved survival in medium alone and enhanced expansion with endogenous or exogenous interleukin 15. Phenotypic and functional data from miR-155 tg NK cells showed constitutive activation and enhanced target cell conjugation, resulting in more potent antitumor activity in vitro and improved survival of lymphoma-bearing mice in vivo when compared with wild type NK cells. The enhanced NK-cell survival, expansion, activation, and tumor control that result from overexpression of miR-155 in NK cells could be explained, in part, via diminished expression of the inositol phosphatase SHIP1 and increased activation of ERK and AKT kinases. Thus, the regulation of miR-155 is important for NK-cell development, homeostasis, and activation.

Eradicating acute myeloid leukemia in a Mll(PTD/wt):Flt3(ITD/wt) murine model: a path to novel therapeutic approaches for human disease.

The coexpression of the MLL partial tandem duplication (PTD) and the FLT3 internal tandem duplication (ITD) mutations associate with a poor outcome in cytogenetically normal acute myeloid leukemia (AML). In mice, a double knock-in (dKI) of Mll(PTD/wt) and Flt3(ITD/wt) mutations induces spontaneous AML with an increase in DNA methyltransferases (Dnmt1, 3a, and 3b) and global DNA methylation index, thereby recapitulating its human AML counterpart. We determined that a regulator of Dnmts, miR-29b, is downregulated in bone marrow of dKI AML mice. Bortezomib exerted a dose-dependent increase in miR-29b expression in AML blasts ex vivo, followed by decreased Dnmts, reduced proliferation, and increased apoptosis. In vivo, bortezomib was not active against dKI AML, yet liposomal-encapsulated bortezomib, as a single agent, reversed downregulation of miR-29b in vivo and induced a long-term (90-day) disease-free remission in 80% of dKI AML mice that exhibited high leukemic burden at the start of therapy, yet showed no signs of relapse at autopsy. Taken together, these data support that liposomal bortezomib, as a single agent, eradicates Mll(PTD/wt):Flt3(ITD/wt) AML in mouse and may represent a powerful and potentially curative approach to high-risk human disease.

IL-12 enhances the antitumor actions of trastuzumab via NK cell IFN-γ production.

The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 µg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.

Effects of epigenetic therapy on natural killer cell function and development in hematologic malignancy.

Epigenetic therapy is an emerging field in the treatment of human cancer, including hematologic malignancies. This class of therapeutic agents approved by the US Food and Drug Administration for cancer treatment includes DNA hypomethylating agents, histone deacetylase inhibitors, IDH1/2 inhibitors, EZH2 inhibitors, and numerous preclinical targets/agents. Most studies measuring the biological effects of epigenetic therapy focus their attention on either their direct cytotoxic effects on malignant cells or their effects on modifying tumor cell antigen expression, exposing them to immune surveillance mechanisms. However, a growing body of evidence suggests that epigenetic therapy also has effects on the development and function of the immune system, including natural killer cells, which can alter their response to cancer cells. In this review, we summarize the body of literature studying the effects of different classes of epigenetic therapy on the development and/or function of natural killer cells.

Deletion of Glycogen Synthase Kinase 3 Beta Reprograms NK Cell Metabolism.

Loss of cytotoxicity and defective metabolism are linked to glycogen synthase kinase 3 beta (GSK3ß) overexpression in natural killer (NK) cells from patients with acute myeloid leukemia or from healthy donors after expansion ex vivo with IL-15. Drug inhibition of GSK3ß in these NK cells improves their maturation and cytotoxic activity, but the mechanisms of GSK3ß-mediated dysfunction have not been well studied. Here, we show that expansion of NK cells with feeder cells expressing membrane-bound IL-21 maintained normal GSK3ß levels, allowing us to study GSK3ß function using CRISPR gene editing. We deleted GSK3B and expanded paired-donor knockout and wild-type (WT) NK cells and then assessed transcriptional and functional alterations induced by loss of GSK3ß. Surprisingly, our data showed that deletion of GSK3B did not alter cytotoxicity, cytokine production, or maturation (as determined by CD57 expression). However, GSK3B-KO cells demonstrated significant changes in expression of genes related to rRNA processing, cell proliferation, and metabolic function, suggesting possible metabolic reprogramming. Next, we found that key genes downregulated in GSK3B-KO NK cells were upregulated in GSK3ß-overexpressing NK cells from AML patients, confirming this correlation in a clinical setting. Lastly, we measured cellular energetics and observed that GSK3B-KO NK cells exhibited 150% higher spare respiratory capacity, a marker of metabolic fitness. These findings suggest a role for GSK3ß in regulating NK cell metabolism.

Single-cell RNA sequencing identifies a population of human liver-type ILC1s.

Group 1 innate lymphoid cells (ILCs) comprise a heterogeneous family of cytotoxic natural killer (NK) cells and ILC1s. We identify a population of "liver-type" ILC1s with transcriptional, phenotypic, and functional features distinct from those of conventional and liver-resident NK cells as well as from other previously described human ILC1 subsets. LT-ILC1s are CD49a+CD94+CD200R1+, express the transcription factor T-BET, and do not express the activating receptor NKp80 or the transcription factor EOMES. Similar to NK cells, liver-type ILC1s produce IFN-γ, TNF-α, and GM-CSF; however, liver-type ILC1s also produce IL-2 and lack perforin and granzyme-B. Liver-type ILC1s are expanded in cirrhotic liver tissues, and they can be produced from blood-derived ILC precursors in vitro in the presence of TGF-ß1 and liver sinusoidal endothelial cells. Cells with similar signature and function can also be found in tonsil and intestinal tissues. Collectively, our study identifies and classifies a population of human cross-tissue ILC1s.