STAT activation in regulatory CD4+ T cells of patients with primary sclerosing cholangitis.

INTRODUCTION: Regulatory CD4+ T cells (Tregs) are pivotal for inhibition of autoimmunity. Primary sclerosing cholangitis (PSC) is an autoimmune cholestatic liver disease of unknown etiology where contribution of Tregs is still unclear. Activation of the JAK-STAT pathway critically modifies functions of Tregs. In PSC, we studied activation of STAT proteins and Treg functions in response to cytokines. METHODS: In 51 patients with PSC, 10 disease controls (chronic replicative hepatitis C), and 36 healthy controls we analyzed frequencies of Foxp3+CD25+CD127lowCD4+ Tregs, their expression of ectonucleotidase CD39, and cytokine-induced phosphorylation of STAT1, 3, 5, and 6 using phospho-flow cytometry. In parallel, we measured cytokines IFN-gamma, interleukin (IL)-6, IL-2, and IL-4 in serum via bead-based immunoassays. RESULTS: In patients with PSC, ex vivo frequencies of peripheral Tregs and their expression of CD39 were significantly reduced (p < .05 each). Furthermore, serum levels of IFN-gamma, IL-6, IL-2, and IL-4 were markedly higher in PSC (p < .05 each). Unlike activation of STAT1, STAT5, and STAT6, IL-6 induced increased phosphorylation of STAT3 in Tregs of PSC-patients (p = .0434). Finally, STAT3 activation in Tregs correlated with leukocyte counts. CONCLUSIONS: In PSC, we observed enhanced STAT3 responsiveness of CD4+ Tregs together with reduced CD39 expression probably reflecting inflammatory activity of the disease.

IL-6-Dependent STAT3 Activation and Induction of Proinflammatory Cytokines in Primary Sclerosing Cholangitis.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease with periductal inflammation and fibrosis. Genetic studies suggest inflammatory cytokines and IL-6-dependent activation of transcription factor STAT3 as pivotal steps in PSC pathogenesis. However, details of inflammatory regulation remain unclear. METHODS: We recruited 50 patients with PSC (36 with inflammatory bowel disease, 14 without inflammatory bowel disease), 12 patients with autoimmune hepatitis, and 36 healthy controls to measure cytokines in the serum, bile, and immune cell supernatant using bead-based immunoassays and flow cytometry and immunohistochemistry to analyze phosphorylation of STATs in immune cells. Finally, we analyzed cytokines and STAT3 phosphorylation of T cells in the presence of JAK1/2 inhibitors. RESULTS: In PSC, IL-6 specifically triggered phosphorylation of STAT3 in CD4 + T cells and lead to enhanced production of interferon (IFN) gamma and interleukin (IL)-17A. Phospho-STAT3-positive CD4 + T cells correlated with systemic inflammation (C-reactive protein serum levels). Combination of immunohistology and flow cytometry indicated that phospho-STAT3-positive cells were enriched in the peribiliary liver stroma and represented CD4 + T cells with prominent production of IFN gamma and IL-17A. JAK1/2 inhibitors blocked STAT3 phosphorylation and production of IFN gamma and IL-6, whereas IL-17A was apparently resistant to this inhibition. DISCUSSION: Our results demonstrate systemic and local activation of the IL-6/STAT3 pathway in PSC. Resistance of IL-17A to STAT3-targeted inhibition points to a more complex immune dysregulation beyond STAT3 activation.

High producer variant of lipoprotein lipase may protect from hepatocellular carcinoma in alcohol-associated cirrhosis.

Background & Aims: Progression of alcohol-associated liver disease (ALD) is driven by genetic predisposition. The rs13702 variant in the lipoprotein lipase (LPL) gene is linked to non-alcoholic fatty liver disease. We aimed at clarifying its role in ALD. Methods: Patients with alcohol-associated cirrhosis, with (n = 385) and without hepatocellular carcinoma (HCC) (n = 656), with HCC attributable to viral hepatitis C (n = 280), controls with alcohol abuse without liver damage (n = 366), and healthy controls (n = 277) were genotyped regarding the LPL rs13702 polymorphism. Furthermore, the UK Biobank cohort was analysed. LPL expression was investigated in human liver specimens and in liver cell lines. Results: Frequency of the LPL rs13702 CC genotype was lower in ALD with HCC in comparison to ALD without HCC both in the initial (3.9% vs. 9.3%) and the validation cohort (4.7% vs. 9.5%; p <0.05 each) and compared with patients with viral HCC (11.4%), alcohol misuse without cirrhosis (8.7%), or healthy controls (9.0%). This protective effect (odds ratio [OR] = 0.5) was confirmed in multivariate analysis including age (OR = 1.1/year), male sex (OR = 3.0), diabetes (OR = 1.8), and carriage of the PNPLA3 I148M risk variant (OR = 2.0). In the UK Biobank cohort, the LPL rs13702 C allele was replicated as a risk factor for HCC. Liver expression of LPL mRNA was dependent on LPL rs13702 genotype and significantly higher in patients with ALD cirrhosis compared with controls and alcohol-associated HCC. Although hepatocyte cell lines showed negligible LPL protein expression, hepatic stellate cells and liver sinusoidal endothelial cells expressed LPL. Conclusions: LPL is upregulated in the liver of patients with alcohol-associated cirrhosis. The LPL rs13702 high producer variant confers protection against HCC in ALD, which might help to stratify people for HCC risk. Impact and implications: Hepatocellular carcinoma is a severe complication of liver cirrhosis influenced by genetic predisposition. We found that a genetic variant in the gene encoding lipoprotein lipase reduces the risk for hepatocellular carcinoma in alcohol-associated cirrhosis. This genetic variation may directly affect the liver, because, unlike in healthy adult liver, lipoprotein lipase is produced from liver cells in alcohol-associated cirrhosis.

Genetic Variation of SAMM50 Is Not an Independent Risk Factor for Alcoholic Hepatocellular Carcinoma in Caucasian Patients.

Hepatocellular carcinoma (HCC) is a severe complication of advanced alcoholic liver disease, which is modulated by genetic predisposition. Identifying new genetic loci might improve screening. Genetic variation of SAMM50 was linked to HCC. We aimed to validate this finding in a large cohort of patients with advanced alcoholic liver disease (ALD). A large, well-characterised cohort of patients with alcoholic cirrhosis without (n = 674) and with (n = 386) HCC, as well as controls with HCC due to viral hepatitis (n = 134), controls with heavy alcohol abuse without liver disease (n = 266) and healthy subjects (n = 237), were genotyped for SAMM50 rs3827385 and rs3761472 and for PNPLA3 rs738409. Genotype frequencies were compared between patients with alcohol-associated cirrhosis with and without HCC by uni- and multivariate analysis. Minor variants in both SAMM50 rs3827385 and rs3761472 were significantly more frequent in patients with alcoholic HCC versus alcoholic cirrhosis and versus the control cohorts. An even stronger association was noted for PNPLA3 rs738409. The univariate analysis resulted in an odds ratio (OR) of 1.8 for carriers of at least one minor variant of SAMM50 rs3827385 and rs3761472 (each p < 0.001), but this association was lost in multivariate analysis with age (OR 1.1/year), male sex (OR 3.2), diabetes (OR 1.9) and carriage of PNPLA3 148M (OR 2.1) remaining in the final model. Although minor variants of both SAMM50 loci are strongly associated with alcoholic HCC, this association is not independent of carriage of the well-known risk variant PNPLA3 148M.

Analysis of antibodies from HCV elite neutralizers identifies genetic determinants of broad neutralization.

The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.

A genetic variant in toll-like receptor 5 is linked to chemokine levels and hepatocellular carcinoma in steatohepatitis.

BACKGROUND & AIMS: Bacterial translocation drives liver disease progression. We investigated whether functional genetic variants in toll-like receptor 5 (TLR5), the receptor for bacterial flagellin, affect the risk for hepatocellular carcinoma (HCC). METHODS: Healthy controls (n = 212), patients with alcohol abuse without liver disease (n = 382), and patients from a discovery cohort of alcohol-associated cirrhosis (n = 372 including 79 HCC cases), a validation cohort of alcohol-associated cirrhosis (n = 355 including 132 HCC cases), and a cohort of cirrhosis due to nonalcoholic steatohepatitis (NASH) (n = 145 including 62 HCC cases) were genotyped for the TLR5 rs5744174 and rs5744168 polymorphisms. Chemokine levels were measured by ELISA in patients' sera and supernatants of flagellin-stimulated healthy monocytes. RESULTS: Frequency of the TLR5 rs5744174 TT genotype was similar in healthy controls (33%), controls with alcohol abuse (34%), and patients with alcohol-associated cirrhosis in the discovery (28%), validation (33%), and NASH cohort (31%). The TT genotype was enriched in patients with versus without HCC in the discovery, validation, and NASH cohort (41% vs 25%; 39% vs 29%; 40% vs 24%; p < .05 each). This genotype remained a risk factor for HCC (OR = 1.9; p = .01) after multivariate correction for age, gender, diabetes, and carriage of the PNPLA3 148M variant. Interleukin-8 induction in monocytes from healthy controls and serum levels of interleukin-8 and CXCL1 from cirrhotic patients with the TT genotype were significantly increased versus C allele carriers. CONCLUSION: The TLR5 rs5744174 polymorphism, affecting immune response to flagellin, is linked to occurrence of HCC in cirrhosis caused by steatohepatitis.

Role of regulatory T cells and checkpoint inhibition in hepatocellular carcinoma.

Immune checkpoint inhibition suggests promising progress for the treatment of advanced hepatocellular carcinoma (HCC). However, the underlying cellular mechanisms remain unclear because liver cancer cells apparently do not upregulate inhibitory checkpoint molecules. Here, we analysed whether regulatory T cells (Tregs) can alternatively trigger checkpoint inhibition pathways in HCC. Using flow cytometry we analysed expression of checkpoint molecules (PD-1, PD-L1, CTLA-4, GITR, Tim-3) on peripheral CD4+CD25+Foxp3+ Tregs and their secretion of inhibitory mediators (IL-10, IL-35, TGF-beta, galectin-9) in 116 individuals (50 patients with HCC, 41 non-tumour bearing liver disease controls, 25 healthy controls). Functional activity of Tregs on T effector cells (IFN-gamma production, cytotoxicity) was characterized in vitro using a lectin-dependent cellular cytotoxicity (LDCC) assay against checkpoint inhibitor-negative P815 target cells. Unlike liver patients without malignancy and healthy controls, the frequency of checkpoint inhibitor-positive Tregs inversely correlated to age of patients with HCC (PD-L1, p = 0.0080; CTLA-4, p = 0.0029) and corresponded to enhanced numbers of Tregs producing IL-10 and IL-35 (p < 0.05 each). Tregs inhibited IFN-gamma secretion and cytotoxicity of CD8+ T cells when added to LDCC against P815 cells. Treg-induced inhibition of IFN-gamma secretion could be partially blocked by neutralizing PD-1 and PD-L1 antibodies specifically in HCC patients. In HCC peripheral Tregs upregulate checkpoint inhibitors and contribute to systemic immune dysfunction and antitumoural activity by several inhibitory pathways, presumably facilitating tumour development at young age. Blocking PD-L1/PD-1 interactions in vitro selectively interfered with inhibitory Treg -T effector cell interactions in the patients with HCC and resulted in improved antitumoural activity also against checkpoint inhibitor-negative tumour cells.

The PNPLA3 I148M variant promotes lipid-induced hepatocyte secretion of CXC chemokines establishing a tumorigenic milieu.

The I148M variant of the Patatin-like phospholipase domain-containing 3 (PNPLA3) protein is associated with an increased risk for liver inflammation and hepatocellular carcinoma (HCC), but the underlying mechanism is unknown. We hypothesized that enhanced CXC chemokine secretion mediates hepatic inflammation that accelerates development of HCC. Expandable primary human (upcyte®) hepatocytes and human PLC/PRF/5 hepatoma cells were lentivirally transduced with both PNPLA3 I148M variants and stimulated with lipids. Cytokine levels in culture supernatant and patient sera (n = 80) were analyzed by ELISA. Supernatants were assessed in transmigration experiments, tube formation, and proliferation assays. In vitro, lipid stimulation of transduced hepatocytes dose-dependently induced the production of interleukin-8 and CXCL1 in hepatocytes carrying the PNPLA3 148M variant. In line, sera from PNPLA3 148M-positive patients with alcoholic liver cirrhosis contained higher levels of interleukin-8 and CXCL1 than patients with wild-type PNPLA3. Supernatants from lipid-stimulated hepatocytes with the PNPLA3 148M variant induced enhanced migration of white blood cells, angiogenesis, and cell proliferation in comparison with supernatants from wild-type hepatocytes via CXC receptors 1 and 2. Increased production of interleukin-8 and CXCL1 by hepatocytes carrying the PNPLA3 148M variant contributes to a pro-inflammatory and tumorigenic milieu in patients with alcoholic liver disease. KEY MESSAGES: The PNPLA3 148M variant is associated with cirrhosis and hepatocellular carcinoma. Lipid stimulation of hepatocytes with this variant induces IL-8 and CXCL1. Supernatants from hepatocytes with this variant promote migration and angiogenesis. Sera from patients with this variant contained enhanced levels of IL-8 and CXCL1. The PNPLA3 148M variant contributes to a tumorigenic milieu via IL-8 and CXCL1.

FOXP3 Inhibitory Peptide P60 Increases Efficacy of Cytokine-induced Killer Cells Against Renal and Pancreatic Cancer Cells.

BACKGROUND/AIM: Cytokine-induced killer (CIK) cells are ex vivo expanded major histocompatibility complex (MHC)-unrestricted cytotoxic cells with promising effects against a variety of cancer types. Regulatory T-cells (T-reg) have been shown to reduce the effectiveness of CIK cells against tumor cells. Peptide P60 has been shown to inhibit the immunosuppressive functions of T-regs. This study aimed at examining the effect of p60 on CIK cells efficacy against renal and pancreatic cancer cells. MATERIALS AND METHODS: The effect of P60 on CIK cytotoxicity was examined using flow cytometry, WST-8-based cell viability assay and interferon γ (IFNγ) ELISA. RESULTS: P60 treatment resulted in a significant decrease in the viability of renal and pancreatic cancer cell lines co-cultured with CIK cells. No increase in IFNγ secretion from CIK cells was detected following treatment with P60. P60 caused no changes in the distribution of major effector cell populations in CIK cell cultures. CONCLUSION: P60 may potentiate CIK cell cytotoxicity against tumor cells.

Increased peripheral CD4+ regulatory T cells persist after successful direct-acting antiviral treatment of chronic hepatitis C.

BACKGROUND & AIMS: CD4+ regulatory T cells (Tregs) expand during chronic hepatitis C virus (HCV) infection, inhibit antiviral immunity and promote fibrosis. Direct-acting antiviral agents (DAA) have revolutionized HCV therapy. However, it is unclear if Tregs are normalized after DAA-induced HCV elimination. METHODS: We analyzed Tregs before (baseline), at end of therapy (EOT), 12 and 24weeks (SVR12, SVR24) and long-term (51±14weeks) after EOT in 26 genotype-1-infected patients who were successfully treated with sofosbuvir (SOF) plus interferon (IFN)/ribavirin (n=12) and IFN-free DAA regimens (SOF plus daclatasvir or simeprevir; n=14). Frequency, phenotype and suppressor function of peripheral Foxp3+ CD25+ CD4+ T cells were studied by multi-color flow cytometry and co-culture inhibition assays. RESULTS: Frequencies and activation status of Foxp3+ CD25+ CD4+ T cells remained elevated above those of normal controls in both treatment groups even long-term after HCV elimination. Co-culture assays indicated a dose-response relationship for functional inhibition of autologous CD4+ effector T cells and confirmed that activation of Tregs remained largely unchanged over the observation period. Unlike IFN-free regimens, SOF plus IFN/ribavirin induced a transiently increased frequency of Foxp3+ CD25+ CD4+ T cells at EOT (5.0% at baseline to 6.1% at EOT; p=0.001). These Foxp3+ CD25+ CD4+ T cells co-expressed the activation markers glycoprotein A repetitions predominant (GARP; p=0.012) and tumor necrosis factor receptor superfamily, member 4 (OX-40; p=0.001) but showed unchanged in vitro inhibitory activity. CONCLUSION: Although IFN-based DAA therapy induced transient expansion of activated Foxp3+ CD25+ CD4+ T cells, neither IFN-based nor IFN-free DAA regimens normalized frequencies and activation status of Tregs one year after viral elimination. Persistence of immunosuppressive Tregs may thus contribute to complications of liver disease even long-term after HCV cure. LAY SUMMARY: In chronic hepatitis C virus (HCV) infection, CD4+ regulatory T cells (Tregs) can reduce antiviral immune responses, promote liver fibrosis and may increase the risk for liver cancer, because they gradually expand during disease. Modern direct-acting antiviral agents (DAA) can "cure" hepatitis C in almost all treated patients. However, our study shows that DAA do not normalize the increased frequency and activation status of Tregs even long-term after HCV elimination. Tregs may persistently modulate functions of the immune system even after "cure" of hepatitis C.

Influence of the CXCL1 rs4074 A allele on alcohol induced cirrhosis and HCC in patients of European descent.

BACKGROUND AND AIMS: CXCL1 (CXC chemokine-ligand-1) is a ligand for CXC chemokine receptor 2 expressed on hepatic stellate cells (HSC). Thus, CXCL1 might contribute to HSC activation and fibrogenesis. In the present study, we investigated the influence of the CXCL1 rs4074 polymorphism on the occurrence of alcohol induced liver cirrhosis and hepatocellular carcinoma (HCC). METHODS: The study involved 458 patients with alcoholic cirrhosis (170 with HCC), 115 alcoholics without liver disease and 342 healthy controls. All subjects were genotyped for the CXCL1 rs4074 polymorphism and CXCL1 serum levels of 132 patients were measured. In vitro CXCL1 secretion in TLR-transfected cell lines were studied by ELISA. RESULTS: Distribution of the CXCL1 genotypes (GG/GA/AA) was 159/219/80 in patients with alcoholic cirrhosis, 52/44/19 in alcoholic controls and 158/140/44 in healthy controls. Patients with alcohol-induced cirrhosis were significantly more often carriers of the CXCL1 rs4074 A allele (65.3%) than alcoholics without liver disease (54.8%, OR=1.55; 95%CI=1.025-2.350; p=0.04) and healthy controls (53.8%, OR=1.62; 95%CI=1.212-2.151; p=0.001). Accordingly, the frequency of the CXCL1 rs4074 A allele was significantly higher in the cirrhotic patients than in the subjects without cirrhosis (41.4% vs. 33.9%, OR=1.38, 95% CI:1.14-1.66, p=0.001). Furthermore cirrhotic carriers of the CXCL1 rs4074 A allele had significantly higher CXCL1 serum levels than carriers of the GG genotype. In contrast to sera from healthy controls, sera from patients with alcoholic cirrhosis induced CXCL1 secretion in TLR2- (p=0.016) and TLR4- (p=0.008) transfected HEK293 cells. This finding indicates that sera from patients with alcoholic cirrhosis contain soluble ligands that can induce CXCL1 production via stimulation of TLRs. CONCLUSION: The enhanced CXCL1 serum levels in carriers of the rs4074 A allele together with their increased frequency in patients with alcohol induced cirrhosis suggest the CXCL1 rs4074 A allele as a genetic risk factor for alcoholic cirrhosis.

Hepatitis C virus core protein induces fibrogenic actions of hepatic stellate cells via toll-like receptor 2.

Hepatic stellate cells (HSCs) represent the main fibrogenic cell type accumulating extracellular matrix in the liver. Recent data suggest that hepatitis C virus (HCV) core protein may directly activate HSCs. Therefore, we examined the influence of recombinant HCV core protein on human HSCs. Primary human HSCs and the human HSC line LX-2 were stimulated with recombinant HCV proteins core and envelope 2 protein. Expression of procollagen type I α-1, α-smooth muscle actin, cysteine- and glycine-rich protein 2, glial fibrillary acidic protein, tissue growth factor ß1, matrix metalloproteinases 2 (MMP2) and 13, tissue inhibitor of metalloproteinases 1 and 2 was investigated by real-time PCR. Intracellular signaling pathways of ERK1/2, p38 and, jun-amino-terminal kinase (JNK) were analyzed by western blot analysis. Recombinant HCV core protein induced upregulation of procollagen type I α-1, α-smooth muscle actin, MMP 2 and 13, tissue inhibitor of metalloproteinases 1 and 2, tissue growth factor ß1, cysteine- and glycine-rich protein 2, and glial fibrillary acidic protein mRNA expression, whereas HCV envelope 2 protein did not exert any significant effect. Blocking of toll-like receptor 2 (TLR2) with a neutralizing antibody prevented mRNA upregulation by HCV core protein confirming that the TLR2 pathway was involved. Furthermore, western blot analysis revealed HCV-induced phosphorylation of the TLR2-dependent signaling molecules ERK1/2, p38 and JNK mitogen-activated kinases. Our in vitro results demonstrate a direct effect of HCV core protein on activation of HSCs toward a profibrogenic state, which is mediated via the TLR2 pathway. Manipulating the TLR2 pathway may thus provide a new approach for antifibrotic therapies in HCV infection.

Interferon-lambda serum levels in hepatitis C.

BACKGROUND & AIMS: Dendritic cells (DCs) trigger adaptive immune responses and are an important source of antiviral cytokines. In hepatitis C virus (HCV) infection DC function is markedly impaired. Thus far, studies have focused on types I and II interferon (IFN). We studied IFN-lambda1 (IL-29) and IFN-lambda2/3 (IL-28A/B) serum levels in patients with different outcomes of HCV infection. METHODS: IFN-lambdas were measured by ELISAs detecting IL-29 or IL-28A and IL-28B, respectively. Results were stratified with respect to the recently discovered rs12979860 T/C polymorphism upstream of the IL-28B gene. RESULTS: In general IL-29 serum levels exceeded IL-28A/B at least twofold, with IL-29 and IL-28A/B levels being significantly higher in carriers of the rs12979860 C allele than in TT homozygous individuals (p<0.02). IL-29 levels were substantially lower in patients with chronic hepatitis C than in healthy controls (p=0.005) and patients with spontaneously resolved hepatitis (p=0.001). Patients with acute hepatitis C showed IL-29 levels intermediate between chronic hepatitis C and normal controls; and IL-29 serum levels were higher in patients who spontaneously resolved hepatitis C than in those who became chronic. In vitro HCV proteins NS3 and E2 directly inhibited IL-29 production in poly I:C-stimulated purified DCs. CONCLUSIONS: Our data suggest that HCV proteins modify IFN-lambda production in DCs. Carriers of the rs12979860 C allele associated with resolution of HCV infection exhibited increased IFN-lambda levels. Moreover, high IFN-lambda levels predisposed to spontaneous resolution of HCV infection. Thus, IFN-lambdas seem to play an important role in the control of hepatitis C.

Regulation of NK cell trafficking by CD81.

NK cells, a heterogeneous sub-population of lymphocytes, are critically involved in the regulation of both innate and adaptive immune responses in humans. Besides their participation in the control of tumors and viral infections, they also regulate inflammatory processes, mediating both beneficial and detrimental effects. To effectively fulfil their role in immune surveillance, proper trafficking of NK cells is essential. However, the mechanisms and factors governing NK cell recruitment are only poorly dissected. Here, we describe the functional role of tetraspanins, a family of evolutionary conserved cell-surface proteins, in modulating migration and transmigration of human NK cells. We demonstrate expression of various tetraspanins on NK cells. Furthermore, we show that stimulation of the NK cell-expressed tetraspanin CD81 induces phosphorylation of ezrin/radixin/moesin proteins and leads to NK cell polarization thereby facilitating NK cell migration toward various chemokines/cytokines. Finally, we provide evidence for a role of CD81 in promoting adhesion of NK cells to components of the extracellular matrix, a prerequisite for extravasation of lymphocytes in inflamed tissues. Thus, our data suggest that the tetraspanin CD81 is importantly involved in the regulation of NK cell recruitment.

Hepatitis C virus E2 and CD81 interaction may be associated with altered trafficking of dendritic cells in chronic hepatitis C.

Dendritic cells (DC) are crucially involved in the induction of immune responses; however, reports on DC functions in chronic hepatitis C are controversial. Function of DC includes proper cell trafficking between sites of infection and lympho-cellular compartments. Thus, we analyzed DC compartmentalization and changes in DC migration in hepatitis C virus (HCV)-infected patients. We found significantly lower numbers of circulating BDCA1+ and BDCA2+ DC in HCV(+) patients (n = 20) than in healthy controls (n = 12) (P < .05). Analyzing liver samples from HCV(+) patients (n = 15), HCV(-) controls (n = 15), and disease controls (n = 10), we demonstrated chronic hepatitis C to be associated with intrahepatic DC enrichment (P < .05). In vitro studies indicated that HCV E2-induced secretion of RANTES efficiently attracts CCR5(+) immature DC. Incubation of DC with sera derived from HCV(+) patients made DC unresponsive to CCL21, the chemokine recruiting DC to lymphoid tissues for T cell priming. Unlike attraction of CCR5+ DCs via RANTES, direct inhibition of DC migration in response to CCL21 was specific for patients with chronic hepatitis C and could be attributed to interaction of HCV E2 with CD81 on DC. In conclusion, migration of DC is markedly affected by interaction of HCV E2 with CD81. Failure of DC to recirculate to lymphoid tissue may be critically involved in impaired T cell priming during HCV infection.

Presentation of the HCV-derived lipopeptide LP20-44 by dendritic cells enhances function of in vitro-generated CD4+ T cells via up-regulation of TLR2.

In vitro immunization with HCV lipopeptide aa 20-44 results in antigen-specific T cells. Here, we studied whether presentation of the lipopeptide by dendritic cells (DC) results in enhanced T cell functions. DC-immunized T cells showed significantly augmented proliferation and IFN-gamma secretion upon HCV-specific re-stimulation. This effect was related to significantly increased expression of TLR2 on DC-immunized CD4+ T cells and required TLR2 triggering during re-stimulation. In contrast, numbers of HLA-A2-pentamer-positive CD8+ T cells and granzyme B-secreting T cells remained unchanged. Thus, up-regulation of TLR2 during immunization with DC enhances functions of CD4+ T cells but not CD8+ T cells.

Comparative study of NK cell-mediated cytotoxicity using radioactive and flow cytometric cytotoxicity assays.

Cell-mediated cytotoxicity is a major effector pathway of the immune system. Thus far, radioactive assays have been widely used, but have significant disadvantages. Meanwhile, flow cytometric assays have been established but have not all been assessed simultaneously relative to the radioactive assays. Here, we have evaluated flow cytometric enumeration of surviving target cells, annexin-V binding and detection of activated caspase-3 and caspase-6 in direct comparison to the 51chromium (51Cr) release assay, and the JAM test. For assay evaluation NKL effector cells Fas-resistant K562 and Fas-sensitive Jurkat target cells were studied. Percent specific lysis measured for each E:T ratio was fitted to a sigmoid dose response curve. Both the flow cytometric and radioactive cytotoxicity assays showed equivalent background lysis (1-13%) but differed considerably with respect to maximum cytotoxicity (11-82% in K562 and 49-75% in Jurkat cells). Half maximum lysis ranged from 4:1 to 28:1 E:T ratios in K562 cells and from 1:3 to 24:1 in Jurkat cells, respectively. Flow cytometric enumeration of surviving target cells was the only assay which permitted detection of cytotoxicity at considerable lower E:T ratios (in K562 cells 1:4 to 2:1 and in Jurkat cells 1:4 to 1:1) than the conventional assays. Prolonged incubation over 24 h did not improve the sensitivity for flow cytometric enumeration of surviving target cells or the JAM test. The observed differences in the lysis of target cells are likely to reflect different sensitivity of cell death-associated changes which are measured by each assay. Thus, the particular choice of a cytotoxicity assay must be carefully adapted to the experimental situation under study.

Cytotoxic capacity of hepatitis C virus (HCV)--specific lymphocytes after in vitro immunization with HCV-derived lipopeptides.

BACKGROUND: Hepatitis C virus (HCV)-derived lipopeptides can induce epitope-specific immune responses in lymphocytes from HCV-naive individuals. We analyzed whether such T cells generated by in vitro immunization with HCV core-derived lipopeptides exert HCV-specific cytolytic activity. METHODS: Using a sensitive flow cytometric cytotoxicity assay we characterized HCV-specific cytotoxicity in T cells generated in vitro with HCV core-derived 25-mer lipopeptides. In addition, we studied expressions of Fas ligand and perforin and interferon-gamma (IFN-gamma) secretion in HLA-A2-HCV(core_35-44) tetramer-positive T cells generated with lipopeptide amino acid 20-44 (LP20-44). RESULTS: CD8+ T cells induced in vitro with HCV core-derived lipopeptides only infrequently exerted HCV-specific cytotoxicity, irrespective of whether antigen-coated T2 cells or autologous B lymphoblasts were used as targets. Detailed analysis of HLA-A2-HCV(core_35-44) tetramer-positive T cells generated with LP20-44 revealed that in vitro immunization resulted in T cells that secreted IFN-gamma after antigen-specific restimulation and that upregulated expression of Fas ligand but not of perforin. CONCLUSIONS: Our data confirm at the functional level that HCV lipopeptides induce antigen-specific T lymphocytes that produce IFN-gamma but exert significant cytotoxicity in only a minority of experiments, probably because expression of cytolytic effector molecules is not enhanced in their granules.

Serum antibodies against the hepatitis C virus E2 protein mediate antibody-dependent cellular cytotoxicity (ADCC).

BACKGROUND/AIMS: The role of antibody dependent cellular cytotoxicity (ADCC) in HCV infection is unclear at present. Antibodies mediating ADCC are usually directed against viral envelope proteins. As cell surface expression of the HCV envelope E2 protein has been shown, the HCV E2 protein is an especially promising candidate target for ADCC. METHODS: Sera from patients with acute (n=6), self-limited (n=11) and chronic (n=19) HCV infection were analyzed in this study. Sera reacting with cell-bound HCV antigens were examined in a flowcytometric cytotoxicity assay using antigen-coated JOK-1 cells as targets. RESULTS: We found that sera from all stages of HCV infection reacted with cells loaded with HCV E2. E2-specific ADCC was observed in patients with acute (n=3/6), self-limited (n=5/11) and chronic (n=13/19) hepatitis C and was closely related to fluorescence intensity in the E2-binding assay (r=0.67, P<0.001). CONCLUSIONS: We conclude that E2-antibodies from all stages of HCV infection can mediate ADCC. Thus, the role of this process in the pathogenesis of chronic hepatitis C should be further elucidated.