Golo Ahlenstiel

IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy

Hepatitis C virus (HCV) infects 3% of the worlds population. Treatment of chronic HCV consists of a combination of PEGylated interferon-α (PEG-IFN-α) and ribavirin (RBV). To identify genetic variants associated with HCV treatment response, we conducted a genome-wide association study of sustained virological response (SVR) to PEG-IFN-α/RBV combination therapy in 293 Australian individuals with genotype 1 chronic hepatitis C, with validation in an independent replication cohort consisting of 555 individuals. We report an association to SVR within the gene region encoding interleukin 28B (IL28B, also called IFNλ3; rs8099917 combined P = 9.25 × 10−9, OR = 1.98, 95% CI = 1.57–2.52). IL28B contributes to viral resistance and is known to be upregulated by interferons and by RNA virus infection. These data suggest that host genetics may be useful for the prediction of drug response, and they also support the investigation of the role of IL28B in the treatment of HCV and in other diseases treated with IFN-α.

Surface expression and cytolytic function of natural killer cell receptors is altered in chronic hepatitis C

Introduction: Impaired activity of natural killer (NK) cells has been proposed as a mechanism contributing to viral persistence in hepatitis C virus (HCV) infection. As the function of NK cells is primarily regulated by NK cell receptors (NKR), we analysed whether decreased NK cell function in hepatitis C may be related to dysregulated NKR expression. Patients and methods: Expression of NK cell was analysed by flow cytometry on lymphocytes from HCV(+) subjects (n = 30), patients who became HCV(−) after antiviral therapy (n = 10), healthy individuals (n = 10), and hepatitis B virus (HBV) infected patients (n = 9). Cytolytic function of lymphocytes was studied in a redirected lysis assay and in a standard 51chromium release cytotoxicity assay, respectively. Results: In patients with chronic hepatitis C, we found a significantly reduced proportion of NKp46 and NKp30 expressing NK cells compared with healthy and HBV infected subjects. Low expression of natural cytotoxicity receptor (NCR) was also confirmed in in vitro activated NK cell populations derived from HCV patients compared with uninfected donors. In contrast, patients who cleared HCV under antiviral therapy showed normal expression of NKp44, NKp30, and NKp46. Reduced NCR expression in chronic hepatitis C was associated with a parallel decrease in NCR mediated target cell killing. Furthermore, we found a significantly increased proportion of NKG2A expressing NK cells and CD8+ T cells in HCV positive patients, resulting in a reduced cytolytic activity against cells incubated with the HLA-E stabilising peptide HCV core35–44. Conclusion: The present study indicates that defective expression of NKR represents a novel mechanism contributing to impaired function of NK cells and CD8+ T cells in chronic hepatitis C.

Natural Killer Cells Are Polarized Toward Cytotoxicity in Chronic Hepatitis C in an Interferon-Alfa-Dependent Manner

BACKGROUND & AIMS Patients with chronic hepatitis C virus (HCV) infection display great variability in disease activity and progression. Although virus-specific adaptive immune responses have been characterized extensively and found to be impaired in chronic hepatitis C, the role of innate immune responses in disease activity and progression of chronic hepatitis C is not well understood. METHODS We studied 42 HCV-infected patients and 12 healthy uninfected controls. RESULTS We found an increased frequency of natural killer (NK) cells expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), NKp44, NKG2C, and CD122 in chronic hepatitis C as compared with healthy controls (P < .05 for all markers) and stronger activation of NK cells in the liver than in the blood (P < .05). This NK cell phenotype was associated with polarization of NK cell function toward CD107a expression as a marker of degranulation, but with not increased interferon (IFN)-gamma production of CD56(dim) NK cells. The polarized NK cell phenotype correlated with alanine aminotransferase levels (r(2) = 0.312, P = .03). To investigate whether in vivo exposure of NK cells to HCV-induced type I IFN was causing this NK cell phenotype, peripheral blood mononuclear cells from 10 healthy controls and 8 HCV-infected patients were stimulated in the presence of IFN-alfa, which resulted in increased NK cell expression of TRAIL and CD107a (P < .001), but not IFN-gamma. CONCLUSIONS Collectively, these results describe a polarized NK cell phenotype induced by chronic exposure to HCV-induced IFN-alfa. This phenotype may contribute to liver injury through TRAIL expression and cytotoxicity, whereas the lacking increase in IFN-gamma production may facilitate the inability to clear HCV.

IL28B, HLA-C, and KIR variants additively predict response to therapy in chronic hepatitis C virus infection in a European Cohort: a cross-sectional study.

Vijayaprakash Suppiah and colleagues show that genotyping hepatitis C patients for the IL28B, HLA-C, and KIR genes improves the ability to predict whether or not patients will respond to antiviral treatment.

Distinct KIR/HLA compound genotypes affect the kinetics of human antiviral natural killer cell responses

Genetic studies suggest a role for killer cell immunoglobulin-like receptor/HLA (KIR/HLA) compound genotypes in the outcome of viral infections, but functional data to explain these epidemiological observations have not been reported. Using an in vitro model of infection with influenza A virus (IAV), we attribute functional differences in human NK cell activity to distinct KIR/HLA genotypes. Multicolor flow cytometry revealed that the HLA-C-inhibited NK cell subset in HLA-C1 homozygous subjects was larger and responded more rapidly in IFN-gamma secretion and CD107a degranulation assays than its counterpart in HLA-C2 homozygous subjects. The differential IFN-gamma response was also observed at the level of bulk NK cells and was independent of KIR3DL1/HLA-Bw4 interactions. Moreover, the differential response was not caused by differences in NK cell maturation status and phenotype, nor by differences in the type I IFN response of IAV-infected accessory cells between HLA-C1 and HLA-C2 homozygous subjects. These results provide functional evidence for differential NK cell responsiveness depending on KIR/HLA genotype and may provide useful insights into differential innate immune responsiveness to viral infections such as IAV.

The HLA-A2 Restricted T Cell Epitope HCV Core35–44 Stabilizes HLA-E Expression and Inhibits Cytolysis Mediated by Natural Killer Cells

Impaired activity of natural killer cells has been proposed as a mechanism contributing to viral persistence in hepatitis C virus (HCV) infection. Natural cytotoxicity is regulated by interactions of HLA-E with inhibitory CD94/NKG2A receptors on natural killer (NK) cells. Here, we studied whether HCV core encodes peptides that bind to HLA-E and inhibit natural cytotoxicity. We analyzed 30 HCV core-derived peptides. Peptide-induced stabilization of HLA-E expression was measured flow cytometrically after incubating HLA-E-transfected cells with peptides. NK cell function was studied with a (51)chromium-release-assay. Intrahepatic HLA-E expression was analyzed by an indirect immunoperoxidase technique and flow cytometry of isolated cells using a HLA-E-specific antibody. We identified peptide aa35-44, a well-characterized HLA-A2 restricted T cell epitope, as a peptide stabilizing HLA-E expression and thereby inhibiting NK cell-mediated lysis. Blocking experiments confirmed that this inhibitory effect of peptide aa35-44 on natural cytotoxicity was mediated via interactions between CD94/NKG2A receptors and enhanced HLA-E expression. In line with these in vitro data we found enhanced intrahepatic HLA-E expression on antigen-presenting cells in HCV-infected patients. Our data indicate the existence of T cell epitopes that can be recognized by HLA-A2 and HLA-E. This dual recognition may contribute to viral persistence in hepatitis C.

Early Changes in Natural Killer Cell Function Indicate Virologic Response to Interferon Therapy for Hepatitis C

BACKGROUND & AIMS Mathematical modeling of hepatitis C virus (HCV) kinetics indicated that cellular immune responses contribute to interferon (IFN)-induced clearance of HCV. We investigated a potential role of natural killer (NK) cells in this process. METHODS Phenotype and function of blood and liver NK cells were studied during the first 12 weeks of treatment with pegylated IFN-alfa and ribavirin, the time period used to define the early virological response. RESULTS Within hours of treatment initiation, NK cells of patients that had an early virological response increased expression of activating receptors NKG2D, NKp30, and CD16 and decreased expression of NKG2C and 2B4, along with inhibitory receptors SIGLEC7 and NKG2A, resulting in NK cell activation. NK cell cytotoxicity, measured by degranulation and tumor necrosis factor-related apoptosis-inducing ligand production, peaked after 24 hours (P<.01), concomitant with an increase in alanine aminotransferase levels (P<.05), whereas IFN-γ production decreased within 6 hours and did not recover for more than 4 weeks (P<.05). NK cells from liver biopsies taken 6 hours after treatment initiation had increased numbers of cytotoxic CD16+NK cells (P<.05) and a trend toward increased production of tumor necrosis factor-related apoptosis-inducing ligand. Degranulation of peripheral blood NK cells correlated with treatment-induced, first-phase decreases in viral load (P<.05) and remained higher in early virological responders than in nonresponders for weeks. CONCLUSIONS IFN activates NK cells early after treatment is initiated. Their cytotoxic function, in particular, is strongly induced, which correlates to virologic response. Therefore, NK cell activation indicates responsiveness to IFN-α-based treatment and suggests the involvement of the innate immune cells in viral clearance.

Natural killer cell function is intact after direct exposure to infectious hepatitis C virions

Although hepatitis C virus (HCV) has been shown to readily escape from virus‐specific T and B cell responses, its effects on natural killer (NK) cells are less clear. Based on two previous reports that recombinant, truncated HCV E2 protein inhibits NK cell functions via crosslinking of CD81, it is now widely believed that HCV impairs NK cells as a means to establish persistence. However, the relevance of these findings has not been verified with HCV E2 expressed as part of intact virions. Here we employed a new cell culture system generating infectious HCV particles with genotype 1a and 2a structural proteins, and analyzed direct and indirect effects of HCV on human NK cells. Antibody‐mediated crosslinking of CD16 stimulated and antibody‐mediated crosslinking of CD81 inhibited NK cell activation and interferon gamma (IFN‐γ) production. However, infectious HCV itself had no effect even at titers that far exceeded HCV RNA and protein concentrations in the blood of infected patients. Consistent with these results, anti‐CD81 but not HCV inhibited NK cell cytotoxicity. These results were independent of the presence or absence of HCV‐binding antibodies and independent of the presence or absence of other peripheral blood mononuclear cell populations. Conclusion: HCV 1a or 2a envelope proteins do not modulate NK cell function when expressed as a part of infectious HCV particles. Without direct inhibition by HCV, NK cells may become activated by cytokines in acute HCV infection and contribute to infection outcome and disease pathogenesis. (HEPATOLOGY 2009;49:12‐21.)

Early changes in interferon signaling define natural killer cell response and refractoriness to interferon‐based therapy of hepatitis C patients

Natural killer (NK) cells exhibit a polarized phenotype with increased cytotoxicity and decreased interferon gamma (IFN‐γ) production in chronic hepatitis C virus (HCV) infection. Here, we asked whether this is caused by type I interferon (IFN)‐induced expression and phosphorylation levels of signal transducer and activator of transcription (STAT) molecules in NK cells and whether it affects the response and refractoriness of NK cells to IFN‐α‐based therapy of HCV. STAT1 levels in NK cells were significantly higher in patients with chronic HCV infection than in uninfected controls. STAT1 levels and induction of phosphorylated STAT1 (pSTAT1) increased further during IFN‐α‐based therapy with preferential STAT1 over STAT4 phosphorylation. Induction of pSTAT1 correlated with increased NK cytotoxicity (tumor necrosis factor–apoptosis‐inducing ligand [TRAIL] expression and degranulation) and decreased IFN‐γ production. NK cells from patients with a greater than 2 log10 first‐phase HCV RNA decline to IFN‐α‐based therapy (>99% IFN effectiveness) displayed strong pSTAT1 induction in vivo and were refractory to further stimulation in vitro. In contrast, NK cells from patients with a less than 2 log10 first‐phase HCV RNA decline exhibited lower pSTAT1 induction in vivo (P = 0.024), but retained greater IFN‐α responsiveness in vitro (P = 0.024). NK cells of all patients became refractory to in vivo and in vitro stimulation by IFN‐α during the second‐phase virological response. Conclusion: These data show that IFN‐α‐induced modulation of STAT1/4 phosphorylation underlies the polarization of NK cells toward increased cytotoxicity and decreased IFN‐γ production in HCV infection, and that NK cell responsiveness and refractoriness correlate to the antiviral effectiveness of IFN‐α‐based therapy. (Hepatology 2012)

Identification of improved IL28B SNPs and haplotypes for prediction of drug response in treatment of hepatitis C using massively parallel sequencing in a cross-sectional European cohort

BackgroundThe hepatitis C virus (HCV) infects nearly 3% of the Worlds population, causing severe liver disease in many. Standard of care therapy is currently pegylated interferon alpha and ribavirin (PegIFN/R), which is effective in less than half of those infected with the most common viral genotype. Two IL28B single nucleotide polymorphisms (SNPs), rs8099917 and rs12979860, predict response to (PegIFN/R) therapy in treatment of HCV infection. These SNPs were identified in genome wide analyses using Illumina genotyping chips. In people of European ancestry, there are 6 common (more than 1%) haplotypes for IL28B, one tagged by the rs8099917 minor allele, four tagged by rs12979860.MethodsWe used massively parallel sequencing of the IL28B and IL28A gene regions generated by polymerase chain reaction (PCR) from pooled DNA samples from 100 responders and 99 non-responders to therapy, to identify common variants. Variants that had high odds ratios and were validated were then genotyped in a cohort of 905 responders and non-responders. Their predictive power was assessed, alone and in combination with HLA-C.ResultsOnly SNPs in the IL28B linkage disequilibrium block predicted drug response. Eighteen SNPs were identified with evidence for association with drug response, and with a high degree of confidence in the sequence call. We found that two SNPs, rs4803221 (homozygote minor allele positive predictive value (PPV) of 77%) and rs7248668 (PPV 78%), predicted failure to respond better than the current best, rs8099917 (PPV 73%) and rs12979860 (PPV 68%) in this cross-sectional cohort. The best SNPs tagged a single common haplotype, haplotype 2. Genotypes predicted lack of response better than alleles. However, combination of IL28B haplotype 2 carrier status with the HLA-C C2C2 genotype, which has previously been reported to improve prediction in combination with IL28B, provides the highest PPV (80%). The haplotypes present alternative putative transcription factor binding and methylation sites.ConclusionsMassively parallel sequencing allowed identification and comparison of the best common SNPs for identifying treatment failure in therapy for HCV. SNPs tagging a single haplotype have the highest PPV, especially in combination with HLA-C. The functional basis for the association may be due to altered regulation of the gene. These approaches have utility in improving diagnostic testing and identifying causal haplotypes or SNPs.