MicroRNA-142 Is Critical for the Homeostasis and Function of Type 1 Innate Lymphoid Cells.

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.

MicroRNA-146a deficiency enhances host protection against murine cytomegalovirus.

Natural killer (NK) cells are innate lymphoid cells that protect a host from viral infections and malignancies. MicroRNA-146a (miR-146a) is an important regulator of immune function that is highly expressed in NK cells and is further upregulated during murine cytomegalovirus (MCMV) infection. Here we utilized mice with a global targeted deletion of miR-146a to understand its impact on the innate immune responses to MCMV infection. MiR-146a-/- mice were protected from lethal MCMV infection, which was intrinsic to the hematopoietic compartment based on bone marrow chimera experiments. NK cell depletion abrogated this protection, implicating NK cells as critical for the miR-146a-/- protection from MCMV. Surprisingly, NK cells from miR-146a-deficient mice were largely similar to control NK cells with respect to development, maturation, trafficking, and effector functions. However, miR-146a-/- mice had increased NK cell numbers and frequency of the most mature Stage IV (CD27-CD11b+) NK cells in the liver at baseline, enhanced STAT1 phosphorylation, and increased selective expansion of Ly49H+ NK cells and T cells during MCMV infection. This study demonstrates a critical role for miR-146a in the host response to MCMV, arising from mechanisms that include increased NK cell numbers and early T-cell expansion.

PTEN regulates natural killer cell trafficking in vivo.

Phosphatase and tensin homolog (PTEN) is a critical negative regulator of the phosphoinositide-3 kinase pathway, members of which play integral roles in natural killer (NK) cell development and function. However, the functions of PTEN in NK cell biology remain unknown. Here, we used an NK cell-specific PTEN-deletion mouse model to define the ramifications of intrinsic NK cell PTEN loss in vivo. In these mice, there was a significant defect in NK cell numbers in the bone marrow and peripheral organs despite increased proliferation and intact peripheral NK cell maturation. Unexpectedly, we observed a significant expansion of peripheral blood NK cells and the premature egress of NK cells from the bone marrow. The altered trafficking of NK cells from peripheral organs into the blood was due to selective hyperresponsiveness to the blood localizing chemokine S1P. To address the importance of this trafficking defect to NK cell immune responses, we investigated the ability of PTEN-deficient NK cells to traffic to a site of tumor challenge. PTEN-deficient NK cells were defective at migrating to distal tumor sites but were more effective at clearing tumors actively introduced into the peripheral blood. Collectively, these data identify PTEN as an essential regulator of NK cell localization in vivo during both homeostasis and malignancy.

Cytokine-Induced Memory-Like Differentiation Enhances Unlicensed Natural Killer Cell Antileukemia and FcγRIIIa-Triggered Responses.

Cytokine-induced memory-like natural killer (NK) cells differentiate after short-term preactivation with IL-12, IL-15, and IL-18 and display enhanced effector function in response to cytokines or tumor targets for weeks after the initial preactivation. Conventional NK cell function depends on a licensing signal, classically delivered by an inhibitory receptor engaging its cognate MHC class I ligand. How licensing status integrates with cytokine-induced memory-like NK cell responses is unknown. We investigated this interaction using killer cell immunoglobulin-like receptor- and HLA-genotyped primary human NK cells. Memory-like differentiation resulted in enhanced IFN-γ production triggered by leukemia targets or FcγRIIIa ligation within licensed NK cells, which exhibited the highest functionality of the NK cell subsets interrogated. IFN-γ production by unlicensed memory-like NK cells was also enhanced to a level comparable with that of licensed control NK cells. Mechanistically, differences in responses to FcγRIIIa-based triggering were not explained by alterations in key signaling intermediates, indicating that the underlying biology of memory-like NK cells is distinct from that of adaptive NK cells in human cytomegalovirus-positive individuals. Additionally, memory-like NK cells responded robustly to cytokine receptor restimulation with no impact of licensing status. These results demonstrate that both licensed and unlicensed memory-like NK cell populations have enhanced functionality, which may be translated to improve leukemia immunotherapy.

Transcriptional and post-transcriptional regulation of NK cell development and function.

Natural killer (NK) cells are specialized innate lymphoid cells that survey against viral infections and malignancy. Numerous advances have improved our understanding of the molecular mechanisms that control NK cell development and function over the past decade. These include both studies on the regulatory effects of transcription factors and translational repression via microRNAs. In this review, we summarize our current knowledge of DNA-binding transcription factors that regulate gene expression and thereby orchestrate NK cell development and activation, with an emphasis on recent discoveries. Additionally, we highlight our understanding of how RNA-binding microRNAs fine tune the NK cell molecular program. We also underscore the large number of open questions in the field that are now being addressed using new technological approaches and genetically engineered model organisms. Ultimately, a deeper understanding of the basic molecular biology of NK cells will facilitate new strategies to manipulate NK cells for the treatment of human disease.

MicroRNA-15/16 Antagonizes Myb To Control NK Cell Maturation.

NK cells develop in the bone marrow and complete their maturation in peripheral organs, but the molecular events controlling maturation are incompletely understood. The miR-15/16 family of microRNA regulates key cellular processes and is abundantly expressed in NK cells. In this study, we identify a critical role for miR-15/16 in the normal maturation of NK cells using a mouse model of NK-specific deletion, in which immature NK cells accumulate in the absence of miR-15/16. The transcription factor c-Myb (Myb) is expressed preferentially by immature NK cells, is a direct target of miR-15/16, and is increased in 15a/16-1 floxed knockout NK cells. Importantly, maturation of 15a/16-1 floxed knockout NK cells was rescued by Myb knockdown. Moreover, Myb overexpression in wild-type NK cells caused a defective NK cell maturation phenotype similar to deletion of miR-15/16, and Myb overexpression enforces an immature NK cell transcriptional profile. Thus, miR-15/16 regulation of Myb controls the NK cell maturation program.

microRNA management of NK-cell developmental and functional programs.

NK cells are innate lymphoid cells that are critical for host defense against infection, and mediate anti-tumor responses. MicroRNAs (miRNAs) are a large family of small noncoding RNAs that target the 3' untranslated region (UTR) of mRNAs, thereby attenuating protein translation. The expression of miRNAs within human peripheral blood and mouse splenic NK cells has been cataloged, with the majority of the miRNA sequence pool represented in the top 60 most abundantly expressed miRNAs. Global miRNA deficiency within NK cells has confirmed their critical role in NK-cell biology, including defects in NK-cell development and altered functionality. Studies using gain- and loss-of-function of individual miRNAs in NK cells have demonstrated the role of specific miRNAs in regulating NK-cell development, maturation, and activation. miRNAs also regulate fundamental NK-cell processes including cytokine production, cytotoxicity, and proliferation. This review provides an update on the intrinsic miRNA regulation of NK cells, including miRNA expression profiles, as well as their impact on NK-cell biology. Additional profiling is needed to better understand miRNA expression within NK-cell developmental intermediates, subsets, tissues, and in the setting of disease. Furthermore, key open questions in the field as well as technical challenges in the study of miRNAs in NK cells are highlighted.

MicroRNA-155 tunes both the threshold and extent of NK cell activation via targeting of multiple signaling pathways.

NK cells are innate lymphocytes important for host defense against viral infections and malignancy. However, the molecular programs orchestrating NK cell activation are incompletely understood. MicroRNA-155 (miR-155) is markedly upregulated following cytokine activation of human and mouse NK cells. Surprisingly, mature human and mouse NK cells transduced to overexpress miR-155, NK cells from mice with NK cell-specific miR-155 overexpression, and miR-155(-/-) NK cells all secreted more IFN-γ compared with controls. Investigating further, we found that activated NK cells with miR-155 overexpression had increased per-cell IFN-γ with normal IFN-γ(+) percentages, whereas greater percentages of miR-155(-/-) NK cells were IFN-γ(+). In vivo murine CMV-induced IFN-γ expression by NK cells in these miR-155 models recapitulated the in vitro phenotypes. We performed unbiased RNA-induced silencing complex sequencing on wild-type and miR-155(-/-) NK cells and found that mRNAs targeted by miR-155 were enriched in NK cell activation signaling pathways. Using specific inhibitors, we confirmed these pathways were mechanistically involved in regulating IFN-γ production by miR-155(-/-) NK cells. These data indicate that miR-155 regulation of NK cell activation is complex and that miR-155 functions as a dynamic tuner for NK cell activation via both setting the activation threshold as well as controlling the extent of activation in mature NK cells. In summary, miR-155(-/-) NK cells are more easily activated, through increased expression of proteins in the PI3K, NF-κB, and calcineurin pathways, and miR-155(-/-) and 155-overexpressing NK cells exhibit increased IFN-γ production through distinct cellular mechanisms.

Preactivation with IL-12, IL-15, and IL-18 induces CD25 and a functional high-affinity IL-2 receptor on human cytokine-induced memory-like natural killer cells.

Natural killer (NK) cells are effector lymphocytes that are under clinical investigation for the adoptive immunotherapy of hematologic malignancies, especially acute myeloid leukemia. Recent work in mice has identified innate memory-like properties of NK cells. Human NK cells also exhibit memory-like properties, and cytokine-induced memory-like (CIML) NK cells are generated via brief preactivation with IL-12, IL-15, and IL-18, which later exhibit enhanced functionality upon restimulation. However, the optimal cytokine receptors and signals for maintenance of enhanced function and homeostasis after preactivation remain unclear. Here, we show that IL-12, IL-15, and IL-18 preactivation induces a rapid and prolonged expression of CD25, resulting in a functional high-affinity IL-2 receptor (IL-2Rαßγ) that confers responsiveness to picomolar concentrations of IL-2. The expression of CD25 correlated with STAT5 phosphorylation in response to picomolar concentrations of IL-2, indicating the presence of a signal-competent IL-2Rαßγ. Furthermore, picomolar concentrations of IL-2 acted synergistically with IL-12 to costimulate IFN-γ production by preactivated NK cells, an effect that was CD25 dependent. Picomolar concentrations of IL-2 also enhanced NK cell proliferation and cytotoxicity via the IL-2Rαßγ. Further, after adoptive transfer into immunodeficient NOD-SCID-γc(-/-) mice, human cytokine-preactivated NK cells expand preferentially in response to exogenous IL-2. Collectively, these data demonstrate that human CIML NK cells respond to IL-2 via IL-2Rαßγ with enhanced survival and functionality, and they provide additional rationale for immunotherapeutic strategies that include brief cytokine preactivation before adoptive NK cell transfer, followed by low-dose IL-2 therapy.

Cytokine activation induces human memory-like NK cells.

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.

MicroRNA-deficient NK cells exhibit decreased survival but enhanced function.

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.

Next-generation sequencing identifies the natural killer cell microRNA transcriptome.

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.

Human NK cells: SET to kill.

In this issue of Blood, Trotta et al define a novel mechanism of human granzyme B and perforin regulation and identify 2 new signaling players involved in modulating NK cytotoxicity.

Natural killer cell regulation by microRNAs in health and disease.

Natural killer (NK) cells are innate immune lymphocytes that are critical for normal host defense against infections and mediate antitumor immune responses. MicroRNAs (miRNAs) are a family of small, noncoding RNAs that posttranscriptionally regulate the majority of cellular processes and pathways. Our understanding of how miRNAs regulate NK cells biology is limited, but recent studies have provided novel insight into their expression by NK cells, and how they contribute to the regulation of NK cell development, maturation, survival, and effector function. Here, we review the expression of miRNAs by NK cells, their contribution to cell intrinsic and extrinsic control of NK cell development and effector response, and their dysregulation in NK cell malignancies.

In vivo role of Flt3 ligand and dendritic cells in NK cell homeostasis.

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.

Management of the American Heart Association's guidelines for orthodontic treatment of patients at risk for infective endocarditis.

INTRODUCTION: For over 50 years, the American Heart Association has made recommendations for the prevention of infective endocarditis. The first guidelines were published in 1955; since then, they have been updated 9 times, most recently in 2007. There is still confusion about which orthodontic procedures are most prone to generate bacteremias and lead to infective endocarditis in susceptible patients. The aim of this study was to conduct a survey to determine orthodontists' knowledge, attitudes, and in-office behaviors regarding the American Heart Association's guidelines. METHODS: A 4-page online survey consisting of 3 sections was sent to members of the American Association of Orthodontists by using a random number generator. The first section consisted of demographic information, the second consisted of questions about the respondents' practice characteristics, and the third included questions about the respondents' knowledge and management of the treatment of patients at risk for infective endocarditis. There were 78 responses. RESULTS AND CONCLUSIONS: Orthodontists are screening for cardiac problems in the patient's medical history but to a lesser extent are requesting written medical clearance from the patient's physician before starting orthodontic treatment. Many of the orthodontists surveyed believed that their knowledge of the American Heart Association's guidelines and management of high-risk patients was in the good-to-excellent range. Orthodontists recommend antibiotic prophylaxis most frequently during band placement and removal. Patients at risk for infective endocarditis are somewhat likely to inquire about possible treatment sequelae associated with previous cardiac problems.

CD56bright NK cells exhibit potent antitumor responses following IL-15 priming.

NK cells, lymphocytes of the innate immune system, are important for defense against infectious pathogens and cancer. Classically, the CD56dim NK cell subset is thought to mediate antitumor responses, whereas the CD56bright subset is involved in immunomodulation. Here, we challenge this paradigm by demonstrating that brief priming with IL-15 markedly enhanced the antitumor response of CD56bright NK cells. Priming improved multiple CD56bright cell functions: degranulation, cytotoxicity, and cytokine production. Primed CD56bright cells from leukemia patients demonstrated enhanced responses to autologous blasts in vitro, and primed CD56bright cells controlled leukemia cells in vivo in a murine xenograft model. Primed CD56bright cells from multiple myeloma (MM) patients displayed superior responses to autologous myeloma targets, and furthermore, CD56bright NK cells from MM patients primed with the IL-15 receptor agonist ALT-803 in vivo displayed enhanced ex vivo functional responses to MM targets. Effector mechanisms contributing to IL-15-based priming included improved cytotoxic protein expression, target cell conjugation, and LFA-1-, CD2-, and NKG2D-dependent activation of NK cells. Finally, IL-15 robustly stimulated the PI3K/Akt/mTOR and MEK/ERK pathways in CD56bright compared with CD56dim NK cells, and blockade of these pathways attenuated antitumor responses. These findings identify CD56bright NK cells as potent antitumor effectors that warrant further investigation as a cancer immunotherapy.

Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia.

Natural killer (NK) cells are an emerging cellular immunotherapy for patients with acute myeloid leukemia (AML); however, the best approach to maximize NK cell antileukemia potential is unclear. Cytokine-induced memory-like NK cells differentiate after a brief preactivation with interleukin-12 (IL-12), IL-15, and IL-18 and exhibit enhanced responses to cytokine or activating receptor restimulation for weeks to months after preactivation. We hypothesized that memory-like NK cells exhibit enhanced antileukemia functionality. We demonstrated that human memory-like NK cells have enhanced interferon-γ production and cytotoxicity against leukemia cell lines or primary human AML blasts in vitro. Using mass cytometry, we found that memory-like NK cell functional responses were triggered against primary AML blasts, regardless of killer cell immunoglobulin-like receptor (KIR) to KIR-ligand interactions. In addition, multidimensional analyses identified distinct phenotypes of control and memory-like NK cells from the same individuals. Human memory-like NK cells xenografted into mice substantially reduced AML burden in vivo and improved overall survival. In the context of a first-in-human phase 1 clinical trial, adoptively transferred memory-like NK cells proliferated and expanded in AML patients and demonstrated robust responses against leukemia targets. Clinical responses were observed in five of nine evaluable patients, including four complete remissions. Thus, harnessing cytokine-induced memory-like NK cell responses represents a promising translational immunotherapy approach for patients with AML.

The IL-15-Based ALT-803 Complex Enhances FcγRIIIa-Triggered NK Cell Responses and In Vivo Clearance of B Cell Lymphomas.

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.

Utilizing cytokines to function-enable human NK cells for the immunotherapy of cancer.

Natural killer (NK) cells are innate lymphoid cells important for host defense against pathogens and mediate antitumor immunity. Cytokine receptors transduce important signals that regulate proliferation, survival, activation status, and trigger effector functions. Here, we review the roles of major cytokines that regulate human NK cell development, survival, and function, including IL-2, IL-12, IL-15, IL-18, and IL-21, and their translation to the clinic as immunotherapy agents. We highlight a recent development in NK cell biology, the identification of innate NK cell memory, and focus on cytokine-induced memory-like (CIML) NK cells that result from a brief, combined activation with IL-12, IL-15, and IL-18. This activation results in long lived NK cells that exhibit enhanced functionality when they encounter a secondary stimulation and provides a new approach to enable NK cells for enhanced responsiveness to infection and cancer. An improved understanding of the cellular and molecular aspects of cytokine-cytokine receptor signals has led to a resurgence of interest in the clinical use of cytokines that sustain and/or activate NK cell antitumor potential. In the future, such strategies will be combined with negative regulatory signal blockade and enhanced recognition to comprehensively enhance NK cells for immunotherapy.