Current treatment of chronic hepatitis C virus (HCV)

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1 GASTROENTEROLOGY 2011;141: Early Changes in Natural Killer Cell Function Indicate Virologic Response to Interferon Therapy for Hepatitis C GOLO AHLENSTIEL,*, BIRGIT EDLICH,*, LEAH J. HOGDAL,*, YARON ROTMAN, MAZEN NOUREDDIN, JORDAN J. FELD, LAUREN E. HOLZ,*, RACHEL H. TITERENCE,*, T. JAKE LIANG, and BARBARA REHERMANN*, *Immunology Section, Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland See editorial on page 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- 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- based treatment and suggests the involvement of the innate immune cells in viral clearance. Keywords: Liver Disease; HCV Treatment; Response to Therapy; Cell Death. Current treatment of chronic hepatitis C virus (HCV) infection consists of a 24- or 48-week course of pegylated interferon-alfa (pegifn) and ribavirin (RBV). It is important to understand the mechanisms of IFN-induced HCV clearance because only about 50% of treated patients achieve a sustained response and IFN will remain an important component of new treatment regimens with direct antiviral agents. 1 Based on mathematical modeling of serum HCV titers during treatment, it has been proposed that an IFN- mediated block of HCV replication and virion release causes the first phase, ie, 48-hour decline in serum HCV titer. 2 This rapid reduction of viremia is followed by a slower second-phase decline thought to represent the elimination of infected hepatocytes. 2 Owing to IFN-alfa s immunomodulatory activities, the elimination of HCV-infected hepatocytes is likely immune-mediated. However, the immune cells responsible for the elimination of infected hepatocytes have not been identified thus far. Multiple studies have concluded that HCV-specific T cells do not contribute to treatment-induced HCV clearance (reviewed in Rehermann 3 ). In fact, HCV-specific CD8 T-cell responses decline during IFN-based therapy and treatment outcomes are associated with neither the strength nor quality of HCV-specific CD8 and CD4 T-cell responses. 3 Several findings prompted us to study NK cells in the context of hepatitis C treatment. First, NK cells are the most prevalent lymphocyte subset in the liver, constituting about 30% of intrahepatic lymphocytes. 4 Second, the current treatment regimen for chronic hepatitis C includes IFN-alfa, a potent activator of NK cells. 5 Third, the genotype for certain receptors regulating NK cell activity is associated with a higher likelihood of spontaneous and treatment-induced HCV clearance. 6,7 NK cells are controlled by the integration of signals from activating and inhibitory cell surface receptors, which include killer cell immunoglobulin-like receptors (KIRs), lectin-like receptors (NKG2A-F), and natural cytotoxicity receptors (NKp30, NKp44, and NKp46). In the absence of inflammation or infection, NK cells receive mostly inhibitory signals. NK cell activation occurs when signaling through activating receptors overcomes inhibition. In addition, NK cells can be activated by inflammatory cytokines, such as type I IFNs (ie, IFN-alfa and IFN-beta) and interleukin (IL)-12, which are commonly released in response to virus infections. 5 Abbreviations used in this paper: ALT, alanine aminotransferase; EVR, early virological response; IL, interleukin; KIR, killer cell immunoglobulinlike receptors; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cells; pegifn, pegylated interferon-alfa; RBV, ribavirin; TRAIL, tumor necrosis factor related apoptosis-inducing ligand by the AGA Institute /$36.00 doi: /j.gastro

2 1232 AHLENSTIEL ET AL GASTROENTEROLOGY Vol. 141, No. 4 With regard to effector functions, NK cells have traditionally been divided into different effector subsets based on expression of CD56 and CD16. CD56 bright NK cells constitute about 10% of the peripheral blood NK cell population, do not express CD16, and are considered the main producers of cytokines and tumor necrosis factor related apoptosisinducing ligand (TRAIL). 8 TRAIL binds to the death receptors DR4 and DR5 on target cells and induces apoptosis. The more mature CD56 dim NK cell subset makes up the remaining 90% of the NK cell population. These cells express high levels of CD16 and exert mostly cytotoxic effector functions by perforin/granzyme. However, the traditional division of NK cells into 2 main subsets with distinct functions has been challenged recently, when CD56dim NK cells were found to produce large amounts of cytokines within the first 2 4 hours after activation. 9,10 In HCV infection, NK cells have been shown to display an activated phenotype with increased expression of receptors, such as NKG2C, NKp30, NKp44, NKp46, and CD Activation is enhanced in the liver as compared to the blood, and cytotoxic NK cell effector functions, as determined by degranulation and TRAIL production, correlate with alanine aminotransferase (ALT) levels, a marker of liver injury. 12 Increased TRAIL production was also observed in spontaneous hepatitis B flares, possibly due to endogenous IFN-, 14 and during IFN- based therapy of chronic hepatitis C. 15 In the current study, we performed a detailed analysis of changes in NK cell phenotype and function during IFN-based treatment of chronic hepatitis C and correlated the results with the virological response. Materials and Methods Study Cohort Peripheral blood NK cells were studied in patients with chronic hepatitis C (Supplementary Table 1). Twenty-two patients achieved an early virological response (EVR) with peginterferon (PegIFN) alfa-2a (180 g/wk subcutaneously) and weight-based RBV (1000 mg for 75 kg bodyweight and 1200 mg for 75 kg bodyweight by mouth daily for HCV genotypes 1 and 4; 800 mg by mouth daily for HCV genotypes 2 and 3). An EVR is defined as serum HCV RNA being either undetectable ( 15 IU/mL) or 2 log 10 lower at week 12 than before treatment. Sampling time points were 4 weeks, 2 weeks, and 0 hours before treatment, and 6 hours, 24 hours, 48 hours, 1 week, 2 weeks, 4 weeks, and 12 weeks after initiation of treatment. One patient discontinued treatment after week 4 and was therefore included in the correlation of NK cell and first-phase virological response only. Fifteen patients with a history of nonresponse to standard PegIFN/RBV therapy (referred to as nonresponders) were studied in a PegIFN/RBV retreatment study 16 to correlate NK cell responses with the first-phase virological decline. These patients were studied 4 weeks and immediately (0 hours) before retreatment and 8 hours, 24 hours, 48 hours, 1 week, and 2 weeks after initiation of PegIFN/ RBV retreatment. They continued pegifn/rbv treatment for a full treatment course, but S-adenosyl methionine was added after the initial 2 weeks 16 and NK cell responses were therefore not studied beyond the initial 2 weeks. Intrahepatic NK cells were studied in 29 patients who were randomized to undergo liver biopsies either immediately before (n 19) or 6 hours after (n 9) initiation of PegIFN/RBV therapy. Six and 4 of these patients, respectively, had been pretreated with RBV for 4 weeks and their intrahepatic NK cell phenotype did not differ from those who had not been pretreated (not shown). All subjects gave written informed consent for research testing under protocols approved by the National Institute of Diabetes and Digestive and Kidney Diseases institutional review board. Lymphocyte Isolation Peripheral blood mononuclear cells (PBMCs) were separated from ACD-anticoagulated blood on Ficoll Histopaque (Mediatech, Manassas, VA) density gradients and washed 3 times with phosphate-buffered saline (Mediatech). Cryopreserved and thawed PBMCs were used for the prospective study and fresh PBMCs were used for the cross-sectional study to compare peripheral blood and liver NK cells. Liver tissue was homogenized, washed with phosphate-buffered saline, and liver-infiltrating lymphocytes were directly stained with the antibodies described in the section entitled NK cell frequency and phenotype. Viral Kinetics HCV RNA levels were measured using Cobas TaqMan real-time PCR (Roche Diagnostics, Palo Alto, CA), with a lower limit of detection of 15 IU/mL. The first-phase virological response was defined as the decline in HCV RNA titer during the first 48 hours of therapy. NK Cell Analysis NK cells were studied as described previously 12 using an LSRII FacsDiva Version (BD Biosciences, San Jose, CA) and FlowJo Version (Tree Star, Ashland, OR) software. NK cell frequency and phenotype. All phenotyping panels included ethidium monoazide, anti-cd19-pecy5 (BD Biosciences, San Jose, CA), and anti-cd14-pecy5 (Serotec, Raleigh, NC) to exclude dead cells, B cells, and monocytes, respectively, and anti-cd56-pecy7, anti-cd3-alexafluor700, and anti-cd16-pacificblue (BD Biosciences) to identify NK cells. In addition, we used fluorescein isothiocyanate conjugated antibodies against CXCR3, CD122 (R&D Systems, Minneapolis, MN), CD69 (BD Biosciences), CD244 (ebiosciences, San Diego, CA), and SIGLEC7 (Lifespan Biosciences, Seattle, WA); phycoerythrin-conjugated antibodies against NKG2A, NKG2D, NKp44 (Beckman Coulter, Brea, CA), and TRAIL (BD Biosciences), and CD300 (Abcam, Cambridge, MA); and allophycocyanin-conjugated antibodies against CCR5 (BD Biosciences), NKp30, NKp46 (Miltenyi, Auburn, CA), NKG2C (R&D Systems), and CD85j (ILT2) (ebiosciences). Liver-infiltrating lymphocytes were stained with anti CXCR3-fluorescein isothiocyanate, anti TRAIL phycoerythrin, anti CD16-PacificBlue, and anti CD69-allophycocyanin (BD Biosciences), in addition to ethidium monoazide and lineage-specific antibodies as described in this section. NK cell degranulation. PBMCs were thawed and cultured overnight at 37 C, 5% CO 2 in RPMI 1640 with 10% fetal calf serum (Serum Source International, Charlotte, NC), 1% penicillin/ streptomycin, 2 mm L-glutamine, and 10 mm HEPES (Cellgro, Manassas, VA) without exogenous IL-2. The next day, PBMCs were counted and stimulated in the presence or absence of K562 cells (ATCC, Manassas, VA) to assess degranulation as described, 12 but without additional cytokines. Cytokine release. PBMCs were incubated with or without IL-12 (0.5 ng/ml; R&D Systems) and IL-15 (20 ng/ml; R&D Systems) for 14 hours, followed by addition of brefeldin A for 4 hours and intracellular staining for IFN-. 12

3 October 2011 NK CELL RESPONSE TO TREATMENT OF HCV INFECTION 1233 Figure 1. Frequency of NK cells and their subsets during PegIFN/RBV therapy. (A) Gating strategy. (B D) Frequency of CD56 CD3 NK cells (B) and their CD56 bright (C) and CD56 dim subsets (D) in PBMCs during PegIFN/RBV therapy. Mean standard error of mean are shown (n 22 patients). ( ) P.001 with repeated measures analysis of variance; P.05; P.01 with paired Student t test comparing the indicated individual time points to the 0-hour time point. ALT Levels Serum ALT levels were determined with a colorimetric assay (Teco Diagnostics, Anaheim, CA) and shown relative to the pretreatment (0 hour) value. Genotyping DNA samples were genotyped for the IL28B rs polymorphism with a TaqMan SNP genotyping assay (Applied Biosystems Inc., Foster City, CA) and for KIR/HLA compound haplotypes as described. 17 Statistical Analysis Statistical analyses were performed with GraphPad Prism Version 5.0 (GraphPad Software Inc, San Diego, CA) and JMP (SAS Inc, Cary, NC). The Mann Whitney nonparametric 2-sample rank test was used to compare liver NK cells and the Wilcoxon matched paired test to compare ALT values. Serial measures analysis of variance was used to compare NK cells from responders and nonresponders throughout the treatment course and changes in NK cell phenotype and function during treatment. Because this test is based on the assumption of a linear model and does not reflect the specific pattern of marker expression (eg, an early increase followed by a plateau or a decrease), we also used paired Student t tests to compare individual treatment time points to the 0-hour value. Pearson correlation analysis was performed to study changes in NK cell degranulation in relation to viral kinetics. A 2-sided P value.05 was considered significant. Results Changes in NK Cell Phenotype of EVR Patients During PegIFN/RBV Therapy To study the effect of IFN- based therapy on NK cells, we assessed the phenotype and function of this lymphocyte subset prospectively in the blood of patients with chronic HCV infection who received PegIFN/RBV therapy. To characterize optimal changes in NK cell phenotype and function, we initially focused on the 22 patients who mounted an EVR. NK cells were identified as CD3 CD56 cells by multicolor flow cytometry after gating on single cells (forward scatter height vs forward scatter area), lymphocytes (forward scatter vs side scatter), and exclusion of CD14 monocytes, CD19 B cells, and ethidium monoazide dead cells (Figure 1A). As shown in Figure 1B, the frequency of NK cells in the blood lymphocyte population increased during the first 2 days of therapy (6.79% 1.29%, mean standard error of mean at 0 hours compared to 9.81% 1.28% 48 hour later; P.01) and was maintained at that level for 2 weeks. This was paralleled by an increase in CD56 bright (Figure 1C) and CD56 dim cells (Figure 1D). CD56 bright cells increased further from week 2 (1.40% 0.40%) to week 12 (2.18% 0.58%), but CD56 dim cells did not. For a detailed NK phenotype analysis, PBMCs were stained for activating and inhibitory NK cell receptors, activation markers, and chemokine receptors (Supplementary

4 1234 AHLENSTIEL ET AL GASTROENTEROLOGY Vol. 141, No. 4 Figure 2. Phenotype of peripheral blood NK cells during PegIFN/RBV therapy. (A E) MFI of NKG2D (A), NKp30 (B), NKG2C (C), SIGLEC7 (D), and CD69 expression (E) on CD56 CD3 NK cells. (F) Frequency of CXCR3 cells in the CD56 CD3 NK cell population. Mean standard error of mean are shown (n 22 patients). ( ) P.001 with repeated measures analysis of variance; P.05; P.001 with paired Student t test comparing the indicated individual time points to the 0-hour time point. Figure 1). Figure 2 details significant changes in the bulk NK cell population during the first 12 weeks of therapy. For example, the expression level of the activating receptor NKG2D peaked as early as 24 hours after initiation of therapy (mean fluorescence intensity [MFI] of at 0 hours; at 24 hours later; P.001, Figure 2A) and the expression level of NKp30 increased steadily until at least week 12 of therapy (MFI of at 0 hours; at week 12; P.0001; Figure 2B), and this observation was most pronounced for the CD56 dim NK cell population (Supplementary Figure 2B). NKp44 and NKp46 expression remained unaltered (not shown), 2B4 expression was slightly down-regulated (MFI of at 0 hours; at week 1; P.01; not shown) and NKG2C expression decreased significantly (MFI of at 0 hours; 49 7 at week 12; P.0004; Figure 2C). Within the group of inhibitory receptors, SIGLEC7 expression decreased significantly during the first 12 weeks of therapy (MFI of at 0 hours; at week 12; P.0001; Figure 2D) and this decrease was most pronounced in the CD56 dim population (Supplementary Figure 2). NKG2A expression was slightly reduced at the 6-hour time point (MFI of at 0 hours, compared to at 6 hours; P.46), but elevated at week 12 of treatment (MFI of ; P.007, not shown) due to the increase in CD56 bright NK cells (Figure 1C). CD85j and CD300 expression remained unaltered (data not shown). Collectively, these data suggest a trend toward increased NK cell activation and less inhibition. Consistent with this interpretation, expression of the activation marker CD69 increased significantly as early as 24 hours after initiation of therapy in the bulk NK cell population (Figure 2E) and both the CD56 bright and CD56 dim subsets (Supplementary Figure 2), and increased expression was maintained throughout the observation period. Finally, we analyzed expression of chemokine receptors associated with lymphocyte recruitment to the liver. Interestingly, frequency of CXCR3 (Figure 2F) and CCR5 NK cells (not shown) in the blood decreased significantly as early as 6 hours after initiation of therapy (CXCR3 NK cells 11.4% 1.8% at 0 hours, 7.7% 0.8% at 24 hours; P.034; CCR5 NK cells 8.8% 0.8% at 0 hours, 6.5% 0.6% at 6 hours; P.001), which might indicate recruitment of chemokine receptor positive NK cells from the blood to the site of infection.

5 October 2011 NK CELL RESPONSE TO TREATMENT OF HCV INFECTION 1235 Figure 3. PegIFN/RBV therapy polarizes NK cell function toward increased degranulation and TRAIL production and decreased IFN- production. (A C) Changes in NK cell CD107a (A), TRAIL (B), and IFN- expression (C) during PegIFN/RBV therapy. Mean standard error of mean of NK cell frequency and MFI are shown for 21, 20, and 22 patients, respectively. Statistical analysis: paired Student t test comparing the indicated individual time points to 0 hours. P.05; P.01; P.001. PegIFN/RBV Therapy Polarizes NK Cell Function Toward Increased Degranulation and TRAIL Production but Decreased IFN- Production NK cells mediate antiviral effects by killing infected cells and by producing antiviral and immunomodulatory cytokines, such as IFN-. To assess changes in NK cell cytotoxicity during therapy, we incubated PBMCs with the major histocompatibility complex negative target K562 and measured degranulation by staining CD107a on the cell surface (Supplementary Figure 1). A significant increase in both frequency of CD107a NK cells and their CD107a expression level was observed within 24 hours of treatment (CD107a NK cells 12.5% 1.4% at 0 hours, 22.8% 2.2% at 24 hours; MFI of at 0 hours, at 24 hours; Figure 3A) and was maintained for at least 4 weeks. Consistent with these results, ex vivo expression of TRAIL, a mediator of NK cell cytotoxicity, increased (TRAIL NK cells 13.8% 2.1% at 0 hours, 24.1% 3.0% at 24 hours; MFI of at 0 hours, at 24 hours, Figure 3B). This was observed for both CD56 dim and CD56 bright NK cells (not shown). In contrast to NK cell cytotoxicity, IFN- production was suppressed as early as 6 hours after initiation of therapy (IFN- NK cells 31.2% 3.9% at 0 hours, 17.7% 2.7% at 6 hours; MFI of at 0 hours, at 6 hours; Figure 3C). Although the frequency of IFN- NK cells and their mean IFN- expression level had slightly recovered by 24 hours and 48 hours, it took at least 12 weeks for both parameters to reach pretreatment levels (Figure 3C). The recovery of IFN- production from week 4 to week 12 of treatment paralleled an increase in the percentage of CD56 bright cells. Increase of Cytotoxic Intrahepatic NK Cells During the Early Phase of PegIFN/RBV Therapy The antiviral function of NK cells is exerted in the liver, the site of HCV replication. We therefore performed a crosssectional analysis of intrahepatic NK cells in patients who were randomized to undergo liver biopsy either immediately before (0 hours) or 6 hours after initiation of PegIFN/RBV therapy. As shown in the left panel of Figure 4A, the size of the intrahepatic CD16 NK cell subset, the main mediators of cytotoxicity,

6 1236 AHLENSTIEL ET AL GASTROENTEROLOGY Vol. 141, No. 4 Figure 4. Changes in the intrahepatic NK cell subset during the early phase of PegIFN/RBV therapy. (A) The size of the CD16 NK cell subset, the percentage of TRAIL-expressing NK cells and the TRAIL MFI in a cross-sectional analysis of patients who were randomized to undergo liver biopsies either immediately before (n 19) or 6 hours after (n 9) initiation of PegIFN/RBV therapy. Lines indicate the median. (B) Relative serum ALT levels during PegIFN/RBV therapy. Statistical analysis: Wilcoxon matched paired test comparing the individual time points to 0 hours. Mean standard error of mean are shown. increased 6 hours after initiation of therapy compared to baseline. Likewise, the percentage of TRAIL-expressing NK cells (Figure 4A, middle panel) and the TRAIL expression level (MFI) increased (Figure 4A, right panel). In contrast, TRAIL expression on T cells was only marginally increased at 6 hours of treatment in liver and blood (data not shown). Collectively, the observed phenotypic and functional changes in circulating and intrahepatic NK cells within the first 6 hours of therapy are consistent with an increase in cytotoxicity. As a clinical correlate of hepatocyte turnover, we measured serum ALT levels. Indeed, serum ALT levels increased slightly within the first 24 hours (P.027 compared to 0 hours) and peaked at the 48-hour time point (Figure 4B). These results suggest that hepatocytes are dying during the first 48 hours of treatment. Degranulation of NK Cells Correlates With Virological Response During PegIFN/RBV Therapy Finally, we investigated a possible relationship between early NK cell response and first-phase virological response to treatment, which is predictive of a sustained Table 1. Epidemiological and Clinical Data of Responder and Nonresponder Patients a Responders (n 10) Nonresponders (n 15) Sex (male/female) 6/4 10/5 Age at start of treatment, y, median (IQR) 54.5 (33 64) 53 (41 61) Ethnicity (Asian/African American/white) 2/4/4 0/2/13 Body mass index, median (IQR) 33.2 ( ) 27.9 ( ) IL-28B rs SNP (CC/CT/TT) 6/2/1 1/7/3 KIR genotype(2dl2/2dl3/2dl2, 2DL2/2DL3, 2DL3) 5/10/0/5 11/12/3/4 HLA genotype (C1/C2/C1C1/C2C2) 11/8/2/2 11/11/4/4 HCV genotype 1/genotype 4 9/1 15/0 HCV RNA titer at start of treatment, IU/mL, median (IQR) (13, ) ( ) ALT level at start of treatment, U/mL, median (IQR) 79 (29 386) 58 (25 150) Ishak inflammatory score, median (IQR) 6.5 (4 13) 7 (5 12) Ishak fibrosis score, median (IQR) 1 (0 3) 2 (0 6) IQR, interquartile range. a As shown in Figure 5C.

7 October 2011 NK CELL RESPONSE TO TREATMENT OF HCV INFECTION 1237 Table 2. Correlation of Changes in NK Cell Phenotype and First-Phase Virological Response Marker Time point, h MFI Percentage of NK cells Pearson r P value Pearson r P value NKG2A 6 NS NS NS SIGLEC7 6 NS NS NS, not significant. NOTE. The first-phase virological response was correlated to relative changes in the frequency of NKG2A- or SIGLEC7-expressing NK cells within the NK cell population and their respective expression level (MFI). Graphs are shown in Supplementary Figure 3. virological response. 18 For this purpose, we included a group of nonresponders to previous PegIFN/RBV treatment and retreatment 16 that was studied for NK cell responses during the first 2 weeks of PegIFN/RBV retreatment (Table 1). Interestingly, an early decrease in the frequency of NK cells that express the inhibitory markers NKG2A and SIGLEC7 correlated to the first-phase virological response (Table 2, Supplementary Figure 3). Along this line, induction of NK cell degranulation during the first 24 hours (Figure 5A, left graph) and 48 hours (Figure 5A, right graph) of therapy also correlated to the first-phase virological response. These observations were more pronounced for the CD56 dim NK subset (Figure 5B), which is the more mature, cytotoxic NK cell population. They were preserved when the analysis was restricted to patients with the CC IL28B rs SNP, and NK cell function did not differ among patient subgroups with IL28B rs genotypes (not shown). This association between changes in NK cell function and virological response prompted us to compare the kinetics of NK cell degranulation during the first 2 weeks of therapy in responders and nonresponders (Figure 5C). Consistent with the correlation between NK cell degranulation and first-phase virological response, NK cells of nonresponders displayed a significantly smaller increase in NK cell degranulation during the first 24 hours of therapy than responders (Figure 5C). Of note, the increase in NK cell degranulation was sustained in the responders for at least 2 weeks, whereas it returned to baseline in the nonresponder group by week 1 (P.003). Similar results were obtained when NK cells of patients with and without EVR were compared (Supplementary Figure 4). Collectively, the results demonstrate a correlation between NK cell responsiveness in particular markers of NK cell cytotoxicity and treatment response. Discussion This study demonstrates a correlation between NK cell responsiveness, specifically the induction of the cytotoxic NK cell function and the first-phase (48 hours) virological response, as well as the early (12 weeks) virological response of IFN- based therapy. Peak changes in NK cell phenotype were most pronounced within 6 hours after initiation of therapy. In particular, expression levels of NKp30, NKG2C, SIGLEC7, CD85j, and CD69 changed significantly during the course of treatment. In contrast, changes in NKG2A and NKG2D expression were only observed within the first 48 hours after IFN injection and lasted less than 1 week. Several observations link these phenotypic changes to the observed increase in NK cell cytotoxicity. For example, NKp30 is one of the natural cytotoxicity receptors and its expression correlates to cytotoxicity. 19 NKp30 levels have been shown to be higher on NK cells from HCV-exposed individuals who remain aviremic than in those who become infected, which may indicate an antiviral effect. 19 Likewise, expression of the activating receptor NKG2D has been directly correlated to NK cell cytotoxicity, and Figure 5. Degranulation of peripheral blood NK cells correlates to treatment response. (A B) Correlation between the 24-hour and 48-hour change in the CD107a expression on either all blood NK cells (A) or their CD56 dim subset (B) and the first-phase virological response (log decline in HCV titer during the first 48 hours of PegIFN/RBV therapy). (C) Comparison between CD107a expression on CD56 dim NK cells of responders (filled circles, n 10) and nonresponders (open circles,n 15). Mean standard error of mean are shown. The P value was calculated by analysis of variance.

8 1238 AHLENSTIEL ET AL GASTROENTEROLOGY Vol. 141, No. 4 NKG2D ligands are up-regulated in the HCV-infected liver. 20 Finally, the observed decrease in expression of the inhibitory receptor NKG2A may indirectly enhance cytotoxic NK cell function because it diminishes signaling in response to HLA-E, which is overexpressed on HCV-infected cells due to stabilization by an endogenously processed HCV core peptide. 21,22 The cytotoxic NK cell phenotype was demonstrated by increased degranulation, a higher frequency of TRAILexpressing NK cells and increased TRAIL expression on NK cells (Figure 3). TRAIL is an important molecule in the context of hepatitis C because HCV enhances the susceptibility of primary human hepatocytes to TRAILmediated killing via increased expression of the death receptors DR4 and DR5. 23 This was also demonstrated in vitro with NK cells killing HCV-infected hepatoma cells in a TRAIL-dependent manner. 15 TRAIL expression was previously shown to increase during IFN-based therapy of hepatitis C, but its peak was observed at late time points, ie, weeks in that study. Furthermore, a statistical analysis of the treatment-induced NK cell response and the decline in viremia was not performed. 15 Here, we had the opportunity to study NK cell phenotype and function not only at early time points in the blood, but also in the liver. We observed an increase of cytotoxic, ie, degranulating and TRAIL-expressing NK cells in the blood, which was most pronounced 24 hours after treatment initiation (Figure 3A and B). The significant increase in the size of the intrahepatic cytotoxic CD16 NK cell subset and the trend of an increase in the percentage and MFI of intrahepatic TRAIL-expressing NK cells 6 hours after treatment initiation support this notion (Figure 4A). The increase in cytotoxic NK cells in the liver may have been caused by either activation of liver-resident NK cells or by recruitment of peripheral blood NK cells. The latter scenario is supported by the decrease in the population of CXCR3 and CCR5 NK cells in the blood (Figure 2). Of note, the peaks of NK cell degranulation and TRAIL production coincided with a small, but significant ALT rise within the first 24 hours of therapy. Although serum ALT levels are considered weak correlates of liver injury, it should be noted that correlations between ALT levels and NK cell activity have been described in earlier studies on patients with chronic hepatitis C. 12,13,24 The observed increase in NK cell cytotoxicity and the decrease in IFN- production are also interesting in light of our earlier data on in vitro exposure of NK cells to IFN-, 12 and a recent report that IFN- production by NK cells is lower in patients with chronic HCV infection than in patients who had been treated successfully. 25 Thus, treatment with IFN- appears to enhance an NK cell phenotype that is already established in chronic HCV infection. 12,13 Exposure to IFN- may increase signal transducer and activator of transcription 1 expression and phosphorylation, 26 which results in increased NK cell cytotoxicity and decreases signal transducer and activator of transcription 4 phosphorylation, causing diminished IFN- production. A possible compensatory mechanism for the suppression of IFN- production might be the observed shift in the NK cell subset proportions toward an increase in the cytokine-producing CD56 bright NK cell subset that we observed later during therapy, which is consistent with published literature (Figure 1C). 15,22,25 27 This study also shows that the immune mechanisms leading to viral clearance differ between spontaneous and treatment-induced resolution of the infection. It is wellestablished that the endogenous type I IFN response does not induce HCV clearance during the natural course of infection (reviewed in Rehermann 3 ). Additional immune components, eg, IFN- producing T cells, are required for spontaneous HCV clearance. This is consistent with published data that IFN- responses of T cells correlate with spontaneous HCV clearance. 3 In contrast, exogenous type I IFN that is used in the treatment protocols at high doses has a strong antiviral effect. NK cells can enhance this antiviral effect and/or serve as biomarkers of the patients IFN responsiveness as suggested by the correlation between early induction of NK cell cytotoxicity and first-phase virological response (Figure 5). Although this correlation is relatively weak (r 0.56 for 24-hour HCV titer decline, r 0.48 for 48-hour HCV titer decline), it is the earliest indicator of differential NK cell cytotoxicity in IFN responders and nonresponders and is maintained for the subsequent weeks (Figure 5C). IFN- production appears to be less important, which is consistent with reports that a treatment response is not associated with an increase in IFN- responses of HCV-specific T cells (reviewed in Rehermann 3 ). Treatment response is also influenced by genetic factors. Although the treatment responder group in Figure 5 had a higher percentage of individuals with the IL28B rs CC SNP (66%) than the nonresponder group (9%; P.016), NK cell phenotype and function did not differ among subgroups with different IL28B genotypes, and the correlation between NK cell response and first-phase virological response was preserved when restricted to patients with IL28B CC rs SNP. This is consistent with the fact that lymphocytes do not respond to type III interferons (including IL28B) because they express a short splice variant of the receptor. 28 A second genetic component of interest is the KIR/HLA compound haplotype (Table 1, Supplementary Table 1), but we found no difference between patient subgroups. A combined effect of KIR and IL28B in the context of IFN treatment is possible, 29 but would require additional studies with a larger number of patients. In conclusion, these results allow at least 2 interpretations. First, the NK cell response in the blood may serve as a biomarker for IFN responsiveness and complement intrahepatic biomarkers, such as the expression level of IFN-induced genes. Second, it is notable that the difference in NK cell responses between responders and nonresponders becomes even more apparent at later time points (Figure 5C), which may reflect differences in second-phase viral decline. Thus, it is conceivable that NK cells become activated early during IFN-based therapy, but that NK cell mediated clearance of HCV-infected cells may take longer to occur. Further follow-up is required to evaluate whether these early NK cell

9 October 2011 NK CELL RESPONSE TO TREATMENT OF HCV INFECTION 1239 responses predict a sustained virological response. If NK cells contribute to virus elimination, targeted enhancement of NK cell function may prove a beneficial option to complement antiviral therapy. 30 Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at and at doi: /j.gastro References 1. Ciesek S, Manns MP. Hepatitis in 2010: the dawn of a new era in HCV therapy. Nat Rev Gastroenterol Hepatol 2011;8: Layden TJ, Mika B, Wiley TE. Hepatitis C kinetics: mathematical modeling of viral response to therapy. Semin Liver Dis 2000;20: Rehermann B. Hepatitis C virus versus innate and adaptive immune responses: a tale of coevolution and coexistence. J Clin Invest 2009;119: Norris S, Collins C, Doherty DG, et al. Resident human hepatic lymphocytes are phenotypically different from circulating lymphocytes. J Hepatol 1998;28: Biron CA, Nguyen KB, Pien GC, et al. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol 1999;17: Khakoo SI, Thio CL, Martin MP, et al. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science 2004;305: Knapp S, Warshow U, Hegazy D, et al. Consistent beneficial effects of killer cell immunoglobulin-like receptor 2DL3 and group 1 human leukocyte antigen-c following exposure to hepatitis C virus. Hepatology 2010;51: Cooper MA, Fehniger TA, Turner SC, et al. Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset. Blood 2001;97: Fauriat C, Long EO, Ljunggren HG, et al. Regulation of human NK-cell cytokine and chemokine production by target cell recognition. Blood 2010;115: De Maria A, Bozzano F, Cantoni C, et al. Revisiting human natural killer cell subset function revealed cytolytic CD56(dim)CD16 NK cells as rapid producers of abundant IFN-gamma on activation. Proc Natl Acad SciUSA2011;108: De Maria A, Fogli M, Mazza S, et al. Increased natural cytotoxicity receptor expression and relevant IL-10 production in NK cells from chronically infected viremic HCV patients. Eur J Immunol 2007; 37: Ahlenstiel G, Titerence RH, Koh C, et al. Natural killer cells are polarized toward cytotoxicity in chronic hepatitis C in an interferonalfa-dependent manner. Gastroenterology 2010;138: Oliviero B, Varchetta S, Paudice E, et al. Natural killer cell functional dichotomy in chronic hepatitis B and chronic hepatitis C virus infections. Gastroenterology 2009;137: , 1160 e Dunn C, Brunetto M, Reynolds G, et al. Cytokines induced during chronic hepatitis B virus infection promote a pathway for NK cell-mediated liver damage. J Exp Med 2007;204: Stegmann KA, Bjorkstrom NK, Veber H, et al. Interferon-alpha-induced TRAIL on natural killer cells is associated with control of hepatitis C virus infection. Gastroenterology 2010;138: Feld JJ, Modi AA, El-Diwany R, et al. S-adenosyl methionine improves early viral responses and interferon-stimulated gene induction in hepatitis C nonresponders. Gastroenterology 2011;140: Ahlenstiel G, Martin MP, Gao X, et al. Distinct KIR/HLA compound genotypes affect the kinetics of human antiviral natural killer cell responses. J Clin Invest 2008;118: Parruti G, Polilli E, Sozio F, et al. Rapid prediction of sustained virological response in patients chronically infected with HCV by evaluation of RNA decay 48h after the start of treatment with pegylated interferon and ribavirin. Antiviral Res 2010;88: Golden-Mason L, Cox AL, Randall JA, et al. Increased natural killer cell cytotoxicity and NKp30 expression protects against hepatitis C virus infection in high-risk individuals and inhibits replication in vitro. Hepatology 2010;52: Sene D, Levasseur F, Abel M, et al. Hepatitis C virus (HCV) evades NKG2D-dependent NK cell responses through NS5A-mediated imbalance of inflammatory cytokines. PLoS Pathog 2010;6:e Nattermann J, Nisschalke HD, Hofmeister V, et al. The HLA-A2 restricted T cell epitope HCV core stabilizes HLA-E expression and inhibits cytolysis mediated by natural killer cells. Am J Pathol 2005;166: Harrison RJ, Ettorre A, Little AM, et al. Association of NKG2A with treatment for chronic hepatitis C virus infection. Clin Exp Immunol 2010;161: Lan L, Gorke S, Rau SJ, et al. Hepatitis C virus infection sensitizes human hepatocytes to TRAIL-induced apoptosis in a caspase 9-dependent manner. J Immunol 2008;181: Bonorino P, Ramzan M, Camous X, et al. Fine characterization of intrahepatic NK cells expressing natural killer receptors in chronic hepatitis B and C. J Hepatol 2009;51: Dessouki O, Kamiya Y, Nagahama H, et al. Chronic hepatitis C viral infection reduces NK cell frequency and suppresses cytokine secretion: reversion by anti-viral treatment. Biochem Biophys Res Commun 2010;393: Miyagi T, Takehara T, Nishio K, et al. Altered interferon-alphasignaling in natural killer cells from patients with chronic hepatitis C virus infection. J Hepatol 2010;53: Lee S, Watson MW, Flexman JP, et al. Increased proportion of the CD56(bright) NK cell subset in patients chronically infected with hepatitis C virus (HCV) receiving interferon-alpha and ribavirin therapy. J Med Virol 2010;82: Witte K, Gruetz G, Volk HD, et al. Despite IFN-lambda receptor expression, blood immune cells, but not keratinocytes or melanocytes, have an impaired response to type III interferons: implications for therapeutic applications of these cytokines. Genes Immun 2009;10: Dring MM, Morrison MH, McSharry BP, et al. Innate immune genes synergize to predict increased risk of chronic disease in hepatitis C virus infection. Proc Natl Acad Sci U S A 2011;108: Vivier E, Raulet DH, Moretta A, et al. Innate or adaptive immunity? The example of natural killer cells. Science 2011;331: Received March 9, Accepted June 24, Reprint requests Address requests for reprints to: Barbara Rehermann, MD, Immunology Section, Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, 10 Center Drive, Building 10, Room 9B16, Bethesda, Maryland Rehermann@nih.gov; fax: (301) Acknowledgments We thank Drs Fuh-Mei Duh, Maureen Martin, and Mary Carrington, SAIC-Frederick, National Cancer Institute-Frederick for KIR and HLA analysis; and Dr Xiongce Zhao, National Institute of Diabetes and Digestive and Kidney Diseases for statistical advice and data analysis. G.A. and B.E. contributed equally to this work. Conflicts of interest The authors disclose no conflicts. Funding This study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health intramural research program.

10 1239.e1 AHLENSTIEL ET AL GASTROENTEROLOGY Vol. 141, No. 4 Supplementary Table 1. Patient Epidemiological and Clinical Data Patient no. EVR SVR Sex Age, y Ethnicity BMI HCV genotype HCV RNA D 0, IU/mL ALT day 0, U/mL 1 Yes Yes M 36 W A e Yes ND F 51 A e Yes No M 53 W B e Yes ND M 50 W B e Yes ND M 40 A A e Yes Yes M 50 W A 2C e Yes ND F 49 AA B e Yes Yes M 54 W B e Yes No M 53 W B e Yes No M 55 W A or C e Yes Yes F 58 W B e Yes Yes M 54 W A e Yes ND M 64 W B e Yes Yes M 33 A B e Yes Yes F 54 AA A e Yes Yes M 55 W A e Yes ND F 54 AA F e Yes ND M 50 W e Yes Yes F 61 W B e Yes ND M 55 AA A e Yes ND F 49 AA A e Yes No M 60 A B e ND ND F 57 W A e No No F 61 AA E Yes No M 54 W e Yes No M 59 W e Yes Yes M 58 W A e No No M 47 W A e Yes No F 48 W A e ND ND M 50 AA A e No No M 49 W e No No M 46 W A e ND ND F 46 W e Yes Yes M 53 W B e Yes No F 59 W A e No No M 53 W e No No M 53 W e No No F 41 W A e Yes No F 43 W A e Yes No M 59 ND e ND ND F 45 AA e Yes ND M 44 W A e Yes ND F 63 AA e Yes Yes M 34 A e Yes Yes M 56 W B e Yes ND F 42 ND B e Yes ND M 24 A A e ND ND M 57 AA A e ND ND F 52 W B e ND ND F 59 W B e ND ND M 59 AA 27.7 ND e ND ND M 33 W e ND ND F 55 W e ND ND F 39 W e No No M 45 AA A e Yes ND M 58 W A e Yes ND F 54 W A e5 38

11 October 2011 NK CELL RESPONSE TO TREATMENT OF HCV INFECTION 1239.e2 Supplementary Table 1. Continued Time of biopsy, h Ishak score IS FS IL-28 rs SNP KIR HLA-C Treatment history Current treatment Included in Figure: CC ND ND Naïve pegifn/rbv 1, 2, 3, 4B CC ND ND Naïve pegifn/rbv 1, 2, 3, 4B CT ND ND Naïve pegifn/rbv 1, 2, 3, 4A, 4B CC ND ND Naïve pegifn/rbv 1, 2, 3, 4B ND ND ND Naïve pegifn/rbv 1, 2, 3, 4B CC ND ND Naïve pegifn/rbv 1, 2, 3, 4A, 4B 6 ND ND CT ND ND Naïve pegifn/rbv 1, 2, 3, 4B CC ND ND Naïve pegifn/rbv 1, 2, 3, 4A, 4B ND ND ND Naïve pegifn/rbv 1, 2, 3, 4B TT ND ND Naïve pegifn/rbv 1, 2, 3, 4A, 4B CC ND ND Naïve pegifn/rbv 1, 2, 3, 4B ND ND ND Naïve pegifn/rbv 1, 2, 3, 4B CT 2DL2/2DL3 C1/C2 Naïve pegifn/rbv 1, 2, 3, 4A, 4B, CC 2DL3/2DL3 C1/C2 Naïve pegifn/rbv 1, 2, 3, 4B, CC 2DL2/2DL3 C1/C2 Naïve pegifn/rbv 1, 2, 3, 4B, CC 2DL2/2DL3 C2/C2 Naïve pegifn/rbv 1, 2, 3, 4B, TT 2DL3/2DL3 C1/C2 Naïve pegifn/rbv 1, 2, 3, 4B, CC 2DL3/2DL3 C1/C1 Naïve pegifn/rbv 1, 2, 3, 4A, 4B, CC 2DL2/2DL3 C1/C2 Naïve pegifn/rbv 1, 2, 3, 4A, 4B, ND 2DL2/2DL3 C1/C2 Naïve pegifn/rbv 1, 2, 3, 4B, CT 2DL3/2DL3 C2/C2 Naïve pegifn/rbv 1, 2, 3, 4B, CC 2DL3/2DL3 C1/C1 Naïve pegifn/rbv 1, 2, 3, 4B, ND 2DL2/2DL2 C1/C1 Naïve pegifn/rbv 1, 2, 3, 4A, 4B, 5A/B Prior 12 6 CT 2DL2/2DL3 C1/C2 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior ND ND TT 2DL2/2DL2 C2/C2 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior 7 2 CT 2DL2/2DL3 C1/C2 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior 9 4 CC 2DL2/2DL3 C1/C1 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior ND 0 TT 2DL2/2DL3 C1/C1 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior ND ND CT 2DL2/2DL2 C2/C2 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior 7 2 CT 2DL2/2DL3 C2/C2 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior ND ND ND 2DL3/2DL3 C1/C1 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior 5 0 ND 2DL3/2DL3 C1/C2 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior 7 0 CT 2DL3/2DL3 C1/C2 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior 7 1 ND 2DL3/2DL3 C1/C1 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior 8 2 CT 2DL2/2DL3 C1/C2 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior ND ND TT 2DL2/2DL3 C1/C2 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior ND 2 CT 2DL2/2DL2 C1/C2 pegifn/rbv pegifn/rbv, pegifn/rbv/same 5 Prior ND ND ND 2DL2/2DL3 C2/C2 pegifn/rbv pegifn/rbv, pegifn/rbv/same ND ND ND 4 wk RBV pegifn/rbv 4A ND ND ND 4 wk RBV pegifn/rbv 4A ND ND ND Naïve pegifn/rbv 4A ND ND ND 4 wk RBV pegifn/rbv 4A ND ND ND Naïve pegifn/rbv 4A ND ND ND 4 wk RBV pegifn/rbv 4A ND ND ND 4 wk RBV pegifn/rbv 4A ND ND ND Naïve pegifn/rbv 4A ND ND ND 4 wk RBV pegifn/rbv 4A ND ND ND Naïve No treatment 4A ND ND ND Naïve No treatment 4A ND ND ND Naïve No treatment 4A ND ND ND Naïve No treatment 4A ND ND ND Naïve No treatment 4A ND ND ND Naïve No treatment 4A ND ND ND Naïve No treatment 4A ND ND ND 4 wk RBV pegifn/rbv 4A ND ND ND 4 wk RBV pegifn/rbv 4A ND ND ND 4 wk RBV pegifn/rbv 4A A, Asian; AA, African American; BMI, body mass index; C1, HLA-C group 1; C2, HLA-C group 2; F, female; FS, fibrosis score; IS, inflammatory score; M, male; ND, not determined; SAMe, S-adeNosyl-methionine; SVR, sustained virological response; W, white.