VDAC1-interacting molecules promote cell death in cancer organoids through mitochondrial-dependent metabolic interference.

The voltage-dependent anion-selective channel isoform 1 (VDAC1) is a pivotal component in cellular metabolism and apoptosis with a prominent role in many cancer types, offering a unique therapeutic intervention point. Through an in-silico-to-in-vitro approach we identified a set of VA molecules (VDAC Antagonists) that selectively bind to VDAC1 and display specificity toward cancer cells. Biochemical characterization showed that VA molecules can directly interact with VDAC1 with micromolar affinity by competing with the endogenous ligand NADH for a partially shared binding site. NADH displacement results in mitochondrial distress and reduced cell proliferation, especially when compared to non-cancerous cells. Experiments performed on organoids derived from intrahepatic cholangiocarcinoma patients demonstrated a dose-dependent reduction in cell viability upon treatment with VA molecules with lower impact on healthy cells than conventional treatments like gemcitabine. VA molecules are chemical entities representing promising candidates for further optimization and development as cancer therapy strategies through precise metabolic interventions.

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.

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.

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.

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.

Defective DNAM-1 Dependent Cytotoxicity in Hepatocellular Carcinoma-Infiltrating NK Cells.

BACKGROUND: Natural killer (NK) cells play a key role in immune surveillance and response to tumors, their function regulated by NK cell receptors and their ligands. The DNAM-1 activating receptor recognizes the CD155 molecule expressed in several tumor cells, such as hepatocellular carcinoma (HCC). This study aims to investigate the role of the DNAM-1/CD155 axis in mediating the NK cell response in patients with HCC. METHODS: Soluble CD155 was measured by ELISA. CD155 expression was sought in HCC cells by immunohistochemistry, qPCR, and flow cytometry. DNAM-1 modulation in NK cells was evaluated in transwell experiments and by a siRNA-mediated knockdown. NK cell functions were examined by direct DNAM-1 triggering. RESULTS: sCD155 was increased in sera from HCC patients and correlated with the parameters of an advanced disease. The expression of CD155 in HCC showed a positive trend toward better overall survival. DNAM-1 downmodulation was induced by CD155-expressing HCC cells, in agreement with lower DNAM-1 expressions in tumor-infiltrating NK (NK-TIL) cells. DNAM-1-mediated cytotoxicity was defective both in circulating NK cells and in NK-TIL of HCC patients. CONCLUSIONS: We provide evidence of alterations in the DNAM-1/CD155 axis in HCC, suggesting a possible mechanism of tumor resistance to innate immune surveillance.

MICA/B-targeted antibody promotes NK cell-driven tumor immunity in patients with intrahepatic cholangiocarcinoma.

The major histocompatibility complex-class I chain related proteins A and B (MICA/B) is upregulated because of cellular stress and MICA/B shedding by cancer cells causes escape from NKG2D recognition favoring the emergence of cancers. Cholangiocarcinoma (CCA) is a relatively rare, though increasingly prevalent, primary liver cancer characterized by a late clinical presentation and a dismal prognosis. We explored the NKG2D-MICA/B axis in NK cells from 41 patients with intrahepatic cholangiocarcinoma (iCCA). The MICA/B-specific 7C6 mAb was used for ex vivo antibody-dependent cytotoxicity (ADCC) experiments using circulating, non tumor liver- and tumor-infiltrating NK cells against the HuCCT-1 cell line and patient-derived primary iCCA cells as targets. MICA/B were more expressed in iCCA than in non-tumoral tissue, MICA transcription being higher in moderately-differentiated compared with poorly-differentiated cancer. Serum MICA was elevated in iCCA patients in line with higher expression of ADAM10 and ADAM17 that are responsible for proteolytic release of MICA/B from tumor. Addition of 7C6 significantly boosted peripheral, liver- and tumor-infiltrating-NK cell degranulation and IFNγ production toward MICA/B-expressing established cell lines and autologous iCCA patient target cells. Our data show that anti-MICA/B drives NK cell anti-tumor activity, and provide preclinical evidence in support of 7C6 as a potential immunotherapeutic tool for iCCA.

Unique immunological profile in patients with COVID-19.

The relationship between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and host immunity is poorly understood. We performed an extensive analysis of immune responses in 32 patients with severe COVID-19, some of whom succumbed. A control population of healthy subjects was included. Patients with COVID-19 had an altered distribution of peripheral blood lymphocytes, with an increased proportion of mature natural killer (NK) cells and low T-cell numbers. NK cells and CD8+ T cells overexpressed T-cell immunoglobulin and mucin domain-3 (TIM-3) and CD69. NK cell exhaustion was attested by increased frequencies of programmed cell death protein 1 (PD-1) positive cells and reduced frequencies of natural killer group 2 member D (NKG2D)-, DNAX accessory molecule-1 (DNAM-1)- and sialic acid-binding Ig-like lectin 7 (Siglec-7)-expressing NK cells, associated with a reduced ability to secrete interferon (IFN)γ. Patients with poor outcome showed a contraction of immature CD56bright and an expansion of mature CD57+ FcεRIγneg adaptive NK cells compared to survivors. Increased serum levels of IL-6 were also more frequently identified in deceased patients compared to survivors. Of note, monocytes secreted abundant quantities of IL-6, IL-8, and IL-1ß which persisted at lower levels several weeks after recovery with concomitant normalization of CD69, PD-1 and TIM-3 expression and restoration of CD8+ T cell numbers. A hyperactivated/exhausted immune response dominate in severe SARS-CoV-2 infection, probably driven by an uncontrolled secretion of inflammatory cytokines by monocytes. These findings unveil a unique immunological profile in COVID-19 patients that will help to design effective stage-specific treatments for this potentially deadly disease.

An Anti-MICA/B Antibody and IL-15 Rescue Altered NKG2D-Dependent NK Cell Responses in Hepatocellular Carcinoma.

Natural killer (NK) cells play a pivotal role in cancer immune surveillance, and activating the receptor/ligand interaction may contribute to control the development and evolution of hepatocellular carcinoma (HCC). We investigated the role of the natural killer group 2 member D (NKG2D) activating receptor and its ligand, the major histocompatibility complex class I chain-related protein A and B (MICA/B) in patients with cirrhosis and HCC subjected to surgical resection, patients with cirrhosis and no HCC, and healthy donors (HD). The NKG2D-mediated function was determined in peripheral blood (PB), in tumor-infiltrating lymphocytes (NK-TIL), and in matched surrounding liver tissue (NK-LIL). A group of patients treated with sorafenib because of clinically advanced HCC was also studied. A humanized anti-MICA/B monoclonal antibody (mAb) was used in in vitro experiments to examine NK cell-mediated antibody-dependent cellular cytotoxicity. Serum concentrations of soluble MICA/B were evaluated by ELISA. IL-15 stimulation increased NKG2D-dependent activity which, however, remained dysfunctional in PB NK cells from HCC patients, in line with the reduced NKG2D expression on NK cells. NK-TIL showed a lower degranulation ability than NK-LIL, which was restored by IL-15 stimulation. Moreover, in vitro IL-15 stimulation enhanced degranulation and interferon-γ production by PB NK from patients at month one of treatment with sorafenib. Anti-MICA/B mAb associated with IL-15 was able to induce PB NK cytotoxicity for primary HCC cells in HD and patients with HCC, who also showed NK-TIL degranulation for autologous primary HCC cells. Our findings highlight the key role of the NKG2D-MICA/B axis in the regulation of NK cell responses in HCC and provide evidence in support of a potentially important role of anti-MICA/B mAb and IL-15 stimulation in HCC immunotherapy.

Natural Killer Cell Responses in Hepatocellular Carcinoma: Implications for Novel Immunotherapeutic Approaches.

Hepatocellular carcinoma (HCC) still represents a significant complication of chronic liver disease, particularly when cirrhosis ensues. Current treatment options include surgery, loco-regional procedures and chemotherapy, according to specific clinical practice guidelines. Immunotherapy with check-point inhibitors, aimed at rescuing T-cells from exhaustion, has been applied as second-line therapy with limited and variable success. Natural killer (NK) cells are an essential component of innate immunity against cancer and changes in phenotype and function have been described in patients with HCC, who also show perturbations of NK activating receptor/ligand axes. Here we discuss the current status of NK cell treatment of HCC on the basis of existing evidence and ongoing clinical trials on adoptive transfer of autologous or allogeneic NK cells ex vivo or after activation with cytokines such as IL-15 and use of antibodies to target cell-expressed molecules to promote antibody-dependent cellular cytotoxicity (ADCC). To this end, bi-, tri- and tetra-specific killer cell engagers are being devised to improve NK cell recognition of tumor cells, circumventing tumor immune escape and efficiently targeting NK cells to tumors. Moreover, the exciting technique of chimeric antigen receptor (CAR)-engineered NK cells offers unique opportunities to create CAR-NK with multiple specificities along the experience gained with CAR-T cells with potentially less adverse effects.

Human Monoclonal Antibodies as Adjuvant Treatment of Chronic Hepatitis B Virus Infection.

Despite the availability of an effective prophylactic vaccine leading to sterilizing immunity, hepatitis B virus (HBV) is responsible for chronic liver disease in more than 250 million individuals, potentially leading to cirrhosis and hepatocellular carcinoma. Antiviral drugs able to completely suppress virus replication are indeed available but they are, by and large, unable to eradicate the virus. Several alternative new treatment approaches are currently being developed but none have so far captured the interest of clinicians for possible clinical development. A constant feature of chronic HBV infection is T-cell exhaustion resulting from persistent exposure to high antigen concentrations as shown by the high expression of programmed cell death protein 1 (PD-1) by HBV-specific CD8 T cells. One way of tackling this problem is to develop HBV-specific neutralizing antibodies that would clear excess envelope proteins from the circulation, allowing for nucleos(t)ide analogs or other antiviral drugs now in preclinical and early clinical development to take advantage of a reconstituted adaptive immunity. Several fully human monoclonal antibodies (mAb) have been developed from HBV-vaccinated and subjects convalescent from acute hepatitis B that show different properties and specificities. It is envisaged that such neutralizing mAb may be used as adjuvant treatment to reduce viral protein load, thus rescuing adaptive immunity in an effort to optimize the effect of antiviral drugs.

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.

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.

Taxanes enhance trastuzumab-mediated ADCC on tumor cells through NKG2D-mediated NK cell recognition.

Recent clinical data indicate a synergistic therapeutic effect between trastuzumab and taxanes in neoadjuvantly treated HER2-positive breast cancer (BC) patients. In HER2+ BC experimental models and patients, we investigated whether this synergy depends on the ability of drug-induced stress to improve NK cell effectiveness and thus trastuzumab-mediated ADCC. HER2+ BC cell lines BT474 and MDAMB361 treated with docetaxel showed up-modulation of NK activator ligands both in vitro and in vivo, accompanied by a 15-40% increase in in vitro trastuzumab-mediated ADCC; antibodies blocking the NKG2D receptor significantly reduced this enhancement. NKG2D receptor expression was increased by docetaxel treatment in circulating and splenic NK cells from mice xenografted with tumor cells, an increase related to expansion of the CD11b+Ly6G+ cell population. Accordingly, NK cells derived from HER2+ BC patients after treatment with taxane-containing therapy expressed higher levels of NKG2D receptor than before treatment. Moreover, plasma obtained from these patients recapitulated the modulation of NKG2D on healthy donors' NK cells, improving their trastuzumab-mediated activity in vitro. This enhancement occurred mainly using plasma from patients with low NKG2D basal expression. Our results indicate that taxanes increase tumor susceptibility to ADCC by acting on tumor and NK cells, and suggest that taxanes concomitantly administered with trastuzumab could maximize the antibody effect, especially in patients with low basal immune effector cytotoxic activity.

Amyloid ß-peptide-dependent activation of human platelets: essential role for Ca2+ and ADP in aggregation and thrombus formation.

Alzheimer's disease is associated with the accumulation of Aß (amyloid ß)-peptides in the brain. Besides their cytotoxic effect on neurons, Aß-peptides are thought to be responsible for the atherothrombotic complications associated with Alzheimer's disease, which are collectively known as cerebrovascular disease. In the present study, we investigated the effect of Aß-peptides on human platelet signal transduction and function. We discovered that the 25-35 domain of Aß-peptides induce an increase in platelet intracellular Ca2+ that stimulates α-granule and dense granule secretion and leads to the release of the secondary agonist ADP. Released ADP acts in an autocrine manner as a stimulant for critical signalling pathways leading to the activation of platelets. This includes the activation of the protein kinases Syk, protein kinase C, Akt and mitogen-activated protein kinases. Ca2+-dependent release of ADP is also the main component of the activation of the small GTPase Rap1b and the fibrinogen receptor integrin αIIbß3, which leads to increased platelet aggregation and increased thrombus formation in human whole blood. Our discoveries complement existing understanding of cerebrovascular dementia and suggest that Aß-peptides can induce vascular complications of Alzheimer's disease by stimulating platelets in an intracellular Ca2+-dependent manner. Despite a marginal ADP-independent component suggested by low levels of signalling activity in the presence of apyrase or P2Y receptor inhibitors, Ca2+-dependent release of ADP by Aß-peptides clearly plays a critical role in platelet activation. Targeting ADP signalling may therefore represent an important strategy to manage the cerebrovascular component of Alzheimer's disease.

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.

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.

Natural killer cell functional dichotomy: a feature of chronic viral hepatitis?

Natural killer (NK) cells are involved in innate immune responses to viral infections either via direct cytotoxicity which destroys virus-infected cells or production of immunoregulatory cytokines which modulate adaptive immunity and directly inhibit virus replication. These functions are mediated by different NK subpopulations, with cytotoxicity being generally performed by CD56(dim) NK cells, whereas CD56(bright) NK cells are mainly involved in cytokine secretion. NK functional defects are usually combined so that impaired degranulation is often associated with deficient cytokine production. Innate immunity is thought to be relevant in the control of hepatitis virus infections such as hepatitis B virus (HBV) and hepatitis C virus (HCV), and recent findings reproducibly indicate that NK cells in chronic viral hepatitis are characterized by a functional dichotomy, featuring a conserved or enhanced cytotoxicity and a reduced production of interferon (IFN)-γ and tumor necrosis factor-α. In chronic HCV infection this appears to be caused by altered IFN-α signaling resulting from increased signal transducer and activator of transcription 1 (STAT1) phosphorylation, which polarizes NK cells toward cytotoxicity, and a concomitantly reduced IFN-α induced STAT4 phosphorylation yielding reduced IFN-γ mRNA levels. These previously unappreciated findings are compatible on the one hand with the inability to clear HCV and HBV from the liver and on the other they may contribute to understand why these patients are often resistant to IFN-α-based therapies.

Enhanced B-cell differentiation and reduced proliferative capacity in chronic hepatitis C and chronic hepatitis B virus infections.

BACKGROUND & AIMS: Chronic microbial infections are frequently associated with B-cell activation and polyclonal proliferation, potentially leading to autoimmunity and lymphoproliferative disorders. We assessed B-cell phenotype and function in chronic hepatitis B (HBV) and chronic hepatitis C (HCV) virus infection. METHODS: We studied 70 patients with chronic HCV infection, 34 with chronic HBV infection and 54 healthy controls. B-cell phenotype was assessed by flow cytometry using monoclonal antibodies specific for CD27, the CD69, CD71, and CD86 activation markers and the chemokine receptor CXCR3. Differentiation into immunoglobulin-producing cells (IPC) was analysed by ELISpot upon stimulation and with CD40 ligand±IL-10 as surrogate bystander T-cell help or CpG oligodeoxynucleotide±IL-2, as innate immunity signal. Proliferation was examined by flow cytometry using carboxyfluorescein diacetate succinimidyl ester (CFSE) after stimulation with CpG. RESULTS: A significantly higher proportion of B cells from both HCV- and HBV-infected patients expressed activation markers compared with controls and a positive correlation was found between CXCR3(+) B cells and HCV RNA values. Memory B cells from patients with chronic HCV and HBV infections showed enhanced differentiation into IPC compared with controls, although this was restricted to IgG and at a lower level in HCV-compared with HBV-infected patients. Moreover, patients' activated B cells displayed significantly lower proliferative ability compared to healthy donors despite low expression of the FcRL4 exhaustion marker. CONCLUSIONS: B-cell activation, but not exhaustion, is common in chronic viral hepatitis. However, enhanced B-cell differentiation and deficient proliferative capacity were not associated with commitment to terminal differentiation.