Divergent roles for STAT4 in shaping differentiation of cytotoxic ILC1 and NK cells during gut inflammation.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) require signal transducer and activator of transcription 4 (STAT4) to elicit rapid effector responses and protect against pathogens. By combining genetic and transcriptomic approaches, we uncovered divergent roles for STAT4 in regulating effector differentiation of these functionally related cell types. Stat4 deletion in Ncr1-expressing cells led to impaired NK cell terminal differentiation as well as to an unexpected increased generation of cytotoxic ILC1 during intestinal inflammation. Mechanistically, Stat4-deficient ILC1 exhibited upregulation of gene modules regulated by STAT5 in vivo and an aberrant effector differentiation upon in vitro stimulation with IL-2, used as a prototypical STAT5 activator. Moreover, STAT4 expression in NCR+ innate lymphocytes restrained gut inflammation in the dextran sulfate sodium-induced colitis model limiting pathogenic production of IL-13 from adaptive CD4+ T cells in the large intestine. Collectively, our data shed light on shared and distinctive mechanisms of STAT4-regulated transcriptional control in NK cells and ILC1 required for intestinal inflammatory responses.

NKG2D engagement on human NK cells leads to DNAM-1 hypo-responsiveness through different converging mechanisms.

Natural killer (NK) cell activation is regulated by activating and inhibitory receptors that facilitate diseased cell recognition. Among activating receptors, NKG2D and DNAM-1 play a pivotal role in anticancer immune responses since they bind ligands upregulated on transformed cells. During tumor progression, however, these receptors are frequently downmodulated and rendered functionally inactive. Of note, NKG2D internalization has been associated with the acquisition of a dysfunctional phenotype characterized by the cross-tolerization of unrelated activating receptors. However, our knowledge of the consequences of NKG2D engagement is still incomplete. Here, by cytotoxicity assays combined with confocal microscopy, we demonstrate that NKG2D engagement on human NK cells impairs DNAM-1-mediated killing through two different converging mechanisms: by the upregulation of the checkpoint inhibitory receptor TIGIT, that in turn suppresses DNAM-1-mediated cytotoxic function, and by direct inhibition of DNAM-1-promoted signaling. Our results highlight a novel interplay between NKG2D and DNAM-1/TIGIT receptors that may facilitate neoplastic cell evasion from NK cell-mediated clearance.

Role of NF-κB Signaling in the Interplay between Multiple Myeloma and Mesenchymal Stromal Cells.

Nuclear factor-κB (NF-κB) transcription factors play a key role in the pathogenesis of multiple myeloma (MM). The survival, proliferation and chemoresistance of malignant plasma cells largely rely on the activation of canonical and noncanonical NF-κB pathways. They are triggered by cancer-associated mutations or by the autocrine and paracrine production of cytokines and growth factors as well as direct interaction with cellular and noncellular components of bone marrow microenvironment (BM). In this context, NF-κB also significantly affects the activity of noncancerous cells, including mesenchymal stromal cells (MSCs), which have a critical role in disease progression. Indeed, NF-κB transcription factors are involved in inflammatory signaling that alters the functional properties of these cells to support cancer evolution. Moreover, they act as regulators and/or effectors of pathways involved in the interplay between MSCs and MM cells. The aim of this review is to analyze the role of NF-κB in this hematologic cancer, focusing on NF-κB-dependent mechanisms in tumor cells, MSCs and myeloma-mesenchymal stromal cell crosstalk.

Nitric Oxide Prevents Glioblastoma Stem Cells' Expansion and Induces Temozolomide Sensitization.

Glioblastoma multiforme (GBM) has high mortality and recurrence rates. Malignancy resilience is ascribed to Glioblastoma Stem Cells (GSCs), which are resistant to Temozolomide (TMZ), the gold standard for GBM post-surgical treatment. However, Nitric Oxide (NO) has demonstrated anti-cancer efficacy in GBM cells, but its potential impact on GSCs remains unexplored. Accordingly, we investigated the effects of NO, both alone and in combination with TMZ, on patient-derived GSCs. Experimentally selected concentrations of diethylenetriamine/NO adduct and TMZ were used through a time course up to 21 days of treatment, to evaluate GSC proliferation and death, functional recovery, and apoptosis. Immunofluorescence and Western blot analyses revealed treatment-induced effects in cell cycle and DNA damage occurrence and repair. Our results showed that NO impairs self-renewal, disrupts cell-cycle progression, and expands the quiescent cells' population. Consistently, NO triggered a significant but tolerated level of DNA damage, but not apoptosis. Interestingly, NO/TMZ cotreatment further inhibited cell cycle progression, augmented G0 cells, induced cell death, but also enhanced DNA damage repair activity. These findings suggest that, although NO administration does not eliminate GSCs, it stunts their proliferation, and makes cells susceptible to TMZ. The resulting cytostatic effect may potentially allow long-term control over the GSCs' subpopulation.

Cereblon regulates NK cell cytotoxicity and migration via Rac1 activation.

Rearrangement of the actin cytoskeleton is critical for cytotoxic and immunoregulatory functions as well as migration of natural killer (NK) cells. However, dynamic reorganization of actin is a complex process, which remains largely unknown. Here, we investigated the role of the protein Cereblon (CRBN), an E3 ubiquitin ligase complex co-receptor and the primary target of the immunomodulatory drugs, in NK cells. We observed that CRBN partially colocalizes with F-actin in chemokine-treated NK cells and is recruited to the immunological synapse, thus suggesting a role for this protein in cytoskeleton reorganization. Accordingly, silencing of CRBN in NK cells results in a reduced cytotoxicity that correlates with a defect in conjugate and lytic synapse formation. Moreover, CRBN depletion significantly impairs the ability of NK cells to migrate and reduces the enhancing effect of lenalidomide on NK cell migration. Finally, we provided evidence that CRBN is required for activation of the small GTPase Rac1, a critical mediator of cytoskeleton dynamics. Indeed, in CRBN-depleted NK cells, chemokine-mediated or target cell-mediated Rac1 activation is significantly reduced. Altogether our data identify a critical role for CRBN in regulating NK cell functions and suggest that this protein may mediate the stimulatory effect of lenalidomide on NK cells.

Granzyme A and CD160 expression delineates ILC1 with graded functions in the mouse liver.

Type 1 innate lymphoid cells (ILC1) are tissue-resident lymphocytes that provide early protection against bacterial and viral infections. Discrete transcriptional states of ILC1 have been identified in homeostatic and pathological contexts. However, whether these states delineate ILC1 with different functional properties is not completely understood. Here, we show that liver ILC1 are heterogeneous for the expression of distinct effector molecules and surface receptors, including granzyme A (GzmA) and CD160, in mice. ILC1 expressing high levels of GzmA are enriched in the liver of adult mice, and represent the main hepatic ILC1 population at birth. However, the heterogeneity of GzmA and CD160 expression in hepatic ILC1 begins perinatally and increases with age. GzmA+ ILC1 differ from NK cells for the limited homeostatic requirements of JAK/STAT signals and the transcription factor Nfil3. Moreover, by employing Rorc(γt)-fate map (fm) reporter mice, we established that ILC3-ILC1 plasticity contributes to delineate the heterogeneity of liver ILC1, with RORγt-fm+ cells skewed toward a GzmA- CD160+ phenotype. Finally, we showed that ILC1 defined by the expression of GzmA and CD160 are characterized by graded cytotoxic potential and ability to produce IFN-γ. In conclusion, our findings help deconvoluting ILC1 heterogeneity and provide evidence for functional diversification of liver ILC1.

NK Cells and Other Cytotoxic Innate Lymphocytes in Colorectal Cancer Progression and Metastasis.

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.

JAK/STAT signaling in regulation of innate lymphoid cells: The gods before the guardians.

Immunity to pathogens is ensured through integration of early responses mediated by innate cells and late effector functions taking place after terminal differentiation of adaptive lymphocytes. In this context, innate lymphoid cells (ILCs) and adaptive T cells represent a clear example of how prototypical effector functions, including polarized expression of cytokines and/or cytotoxic activity, can occur with overlapping modalities but different timing. The ability of ILCs to provide early protection relies on their poised epigenetic state, which determines their propensity to quickly respond to cytokines and to activate specific patterns of signal-dependent transcription factors. Cytokines activating the Janus kinases (JAKs) and members of the signal transducer and activator of transcription (STAT) pathway are key regulators of lymphoid development and sustain the processes underlying T-cell activation and differentiation. The role of the JAK/STAT pathway has been recently extended to several aspects of ILC biology. Here, we discuss how JAK/STAT signals affect ILC development and effector functions in the context of immune responses, highlighting the molecular mechanisms involved in regulation of gene expression as well as the potential of targeting the JAK/STAT pathway in inflammatory pathologies.

Role of Aiolos and Ikaros in the Antitumor and Immunomodulatory Activity of IMiDs in Multiple Myeloma: Better to Lose Than to Find Them.

The Ikaros zing-finger family transcription factors (IKZF TFs) are important regulators of lymphocyte development and differentiation and are also highly expressed in B cell malignancies, including Multiple Myeloma (MM), where they are required for cancer cell growth and survival. Moreover, IKZF TFs negatively control the functional properties of many immune cells. Thus, the targeting of these proteins has relevant therapeutic implications in cancer. Indeed, accumulating evidence demonstrated that downregulation of Ikaros and Aiolos, two members of the IKZF family, in malignant plasma cells as well as in adaptative and innate lymphocytes, is key for the anti-myeloma activity of Immunomodulatory drugs (IMiDs). This review is focused on IKZF TF-related pathways in MM. In particular, we will address how the depletion of IKZF TFs exerts cytotoxic effects on MM cells, by reducing their survival and proliferation, and concomitantly potentiates the antitumor immune response, thus contributing to therapeutic efficacy of IMiDs, a cornerstone in the treatment of this neoplasia.

CD155: A Multi-Functional Molecule in Tumor Progression.

CD155 is an adhesion molecule belonging to the Nectin/Nectin-like family often overexpressed on tumor cells and involved in many different processes such as cell adhesion, migration and proliferation. In contrast to these pro-tumorigenic functions, CD155 is also a ligand for the activating receptor DNAM-1 expressed on cytotoxic lymphocytes including Natural Killer (NK) cells and involved in anti-tumor immune response. However, during tumor progression inhibitory receptors for CD155 are up-regulated on the surface of effector cells, contributing to an impairment of their cytotoxic capacity. In this review we will focus on the roles of CD155 as a ligand for the activating receptor DNAM-1 regulating immune surveillance against cancer and as pro-oncogenic molecule favoring tumor proliferation, invasion and immune evasion. A deeper understanding of the multiple roles played by CD155 in cancer development contributes to improving anti-tumor strategies aimed to potentiate immune response against cancer.

Multifunctional human CD56 low CD16 low natural killer cells are the prominent subset in bone marrow of both healthy pediatric donors and leukemic patients.

We phenotypically and functionally characterized a distinct CD56(low) natural killer cell subset based on CD16 expression levels in bone marrow and peripheral blood of healthy children and pediatric patients with acute lymphoblastic leukemia. Our findings demonstrate for the first time that CD56(low)CD16(low) natural killer cells are more abundant in bone marrow than in peripheral blood and that their frequency is further increased in children with acute lymphoblastic leukemia. Bone marrow and peripheral blood CD56(low)CD16(low) natural killer cells compared with CD56(low)CD16(high) natural killer cells express lower levels of killer inhibitory receptors, higher levels of CD27, CD127, CD122, CD25, but undetectable levels of CD57, suggesting that they have a higher proliferative and differentiation potential. Moreover, CD56(low)CD16(low) natural killer cells display higher levels of CXCR4 and undetectable levels of CX3CR1 and can be consistently and rapidly mobilized in peripheral blood in response to CXCR4 antagonist. Unlike CD56(low)CD16(high), both bone marrow and peripheral blood CD56(low)CD16(low) natural killer cells release IFNγ following cytokine stimulation, and represent the major cytotoxic natural killer cell population against K562 or acute lymphoblastic leukemia target cells. All these data suggest that CD56(low)CD16(low) natural killer cells are multifunctional cells, and that the presence of hematologic malignancies affects their frequency and functional ability at both tumor site and in the periphery.

A low-cost, label-free microfluidic scanning flow cytometer for high-accuracy quantification of size and refractive index of particles.

Flow cytometers and fluorescence activated cells sorters (FCM/FACS) represent the gold standard for high-throughput single-cell analysis, but their usefulness for label-free applications is limited by the unreliability of forward and side scatter measurements. Scanning flow cytometers represent an appealing alternative, as they exploit measurements of the angle-resolved scattered light to provide accurate and quantitative estimates of cellular properties, but the requirements of current setups are unsuitable for integration with other lab-on-chip technologies or for point-of-care applications. Here we present the first microfluidic scanning flow cytometer (µSFC), able to achieve accurate angle-resolved scattering measurements within a standard polydimethylsiloxane microfluidic chip. The system exploits a low cost linearly variable optical density (OD) filter to reduce the dynamic range of the signal and to increase its signal-to-noise ratio. We present a performance comparison between the µSFC and commercial machines for the label free characterization of polymeric beads with different diameters and refractive indices. In contrast to FCM and FACS, the µSFC yields size estimates linearly correlated with nominal particle sizes (R2 = 0.99) and quantitative estimates of particle refractive indices. The feasibility of using the µSFC for the characterization of biological samples is demonstrated by analyzing a population of monocytes identified based on the morphology of a peripheral blood mononuclear cells sample, which yields values in agreement with the literature. The proposed µSFC combines low setup requirements with high performance, and has great potential for integration within other lab-on-chip systems for multi-parametric cell analysis and for next-generation point-of-care diagnostic applications.

Impaired NK-cell migration in WAS/XLT patients: role of Cdc42/WASp pathway in the control of chemokine-induced beta2 integrin high-affinity state.

We analyzed the involvement of Wiskott-Aldrich syndrome protein (WASp), a critical regulator of actin cytoskeleton remodeling, in the control of natural killer (NK)-cell migration. NK cells derived from patients with Wiskott-Aldrich syndrome/X-linked thrombocytopenia (WAS/XLT), carrying different mutations in the WASP coding gene, displayed reduced migration through intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or endothelial cells in response to CXCL12/stromal cell-derived factor-1 and CX3CL1/fractalkine. Inhibition of WAS/XLT NK-cell migration was associated with reduced ability of these cells to up-regulate the expression of CD18 activation neoepitope and to adhere to ICAM-1 or VCAM-1 following chemokine stimulation. Moreover, chemokine receptor or beta1 or beta2 integrin engagement on NK cells rapidly resulted in Cdc42 activation and WASp tyrosine phosphorylation as well as in WASp association with Fyn and Pyk-2 tyrosine kinases. NK-cell pretreatment with wiskostatin, to prevent Cdc42/WASp association, impaired chemokine-induced NK-cell migration through ICAM-1 and beta2 integrin activation-dependent neoepitope expression. These results show that the Cdc42/WASp pathway plays a crucial role in the regulation of NK-cell migration by acting as a critical component of the chemokine-induced inside-out signaling that regulates lymphocyte function-associated antigen-1 function and suggest that after integrin or chemokine receptor engagement WASp function is regulated by the coordinate action of both Cdc42 and tyrosine kinases.

An alternative role of C1q in cell migration and tissue remodeling: contribution to trophoblast invasion and placental development.

Fetal trophoblast cells invading the decidua in the early phase of pregnancy establish complex interaction with the maternal extracellular matrix. We discovered that C1q was widely distributed in human decidual stroma in the absence of C4 and C3 and was actively synthesized by migrating extravillous trophoblasts. The cells expressed the messages for the three chains of C1q and secreted this complement component that interacted with the proteins of the decidual extracellular matrix. Solid phase-bound C1q promoted trophoblast adhesion and migration, and cell binding to C1q resulted in activation of ERK1/2 MAPKs. Ab inhibition experiments showed that the receptors for the globular head of C1q/p33 and α(4)ß(1) integrin were both involved in this process and were colocalized on the cell surface following binding of C1q to trophoblasts. We also found that C1q(-/-) mice manifested increased frequency of fetal resorption, reduced fetal weight, and smaller litter sizes compared with wild-type mice. C1q deficiency was associated with impaired labyrinth development and decidual vessel remodeling. Collectively, these data suggest that C1q plays an important role in promoting trophoblast invasion of decidua and that defective local production of C1q may be involved in pregnancy disorders, such as pre-eclampsia, characterized by poor trophoblast invasion.

GAS6/TAM signaling pathway controls MICA expression in multiple myeloma cells.

NKG2D ligands play a relevant role in Natural Killer (NK) cell -mediated immune surveillance of multiple myeloma (MM). Different levels of regulation control the expression of these molecules at cell surface. A number of oncogenic proteins and miRNAs act as negative regulators of NKG2D ligand transcription and translation, but the molecular mechanisms sustaining their basal expression in MM cells remain poorly understood. Here, we evaluated the role of the growth arrest specific 6 (GAS6)/TAM signaling pathway in the regulation of NKG2D ligand expression and MM recognition by NK cells. Our data showed that GAS6 as well as MERTK and AXL depletion in MM cells results in MICA downregulation and inhibition of NKG2D-mediated NK cell degranulation. Noteworthy, GAS6 derived from bone marrow stromal cells (BMSCs) also increases MICA expression at both protein and mRNA level in human MM cell lines and in primary malignant plasma cells. NF-kB activation is required for these regulatory mechanisms since deletion of a site responsive for this transcription factor compromises the induction of mica promoter by BMSCs. Accordingly, knockdown of GAS6 reduces the capability of BMSCs to activate NF-kB pathway as well as to enhance MICA expression in MM cells. Taken together, these results shed light on molecular mechanism underlying NKG2D ligand regulation and identify GAS6 protein as a novel autocrine and paracrine regulator of basal expression of MICA in human MM cells.

Age-dependent NK cell dysfunctions in severe COVID-19 patients.

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-ß-dependent manner. This inhibitory effect was associated with a shorter hospitalization time of adult patients suggesting a role for TGF-ß 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.

NK cell and ILC heterogeneity in colorectal cancer. New perspectives from high dimensional data.

Innate lymphoid cells (ILCs) and tissue-resident natural killer (NK) cells ensure immunity at environmental interfaces and help maintain barrier integrity of the intestinal tract. This wide range of innate lymphocytes is able to provide fast and potent inflammatory responses that, when deregulated, have been associated with pathogenesis of inflammatory bowel disease (IBD) and colorectal cancer (CRC). While the presence of tumor-infiltrating NK cells is generally associated with a favorable outcome in CRC patients, emerging evidence reveals distinct roles for ILCs in regulating CRC pathogenesis and progression. Advances in next generation sequencing technology, and in particular of single-cell RNA-seq approaches, along with multidimensional flow cytometry analysis, have helped to deconvolute the complexity and heterogeneity of the ILC system both in homeostatic and pathological contexts. In this review, we discuss the protective and detrimental roles of NK cells and ILCs in the pathogenesis of CRC, focusing on the phenotypic and transcriptional modifications these cells undergo during CRC development and progression.

The Regulatory Activity of Noncoding RNAs in ILCs.

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.

Hitting More Birds with a Stone: Impact of TGF-ß on ILC Activity in Cancer.

Transforming growth factor (TGF)-ß is a central immunosuppressive cytokine within tumor microenvironment inhibiting the expansion and function of major cellular components of adaptive and innate immune system. Among them, compelling evidence has demonstrated that TGF-ß is a key regulator of natural killer (NK) cells, innate lymphoid cells (ILCs) with a critical role in immunosurveillance against different kinds of cancer cells. A TGF-ß rich tumor microenvironment blocks NK cell activity at multiple levels. This immunosuppressive factor exerts direct regulatory effects on NK cells including inhibition of cytokine production, alteration of activating/inhibitory receptor expression, and promotion of the conversion into non cytotoxic group I ILC (ILC1). Concomitantly, TGF-ß can render tumor cells less susceptible to NK cell-mediated recognition and lysis. Indeed, accumulating evidence suggest that changes in levels of NKG2D ligands, mainly MICA, as well as an increase of immune checkpoint inhibitors (e.g., PD-L1) and other inhibitory ligands on cancer cells significantly contribute to TGF-ß-mediated suppression of NK cell activity. Here, we will take into consideration two major mechanisms underlying the negative regulation of ILC function by TGF-ß in cancer. First, we will address how TGF-ß impacts the balance of signals governing NK cell activity. Second, we will review recent advances on the role of this cytokine in driving ILC plasticity in cancer. Finally, we will discuss how the development of therapeutic approaches blocking TGF-ß may reverse the suppression of host immune surveillance and improve anti-tumor NK cell response in the clinic.