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.

New Insight into Intestinal Mast Cells Revealed by Single-Cell RNA Sequencing.

Mast cells (MCs) are tissue-resident immune cells distributed in all tissues and strategically located close to blood and lymphatic vessels and nerves. Thanks to the expression of a wide array of receptors, MCs act as tissue sentinels, able to detect the presence of bacteria and parasites and to respond to different environmental stimuli. MCs originate from bone marrow (BM) progenitors that enter the circulation and mature in peripheral organs under the influence of microenvironment factors, thus differentiating into heterogeneous tissue-specific subsets. Even though MC activation has been traditionally linked to IgE-mediated allergic reactions, a role for these cells in other pathological conditions including tumor progression has recently emerged. However, several aspects of MC biology remain to be clarified. The advent of single-cell RNA sequencing platforms has provided the opportunity to understand MCs' origin and differentiation as well as their phenotype and functions within different tissues, including the gut. This review recapitulates how single-cell transcriptomic studies provided insight into MC development as well as into the functional role of intestinal MC subsets in health and disease.

CD155 and Its Receptors as Targets for Cancer Therapy.

CD155, also known as the poliovirus receptor, is an adhesion molecule often overexpressed in tumors of different origins where it promotes cell migration and proliferation. In addition to this pro-tumorigenic function, CD155 plays an immunomodulatory role during tumor progression since it is a ligand for both the activating receptor DNAM-1 and the inhibitory receptor TIGIT, expressed on cytotoxic innate and adaptative lymphocytes. DNAM-1 is a well-recognized receptor involved in anti-tumor immune surveillance. However, in advanced tumor stages, TIGIT is up-regulated and acts as an immune checkpoint receptor, counterbalancing DNAM-1-mediated cancer cell clearance. Pre-clinical studies have proposed the direct targeting of CD155 on tumor cells as well as the enhancement of DNAM-1-mediated anti-tumor functions as promising therapeutic approaches. Moreover, immunotherapeutic use of anti-TIGIT blocking antibody alone or in combined therapy has already been included in clinical trials. The aim of this review is to summarize all these potential therapies, highlighting the still controversial role of CD155 during tumor progression.

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.

SAMHD1 phosphorylation and cytoplasmic relocalization after human cytomegalovirus infection limits its antiviral activity.

SAMHD1 is a host restriction factor that functions to restrict both retroviruses and DNA viruses, based on its nuclear deoxynucleotide triphosphate (dNTP) hydrolase activity that limits availability of intracellular dNTP pools. In the present study, we demonstrate that SAMHD1 expression was increased following human cytomegalovirus (HCMV) infection, with only a modest effect on infectious virus production. SAMHD1 was rapidly phosphorylated at residue T592 after infection by cellular cyclin-dependent kinases, especially Cdk2, and by the viral kinase pUL97, resulting in a significant fraction of phosho-SAMHD1 being relocalized to the cytoplasm of infected fibroblasts, in association with viral particles and dense bodies. Thus, our findings indicate that HCMV-dependent SAMHD1 cytoplasmic delocalization and inactivation may represent a potential novel mechanism of HCMV evasion from host antiviral restriction activities.

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.

The Ubiquitin-proteasome pathway regulates Nectin2/CD112 expression and impairs NK cell recognition and killing.

Nectin2 is a member of immunoglobulin-like cell adhesion molecules and plays a prominent role in the establishment of adherens and tight junctions. It is also upregulated on the surface of tumor and virus-infected cells where it functions as a ligand for the activating receptor CD226, thus contributing to cytotoxic lymphocyte-mediated recognition and killing of damaged cells. Little is currently known about the regulation of Nectin2 expression and, in particular, whether posttranscriptional and posttranslational mechanisms are involved. Here, we analyzed Nectin2 expression on a panel of human tumor cell lines and primary cultures and we found that Nectin2 is mainly expressed in cytoplasmic pools. Moreover, we demonstrated that ubiquitination of Nectin2 promotes its degradation and is responsible for protein intracellular retention. Indeed, inhibition of the ubiquitin pathway results in increased Nectin2 surface expression and enhances tumor cell susceptibility to NK cell cytotoxicity. Our results demonstrate a previously unknown mechanism of Nectin2 regulation revealing that the ubiquitin pathway represents a potential target of intervention in order to increase susceptibility to NK cell-mediated lysis.

Activation of liver X receptor up-regulates the expression of the NKG2D ligands MICA and MICB in multiple myeloma through different molecular mechanisms.

NK cells have an important role in immunosurveillance of multiple myeloma (MM) progression, and their activity is enhanced by combination therapies able to regulate the expression of specific activating ligands. Liver X receptors (LXRs) are nuclear receptors and important regulators of intracellular cholesterol and lipid homeostasis. Moreover, they have regulatory roles in both cancer and immune response. Indeed, they can regulate inflammation and innate and acquired immunity. Furthermore, LXR activation directly acts in cancer cells (e.g., prostate, breast, melanoma, colon cancer, hepatocarcinoma, glioblastoma, and MM) that show an accumulation of cholesterol and alteration of LXR-mediated metabolic pathways. Here, we investigated the role of LXR and cholesterol on the expression of the NK cell-activating ligands major histocompatibility complex class I chain-related molecule A and B (MICA and MICB) in MM cells. The results shown in this work indicate that MM cells are responsive to LXR activation, which induces changes in the intracellular cholesterol content. These changes correlate with an enhanced expression of MICA and MICB in human MM cell lines and in primary malignant plasma cells, 2 ligands of the NK group 2D receptor (NKG2D)/CD314 activating receptor expressed in cytotoxic lymphocytes, rendering MM cells more sensitive to recognition, degranulation, and killing by NK cells. Mechanistically, we observed that LXR activation regulates MICA and MICB expression at different levels: MICA at the transcriptional level, enhancing mica promoter activity, and MICB by inhibiting its degradation in lysosomes. The present study provides evidence that activation of LXR, by enhancing NKG2D ligand expression, can promote NK cell-mediated cytotoxicity and suggests a novel immune-mediated mechanism involving modulation of intracellular cholesterol levels in cancer cells.-Bilotta, M. T., Abruzzese, M. P., Molfetta, R., Scarno, G., Fionda, C., Zingoni, A., Soriani, A., Garofalo, T., Petrucci, M. T., Ricciardi, M. R., Paolini, R., Santoni, A., Cippitelli, M. Activation of liver X receptor up-regulates the expression of the NKG2D ligands MICA and MICB in multiple myeloma through different molecular mechanisms.

FcεRI Signaling in the Modulation of Allergic Response: Role of Mast Cell-Derived Exosomes.

Mast cells (MCs) are immune cells that act as environment resident sentinels playing a crucial role in Th2-mediated immune responses, including allergic reactions. Distinguishing features of MCs are the presence of numerous cytoplasmic granules that encapsulate a wide array of preformed bio-active molecules and the constitutive expression of the high affinity receptor of IgE (FcεRI). Upon FcεRI engagement by means of IgE and multivalent antigens, aggregated receptors trigger biochemical pathways that ultimately lead to the release of granule-stored and newly synthesized pro-inflammatory mediators. Additionally, MCs are also able to release exosomes either constitutively or upon stimulation. Exosomes are nanosized vesicles of endocytic origin endowed with important immunoregulatory properties, and represent an additional way of intercellular communication. Interestingly, exosomes generated upon FcεRI engagement contain co-stimulatory and adhesion molecules, lipid mediators, and MC-specific proteases, as well as receptor subunits together with IgE and antigens. These findings support the notion that FcεRI signaling plays an important role in influencing the composition and functions of exosomes derived by MCs depending on their activation status.

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.

Regulation of NKG2D Expression and Signaling by Endocytosis.

NKG2D is an activating receptor that can bind to a large number of stress-induced ligands that are expressed in the context of cancer or viral infection. This receptor is expressed on many cytotoxic lymphocytes, and plays a crucial role in antitumor and antiviral immune responses. However, exposure to NKG2D ligand-expressing target cells promotes receptor endocytosis, ultimately leading to lysosomal receptor degradation and impairment of NKG2D-mediated functions. Interestingly, before being degraded, internalized receptors can signal from the endosomal compartment, leading to the appropriate activation of cellular functional programs. This review summarizes recent findings on ligand-induced receptor internalization, with particular emphasis on the role of endocytosis in the control of both NKG2D-mediated intracellular signaling and receptor degradation.

Genotoxic Stress Induces Senescence-Associated ADAM10-Dependent Release of NKG2D MIC Ligands in Multiple Myeloma Cells.

Genotoxic stress can promote antitumor NK cell responses by upregulating the surface expression of activating ligands on cancer cells. Moreover, a number of studies suggested a role for soluble NK group 2D ligands in the impairment of NK cell tumor recognition and killing. We investigated whether genotoxic stress could promote the release of NK group 2D ligands (MHC class I-related chain [MIC]A and MICB), as well as the molecular mechanisms underlying this event in human multiple myeloma (MM) cells. Our results show that genotoxic agents used in the therapy of MM (i.e., doxorubicin and melphalan) selectively affect the shedding of MIC molecules that are sensitive to proteolytic cleavage, whereas the release of the short MICA*008 allele, which is frequent in the white population, is not perturbed. In addition, we found that a disintegrin and metalloproteinase 10 expression is upregulated upon chemotherapeutic treatment both in patient-derived CD138(+)/CD38(+) plasma cells and in several MM cell lines, and we demonstrate a crucial role for this sheddase in the proteolytic cleavage of MIC by means of silencing and pharmacological inhibition. Interestingly, the drug-induced upregulation of a disintegrin and metalloproteinase 10 on MM cells is associated with a senescent phenotype and requires generation of reactive oxygen species. Moreover, the combined use of chemotherapeutic drugs and metalloproteinase inhibitors enhances NK cell-mediated recognition of MM cells, preserving MIC molecules on the cell surface and suggesting that targeting of metalloproteinases in conjunction with chemotherapy could be exploited for NK cell-based immunotherapeutic approaches, thus contributing to avoid the escape of malignant cells from stress-elicited immune responses.

Regulation of NKG2D-Dependent NK Cell Functions: The Yin and the Yang of Receptor Endocytosis.

Natural-killer receptor group 2, member D (NKG2D) is a well characterized natural killer (NK) cell activating receptor that recognizes several ligands poorly expressed on healthy cells but up-regulated upon stressing stimuli in the context of cancer or viral infection. Although NKG2D ligands represent danger signals that render target cells more susceptible to NK cell lysis, accumulating evidence demonstrates that persistent exposure to ligand-expressing cells causes the decrease of NKG2D surface expression leading to a functional impairment of NKG2D-dependent NK cell functions. Upon ligand binding, NKG2D is internalized from the plasma membrane and sorted to lysosomes for degradation. However, receptor endocytosis is not only a mechanism of receptor clearance from the cell surface, but is also required for the proper activation of signalling events leading to the functional program of NK cells. This review is aimed at providing a summary of current literature relevant to the molecular mechanisms leading to NKG2D down-modulation with particular emphasis given to the role of NKG2D endocytosis in both receptor degradation and signal propagation. Examples of chronic ligand-induced down-regulation of NK cell activating receptors other than NKG2D, including natural cytotoxicity receptors (NCRs), DNAX accessory molecule-1 (DNAM1) and CD16, will be also discussed.

c-Cbl regulates MICA- but not ULBP2-induced NKG2D down-modulation in human NK cells.

The NKG2D activating receptor on human NK cells mediates "altered self" recognition, as its ligands (NKG2DLs) are upregulated on target cells in a variety of stress conditions. Evidence collected in the past years shows that, even though expression of NKG2DLs acts as a danger signal that renders tumor cells susceptible to cytotoxicity, chronic exposure to soluble or membrane-bound NKG2DLs can lead to down-modulation of receptor expression and impairment of NKG2D-mediated cell functions. Here, we evaluated whether different cell-bound NKG2DLs, namely MICA and ULBP2, are equivalently able to induce NKG2D down-modulation on human NK cells. We found that although both ligands reduce NKG2D surface expression, MICA promotes a stronger receptor down-modulation than ULBP2, leading to a severe impairment of NKG2D-dependent NK-cell cytotoxicity. We also provide evidence that the ubiquitin pathway and c-Cbl direct MICA-induced but not ULBP2-induced NKG2D internalization and degradation, thus identifying a molecular mechanism to explain the differential effects of MICA and ULBP2 on NKG2D expression. A better understanding of the molecular mechanisms employed by the different NKG2DLs to control NKG2D surface expression could be useful for the development of anti-tumor strategies to restore a normal level of NKG2D receptors on human NK cells.

Nitric oxide donors increase PVR/CD155 DNAM-1 ligand expression in multiple myeloma cells: role of DNA damage response activation.

BACKGROUND: DNAX accessory molecule-1 (DNAM-1) is an activating receptor constitutively expressed by macrophages/dendritic cells and by T lymphocytes and Natural Killer (NK) cells, having an important role in anticancer responses; in this regard, combination therapies able to enhance the expression of DNAM-1 ligands on tumor cells are of therapeutic interest. In this study, we investigated the effect of different nitric oxide (NO) donors on the expression of the DNAM-1 ligand Poliovirus Receptor/CD155 (PVR/CD155) in multiple myeloma (MM) cells. METHODS: Six MM cell lines, SKO-007(J3), U266, OPM-2, RPMI-8226, ARK and LP1 were used to investigate the activity of different nitric oxide donors [DETA-NO and the NO-releasing prodrugs NCX4040 (NO-aspirin) and JS-K] on the expression of PVR/CD155, using Flow Cytometry and Real-Time PCR. Western-blot and specific inhibitors were employed to investigate the role of soluble guanylyl cyclase/cGMP and activation of the DNA damage response (DDR). RESULTS: Our results indicate that increased levels of nitric oxide can upregulate PVR/CD155 cell surface and mRNA expression in MM cells; in addition, exposure to nitric oxide donors renders myeloma cells more efficient to activate NK cell degranulation and enhances their ability to trigger NK cell-mediated cytotoxicity. We found that activation of the soluble guanylyl cyclase and increased cGMP concentrations by nitric oxide is not involved in the up-regulation of ligand expression. On the contrary, treatment of MM cells with nitric oxide donors correlated with the activation of a DNA damage response pathway and inhibition of the ATM /ATR/Chk1/2 kinase activities by specific inhibitors significantly abrogates up-regulation. CONCLUSIONS: The present study provides evidence that regulation of the PVR/CD155 DNAM-1 ligand expression by nitric oxide may represent an additional immune-mediated mechanism and supports the anti-myeloma activity of nitric oxide donors.

PIP2-dependent regulation of Munc13-4 endocytic recycling: impact on the cytolytic secretory pathway.

Cytotoxic lymphocytes clear infected and transformed cells by releasing the content of lytic granules at cytolytic synapses, and the ability of cytolytic effectors to kill in an iterative manner has been documented previously. Although bidirectional trafficking of cytolytic machinery components along the endosomal pathway has begun to be elucidated, the molecular mechanisms coordinating granule retrieval remain completely unexplored. In the present study, we focus on the lytic granule priming factor Munc13-4, the mutation of which in familial hemophagocytic lymphohistiocytosis type 3 results in a profound defect of cytotoxic function. We addressed the role of phosphatidylinositol (4,5)-bisphosphate (PIP2) in the regulation of Munc13-4 compartmentalization. We observed that in human natural killer cells, PIP2 is highly enriched in membrane rafts. Granule secretion triggering induces a transient Munc13-4 raft recruitment, followed by AP-2/clathrin-dependent internalization. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) γ gene silencing leads to the impairment of granule secretion associated with increased levels of raft-associated Munc13-4, which is attributable to a defect in AP-2 membrane recruitment. In such conditions, the ability to subsequently kill multiple targets was significantly impaired. These observations indicate that Munc13-4 reinternalization is required for the maintenance of an intracellular pool that is functional to guarantee the serial killing potential.

Optimizing Transfection Efficiency in CAR-T Cell Manufacturing through Multiple Administrations of Lipid-Based Nanoparticles.

The existing manufacturing protocols for CAR-T cell therapies pose notable challenges, particularly in attaining a transient transfection that endures for a significant duration. To address this gap, this study aims to formulate a transfection protocol utilizing multiple lipid-based nanoparticles (LNPs) administrations to enhance transfection efficiency (TE) to clinically relevant levels. By systematically fine-tuning and optimizing our transfection protocol through a series of iterative refinements, we have accomplished a remarkable one-order-of-magnitude augmentation in TE within the immortalized T-lymphocyte Jurkat cell line. This enhancement has been consistently observed over 2 weeks, and importantly, it has been achieved without any detrimental impact on cell viability. In the subsequent phase of our study, we aimed to optimize the gene delivery system by evaluating three lipid-based formulations tailored for DNA encapsulation using our refined protocol. These formulations encompassed two LNPs constructed from ionizable lipids and featuring systematic variations in lipid composition (iLNPs) and a cationic lipoplex (cLNP). Our findings showcased a notable standout among the three formulations, with cLNP emerging as a frontrunner for further refinement and integration into the production pipeline of CAR-T therapies. Consequently, cLNP was scrutinized for its potential to deliver CAR-encoding plasmid DNA to the HEK-293 cell line. Confocal microscopy experiments demonstrated its efficiency, revealing substantial internalization compared to iLNPs. By employing a recently developed confocal image analysis method, we substantiated that cellular entry of cLNP predominantly occurs through macropinocytosis. This mechanism leads to heightened intracellular endosomal escape and mitigates lysosomal accumulation. The successful expression of anti-CD19-CD28-CD3z, a CAR engineered to target CD19, a protein often expressed on the surface of B cells, was confirmed using a fluorescence-based assay. Overall, our results indicated the effectiveness of cLNP in gene delivery and suggested the potential of multiple administration transfection as a practical approach for refining T-cell engineering protocols in CAR therapies. Future investigations may focus on refining outcomes by adjusting transfection parameters like nucleic acid concentration, lipid-to-DNA ratio, and incubation time to achieve improved TE and increased gene expression levels.

Syk-dependent regulation of Hrs phosphorylation and ubiquitination upon FcεRI engagement: impact on Hrs membrane/cytosol localization.

Several lines of evidence suggest that Syk controls immune receptor endocytic trafficking. However, the Syk substrates that regulate this process are not currently known. Here, we demonstrate that Syk knockdown prevents the trafficking of engaged high affinity IgE receptor (FcεRI) to a degradative compartment in mast cells. We then concentrate our attention on hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) as potential Syk substrate, since it serves as critical regulator for FcεRI entry into lysosomes. We show that Hrs undergoes antigen-dependent tyrosine phosphorylation and ubiquitination, and identify Syk as the kinase responsible for Hrs phosphorylation. Syk was also required for Hrs ubiquitination catalyzed by c-Cbl E3 ligase. Syk-dependent regulation of Hrs covalent modifications, without affecting protein stability, controlled Hrs localization. The majority of phosphorylated Hrs forms were observed only in membrane compartments, whereas ubiquitinated Hrs was predominantly cytosolic, suggesting that both modifications might impact on Hrs function. Together, these findings provide a major step forward in understanding how Syk orchestrates endocytosis of engaged immune receptors.

The Controversial Role of Intestinal Mast Cells in Colon Cancer.

Mast cells are tissue-resident sentinels involved in large number of physiological and pathological processes, such as infection and allergic response, thanks to the expression of a wide array of receptors. Mast cells are also frequently observed in a tumor microenvironment, suggesting their contribution in the transition from chronic inflammation to cancer. In particular, the link between inflammation and colorectal cancer development is becoming increasingly clear. It has long been recognized that patients with inflammatory bowel disease have an increased risk of developing colon cancer. Evidence from experimental animals also implicates the innate immune system in the development of sporadically occurring intestinal adenomas, the precursors to colorectal cancer. However, the exact role of mast cells in tumor initiation and growth remains controversial: mast cell-derived mediators can either exert pro-tumorigenic functions, causing the progression and spread of the tumor, or anti-tumorigenic functions, limiting the tumor's growth. Here, we review the multifaceted and often contrasting findings regarding the role of the intestinal mast cells in colon cancer progression focusing on the molecular pathways mainly involved in the regulation of mast cell plasticity/functions during tumor progression.