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In vivo establishment and long-term persistence of a heterogeneous memory or an adaptive NK cell pool represents a functional adaptation to human cytomegalovirus (HCMV) infection in humans. Memory NK cells are commonly identified by lack of the FcεRIγ signalling chain, variably associated to the preferential but not completely overlapping expression of the HLA-E receptor NKG2C and CD57 maturation marker. Although characterized by selective hyperresponsiveness to IgG stimulation, the impact of the CD16/antibody interaction in regulating the establishment/maintenance and size, and in determining the relative abundance of this population, is still under investigation. Memory NK cell subset ex vivo profile and in vitro responsiveness to CD16 stimulation was evaluated in HCMV+ healthy donors and in patients affected by immune thrombocytopenia (ITP), an antibody-mediated autoimmune disease. We identified the FcεRIγ− NKG2C+CD57+ memory NK cell subset, whose abundance is uniquely associated with anti-HCMV antibody levels in healthy seropositive donors, and which is significantly expanded in ITP patients. This fully mature memory subset robustly and selectively expands in vitro in response to mAb-opsonized targets or ITP-derived platelets and displays superior CD16-dependent IFNγ production. Our work identifies opsonizing antibodies as a host-dependent factor that shapes HCMV-driven memory NK cell compartment. We first demonstrate that chronic exposure to auto-antibodies contributes to the establishment/expansion of a highly specialized and unique memory NK cell subset with distinct CD16-dependent functional capabilities. We also identify the specific contribution of the lack of FcεRIγ chain in conferring to NKG2C+CD57+ memory cells a higher responsivity to CD16 engagement.

Janus kinase (JAK) inhibitors are widely used in the treatment of multiple autoimmune and inflammatory diseases. Immunologic and transcriptomic profiling have revealed major alterations on natural killer (NK) cell homeostasis associated with JAK inhibitions, while information on other innate lymphoid cells (ILCs) is still lacking. Herein, we observed that, in mice, the homeostatic pool of liver ILC1 was less affected by JAK inhibitors compared to the pool of NK cells present in the liver, spleen and bone marrow. JAK inhibition had overlapping effects on the transcriptome of both subsets, mainly affecting genes regulating cell cycle and apoptosis. However, the differential impact of JAK inhibition was linked to the high levels of the antiapoptotic gene Bcl2 expressed by ILC1. Our findings provide mechanistic explanations for the effects of JAK inhibitors on NK cells and ILC1 which could be of major clinically relevance.

Differentiation of Natural Killer (NK) cells is a stepwise process having its origin in the bone marrow and proceeding in the periphery, where these cells follow organ specific trajectories. Several soluble factors and cytokines regulate the distinct stages of NK cell differentiation, and ultimately, their functional properties. Cytokines activating the Janus kinases (JAKs) and members of the signal transducer and activator of transcription (STAT) pathway control distinct aspects of NK cell biology, ranging from development, terminal differentiation, activation, and generation of cells with adaptive properties. Here, we discuss how the recent advances of next generation sequencing (NGS) technology have led to unravel novel molecular aspects of gene regulation, with the aim to provide genomic views of how STATs regulate transcriptional and epigenetic features of NK cells during the different functional stages.

NK cell maturation is a continuous process, which initiates in the bone marrow and proceeds in peripheral tissues, where NK cells follow distinct differentiation routes. Drastic phenotypic changes are observed during progression from precursors to mature NK cells, including changes of expression and functionalities of several chemoattractant receptors. Upon differentiation, mature NK cells migrate outside the bone marrow; as well, peculiar subsets of NK cells can also home back to or localize in this anatomic compartment to play specific functions. In humans, NK cells with a tissue resident phenotype have been identified in bone marrow, sharing similarities with tissue resident memory CD8+ T cells; while in mouse, long-lived NK cells undergo homeostatic proliferation in this site during viral infections. The mechanisms underlying NK cell subset localization in the bone marrow have only recently started to be investigated, especially in pathological settings such as tumors or infections. In this review, we discuss the phenotype and function of NK cells as well as their requirements for bone marrow maintenance and/or homing.

The discovery of innate lymphoid cells (ILCs) with selective production of cytokines typically attributed to subsets of T helper cells forces immunologists to reassess the mechanisms by which selective effector functions arise. The parallelism between ILCs and T cells extends beyond these two cell types and comprises other innate-like T lymphocytes. Beyond the recognition of specialized effector functionalities in diverse lymphocytes, features typical of T cells, such as plasticity and memory, are also relevant for innate lymphocytes. Herein, we review what we have learned in terms of the molecular mechanisms underlying these shared functions, focusing on insights provided by next generation sequencing technologies. We review data on the role of lineage-defining- and signal-dependent transcription factors (TFs). ILC regulomes emerge developmentally whereas the much of the open chromatin regions of T cells are generated acutely, in an activation-dependent manner. And yet, these regions of open chromatin in T cells and ILCs have remarkable overlaps, suggesting that though accessibility is acquired by distinct modes, the end result is that convergent signaling pathways may be involved. Although much is left to be learned, substantial progress has been made in understanding how TFs and epigenomic status contribute to ILC biology in terms of differentiation, specification, and plasticity.

Mucosal epithelia encounter both physicochemical and biological stress during their life and have evolved several mechanisms to deal with them, including regulation of immune cell functions. Stressed and damaged cells need to be cleared to control local inflammation and trigger tissue healing. Engagement of the activating NKG2D receptor is one of the most direct mechanisms involved in the recognition of stressed cells by the immune system. Indeed, injured cells promptly express NKG2D ligands that in turn mediate the activation of lymphocytes of both innate and adaptive arms of the immune system. This review focuses on different conditions that are able to modulate NKG2D ligand expression on the epithelia. Special attention is given to the mechanisms of immunosurveillance mediated by natural killer cells, which are finely tuned by NKG2D. Different types of stress, including viral and bacterial infections, chronic inflammation, and cigarette smoke exposure, are discussed as paradigmatic conditions for NKG2D ligand modulation, and the implications for tissue homeostasis are discussed.

The advent of blockchain technology allows the raise of new business models for the electricity market, opening the way also to end-users and letting them offer regulation services to the power grid. Thanks to the characteristic of being distributed, the blockchain technology could be a solution to balancing problems caused by the penetration of renewable sources, implementing a platform for Demand-Response programs delivery. Demand-Response allows consumers to respond to market signals by increasing or reducing their energy consumption, contributing to greater flexibility and stability of the grid and to a more efficient use of infrastructures and energy resources. Currently, Demand-Response is carried out by controlling aggregates of loads, storage or generating units managed by centralized Supervisory Control and Data Acquisition systems such as SCADA. Regulatory changes and the increasing penetration of renewable sources distributed over the territory are turning the whole electricity system into a smart-grid. More recently and with reference to the end-users participation in regulation services, smartness is achieved through the so-called Internet of Things, which can be considered the modern equivalent of SCADA, but with the possibility of to being applied to distributed and diversified assets. For this reason, great efforts have been made to study the interoperability and coexistence between Internet of Things and blockchain, two emerging paradigms that are gaining popularity in the energy world. Limited or no contribution can instead be found in the literature on the integration of SCADA systems and blockchain. Indeed, in order to ensure an easier and faster widespread application of blockchain in the context of power systems, it is interesting to study its possible coexistence with legacy and more established industrial technologies such as OpenADR or SCADA. In Europe, the prevailing technology is the latter one. For this reason, in this paper, the coexistence of blockchain technology with SCADA systems is discussed. In particular, both Hyperledger Fabric blockchain and SCADA systems are considered together to assess the feasibility of aggregation of energy resources for Demand-Response, as well as the relevant measured data. The analysis is carried out by first presenting the two different paradigms: the centralized data acquisition in trusted environments and analysis via OpenADR and SCADA, and the global, distributed and secured ones with the blockchain. Then an architecture for the integration of SCADA and blockchain technology is proposed and the related challenges within the frame of a project for innovative technologies DR programs implementation are outlined.

The use of Distributed Ledger Technologies such as Blockchain for certifying Demand Response services allows for the creation of a distributed system in which customers can communicate with the system operator to provide their flexibility, in a secure, transparent and traceable way. Blockchain technology also supports incentive mechanisms for users taking part in the service through the generation of utility tokens to recognize the user's contribution. This paper presents the experimental test of a novel methodology for Demand Response programs implementation by using the Blockchain technology. The latter is employed for defining a distributed Demand Response service and a new system for its tracing and certification. For this work, a Smart Contract has been conceived and written to execute Demand Response events, calculate users' baseline, compute the support provided by each user towards the fulfilment of the requested load curve modification and remunerate each user with utility tokens proportionally to their contribution. To test the methodology, a Hyperledger Fabric network and a Smart Contract were deployed on four nodes of the Microgrid Laboratory of the Department of Energy Technology at Aalborg University (DK). Subsequently, a realistic scenario comprising two consumer nodes was developed using power electronic converters for generating the household profiles and Smart Meters for the measurement of the consumption profiles. Theoretical and experimental results show the feasibility of Distributed Ledger Technologies in smart grids management with a minimum investment in new hardware while enabling the active participation of customers in Demand Response more transparently and fairly.

IntroductionChemotherapy remains the mainstay treatment for triple-negative breast cancer (TNBC) due to the lack of specific targets. Given a modest response of immune checkpoint inhibitors in TNBC patients, improving immunotherapy is an urgent and crucial task in this field. CD73 has emerged as a novel immunotherapeutic target, given its elevated expression on tumor, stromal, and specific immune cells, and its established role in inhibiting anti-cancer immunity. CD73-generated adenosine suppresses immunity by attenuating tumor-infiltrating T- and NK-cell activation, while amplifying regulatory T cell activation. Chemotherapy often leads to increased CD73 expression and activity, further suppressing anti-tumor immunity. While debulking the tumor mass, chemotherapy also enriches heterogenous cancer stem cells (CSC), potentially leading to tumor relapse. Therefore, drugs targeting both CD73, and CSCs hold promise for enhancing chemotherapy efficacy, overcoming treatment resistance, and improving clinical outcomes. However, safe and effective inhibitors of CD73 have not been developed as of now.MethodsWe used in silico docking to screen compounds that may be repurposed for inhibiting CD73. The efficacy of these compounds was investigated through flow cytometry, RT-qPCR, CD73 activity, cell viability, tumorsphere formation, and other in vitro functional assays. For assessment of clinical translatability, TNBC patient-derived xenograft organotypic cultures were utilized. We also employed the ovalbumin-expressing AT3 TNBC mouse model to evaluate tumor-specific lymphocyte responses.ResultsWe identified quercetin and luteolin, currently used as over-the-counter supplements, to have high in silico complementarity with CD73. When quercetin and luteolin were combined with the chemotherapeutic paclitaxel in a triple-drug regimen, we found an effective downregulation in paclitaxel-enhanced CD73 and CSC-promoting pathways YAP and Wnt. We found that CD73 expression was required for the maintenance of CD44highCD24low CSCs, and co-targeting CD73, YAP, and Wnt effectively suppressed the growth of human TNBC cell lines and patient-derived xenograft organotypic cultures. Furthermore, triple-drug combination inhibited paclitaxel-enriched CSCs and simultaneously improved lymphocyte infiltration in syngeneic TNBC mouse tumors.DiscussionConclusively, our findings elucidate the significance of CSCs in impairing anti-tumor immunity. The high efficacy of our triple-drug regimen in clinically relevant platforms not only underscores the importance for further mechanistic investigations but also paves the way for potential development of new, safe, and cost-effective therapeutic strategies for TNBC.

Abstract Mast cells (MCs) are multifaceted innate immune cells often present in the tumor microenvironment (TME). Several recent findings support their contribution to the transition from chronic inflammation to cancer. However, MC-derived mediators can either favor tumor progression, inducing the spread of the tumor, or exert anti-tumorigenic functions, limiting tumor growth. This apparent controversial role likely depends on the plastic nature of MCs that under different microenvironmental stimuli can rapidly change their phenotype and functions. Thus, the exact effect of unique MC subset(s) during tumor progression is far from being understood. Using a murine model of colitis-associated colorectal cancer, we initially characterized the MC population within the TME and in non-lesional colonic areas, by multicolor flow cytometry and confocal microscopy. Our results demonstrated that tumor-associated MCs harbor a main connective tissue phenotype and release high amounts of Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-α. This MC phenotype correlates with the presence of high levels of Stem Cell Factor (SCF) and IL-33 inside the tumor. Thus, we investigated the effect of SCF and IL-33 on primary MC cultures and underscored their ability to shape MC phenotype eliciting the production of pro-inflammatory cytokines. Our findings support the conclusion that during colonic transformation a sustained stimulation by SCF and IL-33 promotes the accumulation of a prevalent connective tissue-like MC subset that through the secretion of IL-6 and TNF-α maintains a pro-inflammatory microenvironment.

Abstract The mechanisms of communication between the brain and the immune cells are still largely unclear. Here, we characterize the populations of resident natural killer (NK) cells and innate lymphoid cells (ILC) 1 in the meningeal dura layer of adult mice. We describe that ILC1/NK cell-derived interferon-γ and acetylcholine can contribute to the modulation of brain homeostatic functions, shaping synaptic neuronal transmission and neurotransmitter levels with effects on mice behavior. In detail, the interferon-γ plays a role in the formation of non-spatial memory, tuning the frequency of GABAergic neurotransmission on cortical pyramidal neurons, while the acetylcholine is a mediator involved in the modulation of brain circuitries that regulate anxiety-like behavior. These findings disclose mechanisms of immune-to-brain communication that modulate brain functions under physiological conditions.

We propose a novel image-informed glioblastoma mathematical model within a reactive multiphase poromechanical framework. Poromechanics offers to model in a coupled manner the interplay between tissue deformation and pressure-driven fluid flows, these phenomena existing simultaneously in cancer disease. The model also relies on two mechano-biological hypotheses responsible for the heterogeneity of the GBM: hypoxia signaling cascade and interaction between extra-cellular matrix and tumor cells. The model belongs to the category of patient-specific image-informed models as it is initialized, calibrated and evaluated by the means of patient imaging data. The model is calibrated with patient data after 6 cycles of concomitant radiotherapy chemotherapy and shows good agreement with treatment response 3 months after chemotherapy maintenance. Sensitivity of the solution to parameters and to boundary conditions is provided. As this work is only a first step of the inclusion of poromechanical framework in image-informed glioblastoma mathematical models, leads of improvement are provided in the conclusion.

Natural Killer (NK) cells are key innate effectors of antiviral immune response, and their activity changes in ageing and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we investigated the age-related changes of NK cell phenotype and function during SARS-CoV-2 infection, by comparing adult and elderly patients both requiring mechanical ventilation. Adult patients had a reduced number of total NK cells, while elderly showed a peculiar skewing of NK cell subsets towards the CD56lowCD16high and CD56neg phenotypes, expressing activation markers and check-point inhibitory receptors. Although NK cell degranulation ability is significantly compromised in both cohorts, IFN-γ production is impaired only in adult patients in a TGF-β−dependent manner. This inhibitory effect was associated with a shorter hospitalization time of adult patients suggesting a role for TGF-β in preventing an excessive NK cell activation and systemic inflammation. Our data highlight an age-dependent role of NK cells in shaping SARS-CoV-2 infection toward a pathophysiological evolution.

Colorectal cancer (CRC) is one of the most common malignancies and leading causes of cancer-related deaths worldwide. Despite its complex pathogenesis and progression, CRC represents a well-fitting example of how the immune contexture can dictate the disease outcome. The presence of cytotoxic lymphocytes, both CD8+ T cells and natural killer (NK) cells, represents a relevant prognostic factor in CRC and is associated with a better overall survival. Together with NK cells, other innate lymphocytes, namely, innate lymphoid cells (ILCs), have been found both in biopsies of CRC patients and in murine models of intestinal cancer, playing both pro- and anti-tumor activities. In particular, several type 1 innate lymphoid cells (ILC1) with cytotoxic functions have been recently described, and evidence in mice shows a role for both NK cells and ILC1 in controlling CRC metastasis. In this review, we provide an overview of the features of NK cells and the expanding spectrum of innate lymphocytes with cytotoxic functions. We also comment on both the described and the potential roles these innate lymphocytes can play during the progression of intestinal cancer leading to metastasis. Finally, we discuss recent advances in the molecular mechanisms underlying the functional regulation of cytotoxic innate lymphocytes in CRC.

The precise control of cytokine production by innate lymphoid cells (ILCs) and their T cell adaptive system counterparts is critical to mounting a proper host defense immune response without inducing collateral damage and autoimmunity. Unlike T cells that differentiate into functionally divergent subsets upon antigen recognition, ILCs are developmentally programmed to rapidly respond to environmental signals in a polarized manner, without the need of T cell receptor (TCR) signaling. The specification of cytokine production relies on dynamic regulation of cis-regulatory elements that involve multi-dimensional epigenetic mechanisms, including DNA methylation, transcription factor binding, histone modification and DNA-DNA interactions that form chromatin loops. How these different layers of gene regulation coordinate with each other to fine tune cytokine production, and whether ILCs and their T cell analogs utilize the same regulatory strategy, remain largely unknown. Herein, we review the molecular mechanisms that underlie cell identity and functionality of helper T cells and ILCs, focusing on networks of transcription factors and cis-regulatory elements. We discuss how higher-order chromatin architecture orchestrates these components to construct lineage- and state-specific regulomes that support ordered immunoregulation.

Spheroids encapsulated within alginate capsules are emerging as suitable in vitro tools to investigate the impact of mechanical forces on tumor growth since the internal tumor pressure can be retrieved from the deformation of the capsule. Here we focus on the particular case of Cellular Capsule Technology (CCT). We show in this contribution that a modeling approach accounting for the triphasic nature of the spheroid (extracellular matrix, tumor cells and interstitial fluid) offers a new perspective of analysis revealing that the pressure retrieved experimentally cannot be interpreted as a direct picture of the pressure sustained by the tumor cells and, as such, cannot therefore be used to quantify the critical pressure which induces stress-induced phenotype switch in tumor cells. The proposed multiphase reactive poro-mechanical model was cross-validated. Parameter sensitivity analyses on the digital twin revealed that the main parameters determining the encapsulated growth configuration are different from those driving growth in free condition, confirming that radically different phenomena are at play. Results reported in this contribution support the idea that multiphase reactive poro-mechanics is an exceptional theoretical framework to attain an in-depth understanding of CCT experiments, to confirm their hypotheses and to further improve their design.

Innate lymphoid cells (ILCs) are innate lymphocytes playing essential functions in protection against microbial infections and participate in both homeostatic and pathological contexts, including tissue remodeling, cancer, and inflammatory disorders. A number of lineage-defining transcription factors concurs to establish transcriptional networks which determine the identity and the activity of the distinct ILC subsets. However, the contribution of other regulatory molecules in controlling ILC development and function is also recently emerging. In this regard, noncoding RNAs (ncRNAs) represent key elements of the complex regulatory network of ILC biology and host protection. ncRNAs mostly lack protein-coding potential, but they are endowed with a relevant regulatory activity in immune and nonimmune cells because of their ability to control chromatin structure, RNA stability, and/or protein synthesis. Herein, we summarize recent studies describing how distinct types of ncRNAs, mainly microRNAs, long ncRNAs, and circular RNAs, act in the context of ILC biology. In particular, we comment on how ncRNAs can exert key effects in ILCs by controlling gene expression in a cell- or state-specific manner and how this tunes distinct functional outputs in ILCs.