The RIG-I agonist M8 triggers cell death and natural killer cell activation in human papillomavirus-associated cancer and potentiates cisplatin cytotoxicity.

Although the activation of innate immunity to treat a wide variety of cancers is gaining increasing attention, it has been poorly investigated in human papillomavirus (HPV)-associated malignancies. Because these tumors harbor a severely impaired cGAS-STING axis, but they still retain a largely functional RIG-I pathway, another critical mediator of adaptive and innate immune responses, we asked whether RIG-I activation by the 5'ppp-RNA RIG-I agonist M8 would represent a therapeutically viable option to treat HPV+ cancers. Here, we show that M8 transfection of two cervical carcinoma-derived cell lines, CaSki and HeLa, both expressing a functional RIG-I, triggers intrinsic apoptotic cell death, which is significantly reduced in RIG-I KO cells. We also demonstrate that M8 stimulation potentiates cisplatin-mediated cell killing of HPV+ cells in a RIG-I dependent manner. This combination treatment is equally effective in reducing tumor growth in a syngeneic pre-clinical mouse model of HPV16-driven cancer, where enhanced expression of lymphocyte-recruiting chemokines and cytokines correlated with an increased number of activated natural killer (NK) cells in the tumor microenvironment. Consistent with a role of RIG-I signaling in immunogenic cell killing, stimulation of NK cells with conditioned medium from M8-transfected CaSki boosted NK cell proliferation, activation, and migration in a RIG-I-dependent tumor cell-intrinsic manner. Given the highly conserved molecular mechanisms of carcinogenesis and genomic features of HPV-driven cancers and the remarkably improved prognosis for HPV+ oropharyngeal cancer, targeting RIG-I may represent an effective immunotherapeutic strategy in this setting, favoring the development of de-escalating strategies.

Cross-Dressing of Multiple Myeloma Cells Mediated by Extracellular Vesicles Conveying MIC and ULBP Ligands Promotes NK Cell Killing.

Natural Killer (NK) cells are innate cytotoxic lymphoid cells that play a crucial role in cancer immunosurveillance. NKG2D is an activating receptor that binds to MIC and ULBP molecules typically induced on damaged, transformed, or infected cells. The secretion of NKG2D ligands (NKG2DLs) through protease-mediated cleavage or in an extracellular vesicle (EV) is a mode to control their cell surface expression and a mechanism used by cancer cells to evade NKG2D-mediated immunosurveillance. EVs are emerging as important players in mediating cell-to-cell communication due to their ability to transfer biological material to acceptor cells. Herein, we investigated the spreading of NKG2DLs of both MIC and ULBP molecules through the EV-mediated cross-dressing on multiple myeloma (MM) cells. We focused our attention on two MICA allelic variants, namely MICA*008 and MICA*019, representing the prototype of short and long MICA alleles, respectively, and on ULBP-1, ULBP-2, and ULBP-3. Our findings demonstrate that both ULBP and MICA ligands can be acquired from tumor cells through EVs enhancing NK cell recognition and killing. Moreover, besides MICA, EVs expressing ULBP-1 but not ULBP-2 and 3 were detected in bone marrow aspirates derived from a cohort of MM patients. Our findings shed light on the role of EV-associated MICA allelic variants and ULBP molecules in the modulation of NKG2D-mediated NK cell immunosurveillance in the tumor microenvironment. Moreover, the EV-mediated transfer of NKG2DLs could suggest novel therapeutic approaches based on the usage of engineered nanoparticles aimed at increasing cancer cell immunogenicity.

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.

Counter-regulation of regulatory T cells by autoreactive CD8+ T cells in rheumatoid arthritis.

The mechanisms whereby autoreactive T cells escape peripheral tolerance establishing thus autoimmune diseases in humans remain an unresolved question. Here, we demonstrate that autoreactive polyfunctional CD8+ T cells recognizing self-antigens (i.e., vimentin, actin cytoplasmic 1, or non-muscle myosin heavy chain 9 epitopes) with high avidity, counter-regulate Tregs by killing them, in a consistent percentage of rheumatoid arthritis (RA) patients. Indeed, these CD8+ T cells express a phenotype and gene profile of effector (eff) cells and, upon antigen-specific activation, kill Tregs indirectly in an NKG2D-dependent bystander fashion in vitro. This data provides a mechanistic basis for the finding showing that AE-specific (CD107a+) CD8+ T killer cells correlate, directly with the disease activity score, and inversely with the percentage of activated Tregs, in both steady state and follow-up studies in vivo. In addition, multiplex immunofluorescence imaging analyses of inflamed synovial tissues in vivo show that a remarkable number of CD8+ T cells express granzyme-B and selectively contact FOXP3+ Tregs, some of which are in an apoptotic state, validating hence the possibility that CD8+ Teff cells can counteract neighboring Tregs within inflamed tissues, by killing them. Alternatively, the disease activity score of a different subset of patients is correlated with the expansion of a peculiar subpopulation of autoreactive low avidity, partially-activated (pa)CD8+ T cells that, despite they conserve the conventional naïve (N) phenotype, produce high levels of tumor necrosis factor (TNF)-α and exhibit a gene expression signature of a progressive activation state. Tregs directly correlate with the expansion of this autoreactive (low avidity) paCD8+ TN cell subset in vivo, and efficiently control their differentiation rather their proliferation in vitro. Interestingly, autoreactive high avidity CD8+ Teff cells or low avidity paCD8+ TN cells are significantly expanded in RA patients who would become non-responders or patients who would become responders to TNF-α inhibitor therapy, respectively. These data provide evidence of a previously undescribed role of such mechanisms in the progression and therapy of RA.

Cancer Exosomes as Conveyors of Stress-Induced Molecules: New Players in the Modulation of NK Cell Response.

Natural killer (NK) cells are innate lymphoid cells that play a pivotal role in tumor surveillance. Exosomes are nanovesicles released into the extracellular environment via the endosomal vesicle pathway and represent an important mode of intercellular communication. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Moreover, the activation of the DNA damage response (DDR) and the induction of senescence represent two crucial modalities aimed at promoting the clearance of drug-treated tumor cells by NK cells. Emerging evidence has shown that stress stimuli provoke an increased release of exosome secretion. Remarkably, tumor-derived exosomes (Tex) produced in response to stress carry distinct type of DAMPs that activate innate immune cell populations. Moreover, stress-induced ligands for the activating receptor NKG2D are transported by this class of nanovesicles. Here, we will discuss how Tex interact with NK cells and provide insight into their potential role in response to chemotherapy-induced stress stimuli. The capability of some "danger signals" carried by exosomes that indirectly affect the NK cell activity in the tumor microenvironment will be also addressed.

Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the Transcriptional Regulation of MICA and PVR/CD155 Expression.

Elimination of virus-infected cells by cytotoxic lymphocytes is triggered by activating receptors, among which NKG2D and DNAM-1/CD226 play an important role. Their ligands, that is, MHC class I-related chain (MIC) A/B and UL16-binding proteins (ULBP)1-6 (NKG2D ligand), Nectin-2/CD112, and poliovirus receptor (PVR)/CD155 (DNAM-1 ligand), are often induced on virus-infected cells, although some viruses, including human CMV (HCMV), can block their expression. In this study, we report that infection of different cell types with laboratory or low-passage HCMV strains upregulated MICA, ULBP3, and PVR, with NKG2D and DNAM-1 playing a role in NK cell-mediated lysis of infected cells. Inhibition of viral DNA replication with phosphonoformic acid did not prevent ligand upregulation, thus indicating that early phases of HCMV infection are involved in ligand increase. Indeed, the major immediate early (IE) proteins IE1 and IE2 stimulated the expression of MICA and PVR, but not ULBP3. IE2 directly activated MICA promoter via its binding to an IE2-responsive element that we identified within the promoter and that is conserved among different alleles of MICA. Both IE proteins were instead required for PVR upregulation via a mechanism independent of IE DNA binding activity. Finally, inhibiting IE protein expression during HCMV infection confirmed their involvement in ligand increase. We also investigated the contribution of the DNA damage response, a pathway activated by HCMV and implicated in ligand regulation. However, silencing of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related protein, and DNA-dependent protein kinase did not influence ligand expression. Overall, these data reveal that MICA and PVR are directly regulated by HCMV IE proteins, and this may be crucial for the onset of an early host antiviral response.

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.

Soluble ligands for the NKG2D receptor are released during HIV-1 infection and impair NKG2D expression and cytotoxicity of NK cells.

In humans, the interaction of the natural killer group 2 member D (NKG2D)-activating receptor on natural killer (NK) and CD8(+) T cells with its major histocompatibility complex class I-related chain (MIC) and UL16 binding protein (ULBP) ligands (NKG2DLs) promotes recognition and elimination of stressed cells, such as tumor or infected cells. Here, we investigated the capacity of HIV-1 to modulate NKG2DL expression and escape NGK2D-mediated immunosurveillance. In CD4(+) T lymphocytes, both cell surface expression and release of MICA, MICB, and ULBP2 were up-regulated >2-fold by HIV-1 infection. In HIV-infected CD4(+) T lymphocytes or Jurkat T-cell lines, increased shedding of soluble NKG2DLs (sNKG2DLs) was impaired by a matrix metalloproteinase inhibitor (MMPI). Moreover, naive HIV(+) patients displayed increased plasma sMICA and sULBP2 levels and reduced NKG2D expression on NK and CD8(+) T cells compared to patients receiving highly active antiretroviral therapy (HAART) or healthy donors. In individual patients, HAART uptake resulted in the drop of sNKG2DL and recovery of NKG2D expression. Finally, sNKG2DLs in patients' plasma down-regulated NKG2D on NK and CD8(+) T cells and impaired NKG2D-mediated cytotoxicity of NK cells. Thus, NKG2D detuning by sNKG2DLs may promote HIV-1 immune evasion and compromise host resistance to opportunistic infections, but HAART and MMPI have the potential to avoid such immune dysfunction.

The human immunodeficiency virus type 1 Nef and Vpu proteins downregulate the natural killer cell-activating ligand PVR.

The human immunodeficiency virus type 1 (HIV-1) evades the immune responses of natural killer (NK) cells through mechanisms that have been partially deciphered. Here we show that in HIV-1-infected T lymphocytes, the early viral Nef protein downmodulates PVR (CD155, Necl-5), a ligand for the activating receptor DNAM-1 (CD226) expressed by all NK cells, CD8(+) T cells, and other cell types. This novel Nef activity is conserved by Nef proteins of laboratory HIV-1 strains (NL4-3, SF2) and of a patient-derived virus, but it is not maintained by HIV-2. Nef uses the same motifs to downregulate PVR and HLA-I molecules, likely by the same mechanisms. Indeed, as previously demonstrated for HLA-I, Nef reduces the total amounts of cell-associated PVR. Optimal downregulation of cell surface PVR by Nef also requires the presence of the late viral factor Vpu. In line with PVR reduction, the NK cell-mediated lysis of T cells infected by a wild-type but not Nef-deficient virus is virtually abrogated upon blocking of both DNAM-1 and another activating receptor, NKG2D, previously shown to mediate killing of HIV-infected cells. Together, these data demonstrate that the PVR downmodulation by Nef and Vpu is a strategy evolved by HIV-1 to prevent NK cell-mediated lysis of infected cells. The PVR downregulation reported here has the potential to affect the immune responses of other DNAM-1-positive cells besides NK cells and to alter multiple PVR-mediated cellular processes, such as adhesion and migration, and may thus greatly influence HIV-1 pathogenesis.

DNAM-1 ligand expression on Ag-stimulated T lymphocytes is mediated by ROS-dependent activation of DNA-damage response: relevance for NK-T cell interaction.

An important role for natural killer (NK) cells in the regulation of T-cell responses is emerging, although the receptor pairs regulating the NK-T-cell interaction have still not been identified. We found that superantigen-stimulated T cells express Nectin-2 (CD112) and poliovirus receptor (PVR; CD155), the ligands of the activating NK receptor DNAX accessory molecule-1 (DNAM-1; CD226). Interestingly, only PVR was present at the T cell surface, particularly on cells in the S and G(2)/M phases of the cell cycle. The up-regulation of PVR expression involves DNA-damage response (DDR)-dependent pathways, because we found that pharmacologic inhibition of ATM and ATR kinases reduced PVR expression and that PVR was almost exclusively induced on cells expressing the DDR marker γH2AX. Oxidative stress contributed to DDR activation, and our results showed impaired PVR levels in the presence of the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC), being monocytes the main ROS source needed for optimal PVR expression on activated T cells. Interestingly, in accordance with ligand expression, NK cells lysed allogeneic proliferating more efficiently than nonproliferating T lymphocytes, with a mechanism requiring the cooperation between DNAM-1 and NKG2D. These results could contribute to unraveling the role of NK cells in the down-regulation of T-cell responses in physiologic and pathologic processes such as autoimmunity or GVHD.

NEDD8-activating enzyme inhibition potentiates the anti-myeloma activity of natural killer cells.

Natural Killer (NK) cells act as important regulators in the development and progression of hematological malignancies and their suppressor activity against Multiple Myeloma (MM) cells has been confirmed in many studies. Significant changes in the distribution of NK cell subsets and dysfunctions of NK cell effector activities were described in MM patients and correlated with disease staging. Thus, restoring or enhancing the functionality of these effectors for the treatment of MM represents a critical need. Neddylation is a post-translational modification that adds a ubiquitin-like molecule, NEDD8, to the substrate protein. One of the outcomes is the activation of the Cullin Ring Ligases (CRLs), a class of ubiquitin-ligases that controls the degradation of about 20% of proteasome-regulated proteins. Overactivation of CRLs has been described in cancer and can lead to tumor growth and progression. Thus, targeting neddylation represents an attractive approach for cancer treatment. Our group has recently described how pharmacologic inhibition of neddylation increases the expression of the NKG2D activating receptor ligands, MICA and MICB, in MM cells, making these cells more susceptible to NK cell degranulation and killing. Here, we extended our investigation to the direct role of neddylation on NK cell effector functions exerted against MM. We observed that inhibition of neddylation enhanced NK cell-mediated degranulation and killing against MM cells and improved Daratumumab/Elotuzumab-mediated response. Mechanistically, inhibition of neddylation increased the expression of Rac1 and RhoA GTPases in NK cells, critical mediators for an efficient degranulation at the immunological synapse of cytotoxic lymphocytes, and augmented the levels of F-actin and perforin polarization in NK cells contacting target cells. Moreover, inhibition of neddylation partially abrogated TGFß-mediated repression of NK cell effector activity. This study describes the role of neddylation on NK cell effector functions and highlights the positive immunomodulatory effects achieved by the inhibition of this pathway in MM.

Interplay between human cytomegalovirus and intrinsic/innate host responses: a complex bidirectional relationship.

The interaction between human cytomegalovirus (HCMV) and its host is a complex process that begins with viral attachment and entry into host cells, culminating in the development of a specific adaptive response that clears the acute infection but fails to eradicate HCMV. We review the viral and cellular partners that mediate early host responses to HCMV with regard to the interaction between structural components of virions (viral glycoproteins) and cellular receptors (attachment/entry receptors, toll-like receptors, and other nucleic acid sensors) or intrinsic factors (PML, hDaxx, Sp100, viperin, interferon inducible protein 16), the reactions of innate immune cells (antigen presenting cells and natural killer cells), the numerous mechanisms of viral immunoevasion, and the potential exploitation of events that are associated with early phases of virus-host interplay as a therapeutic strategy.

Self or Non-Self? It Is also a Matter of RNA Recognition and Editing by ADAR1.

A-to-I editing is a post-transcriptional mechanism affecting coding and non-coding dsRNAs, catalyzed by the adenosine deaminases acting on the RNA (ADAR) family of enzymes. A-to-I modifications of endogenous dsRNA (mainly derived from Alu repetitive elements) prevent their recognition by cellular dsRNA sensors, thus avoiding the induction of antiviral signaling and uncontrolled IFN-I production. This process, mediated by ADAR1 activity, ensures the activation of an innate immune response against foreign (non-self) but not self nucleic acids. As a consequence, ADAR1 mutations or its de-regulated activity promote the development of autoimmune diseases and strongly impact cell growth, also leading to cancer. Moreover, the excessive inflammation promoted by Adar1 ablation also impacts T and B cell maturation, as well as the development of dendritic cell subsets, revealing a new role of ADAR1 in the homeostasis of the immune system.

Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle-associated MICA: Dual role in cancer immunosurveillance.

Natural killer (NK) cells are innate cytotoxic lymphocytes that play a key role in cancer immunosurveillance thanks to their ability to recognize and kill cancer cells. NKG2D is an activating receptor that binds to MIC and ULBP molecules typically induced on damaged, transformed or infected cells. The release of NKG2D ligands (NKG2DLs) in the extracellular milieu through protease-mediated cleavage or by extracellular vesicle (EV) secretion allows cancer cells to evade NKG2D-mediated immunosurveillance. In this work, we investigated the immunomodulatory properties of the NKG2D ligand MICA*008 associated to distinct populations of EVs (i.e., small extracellular vesicles [sEVs] and medium size extracellular vesicles [mEVs]). By using as model a human MICA*008-transfected multiple myeloma (MM) cell line, we found that this ligand is present on both vesicle populations. Interestingly, our findings reveal that NKG2D is specifically involved in the uptake of vesicles expressing its cognate ligand. We provide evidence that MICA*008-expressing sEVs and mEVs are able on one hand to activate NK cells but, following prolonged stimulation induce a sustained NKG2D downmodulation leading to impaired NKG2D-mediated functions. Moreover, our findings show that MICA*008 can be transferred by vesicles to NK cells causing fratricide. Focusing on MM as a clinically and biologically relevant model of tumour-NK cell interactions, we found enrichment of EVs expressing MICA in the bone marrow of a cohort of patients. All together our results suggest that the accumulation of NKG2D ligands associated to vesicles in the tumour microenvironment could favour the suppression of NK cell activity either by NKG2D down-modulation or by fratricide of NK cell dressed with EV-derived NKG2D ligands.

Detuning CD8+ T lymphocytes by down-regulation of the activating receptor NKG2D: role of NKG2D ligands released by activated T cells.

NKG2D is an activating receptor expressed on CD8(+)alphabeta(+) T cells, gammadelta(+) T cells, natural killer (NK) cells, and some CD4(+) T cells. For a long time, the interaction of NKG2D with its ligands (NKG2DLs) MICA, MICB, and ULBP1-3 has been considered a mechanism for recognition and elimination of tumor, infected, or otherwise "stressed" cells. However, a new role for NKG2D as an immunoregulatory receptor is emerging. Here, we show that NKG2D is strongly down-modulated on antigen-activated CD8(+) T cells but only if CD4(+) T cells are present. Down-modulation was caused by soluble factors produced by CD4(+) T cells, and in particular soluble NKG2DLs were found in the supernatants of antigen-activated T-cell cultures. MICB was the ligand released at higher levels when CD4(+) T cells were present in the cell cultures, suggesting that it could be the major player of NKG2D down-modulation. CD8(+) T cells expressing low levels of NKG2D had impaired effector functions, as evaluated by proliferation, cytokine production, and cytotoxicity assays after combined triggering of NKG2D and TCR-CD3 complex. These findings show that activated CD4(+) T cells expressing NKG2DLs can efficiently prevent NKG2D-mediated CD8(+) T-cell functions, and suggest that the NKG2D/NKG2DL interaction can regulate immune responses.

ATM-ATR-dependent up-regulation of DNAM-1 and NKG2D ligands on multiple myeloma cells by therapeutic agents results in enhanced NK-cell susceptibility and is associated with a senescent phenotype.

There is much evidence to support a role for natural killer (NK) cells in controlling the progression of multiple myeloma (MM), a malignancy characterized by an abnormal plasma cell proliferation in the bone marrow (BM). Induction of DNA damage response has been recently shown capable of enhancing NKG2D ligand (NKG2DL) expression, but nothing is known about DNAM-1 ligand (DNAM-1L) regulation. In this study, we show that myeloma cells treated with low doses of therapeutic agents commonly used in the management of patients with MM, such as doxorubicin, melphalan, and bortezomib, up-regulate DNAM-1 and NKG2D ligands. Accordingly, therapeutic drug treatment of MM cells increases NK-cell degranulation, the NKG2D and DNAM-1 receptors being the major triggering molecules. Similar data were also obtained using ex vivo primary plasma cells derived from MM patients. Drug-induced DNAM-1 and NKG2D ligand expression was abolished after treatment with the ATM (ataxia telangiectasia mutated) and ATR (ATM- and RAD3-related) pharmacologic inhibitors caffeine and KU-55933, and was preferentially associated with senescent cells arrested in the G2 phase of the cell cycle. Altogether, our findings have identified a common pathway that can trigger the up-regulation of different NK cell-activating ligands and suggest that NK cells represent an immunosurveillance mechanism toward cells undergoing stress-induced senescent programs.

Genetic Variability of Human Cytomegalovirus Clinical Isolates Correlates With Altered Expression of Natural Killer Cell-Activating Ligands and IFN-γ.

Human cytomegalovirus (HCMV) infection often leads to systemic disease in immunodeficient patients and congenitally infected children. Despite its clinical significance, the exact mechanisms contributing to HCMV pathogenesis and clinical outcomes have yet to be determined. One of such mechanisms involves HCMV-mediated NK cell immune response, which favors viral immune evasion by hindering NK cell-mediated cytolysis. This process appears to be dependent on the extent of HCMV genetic variation as high levels of variability in viral genes involved in immune escape have an impact on viral pathogenesis. However, the link between viral genome variations and their functional effects has so far remained elusive. Thus, here we sought to determine whether inter-host genetic variability of HCMV influences its ability to modulate NK cell responses to infection. For this purpose, five HCMV clinical isolates from a previously characterized cohort of pediatric patients with confirmed HCMV congenital infection were evaluated by next-generation sequencing (NGS) for genetic polymorphisms, phylogenetic relationships, and multiple-strain infection. We report variable levels of genetic characteristics among the selected clinical strains, with moderate variations in genome regions associated with modulation of NK cell functions. Remarkably, we show that different HCMV clinical strains differentially modulate the expression of several ligands for the NK cell-activating receptors NKG2D, DNAM-1/CD226, and NKp30. Specifically, the DNAM-1/CD226 ligand PVR/CD155 appears to be predominantly upregulated by fast-replicating ("aggressive") HCMV isolates. On the other hand, the NGK2D ligands ULBP2/5/6 are downregulated regardless of the strain used, while other NK cell ligands (i.e., MICA, MICB, ULBP3, Nectin-2/CD112, and B7-H6) are not significantly modulated. Furthermore, we show that IFN-γ; production by NK cells co-cultured with HCMV-infected fibroblasts is directly proportional to the aggressiveness of the HCMV clinical isolates employed. Interestingly, loss of NK cell-modulating genes directed against NK cell ligands appears to be a common feature among the "aggressive" HCMV strains, which also share several gene variants across their genomes. Overall, even though further studies based on a higher number of patients would offer a more definitive scenario, our findings provide novel mechanistic insights into the impact of HCMV genetic variability on NK cell-mediated immune responses.

Pivotal role of KARAP/DAP12 adaptor molecule in the natural killer cell-mediated resistance to murine cytomegalovirus infection.

Natural killer (NK) cells are major contributors to early defense against infections. Their effector functions are controlled by a balance between activating and inhibiting signals. To date, however, the involvement of NK cell activating receptors and signaling pathways in the defense against pathogens has not been extensively investigated. In mice, several NK cell activating receptors are coexpressed with and function through the immunoreceptor tyrosine-based activation motif (ITAM)-bearing molecule KARAP/DAP12. Here, we have analyzed the role of KARAP/DAP12 in the early antiviral response to murine cytomegalovirus (MCMV). In KARAP/DAP12 mutant mice bearing a nonfunctional ITAM, we found a considerable increase in viral titers in the spleen (30-40-fold) and in the liver (2-5-fold). These effects were attributed to NK cells. The formation of hepatic inflammatory foci appeared similar in wild-type and mutant mice, but the latter more frequently developed severe hepatitis with large areas of focal necrosis. Moreover, the percentage of hepatic NK cells producing interferon gamma was reduced by 56 +/- 22% in the absence of a functional KARAP/DAP12. This is the first study that shows a crucial role for a particular activating signaling pathway, in this case the one induced through KARAP/DAP12, in the NK cell-mediated resistance to an infection. Our results are discussed in relation to recent reports demonstrating that innate resistance to MCMV requires the presence of NK cells expressing the KARAP/DAP12-associated receptor Ly49H.

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.

Tuning the Orchestra: HCMV vs. Innate Immunity.

Understanding how the innate immune system keeps human cytomegalovirus (HCMV) in check has recently become a critical issue in light of the global clinical burden of HCMV infection in newborns and immunodeficient patients. Innate immunity constitutes the first line of host defense against HCMV as it involves a complex array of cooperating effectors - e.g., inflammatory cytokines, type I interferon (IFN-I), natural killer (NK) cells, professional antigen-presenting cells (APCs) and phagocytes - all capable of disrupting HCMV replication. These factors are known to trigger a highly efficient adaptive immune response, where cellular restriction factors (RFs) play a major gatekeeping role. Unlike other innate immunity components, RFs are constitutively expressed in many cell types, ready to act before pathogen exposure. Nonetheless, the existence of a positive regulatory feedback loop between RFs and IFNs is clear evidence of an intimate cooperation between intrinsic and innate immunity. In the course of virus-host coevolution, HCMV has, however, learned how to manipulate the functions of multiple cellular players of the host innate immune response to achieve latency and persistence. Thus, HCMV acts like an orchestra conductor able to piece together and rearrange parts of a musical score (i.e., innate immunity) to obtain the best live performance (i.e., viral fitness). It is therefore unquestionable that innovative therapeutic solutions able to prevent HCMV immune evasion in congenitally infected infants and immunocompromised individuals are urgently needed. Here, we provide an up-to-date review of the mechanisms regulating the interplay between HCMV and innate immunity, focusing on the various strategies of immune escape evolved by this virus to gain a fitness advantage.