Long-term dynamics of natural killer cells in response to SARS-CoV-2 vaccination: Persistently enhanced activity postvaccination.

Natural Killer (NK) cells play a significant role in the early defense against virus infections and cancer. Recent studies have demonstrated the involvement of NK cells in both the induction and effector phases of vaccine-induced immunity in various contexts. However, their role in shaping immune responses following SARS-CoV-2 vaccination remains poorly understood. To address this matter, we conducted a comprehensive analysis of NK cell phenotype and function in SARS-CoV-2 unexposed individuals who received the BNT162b2 vaccine. We employed a longitudinal study design and utilized a panel of 53 15-mer overlapping peptides covering the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein to assess NK cell function at 0 and 20 days following the first vaccine, and 30 and 240 days following booster. Additionally, we evaluated the levels of total IgG anti-Spike antibodies and their potential neutralizing ability. Our findings revealed an increased NK cell activity upon re-exposure to RBD when combined with IL12 and IL18 several months after booster. Concurrently, we observed that the frequencies of NKG2A + NK cells declined over the course of the follow-up period, while NKG2C increased only in CMV positive subjects. The finding that NK cell functions are inducible 9 months after vaccination upon re-exposure to RBD and cytokines, sheds light on the role of NK cells in contributing to SARS-CoV-2 vaccine-induced immune protection and pave the way to further studies in the field.

TLRs: Innate Immune Sentries against SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been responsible for a devastating pandemic since March 2020. Toll-like receptors (TLRs), crucial components in the initiation of innate immune responses to different pathogens, trigger the downstream production of pro-inflammatory cytokines, interferons, and other mediators. It has been demonstrated that they contribute to the dysregulated immune response observed in patients with severe COVID-19. TLR2, TLR3, TLR4 and TLR7 have been associated with COVID-19 severity. Here, we review the role of TLRs in the etiology and pathogenesis of COVID-19, including TLR7 and TLR3 rare variants, the L412F polymorphism in TLR3 that negatively regulates anti-SARS-CoV-2 immune responses, the TLR3-related cellular senescence, the interaction of TLR2 and TLR4 with SARS-CoV-2 proteins and implication of TLR2 in NET formation by SARS-CoV-2. The activation of TLRs contributes to viral clearance and disease resolution. However, TLRs may represent a double-edged sword which may elicit dysregulated immune signaling, leading to the production of proinflammatory mediators, resulting in severe disease. TLR-dependent excessive inflammation and TLR-dependent antiviral response may tip the balance towards the former or the latter, altering the equilibrium that drives the severity of disease.

Hepatitis C Virus and the Host: A Mutual Endurance Leaving Indelible Scars in the Host's Immunity.

Hepatitis C virus (HCV) has spread worldwide, and it is responsible for potentially severe chronic liver disease and primary liver cancer. Chronic infection remains for life if not spontaneously eliminated and viral persistence profoundly impairs the efficiency of the host's immunity. Attempts have been made to develop an effective vaccine, but efficacy trials have met with failure. The availability of highly efficacious direct-acting antivirals (DAA) has created hope for the progressive elimination of chronic HCV infections; however, this approach requires a monumental global effort. HCV elicits a prompt innate immune response in the host, characterized by a robust production of interferon-α (IFN-α), although interference in IFN-α signaling by HCV proteins may curb this effect. The late appearance of largely ineffective neutralizing antibodies and the progressive exhaustion of T cells, particularly CD8 T cells, result in the inability to eradicate the virus in most infected patients. Moreover, an HCV cure resulting from DAA treatment does not completely restore the normal immunologic homeostasis. Here, we discuss the main immunological features of immune responses to HCV and the epigenetic scars that chronic viral persistence leaves behind.

Humoral and Cellular Immune Response Elicited by the BNT162b2 COVID-19 Vaccine Booster in Elderly.

Although the safety and efficacy of COVID-19 vaccines in older people are critical to their success, little is known about their immunogenicity among elderly residents of long-term care facilities (LTCFs). A single-center prospective cohort study was conducted: a total IgG antibody titer, neutralizing antibodies against Wild-type, Delta Plus, and Omicron BA.2 variants and T cell response, were measured eight months after the second dose of BNT162b2 vaccine (T0) and at least 15 days after the booster (T1). Forty-nine LTCF residents, with a median age of 84.8 ± 10.6 years, were enrolled. Previous COVID-19 infection was documented in 42.9% of the subjects one year before T0. At T1, the IgG titers increased up to 10-fold. This ratio was lower in the subjects with previous COVID-19 infection. At T1, IgG levels were similar in both groups. The neutralizing activity against Omicron BA.2 was significantly lower (65%) than that measured against Wild-type and Delta Plus (90%). A significant increase of T cell-specific immune response was observed after the booster. Frailty, older age, sex, cognitive impairment, and comorbidities did not affect antibody titers or T cell response. In the elderly sample analyzed, the BNT162b2 mRNA COVID-19 vaccine produced immunogenicity regardless of frailty.

Adaptive Natural Killer Cell Functional Recovery in Hepatitis C Virus Cured Patients.

BACKGROUND AND AIMS: Current evidence suggests that dysfunctional natural killer (NK) cell responses during hepatitis C virus (HCV) infection can be restored after viral eradication with direct acting antivirals (DAAs). However, the fate of the recently described adaptive NK cell population, endowed with increased ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), during HCV infection is poorly defined, while no study has explored the effects of DAAs on this NK subset. APPROACH AND RESULTS: We performed multicolor flow cytometry to investigate CD57+ FcεRIγneg adaptive and FcεRIγpos conventional NK cell phenotype and function before and after DAA treatment in 59 patients chronically infected with HCV, 39 with advanced liver fibrosis, and 20 with mild-moderate liver fibrosis. Moreover, bulk NK cell phenotype and function were analyzed after cytokine activation following contact with K562 target cells. The proportion of FcεRIγneg NK cells in patients with HCV was associated with increased HCV load at baseline, and it was significantly reduced after treatment. Patients with an advanced fibrosis stage displayed increased NK cell activation and exhaustion markers that normalized after therapy. Of note, adaptive NK cells from patients with HCV were characterized by increased programmed death receptor 1 expression and reduced ADCC activity at baseline. DAA treatment restored ADCC ability and reduced programmed death receptor 1 expression. CONCLUSIONS: HCV profoundly affects the frequency, phenotype, and function of adaptive NK cells. DAA therapy restores a normal adaptive NK phenotype and enhances interferon-gamma production by this cell subset.

Early and Polyantigenic CD4 T Cell Responses Correlate with Mild Disease in Acute COVID-19 Donors.

We assessed SARS-CoV-2-specific CD4+ and CD8+ T cell responses in samples from 89 acute COVID-19 patients, utilizing blood samples collected during the first wave of COVID-19 in Italy. The goal of the study was to examine correlations between SARS-CoV-2-specific T cell responses in the early phase comparing mild, moderate, or severe COVID-19 disease outcomes. T cell responses to the spike (S) and non-S proteins were measured in a combined activation-induced marker (AIM) and intracellular cytokine staining (ICS) assay. Early CD4+ T cell responses to SARS-CoV-2 S correlated with milder disease by both AIM and IFNγ ICS readouts. The correlation of S-specific CD4+ T cell responses with milder disease severity was most striking within the first two weeks of symptom onset compared to later time points. Furthermore, donors with milder disease were associated with polyantigenic CD4+ T cell responses that recognized more prominently non-S proteins in addition to S, while severe acute COVID-19 was characterized by lower magnitudes of CD4+ T cell responses and a narrower repertoire. In conclusion, this study highlights that both the magnitude and breadth of early SARS-CoV-2-specific CD4+ T cell responses correlated with milder disease outcomes in acute COVID-19 patients.

A new algorithm shows superior ability to discriminate liver fibrosis stages in chronic hepatitis C.

Previous evidence suggests that sialic acid-binding Ig-like lectin 7 (Siglec-7) protein is significantly increased in patients with chronic hepatitis C virus (HCV) infection and directly correlates with clinical parameters of liver inflammation and fibrosis. The aim of this study was to determine the diagnostic value of Siglec-7 as a non-invasive tool to assess liver fibrosis in patients with chronic hepatitis C in a cross-sectional study. Serum levels of Siglec-7 were retrospectively tested in 1007 consecutive patients with chronic HCV infection recruited at three different European sites and data examined by the 'imperfect gold-standard' statistical analysis. Liver stiffness obtained by transient elastography (TE) was considered the standard reference. Liver fibrosis was staged according to published cut-offs of liver stiffness measurement by TE. Accuracy of detection of liver fibrosis stage was not increased by Siglec-7 alone. However, we developed a new index (SiGAP) including Siglec-7, γ-glutamyl transferase, age and platelet count which showed increased sensitivity and specificity in predicting fibrosis compared with APRI or FIB4 indices. The AUROC of SiGAP for the diagnosis of significant (≥F2) and advanced liver fibrosis (≥F3) showed significantly higher values than those of APRI and FIB-4. Siglec-7 may be useful as a complementary tool to assess liver fibrosis stage in patients with chronic hepatitis C when included in a specifically designed algorithm, which showed high level of accuracy in the detection of F2 and F3 fibrosis stage.

Deficient Natural Killer Cell NKp30-Mediated Function and Altered NCR3 Splice Variants in Hepatocellular Carcinoma.

The activating natural cytotoxicity receptor NKp30 is critical for natural killer (NK) cell function and tumor immune surveillance. The natural cytotoxicity receptor-3 (NCR3) gene is transcribed into several splice variants whose physiological relevance is still incompletely understood. In this study, we investigated the role of NKp30 and its major ligand B7 homolog 6 (B7-H6) in patients with hepatocellular carcinoma (HCC). Peripheral blood NK cell phenotype was skewed toward a defective/exhausted immune profile with decreased frequencies of cells expressing NKp30 and natural killer group 2, member D and an increased proportion of cells expressing T-cell immunoglobulin and mucin-domain containing-3. Moreover, NKp30-positive NK cells had a reduced expression of NCR3 immunostimulatory splice variants and an increased expression of the inhibitory variant in patients with advanced tumor, resulting in deficient NKp30-mediated functionality. Tumor-infiltrating lymphocytes showed a prevalent inhibitory NKp30 isoform profile, consistent with decreased NKp30-mediated function. Of note, there were significant differences in the cytokine milieu between the neoplastic and the surrounding non-neoplastic tissue, which may have further influenced NKp30 function. Exposure of NK cells to B7-H6-expressing HCC cells significantly down-modulated NKp30, that was prevented by small interfering RNA-mediated knockdown, suggesting a role for this ligand in inhibiting NKp30-mediated responses. Interestingly, B7-H6 expression was reduced in HCC tissue and simultaneously augmented as a soluble form in HCC patients, particularly those with advanced staging or larger nodule size. Conclusion: These findings provide evidence in support of a role of NKp30 and its major ligand in HCC development and evolution.

Decreased interferon-γ production by NK cells from KIR haplotype B carriers in hepatitis C virus infection.

BACKGROUND AND AIMS: Different population genetics studies showed that interactions between killer-cell immunoglobulin-like receptors (KIR) and HLA play a role in viral disease outcome, but functional correlates are missing. Building upon our previous work pointing to a regulatory role for KIR3DL1/DS1 in hepatitis C virus (HCV) infection, we analysed whether its expression may affect natural killer (NK) cell function in the presence or absence of its principal ligand HLA-Bw4 in KIR haplotype A and B carriers, which are characterized by a different representation of activating and inhibitory KIRs. METHODS: We performed KIR and HLA class I genotypic analysis in 54 healthy donors (HD) and 50 HCV+ subjects and examined NK cell cytokine secretion and degranulation in the context of KIR3DL1-HLA-Bw4 match stratified by KIR haplotype. RESULTS: KIR3DL1-HLA-Bw4 match induced functional NK cell modulation, reflected by reduced interferon (IFN)γ production in haplotype B HCV+ patients compared to HD. This functional impairment could be ascribed to the KIR3DS1 negative HCV-infected patient population, whose NK cells also showed a significantly decreased proportion of KIR3DL1. Haplotype A HCV-infected patients showed increased NK cell degranulation compared with HD in the absence of KIR-HLA-Bw4 match and this activity was associated with increased phosphorylation of signal transducer and activator of transcription (STAT) 1. CONCLUSIONS: Our data show that NK cells from HCV+ patients have an unbalanced ability to produce IFNγ and to kill target cells in haplotype A and B carriers, suggesting the existence of complex functional differences governed by KIR-HLA interaction, particularly on KIR3DL1 expressing NK cells.

Altered natural killer cell cytokine profile in type 2 autoimmune hepatitis.

Type 2 autoimmune hepatitis (AIH-2) is a rare disease presenting in early childhood. The immunopathogenetic mechanisms are poorly characterized, although a defect of regulatory T cells (Treg) has been shown. There is virtually no information on innate immune responses and natural killer (NK) cells in particular. We have performed an extended immunophenotypic and functional analysis of NK cells in children with AIH-2. We show that NK cell frequency is reduced in this setting and that the balance between NK activating and inhibitory receptors is skewed toward activation. More importantly, NK cells display an altered cytokine pattern characterized by increased IFNγ and reduced IL2 production which could contribute to impaired Treg function. Exposure of mononuclear cells to IL2 resulted in normalization of NK IFNγ production. Thus, our findings support treatment of AIH-2 with low-dose IL2, which would result in normalization of NK cell function and expansion of the Treg cell subset.

Hepatitis C virus-induced NK cell activation causes metzincin-mediated CD16 cleavage and impaired antibody-dependent cytotoxicity.

BACKGROUND & AIMS: The Fc receptor family for immunoglobulin (Ig)G type III (FcγRIII, CD16) is an activating receptor on natural killer (NK) cells and an essential mediator of antibody-dependent cellular cytotoxicity (ADCC). There is only limited information on its role during chronic hepatitis C virus (HCV) infection. We studied CD16 expression in relation to NK cell functional activity in HCV-infected patients and sought mechanistic insights into virus-induced modulation. METHODS: NK cell CD16 expression and activation status were evaluated ex vivo by flow cytometry in HCV-infected patients and healthy controls (HC) as well as in vitro after co-culture with HCV-infected HuH7.5 cells. Rituximab-mediated ADCC was assessed in HC and HCV-infected patients using Daudi cells as a target. The role of metzincins in CD16 down-modulation was assessed using specific inhibitory molecules and by evaluating intracellular mRNA levels. RESULTS: HCV-infected patients exhibited increased frequencies of ex vivo activated NK cells and a concomitantly decreased NK CD16 expression, which resulted in impaired ADCC activity. Moreover, exposure of NK cells to culture-derived HCV recapitulated the ex vivo findings of decreased CD16 expression and increased NK cell activation. Importantly, blockade of metzincin-mediated shedding activity, including selective a disintegrin and metalloproteinase 17 (ADAM-17) inhibition, restored NK CD16 expression. Successful treatment with direct-acting antivirals partially improved NK ADCC function despite delayed CD16 reconstitution. CONCLUSION: Chronic HCV infection induces NK cell activation resulting in ADAM-17-dependent CD16 shedding and consequent impaired ADCC function. Altered ADCC may contribute to failure to eradicate HCV-infected hepatocytes. LAY SUMMARY: We show here that hepatitis C virus (HCV) activates natural killer (NK) lymphocytes which, as a consequence, loose their Fc receptor for IgG (CD16), an essential molecule for antibody binding. We show that this occurs through the action of enzymes named metzincins, resulting in altered NK-mediated antibody-dependent killing (ADCC) of target cells. This mechanism may contribute to HCV persistence and may represent a general phenomenon whereby some viruses can escape host's immune responses.

Monocytes inhibit hepatitis C virus-induced TRAIL expression on CD56bright NK cells.

BACKGROUND & AIMS: Natural killer (NK) cells play an important role in the pathogenesis of hepatitis C virus (HCV) infection. We have previously shown that culture-derived HCV (HCVcc) enhance tumor necrosis-factor-related apoptosis-inducing ligand (TRAIL) expression on healthy NK cells, but not on those from patients infected with HCV, which was likely dependent on accessory cells. Here we sought to elucidate the mechanisms involved in altered TRAIL upregulation in this setting. METHODS: Peripheral blood mononuclear cells (PBMC) from controls and patients infected with HCV were exposed to HCVcc. Cell depletions were performed to identify cells responsible for NK cell activation. Flow cytometry and ELISA were used to identify the cytokines involved in the NK activation process. RESULTS: In patients infected with HCV, soluble factors secreted by control PBMC restored the ability of NK cells to express TRAIL. Of note, CD14+ cell depletion had identical effects upon virus exposure and promoted increased degranulation. Moreover, increased concentrations of interleukin (IL)-18 binding protein a (IL-18BPa) and IL-36 receptor antagonist (IL-36RA) were observed after PBMC exposure to HCVcc in patients with HCV. HCVcc-induced NK cell TRAIL expression was inhibited by IL-18BPa and IL-36RA in control subjects. There were statistically significant correlations between IL-18BPa and indices of liver inflammation and fibrosis, supporting a role for this protein in the pathogenesis of chronic HCV infection. CONCLUSIONS: During chronic HCV infection, monocytes play a key role in negative regulation of NK cell activation, predominantly via secretion of inhibitors of IL-18 and IL-36. LAY SUMMARY: Coordination and collaboration between immune cells are essential to fight pathogens. Herein we show that during HCV infection monocytes secrete IL-18 and IL-36 inhibitory proteins, reducing NK cell activation, and consequently inhibiting their ability to express TRAIL and kill target cells.

Lack of Siglec-7 expression identifies a dysfunctional natural killer cell subset associated with liver inflammation and fibrosis in chronic HCV infection.

OBJECTIVE: Sialic-acid-binding immunoglobulin-like lectin-7 (Siglec-7) is a natural killer (NK) cell inhibitory receptor associated with NK phenotypic and functional abnormalities in HIV-1 infection. We investigated the significance of NK-expressed and serum soluble Siglec-7 in relation to NK functional ability and parameters of liver necroinflammation and fibrosis in chronic HCV infection. DESIGN: NK-expressed and serum Siglec-7 were evaluated in 130 and 166 HCV-infected individuals by flow cytometry and ELISA, respectively. NK cell degranulation and cytokine secretion were determined by flow cytometry. 65 patients with chronic HBV infection, 84 with chronic biliary disorders and 168 healthy donors served as controls. RESULTS: Expression of Siglec-7 was significantly decreased on NK cells from HCV-infected and HBV-infected patients and, conversely, serum Siglec-7 was significantly increased in these patients compared with controls. The frequency of Siglec-7pos NK cells was significantly higher at baseline in sustained virological responders to pegylated interferon-α/ribavirin treatment than in non-responders. Activating receptor expression was significantly higher in Siglec-7pos NK cells and was associated with increased degranulation and cytokine secretion compared with Siglec-7neg cells. In chronic HCV infection, there was an inverse correlation between Siglec-7 expression and serum aminotransferases, γ-glutamyl transpeptidase, liver stiffness, aspartate aminotransferase to platelet ratio index and fibrosis-4 scores, and a positive correlation between serum Siglec-7 and the same clinical parameters, including histological staging. CONCLUSIONS: These findings identify Siglec-7neg NK cells as a dysfunctional subpopulation associated with severe liver disease in chronic HCV infection.

NKp30 isoforms in patients with chronic hepatitis C virus infection.

Natural killer (NK) cells play an important role in virus infection, their action being regulated by several activating and inhibitory receptors. The NKp30 activating receptor and its isoforms have recently emerged as important determinants of efficient NK cell responses. We determined the relative proportions of NKp30 isoforms in patients with chronic hepatitis C virus (HCV) infection and healthy donors (HD). NK cell function (degranulation and cytokine production) and correlations with clinical parameters were assessed following unsupervised hierarchical clustering of patients according to isoform expression. NKp30 receptor expression on NK cells and all isoforms were reduced in HCV-infected patients. Patients were clustered into two groups: the HCV-1 group had similar isoform expression to the HD group, whereas the HCV-2 group had lower expression. The latter showed a better functional activity, and a higher proportion of the activating a isoform and of the NKp30 isoform a/c ratio compared with the HCV-1 cluster. There was a positive correlation between the activating a isoform and liver stiffness and an inverse relationship between the immunosuppressive c isoform and the fibrosis 4 score, suggesting a potentially important role of NKp30 isoforms in influencing liver damage and ensuing fibrosis.

Sialic acid-binding Ig-like lectin-7 interacts with HIV-1 gp120 and facilitates infection of CD4pos T cells and macrophages.

BACKGROUND: Sialic acid-binding Ig-like lectin-7 (Siglec-7) expression is strongly reduced on natural killer (NK) cells from HIV-1 infected viremic patients. To investigate the mechanism(s) underlying this phenomenon, we hypothesized that Siglec-7 could contribute to the infection of CD4pos target cells following its interaction with HIV-1 envelope (Env) glycoprotein 120 (gp120). RESULTS: The ability of Siglec-7 to bind gp120 Env in a sialic acid-dependent manner facilitates the infection of both T cells and monocyte-derived macrophages (MDMs). Indeed, pre-incubation of HIV-1 with soluble Siglec-7 (sSiglec-7) increases the infection rate of CD4pos T cells, which do not constitutively express Siglec-7. Conversely, selective blockade of Siglec-7 markedly reduces the degree of HIV-1 infection in Siglec-7pos MDMs. Finally, the sSiglec-7 amount is increased in the serum of AIDS patients with high levels of HIV-1 viremia and inversely correlates with CD4pos T cell counts. CONCLUSIONS: Our results show that Siglec-7 binds HIV-1 and contributes to enhance the susceptibility to infection of CD4pos T cells and MDMs. This phenomenon plays a role in HIV-1 pathogenesis and in disease progression, as suggested by the inverse correlation between high serum level of sSiglec-7 and the low CD4pos T cell count observed in AIDS patients in the presence of chronic viral replication.

Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection.

BACKGROUND & AIMS: A substantial proportion of patients with chronic hepatitis C virus infection treated with pegylated interferon α/ribavirin fail to achieve sustained virological response (SVR). Since growing evidence suggests that innate immunity may influence treatment responses, we examined natural killer (NK) cell phenotypic and functional changes during standard antiviral therapy. METHODS: Expression of several NK-cell regulatory molecules was evaluated by flow cytometry in 37 consecutive patients with chronic HCV infection at baseline and at different time points during and after discontinuation of treatment. Cytokine production was evaluated by intracellular staining. Cytolytic potential was assessed as degranulation and as antibody-dependent cytotoxicity. RESULTS: Baseline frequencies of CD56(dim) NK cells and perforin content were significantly higher, whereas CD16 expression was lower in SVR vs. non-responder subjects. Analysis by linear regression for repeated measures during the first 12 weeks showed significantly increased frequencies of activated (CD69(+)) NK cells in rapid virological responders (RVR) and identified a typical NK cell profile associated with SVR, featuring higher NK perforin content, lower CD16 expression, and higher proportion of CD56(dim)/CD16(-) cells. Moreover, SVR patients displayed higher natural and antibody-dependent NK cell cytotoxicity. IL28B rs12979860 CC homozygosis was significantly associated with SVR, independently of NK-cell phenotype and function. CONCLUSIONS: Different NK-cell phenotypic and functional features, in patients with chronic hepatitis C treated with standard therapy, were observed between non-responder vs. SVR patients, suggesting a potential role of NK cells in the response to treatment.

Impaired intrahepatic natural killer cell cytotoxic function in chronic hepatitis C virus infection.

UNLABELLED: Hepatitis C virus (HCV) persistence in the host results from inefficiencies of innate and adaptive immune responses. Most studies addressing the role of innate immunity concentrated on peripheral blood (PB) natural killer (NK) cells, whereas only limited information is available on intrahepatic (IH) NK cells. We therefore examined phenotypic and functional features of IH and PB NK cells in paired liver biopsy and venous blood samples from 70 patients with chronic HCV infection and 26 control persons subjected to cholecystectomy for gallstones as controls. Ex vivo isolated IH NK cells from HCV-infected patients displayed unique phenotypic features, including increased expression of NKp46-activating receptor in the face of reduced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cluster of differentiation (CD) 107a expression, which resulted in impaired degranulation compared with controls. To gain insights into the effect of HCV on NK cells, we exposed peripheral blood mononuclear cells (PBMCs) from patients and healthy donors to cell-culture-derived HCV (HCVcc) and measured NK cell degranulation, TRAIL, and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) expression. Exposure of PBMCs to HCVcc significantly boosted NK degranulation, pERK1/2, and TRAIL expression in healthy donors, but not in patients with chronic HCV infection, a defect that was completely reversed by interferon-alpha. Purified NK cells showed a minimal, though significant, increase in degranulation and TRAIL expression, both in patients and controls, after exposure to HCVcc. CONCLUSIONS: These findings indicate dysfunctional IH NK cell cytotoxicity associated with TRAIL down-regulation in chronic HCV infection, which may contribute to virus persistence. PB NK cell impairment upon exposure to HCVcc suggests the existence of an accessory cell-dependent NK cell lytic defect in chronic HCV infection predominantly involving the TRAIL pathway.

Different combinations of cytokines and activating receptor stimuli are required for human natural killer cell functional diversity.

Although cytokine induced NK cell activation protocols are commonly used in many laboratories worldwide, a systematic study of the effect of different cytokines either alone or in combination on NK cell function is lacking. In this study we performed a comparative evaluation of several cytokines potentially important for NK cell stimulation, focusing particularly on IL21 because of its promising role in anti-tumor therapy. To simulate in vivo physiological condition, we evaluated cytokine stimulation in total peripheral blood mononuclear cells (PBMCs), as accessory cells are responsible for the secretion of many soluble factors and can simultaneously trigger multiple activation signals through engagement of NK cell activating receptors. We show here that NK cell responses are finely regulated by several incoming stimuli and that combinations of IL21+IL2 or IL21+IL15 strongly induced NK cell function. Cytokine stimulation combined with NK receptor engagement can be helpful in the dissection of NK cell responses in health and disease.

Ceramide present in cholangiocarcinoma-derived extracellular vesicle induces a pro-inflammatory state in monocytes.

Cholangiocarcinoma (CCA) is a rare cancer characterized by a global increasing incidence. Extracellular vesicles (EV) contribute to many of the hallmarks of cancer through transfer of their cargo molecules. The sphingolipid (SPL) profile of intrahepatic CCA (iCCA)-derived EVs was characterized by liquid chromatography-tandem mass spectrometry analysis. The effect of iCCA-derived EVs as mediators of inflammation was assessed on monocytes by flow cytometry. iCCA-derived EVs showed downregulation of all SPL species. Of note, poorly-differentiated iCCA-derived EVs showed a higher ceramide and dihydroceramide content compared with moderately-differentiated iCCA-derived EVs. Of note, higher dihydroceramide content was associated with vascular invasion. Cancer-derived EVs induced the release of pro-inflammatory cytokines in monocytes. Inhibition of synthesis of ceramide with Myriocin, a specific inhibitor of the serine palmitoyl transferase, reduced the pro-inflammatory activity of iCCA-derived EVs, demonstrating a role for ceramide as mediator of inflammation in iCCA. In conclusion, iCCA-derived EVs may promote iCCA progression by exporting the excess of pro-apoptotic and pro-inflammatory ceramides.

Impaired intratumoral natural killer cell function in head and neck carcinoma.

Natural killer (NK) cells are emerging as unique players in the immune response against cancer; however, only limited data are available on tumor infiltrating NK cells in head and neck squamous cell carcinoma (HNSCC), one of the most common cancer. Occurrence of HNSCC is closely related to the immune microenvironment, and immunotherapy is increasingly being applied to this setting. However, the limited success of this type of treatment in this tumor calls for further investigation in the field. Surgical HNSSC specimens of 32 consecutive patients were mechanically and enzymatically dissociated. Tumor cells were separated from infiltrating cells by short centrifugation and infiltrating NK cells were phenotypically and functionally characterized by multiple antibody staining and flow cytometry. Tumor infiltrating NK cells in HNSCC showed a peculiar phenotype predominantly characterized by increased NKG2A and reduced Siglec-7, NKG2D, NKp30 and CD16 expression. This phenotype was associated with a decreased ability to perform antibody-dependent cellular cytotoxicity (ADCC). However, NK, CD4 and CD8 shared an increment of glucocorticoid-induced tumor necrosis factor-related (GITR) costimulatory receptor which could be exploited for immunotherapy with agonistic anti-GITR antibodies combined with checkpoint inhibitors.