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.

Neurodesk: an accessible, flexible and portable data analysis environment for reproducible neuroimaging.

Neuroimaging research requires purpose-built analysis software, which is challenging to install and may produce different results across computing environments. The community-oriented, open-source Neurodesk platform ( https://www.neurodesk.org/ ) harnesses a comprehensive and growing suite of neuroimaging software containers. Neurodesk includes a browser-accessible virtual desktop, command-line interface and computational notebook compatibility, allowing for accessible, flexible, portable and fully reproducible neuroimaging analysis on personal workstations, high-performance computers and the cloud.

Genetic and Epigenetic Regulation in Lingo-1: Effects on Cognitive Function and White Matter Microstructure in a Case-Control Study for Schizophrenia.

Leucine-rich repeat and immunoglobulin domain-containing protein (Lingo-1) plays a vital role in a large number of neuronal processes underlying learning and memory, which are known to be disrupted in schizophrenia. However, Lingo-1 has never been examined in the context of schizophrenia. The genetic association of a single-nucleotide polymorphism (SNP, rs3144) and methylation (CpG sites) in the Lingo-1 3'-UTR region was examined, with the testing of cognitive dysfunction and white matter (WM) integrity in a schizophrenia case-control cohort (n = 268/group). A large subset of subjects (97 control and 161 schizophrenia subjects) underwent structural magnetic resonance imaging (MRI) brain scans to assess WM integrity. Frequency of the rs3144 minor allele was overrepresented in the schizophrenia population (p = 0.03), with an odds ratio of 1.39 (95% CI 1.016-1.901). CpG sites surrounding rs3144 were hypermethylated in the control population (p = 0.032) compared to the schizophrenia group. rs3144 genotype was predictive of membership to a subclass of schizophrenia subjects with generalized cognitive deficits (p < 0.05), in addition to having associations with WM integrity (p = 0.018). This is the first study reporting a potential implication of genetic and epigenetic risk factors in Lingo-1 in schizophrenia. Both of these genetic and epigenetic alterations may also have associations with cognitive dysfunction and WM integrity in the context of the schizophrenia pathophysiology.

The Stark Effect: A Tool for the Design of High-Performance Molecular Rectifiers.

Molecular electronic devices offer a path to the miniaturization of electronic circuits and could potentially facilitate novel functionalities that can be embedded into the molecular structure. Given their nanoscale dimensions, device properties are strongly influenced by quantum effects, yet many of these phenomena have been largely overlooked. We investigated the mechanism responsible for current rectification in molecular diodes and found that efficient rectification is achieved by enhancing the Stark effect strength and enabling a large number of molecules to participate in transport. These findings provided insights into the operation of molecular rectifiers and guided the development of high-performance devices via the design of molecules containing polarizable aromatic rings. Our results are consistent for different molecular structures and are expected to have broad applicability to all molecular devices by answering key questions related to charge transport mechanisms in such systems.

Neurodesk: An accessible, flexible, and portable data analysis environment for reproducible neuroimaging.

Neuroimaging data analysis often requires purpose-built software, which can be challenging to install and may produce different results across computing environments. Beyond being a roadblock to neuroscientists, these issues of accessibility and portability can hamper the reproducibility of neuroimaging data analysis pipelines. Here, we introduce the Neurodesk platform, which harnesses software containers to support a comprehensive and growing suite of neuroimaging software (https://www.neurodesk.org/). Neurodesk includes a browser-accessible virtual desktop environment and a command line interface, mediating access to containerized neuroimaging software libraries on various computing platforms, including personal and high-performance computers, cloud computing and Jupyter Notebooks. This community-oriented, open-source platform enables a paradigm shift for neuroimaging data analysis, allowing for accessible, flexible, fully reproducible, and portable data analysis pipelines.

Humidity sensors based on molecular rectifiers.

Ambient humidity plays a key role in the health and well-being of us and our surroundings, making it necessary to carefully monitor and control it. To achieve this goal, several types of instruments based on various materials and operating principles have been developed. Reducing the production costs for such systems without affecting their sensitivity and reliability would allow for broader use and greater efficiency. Organic materials are prime candidates for incorporation in humidity sensors given their extraordinary chemical diversity, low cost, and ease of processing. Here, we designed, assembled and tested humidity sensors based on molecular rectifiers that can electrically transduce the changes in the ambient humidity to offer accurate quantitative information in the range of 0 to 70% relative humidity. Their operation relies on the changes occurring in the electric field experienced by the molecular layer upon absorption of the polar water molecules, resulting in modifications in the height and shape of the tunneling barrier. The response is reversible and reproducible upon multiple cycles and, coupled with the simplicity of the device architecture and manufacturing, makes these nanoscale sensors attractive for incorporation in various applications.

Intermolecular charge transfer enhances the performance of molecular rectifiers.

Molecular-scale diodes made from self-assembled monolayers (SAMs) could complement silicon-based technologies with smaller, cheaper, and more versatile devices. However, advancement of this emerging technology is limited by insufficient electronic performance exhibited by the molecular current rectifiers. We overcome this barrier by exploiting the charge-transfer state that results from co-assembling SAMs of molecules with strong electron donor and acceptor termini. We obtain a substantial enhancement in current rectification, which correlates with the degree of charge transfer, as confirmed by several complementary techniques. These findings provide a previously enexplored method for manipulating the properties of molecular electronic devices by exploiting donor/acceptor interactions. They also serve as a model test platform for the study of doping mechanisms in organic systems. Our devices have the potential for fast widespread adoption due to their low-cost processing and self-assembly onto silicon substrates, which could allow seamless integration with current technologies.

A Fusion Protein Complex that Combines IL-12, IL-15, and IL-18 Signaling to Induce Memory-Like NK Cells for Cancer Immunotherapy.

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)-grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.

Self-Replicating RNAs Drive Protective Anti-tumor T Cell Responses to Neoantigen Vaccine Targets in a Combinatorial Approach.

Historically poor clinical results of tumor vaccines have been attributed to weakly immunogenic antigen targets, limited specificity, and vaccine platforms that fail to induce high-quality polyfunctional T cells, central to mediating cellular immunity. We show here that the combination of antigen selection, construct design, and a robust vaccine platform based on the Synthetically Modified Alpha Replicon RNA Technology (SMARRT), a self-replicating RNA, leads to control of tumor growth in mice. Therapeutic immunization with SMARRT replicon-based vaccines expressing tumor-specific neoantigens or tumor-associated antigen were able to generate polyfunctional CD4+ and CD8+ T cell responses in mice. Additionally, checkpoint inhibitors, or co-administration of cytokine also expressed from the SMARRT platform, synergized to enhance responses further. Lastly, SMARRT-based immunization of non-human primates was able to elicit high-quality T cell responses, demonstrating translatability and clinical feasibility of synthetic replicon technology for therapeutic oncology vaccines.

Stage-Specific Requirement for Eomes in Mature NK Cell Homeostasis and Cytotoxicity.

Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) that mediate antiviral and antitumor responses and require the transcriptional regulator Eomesodermin (Eomes) for early development. However, the role of Eomes and its molecular program in mature NK cell biology is unclear. To address this, we develop a tamoxifen-inducible, type-1-ILC-specific (Ncr1-targeted) cre mouse and combine this with Eomes-floxed mice. Eomes deletion after normal NK cell ontogeny results in a rapid loss of NK cells (but not ILC1s), with a particularly profound effect on penultimately mature stage III NK cells. Mechanisms responsible for stage III reduction include increased apoptosis and impaired maturation from stage II precursors. Induced Eomes deletion also decreases NK cell cytotoxicity and abrogates in vivo rejection of major histocompatibility complex (MHC)-class-I-deficient cells. However, other NK cell functional responses, and stage IV NK cells, are largely preserved. These data indicate that mature NK cells have distinct Eomes-dependent and -independent stages.

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.

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.

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.

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.

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.

Ink-on-probe hydrodynamics in atomic force microscope deposition of liquid inks.

The controlled deposition of attolitre volumes of liquids may engender novel applications such as soft, nano-tailored cell-material interfaces, multi-plexed nano-arrays for high throughput screening of biomolecular interactions, and localized delivery of reagents to reactions confined at the nano-scale. Although the deposition of small organic molecules from an AFM tip, known as dip-pen nanolithography (DPN), is being continually refined, AFM deposition of liquid inks is not well understood, and is often fraught with inconsistent deposition rates. In this work, the variation in feature-size over long term printing experiments for four model inks of varying viscosity is examined. A hierarchy of recurring phenomena is uncovered and there are attributed to ink movement and reorganisation along the cantilever itself. Simple analytical approaches to model these effects, as well as a method to gauge the degree of ink loading using the cantilever resonance frequency, are described. In light of the conclusions, the various parameters which need to be controlled in order to achieve uniform printing are dicussed. This work has implications for the nanopatterning of viscous liquids and hydrogels, encompassing ink development, the design of probes and printing protocols.

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.

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.