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Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection
Research Article
Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infectionq Barbara Oliviero1, Dalila Mele1, Elisabetta Degasperi4, Alessio Aghemo4, Eleonora Cremonesi1, Maria Grazia Rumi5, Carmine Tinelli2, Stefania Varchetta1, Stefania Mantovani1, Massimo Colombo4, Mario U. Mondelli1,3,⇑ 1
Research Laboratories, Department of Infectious Diseases, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 2Statistics and Clinical Epidemiology Service, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 3Department of Internal Medicine, University of Pavia, Pavia, Italy; 4Division of Gastroenterology, Department of Medicine, Fondazione IRCCS Ospedale Maggiore Mangiagalli Regina Elena, University of Milan, Milan, Italy; 5Hepatology Unit, Ospedale San Giuseppe, University of Milan, Milan, Italy
Background & Aims: A substantial proportion of patients with chronic hepatitis C virus infection treated with pegylated interferon a/ribavirin fail to achieve sustained virological response (SVR). Since growing evidence suggests that innate immunity may influence treatment responses, we examined natural killer (NK) cell phenotypic and functional changes during standard antiviral therapy. Methods: Expression of several NK-cell regulatory molecules was evaluated by flow cytometry in 37 consecutive patients with chronic HCV infection at baseline and at different time points during and after discontinuation of treatment. Cytokine production was evaluated by intracellular staining. Cytolytic potential was assessed as degranulation and as antibody-dependent cytotoxicity. Results: Baseline frequencies of CD56dim NK cells and perforin content were significantly higher, whereas CD16 expression was lower in SVR vs. non-responder subjects. Analysis by linear regression for repeated measures during the first 12 weeks showed significantly increased frequencies of activated (CD69+)
Keywords: Hepatitis C virus; Natural killer cells; Interferon alpha; Antiviral therapy. Received 30 July 2012; received in revised form 27 February 2013; accepted 2 March 2013 q Financial support: This work was supported by Research Funds of the Italian Ministry of Health (Ricerca Corrente, Fondazione IRCCS Policlinico San Matteo), by a grant from the Italian Ministry of Education, University and Research MiUR (Fondi di Investimento per la Ricerca di Base, FIRB, Protocollo: RBAP10TPXK), and by COPEV Associazione per la Prevenzione e Cura dell’Epatite Virale Beatrice Vitiello ONLUS. ⇑ Corresponding author. Address: Department of Infectious Diseases, Research Laboratories, Fondazione IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy. Tel. +39 0382 502636; fax: +39 0382 526450. E-mail address: [email protected] (M.U. Mondelli). Abbreviations: NK, natural killer; HCV, hepatitis C virus; IFNc, interferon gamma; TNFa, tumour necrosis factor alpha; DAAs, directly-acting antiviral; PegIFNa, pegylated interferon alpha; RBV, ribavirin; SOC, standard of care; SVR, sustained virological response; NR, non-responders; REL, relapsers; PBMC, peripheral blood mononuclear cells; mAbs, monoclonal antibodies; ICS, intracellular staining; PFN, perforin; IL, interleukin; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand; SNP, single nucleotide polymorphism; MFI, mean fluorescence intensity; 7-AAD, 7-aminoactinomycin D; ADCC, antibody-dependent cell-mediated cytotoxicity; EGTA, ethylene glycol tetra-acetic acid.
NK cells in rapid virological responders (RVR) and identified a typical NK cell profile associated with SVR, featuring higher NK perforin content, lower CD16 expression, and higher proportion of CD56dim/CD16 cells. Moreover, SVR patients displayed higher natural and antibody-dependent NK cell cytotoxicity. IL28B rs12979860 CC homozygosis was significantly associated with SVR, independently of NK-cell phenotype and function. Conclusions: Different NK-cell phenotypic and functional features, in patients with chronic hepatitis C treated with standard therapy, were observed between non-responder vs. SVR patients, suggesting a potential role of NK cells in the response to treatment. Ó 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Natural killer (NK) cells represent the principal effector cell population involved in innate immune responses to viral infections and exert their antiviral activity through a direct cytotoxic effect which destroys virus-infected target cells, and via production of immunoregulatory cytokines, which may influence adaptive immune responses as well as directly inhibiting virus replication [1]. NK-cell function is controlled by a complex network of signals which interact with membraneexpressed inhibitory and activating receptors and which affect their balance. In patients with chronic hepatitis C virus (HCV) infection, the frequency of circulating NK cells is reduced compared with controls [2] and is accompanied by skewing in subset distribution, with an enrichment of both CD56bright [3–6] and of a terminally differentiated hypofunctional CD56–CD16+ NK subset [7], although the latter has not been confirmed by all groups. Moreover, NK-cell phenotype is altered, showing variations in the expression of several activating and inhibitory receptors [2,8,9]. Finally, NK cells of HCV-infected patients display a functional dichotomy, characterised by normal or
Journal of Hepatology 2013 vol. xxx j xxx–xxx
Please cite this article in press as: Oliviero B et al. Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection. J Hepatol (2013), http://dx.doi.org/10.1016/j.jhep.2013.03.003
Research Article
NK-cell phenotypic and functional characterization NK-cell phenotype was examined as previously described [15]. Further technical details, including the analysis of the expression of activating and inhibitory receptors and the monoclonal antibodies (mAbs) used for cytofluorimetric analyses, are provided in Supplementary Materials and methods. NK-cell IFNc and TNFa production analysis was carried out by intracellular staining (ICS) as previously described [2]. CD107a degranulation assay was also performed as described [2], using HuH7.5 instead of K562 cells as targets.
2
Statistical analysis This is described in detail in Supplementary Materials and methods.
Results Analysis of NK-cell phenotype and function at baseline identifies features associated with treatment response Before institution of treatment, we observed a reduced proportion of CD56dim and a concomitantly increased proportion of CD56bright NK cells in NR compared with SVR patients (Fig. 1A–B). Moreover, NK cells of NR patients displayed a lower perforin (PFN) content than those of SVR (Fig. 1C). CD16 expression was instead significantly higher in NR than in SVR patients (Fig. 1D), which was seemingly caused by a significantly increased prevalence of a CD56dim/CD16 NK subset in SVR subjects (Fig. 1E). Representative dot plots and histograms are shown in Supplementary Fig. 1. There were no other statistically significant differences among groups. Antiviral treatment induces significant NK phenotypic changes We examined the effects of PegIFNa/RBV therapy on the kinetics of NK-cell phenotype in NR and SVR patients, by comparing trends in expression of each molecule with the corresponding
B
p = 0.02
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20 p = 0.017
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Peripheral blood mononuclear cells (PBMC) were obtained from 37 consecutive patients with chronic HCV infection undergoing treatment at predefined time points and cryopreserved in liquid N2 until use. All patients were recruited at the Division of Gastroenterology, Department of Medicine, Fondazione IRCCS Ospedale Maggiore, Mangiagalli, Regina Elena of Milan, Italy, and treated with PegIFNa-2a and weight-based RBV as per current clinical practice guidelines [11]. Demographic, biochemical, and virological features of the patients are listed in Supplementary Table 1. Twenty-five patients were SVR, 7 were NR and discontinued treatment according to established futility rules [11], and 5 relapsed after cessation of therapy. Informed consent in writing was obtained from each individual. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Review Board and Ethical Committee of Fondazione I.R.C.C.S. Policlinico San Matteo. PBMC were collected at baseline, and 2, 4, 12, 24, 26, 28, 48 weeks after treatment commencement for genotype 2-infected patients, and 2, 4, 12, 24, 48, 50, 52, 72 weeks for genotype 1/4-infected patients. NR patients discontinued treatment between 16 and 24 weeks after initiation.
These are described in Supplementary Materials and methods.
% NK CD56bright
Patients and methods
7-Aminoactinomycin D (7-AAD) cytotoxicity assay, inhibition of perforin polymerization, antibody-dependent cell-mediated cytotoxicity (ADCC) assay
PFN/isotype MFI
enhanced cytotoxicity [2,5,8,10] and reduced production of interferon c (IFNc) [2,10] and tumour necrosis factor a (TNFa) [2]. Despite the availability of several new directly-acting antiviral agents (DAAs) which specifically inhibit HCV replication, pegylated interferon-a (PegIFNa) and ribavirin (RBV) still represent the current standard of care (SOC) treatment for chronic HCV infection [11]. However, a sustained virological response (SVR), defined as undetectable HCV RNA 6 months after treatment discontinuation, is achieved in approximately 40–75% of cases, depending on several environmental, genetic, and viral factors [12]. In spite of the potentially important role played by innate immunity, and particularly NK cells, in treatment outcome in chronic HCV infection, only few studies were conducted on this topic. Recently, two studies pointed to the importance of IFNa-induced early NK-cell activation [13] and IFN signaling [14] in response to treatment. However, no specific profiles predictive of response to therapy were so far described and NK-cell responses were not examined throughout completion of treatment. Identification of patients responding to SOC is an unmet medical need since determination of IL28B polymorphism is insufficient alone to clearly discriminate between sustained virological responders (SVR), non-responders (NR), and relapsers (REL) to treatment. This is particularly relevant since PegIFNa and RBV will remain in the HCV pharmacopoeia for quite some time, because of their important role in preventing the emergence of DAA-resistant mutants, the well-defined, controllable adverse effects, and the definitely lower cost of a complete course of treatment compared to the newly available DAAs. In this study, we performed an in-depth prospective analysis of the kinetics of NK-cell phenotype and function in patients with chronic hepatitis C, during and after discontinuation of SOC therapy, in order to identify possible biomarkers associated with treatment outcome.
0
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p = 0.049
100 80 60 40 20 0 SVR
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Fig. 1. Baseline NK-cell phenotype in patients with different treatment outcomes. There was a (A) reduced frequency of CD56bright cells and (B) increased frequency of CD56dim cells in SVR compared with NR patients. SVR patients also show (C) significantly higher NK PFN content and (D) lower expression of CD16. (E) There was also an increased frequency of CD56dim/CD16– NK cells in SVR compared with NR patients.
Journal of Hepatology 2013 vol. xxx j xxx–xxx
Please cite this article in press as: Oliviero B et al. Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection. J Hepatol (2013), http://dx.doi.org/10.1016/j.jhep.2013.03.003
JOURNAL OF HEPATOLOGY
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Fig. 2. Statistically significant changes from baseline in NK phenotype of (A, C, E) SVR and (B, D, F) NR patients during SOC therapy. Asterisks indicate the level of statistical significance. EOT, end of treatment.
baseline value (Fig. 2 and Supplementary Figs. 2 and 3). Early NKcell activation, indicated by an increased percentage of CD69expressing NK cells, was observed at week 2 on treatment in all patient groups. Significantly higher than baseline frequencies of CD69+ NK cells were maintained until therapy discontinuation only in the SVR group at selected time points (Fig. 2A and B). Further, starting from week 2 to end of treatment, an increase in NKp30 expression was observed in both SVR and NR patients (Supplementary Fig. 2A and B), whereas NKp46 expression rose later, at 12 weeks, and remained higher than baseline during follow-up in SVR and NR subjects, although this finding reached statistical significance in SVR patients only (Supplementary Fig. 2C and D). Compared to baseline, there was a statistically significant progressive decline in the expression of CD16 on NK cells of SVR patients starting at week 4 on therapy, which remained lower until 4 weeks after treatment discontinuation (Fig. 2C). The same trend was found in NR subjects, even though the kinetics was delayed (Fig. 2D). Moreover, there was a decrease in PFN content (Fig. 2E and F) and Siglec-7 expression (Supplementary Fig. 2E and F), observed from the very early phases of therapy to the subsequent weeks, which reached significantly lower values than baseline in SVR patients only. Interestingly, there was a progressive increase in the proportion of a CD56dim/CD16 NK subpopulation in both SVR and NR patients, which was delayed in the latter, since statistically significant differences from baseline were only detected at week 12 on treatment, whereas in the former group this was already observed at week 4 (Supplementary Fig. 3A and B). Additional notable findings included a statistically
On treatment NK-cell phenotypic and functional changes define a non-responder profile To identify early biomarkers associated with treatment outcome, we elected to analyse the NK-cell phenotype during the initial 12 weeks on treatment, since this is a conventional time frame universally regarded as predictive of response to SOC therapy. Trends of expression of each molecule analysed on NK cells of NR were compared with those of SVR patients. Linear regression analysis for repeated measures revealed that CD16 expression in NR subjects was maintained at significantly higher levels than SVR patients (Fig. 3A) whereas the PFN content was significantly lower (Fig. 3B). Interestingly, the proportion of CD56dim/CD16 NK cells, a subset appearing as a result of exposure to target cells [16], was significantly higher over time in SVR compared with NR patients (Fig. 3C). Similar time trends of molecule expression on NK cells could also be identified when the analysis was restricted to genotype 1 patients, even though statistical significance was
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significant increase in the percentage of NKG2A+ NK cells from 12 weeks to the end of treatment in both groups of patients (Supplementary Fig. 3C and D), and an early significant reduction in NKG2D+ NK cells persisting through week 12, in SVR patients only (Supplementary Fig. 3F). No other significant changes were observed during and after therapy (data not shown).
EOT
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Fig. 3. Linear regression for repeated measures during the first 12 weeks of therapy identifies NK profiles associated with treatment outcome. The following parameters were significantly different between SVR and NR patients: expression of (A) CD16, (B) PFN; and (C) frequency of CD56dim/CD16– cells. (D) RVR had significantly higher CD69+ NK cells throughout the observation period compared to non-RVR. Further, NK from SVR patients showed statistically higher degranulation ability than NR at selected time points in the presence of (E) IL-2 + IL-21 or (F) IL-2 + IL-12.
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Please cite this article in press as: Oliviero B et al. Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection. J Hepatol (2013), http://dx.doi.org/10.1016/j.jhep.2013.03.003
Research Article
In consideration of the aforementioned, we felt that the most consistent difference between SVR and NR patients was the baseline and dynamic profile of the NK-cell perforin content. We therefore asked whether cytotoxicity was important in inhibiting HCV infection in vitro. As expected, inhibition of perforin polymerization resulted in reduced target cell lysis (Supplementary Fig. 5A), but no effect was seen on degranulation (Supplementary Fig. 5B), perforin content (Supplementary Fig. 5C) and, more importantly, on target cell infection (Supplementary Fig. 5D), suggesting that cytokine (e.g., IFNc) secretion, rather than single-target cell lysis, is more efficient in preventing HCV infection, as shown by others [17]. However, when we explored the NK cytotoxic capacity, we showed that SVR patients had higher baseline natural or antibody-dependent cytolytic activity than NR patients, providing additional evidence in favour of this being an important feature associated with favourable response to IFNa-based therapy (Supplementary Fig. 6). The data of reduced ADCC is apparently in contrast with findings of consistently increased FccRIII expression in NR patients, suggesting that this receptor is poorly functional in this patient group. Relapser patients display a unique baseline and on treatment increase in NKp30 A small group of 5 patients in this study relapsed after therapy. Despite the low number of subjects, we decided to consider them as a separate category since they showed peculiar NK-cell phenotypic features, which did not allow unification with other groups. Thus, before therapy, we observed an NK-cell phenotype comparable with that of SVR patients, except for a higher percentage of NKp30+ NK cells and a higher expression of this activating receptor compared with both SVR and NR patients (Fig. 4A and B); however, there were no noticeable differences in NK function. Enhanced expression of NKp30 together with increased proportions of 4
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only maintained for PFN expression (p = 0.05, not shown). Moreover, rapid virological responders (RVR), 16 patients, virtually all of whom infected with genotype 2, showed significantly higher frequencies of CD69+ (activated) NK cells compared with the remainder, including non-RVR, SVR patients (Fig. 3D). Consistent with phenotypic data, NK-cell degranulation in the presence of HuH7.5 cells was significantly enhanced by PegIFNa/ RBV treatment at selected time points, in SVR patients from baseline to week 12 on treatment (Fig. 3E–F). In contrast, NR subjects showed a NK-cell cytotoxic activity comparable to baseline levels using interleukin (IL)-2 + IL-21 (Fig. 3E) stimulation, but statistically lower values at weeks 2 and 4 on therapy when stimulated with IL-2 and IL-12 (Fig. 3F). IFNc and TNFa production was evaluated by ICS with or without different cytokine combinations. In SVR patients, there was a reduced production of both IFNc and TNFa by unstimulated NK cells, which reached significantly lower than baseline values at week 12 (Supplementary Fig. 4), concomitant with the decreasing trend in the expression of CD16. Unstimulated cytokine secretion at week 12 was significantly higher in NR compared with SVR subjects. Stimulation with cytokine combinations as reported above did not show clearly significant differences as with unstimulated NK cells (data not shown).
0
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12
p = 0.037
80 60 40 20
SVR patients REL patients
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Fig. 4. REL patients show a unique NK phenotypic profile characterised by high NKp30 expression at baseline and during the early phases of treatment. Baseline data are shown in (A) (frequency) and (B) (MFI). (C) Representative histogram of NKp30 expression in an REL, SVR, and NR patient at baseline. Temporal trends of NKp30 expression, NKp30+ NK cell frequencies, and NKp46 expression in REL vs. SVR patients are shown in (D), (E), and (F), respectively.
NKp30-expressing NK cells, compared with SVR patients, persisted through week 12 (Fig. 4D and E) and was accompanied by a less durable but significant increase in NKp46-expressing NK cells (Fig. 4F). Persistent upregulation of NKp30 from baseline to week 12 appeared to be a unique feature of REL patients. Time trends of the different molecules analysed were similar to the other groups and are shown in Supplementary Fig. 7. Additional host and viral factors associated with response to SOC therapy As expected, genotype 2 infection was significantly associated with SVR (p = 0.004, by Fisher’s exact test) and 15 of 16 patients in this group were RVR. Moreover, infection with genotype 2 was associated with a significantly higher proportion of CD69+ NK cells (p = 0.005) during treatment. We also examined the prevalence of the single nucleotide polymorphism near the IL28B encoding gene, most commonly associated with response to SOC therapy (rs12979860) in chronic HCV infection [18], confirming that CC homozygosis was significantly associated with SVR (p = 0.005, by Fisher’s exact test) (Supplementary Table 1). There was no other association between host and viral factors (including sex, age, HCV genotype, and IL28B polymorphism) and NK phenotype and function by multivariate regression analysis.
Journal of Hepatology 2013 vol. xxx j xxx–xxx
Please cite this article in press as: Oliviero B et al. Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection. J Hepatol (2013), http://dx.doi.org/10.1016/j.jhep.2013.03.003
JOURNAL OF HEPATOLOGY Discussion In this study we performed an in-depth analysis of phenotypic and functional characteristics of NK cells in patients with chronic HCV infection treated with a standard combination of PegIFNa-2a and RBV, in order to identify immunological profiles associated with treatment outcome. The choice of studying innate immune responses originated from previous work which suggested that adaptive immunity does not appear to play a major role in treatment-induced viral eradication [19,20]. Moreover, because NK cells are rapidly activated by IFNa in vitro [10,21] and in vivo [13], we reasoned that their stimulation during treatment may contribute to HCV clearance. To this end, unselected consecutive patients were enrolled in a prospective fashion and changes in NK-cell phenotype and function analysed at several predefined time points during and after cessation of SOC treatment. With this approach, we showed that important NK phenotypic differences between SVR and NR were already present prior to treatment commencement. Indeed, NR patients were characterised by an enrichment in CD56bright and, conversely, by a lower frequency of CD56dim NK cells. These findings are in agreement with those of Bozzano and associates [22] who also found increased frequencies of CD56bright/CD16+/ NK cells in NR patients and increased frequencies of CD56dim/CD16+ NK cells in SVR patients. The CD56bright subset is thought to contain relatively immature NK cells [23,24] with prominent proliferative capacity and immunoregulatory function, as shown by their ability to secrete cytokines, together with relatively poor cytotoxic activity [25]. In contrast, the more differentiated CD56dim subset features a predominant cytolytic function [25]. This prospective study suggests that NK cytolytic function could be one mechanism of elimination of HCV-infected hepatocytes, which would be supported by in vitro studies showing that NK cells can kill HCV-infected hepatocytes via perforin/granzyme [26], as well as tumour necrosis factorrelated apoptosis-inducing ligand (TRAIL)-mediated mechanisms [21]. More importantly, we showed that the NK PFN content remained significantly higher until week 12 in patients developing SVR compared with NR patients, corroborating the hypothesis that an efficient NK cytolytic function is required for optimal clearance of HCV-infected cells, and suggesting that a reduced PFN content is a characteristic feature of patients not responding to IFN-based therapy. However, from our own and previous data obtained with CD8 T cells [17], it would appear that single-target cell killing is rather inefficient in preventing HCV infection, at least in vitro, suggesting that IFNc-mediated non-cytolytic mechanisms provide a stronger antiviral effect [27]. In line with this, it may be inferred that IFNc release would be a more powerful mechanism of protection from infection, whereas optimal NK killing activity would be required for IFNa-induced HCV clearance. It is noteworthy that a recently published study showed upregulation of several NK-cell activating receptors, including the CD69 early activation antigen, within hours from the first IFNa injection [13]. Although our protocol did not allow us to perform early liver biopsies nor we were permitted to recall patients for venepuncture before the second week on therapy, our data are compatible with those of Ahlenstiel [13], since the proportion of CD69+ cells was upregulated in all patients, early after initiation of treatment. However, while this activated NK phenotype persisted until treatment discontinuation in the subjects who achieved SVR, it was only transient and limited to week 2 on therapy in NR patients, suggesting that an early and sustained NK-cell
activation is of paramount importance for a successful treatment outcome. These findings are further corroborated by evidence that CD69 expression is statistically and consistently higher in RVR patients compared to the remainder. Interestingly, the frequency of CD56dim/CD16 NK cells, a reportedly activated NK subpopulation [28], was increased in the first 12 weeks of treatment in the SVR group. The role of CD56dim/CD16 NK cells is still poorly defined, although previous studies suggested that these cells represent the only subset able to degranulate when exposed to tumour targets, whereas CD56dim/CD16+ cells would be involved in antibody-dependent cytotoxicity [29]. Further studies actually showed that CD56dim/ CD16 NK cells are mature cytotoxic CD16+ cells that have downregulated CD16 following exposure to targets (16 and our own data not shown). In line with these findings, a progressive proportional increase of these cells in SVR patients would suggest CD16 downmodulation as a result of contact with, and subsequent successful killing of HCV-infected hepatocytes in patients eventually developing SVR. Progressive reduction of Siglec-7 expression to lower than baseline values appeared to be associated with antiviral treatment in all patient groups during and after discontinuation of treatment akin to Ahlenstiel’s study [13]. However, the observation that levels of NK Siglec-7 expression over time were statistically lower than baseline in SVR patients only, suggests that reduced expression of this inhibitory receptor [30,31] would contribute to maintain a stronger and sustained NK-cell activated status in this group. One important issue was to determine whether IL28B polymorphism was associated with specific NK profiles in this study. Indeed, it is possible that members of the IFNk family can modulate NK activity and that IL28B polymorphism is a marker for this interaction. Interestingly, it has recently been reported that the unfavourable rs12979860 IL28B TT homozygosis was associated with increased expression of the NKG2A inhibitory receptor and reduced expression of TRAIL on CD56dim NK cells [32], providing evidence in support of IL28B polymorphism regulating innate immune responses in HCV infection. As extensively reported in recent studies, reviewed in [33], IL28B CC homozygosis was significantly associated with SVR also in our series of patients, but no correlation was seen between NK-cell phenotype and function and IL28B polymorphism with respect to response to treatment. These findings are in agreement with those of a recent study, in which analysis of the same IL28B SNP was shown to be associated with the favourable CC SNP in early virological responders [13]. The difference in NK-cell activation between responders and non-reponders was maintained even when the analysis was restricted to CC homozygotes, suggesting that early NK activation during IFNa therapy is independent of IL28B polymorphism. This interpretation is further supported by our data confirming that IL28B polymorphism is associated with treatment outcome independently from the patients’ NK dynamic profile. REL patients exhibited a peculiar phenotypic pattern characterised by a consistently increased expression of NKp30 from baseline to week 12 on treatment and a transient (until week 4) increase of NKp46, which prompted us to analyse this small group separately. We have no simple explanation for such findings, but it is possible that NKp30, which correlates with cytotoxicity [34], plays a specific role in the relapse process, since upregulation is already present at baseline. Interestingly, lower frequencies of NKp30+ and NKp46+ NK cells have been recently
Journal of Hepatology 2013 vol. xxx j xxx–xxx
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Research Article associated with the ability to clear HCV following acute infection [35], suggesting that NCR upregulation may reflect incomplete NK-cell activation in individuals who do not clear infection either spontaneously or following successful antiviral treatment. In summary, we have identified specific NK-cell phenotypic and functional profiles that may help distinguish patients according to treatment outcome. Early NK-cell activation and robust cytolytic function appear to be fundamentally important in the rapid IFNa-induced elimination of HCV-infected hepatocytes. Even though the standard of care for chronic HCV infection is rapidly changing, we submit that NK dynamic profiling could still provide valuable information on dual therapy outcome in this clinical setting.
Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Acknowledgements We thank Dr. E. Tagliabue, Molecular Targeting Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, for providing the BT474 HER2-positive breast cancer cell line.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2013.03. 003.
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[9] Nattermann J, Feldmann G, Ahlenstiel G, Langhans B, Sauerbruch T, Spengler U. Surface expression and cytolytic function of natural killer cell receptors is altered in chronic hepatitis C. Gut 2006;55:869–877. [10] Ahlenstiel G, Titerence RH, Koh C, Edlich B, Feld JJ, Rotman Y, et al. Natural killer cells are polarized toward cytotoxicity in chronic hepatitis C in an interferon-alfa-dependent manner. Gastroenterology 2010;138:325–335. [11] European Association for the Study of the L. EASL Clinical Practice Guidelines: management of hepatitis C virus infection. J Hepatol 2011;55:245–264. [12] Feld JJ, Hoofnagle JH. Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature 2005;436:967–972. [13] Ahlenstiel G, Edlich B, Hogdal LJ, Rotman Y, Noureddin M, Feld JJ, et al. Early changes in natural killer cell function indicate virologic response to interferon therapy for hepatitis C. Gastroenterology 2011; 141:1231–1239. [14] Edlich B, Ahlenstiel G, Azpiroz AZ, Stoltzfus J, Noureddin M, Serti E, et al. Early changes in interferon signaling define natural killer cell response and refractoriness to interferon-based therapy of hepatitis C patients. Hepatology 2012;55:39–48. [15] Varchetta S, Mele D, Mantovani S, Oliviero B, Cremonesi E, Ludovisi S, et al. Impaired intrahepatic natural killer cell cytotoxic function in chronic hepatitis C virus infection. Hepatology 2012;56:841–849. [16] Grzywacz B, Kataria N, Verneris MR. CD56(dim)CD16(+) NK cells downregulate CD16 following target cell induced activation of matrix metalloproteinases. Leukemia 2007;21:356–359. [17] Jo J, Bengsch B, Seigel B, Rau SJ, Schmidt J, Bisse E, et al. Low perforin expression of early differentiated HCV-specific CD8+ T cells limits their hepatotoxic potential. J Hepatol 2012;57:9–16. [18] Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV, Urban TJ, et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 2009;461:399–401. [19] Pembroke T, Rees I, Gallagher K, Jones E, Mizen P, Navruzov T, et al. Rapid early innate control of hepatitis C virus during IFN-alpha treatment compromises adaptive CD4(+) T-cell immunity. Eur J Immunol 2012;42:2383–2394. [20] Rehermann B, Hepatitis C. Virus versus innate and adaptive immune responses: a tale of coevolution and coexistence. J Clin Invest 2009;119:1745–1754. [21] Stegmann KA, Bjorkstrom NK, Veber H, Ciesek S, Riese P, Wiegand J, et al. Interferon-alpha-induced TRAIL on natural killer cells is associated with control of hepatitis C virus infection. Gastroenterology 2010;138:1885–1897. [22] Bozzano F, Picciotto A, Costa P, Marras F, Fazio V, Hirsch I, et al. Activating NK cell receptor expression/function (NKp30, NKp46, DNAM-1) during chronic viraemic HCV infection is associated with the outcome of combined treatment. Eur J Immunol 2011;41:2905–2914. [23] Chan A, Hong DL, Atzberger A, Kollnberger S, Filer AD, Buckley CD, et al. CD56bright human NK cells differentiate into CD56dim cells: role of contact with peripheral fibroblasts. J Immunol 2007;179:89–94. [24] Romagnani C, Juelke K, Falco M, Morandi B, D’Agostino A, Costa R, et al. CD56brightCD16 killer Ig-like receptor – NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation. J Immunol 2007;178:4947–4955. [25] Cooper MA, Fehniger TA, Caligiuri MA. The biology of human natural killercell subsets. Trends Immunol 2001;22:633–640. [26] Larkin J, Bost A, Glass JI, Tan SL. Cytokine-activated natural killer cells exert direct killing of hepatoma cells harboring hepatitis C virus replicons. J Interferon Cytokine Res 2006;26:854–865. [27] Jo J, Aichele U, Kersting N, Klein R, Aichele P, Bisse E, et al. Analysis of CD8+ T-cell-mediated inhibition of hepatitis C virus replication using a novel immunological model. Gastroenterology 2009;136:1391–1401. [28] Dessouki O, Kamiya Y, Nagahama H, Tanaka M, Suzu S, Sasaki Y, 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;393:331–337. [29] Penack O, Gentilini C, Fischer L, Asemissen AM, Scheibenbogen C, Thiel E, et al. CD56dimCD16neg cells are responsible for natural cytotoxicity against tumor targets. Leukemia 2005;19:835–840. [30] Falco M, Biassoni R, Bottino C, Vitale M, Sivori S, Augugliaro R, et al. Identification and molecular cloning of p75/AIRM1, a novel member of the sialoadhesin family that functions as an inhibitory receptor in human natural killer cells. J Exp Med 1999;190:793–802. [31] Nicoll G, Ni J, Liu D, Klenerman P, Munday J, Dubock S, et al. Identification and characterization of a novel siglec, siglec-7, expressed by human natural killer cells and monocytes. J Biol Chem 1999;274:34089–34095.
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JOURNAL OF HEPATOLOGY [32] Golden-Mason L, Bambha KM, Cheng L, Howell CD, Taylor MW, Clark PJ, et al. Natural killer inhibitory receptor expression associated with treatment failure and interleukin-28B genotype in patients with chronic hepatitis C. Hepatology 2011;54:1559–1569. [33] Balagopal A, Thomas DL, Thio CL. IL28B and the control of hepatitis C virus infection. Gastroenterology 2010;139:1865–1876.
[34] Golden-Mason L, Cox AL, Randall JA, Cheng L, Rosen HR. 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;52:1581–1589. [35] Alter G, Jost S, Rihn S, Reyor LL, Nolan BE, Ghebremichael M, et al. Reduced frequencies of NKp30+NKp46+, CD161+, and NKG2D+ NK cells in acute HCV infection may predict viral clearance. J Hepatol 2011;55:278–288.
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Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infectionq Barbara Oliviero1, Dalila Mele1, Elisabetta Degasperi4, Alessio Aghemo4, Eleonora Cremonesi1, Maria Grazia Rumi5, Carmine Tinelli2, Stefania Varchetta1, Stefania Mantovani1, Massimo Colombo4, Mario U. Mondelli1,3,⇑ 1
Research Laboratories, Department of Infectious Diseases, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 2Statistics and Clinical Epidemiology Service, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 3Department of Internal Medicine, University of Pavia, Pavia, Italy; 4Division of Gastroenterology, Department of Medicine, Fondazione IRCCS Ospedale Maggiore Mangiagalli Regina Elena, University of Milan, Milan, Italy; 5Hepatology Unit, Ospedale San Giuseppe, University of Milan, Milan, Italy
Background & Aims: A substantial proportion of patients with chronic hepatitis C virus infection treated with pegylated interferon a/ribavirin fail to achieve sustained virological response (SVR). Since growing evidence suggests that innate immunity may influence treatment responses, we examined natural killer (NK) cell phenotypic and functional changes during standard antiviral therapy. Methods: Expression of several NK-cell regulatory molecules was evaluated by flow cytometry in 37 consecutive patients with chronic HCV infection at baseline and at different time points during and after discontinuation of treatment. Cytokine production was evaluated by intracellular staining. Cytolytic potential was assessed as degranulation and as antibody-dependent cytotoxicity. Results: Baseline frequencies of CD56dim NK cells and perforin content were significantly higher, whereas CD16 expression was lower in SVR vs. non-responder subjects. Analysis by linear regression for repeated measures during the first 12 weeks showed significantly increased frequencies of activated (CD69+)
Keywords: Hepatitis C virus; Natural killer cells; Interferon alpha; Antiviral therapy. Received 30 July 2012; received in revised form 27 February 2013; accepted 2 March 2013 q Financial support: This work was supported by Research Funds of the Italian Ministry of Health (Ricerca Corrente, Fondazione IRCCS Policlinico San Matteo), by a grant from the Italian Ministry of Education, University and Research MiUR (Fondi di Investimento per la Ricerca di Base, FIRB, Protocollo: RBAP10TPXK), and by COPEV Associazione per la Prevenzione e Cura dell’Epatite Virale Beatrice Vitiello ONLUS. ⇑ Corresponding author. Address: Department of Infectious Diseases, Research Laboratories, Fondazione IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy. Tel. +39 0382 502636; fax: +39 0382 526450. E-mail address: [email protected] (M.U. Mondelli). Abbreviations: NK, natural killer; HCV, hepatitis C virus; IFNc, interferon gamma; TNFa, tumour necrosis factor alpha; DAAs, directly-acting antiviral; PegIFNa, pegylated interferon alpha; RBV, ribavirin; SOC, standard of care; SVR, sustained virological response; NR, non-responders; REL, relapsers; PBMC, peripheral blood mononuclear cells; mAbs, monoclonal antibodies; ICS, intracellular staining; PFN, perforin; IL, interleukin; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand; SNP, single nucleotide polymorphism; MFI, mean fluorescence intensity; 7-AAD, 7-aminoactinomycin D; ADCC, antibody-dependent cell-mediated cytotoxicity; EGTA, ethylene glycol tetra-acetic acid.
NK cells in rapid virological responders (RVR) and identified a typical NK cell profile associated with SVR, featuring higher NK perforin content, lower CD16 expression, and higher proportion of CD56dim/CD16 cells. Moreover, SVR patients displayed higher natural and antibody-dependent NK cell cytotoxicity. IL28B rs12979860 CC homozygosis was significantly associated with SVR, independently of NK-cell phenotype and function. Conclusions: Different NK-cell phenotypic and functional features, in patients with chronic hepatitis C treated with standard therapy, were observed between non-responder vs. SVR patients, suggesting a potential role of NK cells in the response to treatment. Ó 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Natural killer (NK) cells represent the principal effector cell population involved in innate immune responses to viral infections and exert their antiviral activity through a direct cytotoxic effect which destroys virus-infected target cells, and via production of immunoregulatory cytokines, which may influence adaptive immune responses as well as directly inhibiting virus replication [1]. NK-cell function is controlled by a complex network of signals which interact with membraneexpressed inhibitory and activating receptors and which affect their balance. In patients with chronic hepatitis C virus (HCV) infection, the frequency of circulating NK cells is reduced compared with controls [2] and is accompanied by skewing in subset distribution, with an enrichment of both CD56bright [3–6] and of a terminally differentiated hypofunctional CD56–CD16+ NK subset [7], although the latter has not been confirmed by all groups. Moreover, NK-cell phenotype is altered, showing variations in the expression of several activating and inhibitory receptors [2,8,9]. Finally, NK cells of HCV-infected patients display a functional dichotomy, characterised by normal or
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Research Article
NK-cell phenotypic and functional characterization NK-cell phenotype was examined as previously described [15]. Further technical details, including the analysis of the expression of activating and inhibitory receptors and the monoclonal antibodies (mAbs) used for cytofluorimetric analyses, are provided in Supplementary Materials and methods. NK-cell IFNc and TNFa production analysis was carried out by intracellular staining (ICS) as previously described [2]. CD107a degranulation assay was also performed as described [2], using HuH7.5 instead of K562 cells as targets.
2
Statistical analysis This is described in detail in Supplementary Materials and methods.
Results Analysis of NK-cell phenotype and function at baseline identifies features associated with treatment response Before institution of treatment, we observed a reduced proportion of CD56dim and a concomitantly increased proportion of CD56bright NK cells in NR compared with SVR patients (Fig. 1A–B). Moreover, NK cells of NR patients displayed a lower perforin (PFN) content than those of SVR (Fig. 1C). CD16 expression was instead significantly higher in NR than in SVR patients (Fig. 1D), which was seemingly caused by a significantly increased prevalence of a CD56dim/CD16 NK subset in SVR subjects (Fig. 1E). Representative dot plots and histograms are shown in Supplementary Fig. 1. There were no other statistically significant differences among groups. Antiviral treatment induces significant NK phenotypic changes We examined the effects of PegIFNa/RBV therapy on the kinetics of NK-cell phenotype in NR and SVR patients, by comparing trends in expression of each molecule with the corresponding
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Peripheral blood mononuclear cells (PBMC) were obtained from 37 consecutive patients with chronic HCV infection undergoing treatment at predefined time points and cryopreserved in liquid N2 until use. All patients were recruited at the Division of Gastroenterology, Department of Medicine, Fondazione IRCCS Ospedale Maggiore, Mangiagalli, Regina Elena of Milan, Italy, and treated with PegIFNa-2a and weight-based RBV as per current clinical practice guidelines [11]. Demographic, biochemical, and virological features of the patients are listed in Supplementary Table 1. Twenty-five patients were SVR, 7 were NR and discontinued treatment according to established futility rules [11], and 5 relapsed after cessation of therapy. Informed consent in writing was obtained from each individual. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Review Board and Ethical Committee of Fondazione I.R.C.C.S. Policlinico San Matteo. PBMC were collected at baseline, and 2, 4, 12, 24, 26, 28, 48 weeks after treatment commencement for genotype 2-infected patients, and 2, 4, 12, 24, 48, 50, 52, 72 weeks for genotype 1/4-infected patients. NR patients discontinued treatment between 16 and 24 weeks after initiation.
These are described in Supplementary Materials and methods.
% NK CD56bright
Patients and methods
7-Aminoactinomycin D (7-AAD) cytotoxicity assay, inhibition of perforin polymerization, antibody-dependent cell-mediated cytotoxicity (ADCC) assay
PFN/isotype MFI
enhanced cytotoxicity [2,5,8,10] and reduced production of interferon c (IFNc) [2,10] and tumour necrosis factor a (TNFa) [2]. Despite the availability of several new directly-acting antiviral agents (DAAs) which specifically inhibit HCV replication, pegylated interferon-a (PegIFNa) and ribavirin (RBV) still represent the current standard of care (SOC) treatment for chronic HCV infection [11]. However, a sustained virological response (SVR), defined as undetectable HCV RNA 6 months after treatment discontinuation, is achieved in approximately 40–75% of cases, depending on several environmental, genetic, and viral factors [12]. In spite of the potentially important role played by innate immunity, and particularly NK cells, in treatment outcome in chronic HCV infection, only few studies were conducted on this topic. Recently, two studies pointed to the importance of IFNa-induced early NK-cell activation [13] and IFN signaling [14] in response to treatment. However, no specific profiles predictive of response to therapy were so far described and NK-cell responses were not examined throughout completion of treatment. Identification of patients responding to SOC is an unmet medical need since determination of IL28B polymorphism is insufficient alone to clearly discriminate between sustained virological responders (SVR), non-responders (NR), and relapsers (REL) to treatment. This is particularly relevant since PegIFNa and RBV will remain in the HCV pharmacopoeia for quite some time, because of their important role in preventing the emergence of DAA-resistant mutants, the well-defined, controllable adverse effects, and the definitely lower cost of a complete course of treatment compared to the newly available DAAs. In this study, we performed an in-depth prospective analysis of the kinetics of NK-cell phenotype and function in patients with chronic hepatitis C, during and after discontinuation of SOC therapy, in order to identify possible biomarkers associated with treatment outcome.
0
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p = 0.049
100 80 60 40 20 0 SVR
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Fig. 1. Baseline NK-cell phenotype in patients with different treatment outcomes. There was a (A) reduced frequency of CD56bright cells and (B) increased frequency of CD56dim cells in SVR compared with NR patients. SVR patients also show (C) significantly higher NK PFN content and (D) lower expression of CD16. (E) There was also an increased frequency of CD56dim/CD16– NK cells in SVR compared with NR patients.
Journal of Hepatology 2013 vol. xxx j xxx–xxx
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JOURNAL OF HEPATOLOGY
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Fig. 2. Statistically significant changes from baseline in NK phenotype of (A, C, E) SVR and (B, D, F) NR patients during SOC therapy. Asterisks indicate the level of statistical significance. EOT, end of treatment.
baseline value (Fig. 2 and Supplementary Figs. 2 and 3). Early NKcell activation, indicated by an increased percentage of CD69expressing NK cells, was observed at week 2 on treatment in all patient groups. Significantly higher than baseline frequencies of CD69+ NK cells were maintained until therapy discontinuation only in the SVR group at selected time points (Fig. 2A and B). Further, starting from week 2 to end of treatment, an increase in NKp30 expression was observed in both SVR and NR patients (Supplementary Fig. 2A and B), whereas NKp46 expression rose later, at 12 weeks, and remained higher than baseline during follow-up in SVR and NR subjects, although this finding reached statistical significance in SVR patients only (Supplementary Fig. 2C and D). Compared to baseline, there was a statistically significant progressive decline in the expression of CD16 on NK cells of SVR patients starting at week 4 on therapy, which remained lower until 4 weeks after treatment discontinuation (Fig. 2C). The same trend was found in NR subjects, even though the kinetics was delayed (Fig. 2D). Moreover, there was a decrease in PFN content (Fig. 2E and F) and Siglec-7 expression (Supplementary Fig. 2E and F), observed from the very early phases of therapy to the subsequent weeks, which reached significantly lower values than baseline in SVR patients only. Interestingly, there was a progressive increase in the proportion of a CD56dim/CD16 NK subpopulation in both SVR and NR patients, which was delayed in the latter, since statistically significant differences from baseline were only detected at week 12 on treatment, whereas in the former group this was already observed at week 4 (Supplementary Fig. 3A and B). Additional notable findings included a statistically
On treatment NK-cell phenotypic and functional changes define a non-responder profile To identify early biomarkers associated with treatment outcome, we elected to analyse the NK-cell phenotype during the initial 12 weeks on treatment, since this is a conventional time frame universally regarded as predictive of response to SOC therapy. Trends of expression of each molecule analysed on NK cells of NR were compared with those of SVR patients. Linear regression analysis for repeated measures revealed that CD16 expression in NR subjects was maintained at significantly higher levels than SVR patients (Fig. 3A) whereas the PFN content was significantly lower (Fig. 3B). Interestingly, the proportion of CD56dim/CD16 NK cells, a subset appearing as a result of exposure to target cells [16], was significantly higher over time in SVR compared with NR patients (Fig. 3C). Similar time trends of molecule expression on NK cells could also be identified when the analysis was restricted to genotype 1 patients, even though statistical significance was
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significant increase in the percentage of NKG2A+ NK cells from 12 weeks to the end of treatment in both groups of patients (Supplementary Fig. 3C and D), and an early significant reduction in NKG2D+ NK cells persisting through week 12, in SVR patients only (Supplementary Fig. 3F). No other significant changes were observed during and after therapy (data not shown).
EOT
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Fig. 3. Linear regression for repeated measures during the first 12 weeks of therapy identifies NK profiles associated with treatment outcome. The following parameters were significantly different between SVR and NR patients: expression of (A) CD16, (B) PFN; and (C) frequency of CD56dim/CD16– cells. (D) RVR had significantly higher CD69+ NK cells throughout the observation period compared to non-RVR. Further, NK from SVR patients showed statistically higher degranulation ability than NR at selected time points in the presence of (E) IL-2 + IL-21 or (F) IL-2 + IL-12.
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Please cite this article in press as: Oliviero B et al. Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection. J Hepatol (2013), http://dx.doi.org/10.1016/j.jhep.2013.03.003
Research Article
In consideration of the aforementioned, we felt that the most consistent difference between SVR and NR patients was the baseline and dynamic profile of the NK-cell perforin content. We therefore asked whether cytotoxicity was important in inhibiting HCV infection in vitro. As expected, inhibition of perforin polymerization resulted in reduced target cell lysis (Supplementary Fig. 5A), but no effect was seen on degranulation (Supplementary Fig. 5B), perforin content (Supplementary Fig. 5C) and, more importantly, on target cell infection (Supplementary Fig. 5D), suggesting that cytokine (e.g., IFNc) secretion, rather than single-target cell lysis, is more efficient in preventing HCV infection, as shown by others [17]. However, when we explored the NK cytotoxic capacity, we showed that SVR patients had higher baseline natural or antibody-dependent cytolytic activity than NR patients, providing additional evidence in favour of this being an important feature associated with favourable response to IFNa-based therapy (Supplementary Fig. 6). The data of reduced ADCC is apparently in contrast with findings of consistently increased FccRIII expression in NR patients, suggesting that this receptor is poorly functional in this patient group. Relapser patients display a unique baseline and on treatment increase in NKp30 A small group of 5 patients in this study relapsed after therapy. Despite the low number of subjects, we decided to consider them as a separate category since they showed peculiar NK-cell phenotypic features, which did not allow unification with other groups. Thus, before therapy, we observed an NK-cell phenotype comparable with that of SVR patients, except for a higher percentage of NKp30+ NK cells and a higher expression of this activating receptor compared with both SVR and NR patients (Fig. 4A and B); however, there were no noticeable differences in NK function. Enhanced expression of NKp30 together with increased proportions of 4
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only maintained for PFN expression (p = 0.05, not shown). Moreover, rapid virological responders (RVR), 16 patients, virtually all of whom infected with genotype 2, showed significantly higher frequencies of CD69+ (activated) NK cells compared with the remainder, including non-RVR, SVR patients (Fig. 3D). Consistent with phenotypic data, NK-cell degranulation in the presence of HuH7.5 cells was significantly enhanced by PegIFNa/ RBV treatment at selected time points, in SVR patients from baseline to week 12 on treatment (Fig. 3E–F). In contrast, NR subjects showed a NK-cell cytotoxic activity comparable to baseline levels using interleukin (IL)-2 + IL-21 (Fig. 3E) stimulation, but statistically lower values at weeks 2 and 4 on therapy when stimulated with IL-2 and IL-12 (Fig. 3F). IFNc and TNFa production was evaluated by ICS with or without different cytokine combinations. In SVR patients, there was a reduced production of both IFNc and TNFa by unstimulated NK cells, which reached significantly lower than baseline values at week 12 (Supplementary Fig. 4), concomitant with the decreasing trend in the expression of CD16. Unstimulated cytokine secretion at week 12 was significantly higher in NR compared with SVR subjects. Stimulation with cytokine combinations as reported above did not show clearly significant differences as with unstimulated NK cells (data not shown).
0
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p = 0.037
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Fig. 4. REL patients show a unique NK phenotypic profile characterised by high NKp30 expression at baseline and during the early phases of treatment. Baseline data are shown in (A) (frequency) and (B) (MFI). (C) Representative histogram of NKp30 expression in an REL, SVR, and NR patient at baseline. Temporal trends of NKp30 expression, NKp30+ NK cell frequencies, and NKp46 expression in REL vs. SVR patients are shown in (D), (E), and (F), respectively.
NKp30-expressing NK cells, compared with SVR patients, persisted through week 12 (Fig. 4D and E) and was accompanied by a less durable but significant increase in NKp46-expressing NK cells (Fig. 4F). Persistent upregulation of NKp30 from baseline to week 12 appeared to be a unique feature of REL patients. Time trends of the different molecules analysed were similar to the other groups and are shown in Supplementary Fig. 7. Additional host and viral factors associated with response to SOC therapy As expected, genotype 2 infection was significantly associated with SVR (p = 0.004, by Fisher’s exact test) and 15 of 16 patients in this group were RVR. Moreover, infection with genotype 2 was associated with a significantly higher proportion of CD69+ NK cells (p = 0.005) during treatment. We also examined the prevalence of the single nucleotide polymorphism near the IL28B encoding gene, most commonly associated with response to SOC therapy (rs12979860) in chronic HCV infection [18], confirming that CC homozygosis was significantly associated with SVR (p = 0.005, by Fisher’s exact test) (Supplementary Table 1). There was no other association between host and viral factors (including sex, age, HCV genotype, and IL28B polymorphism) and NK phenotype and function by multivariate regression analysis.
Journal of Hepatology 2013 vol. xxx j xxx–xxx
Please cite this article in press as: Oliviero B et al. Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection. J Hepatol (2013), http://dx.doi.org/10.1016/j.jhep.2013.03.003
JOURNAL OF HEPATOLOGY Discussion In this study we performed an in-depth analysis of phenotypic and functional characteristics of NK cells in patients with chronic HCV infection treated with a standard combination of PegIFNa-2a and RBV, in order to identify immunological profiles associated with treatment outcome. The choice of studying innate immune responses originated from previous work which suggested that adaptive immunity does not appear to play a major role in treatment-induced viral eradication [19,20]. Moreover, because NK cells are rapidly activated by IFNa in vitro [10,21] and in vivo [13], we reasoned that their stimulation during treatment may contribute to HCV clearance. To this end, unselected consecutive patients were enrolled in a prospective fashion and changes in NK-cell phenotype and function analysed at several predefined time points during and after cessation of SOC treatment. With this approach, we showed that important NK phenotypic differences between SVR and NR were already present prior to treatment commencement. Indeed, NR patients were characterised by an enrichment in CD56bright and, conversely, by a lower frequency of CD56dim NK cells. These findings are in agreement with those of Bozzano and associates [22] who also found increased frequencies of CD56bright/CD16+/ NK cells in NR patients and increased frequencies of CD56dim/CD16+ NK cells in SVR patients. The CD56bright subset is thought to contain relatively immature NK cells [23,24] with prominent proliferative capacity and immunoregulatory function, as shown by their ability to secrete cytokines, together with relatively poor cytotoxic activity [25]. In contrast, the more differentiated CD56dim subset features a predominant cytolytic function [25]. This prospective study suggests that NK cytolytic function could be one mechanism of elimination of HCV-infected hepatocytes, which would be supported by in vitro studies showing that NK cells can kill HCV-infected hepatocytes via perforin/granzyme [26], as well as tumour necrosis factorrelated apoptosis-inducing ligand (TRAIL)-mediated mechanisms [21]. More importantly, we showed that the NK PFN content remained significantly higher until week 12 in patients developing SVR compared with NR patients, corroborating the hypothesis that an efficient NK cytolytic function is required for optimal clearance of HCV-infected cells, and suggesting that a reduced PFN content is a characteristic feature of patients not responding to IFN-based therapy. However, from our own and previous data obtained with CD8 T cells [17], it would appear that single-target cell killing is rather inefficient in preventing HCV infection, at least in vitro, suggesting that IFNc-mediated non-cytolytic mechanisms provide a stronger antiviral effect [27]. In line with this, it may be inferred that IFNc release would be a more powerful mechanism of protection from infection, whereas optimal NK killing activity would be required for IFNa-induced HCV clearance. It is noteworthy that a recently published study showed upregulation of several NK-cell activating receptors, including the CD69 early activation antigen, within hours from the first IFNa injection [13]. Although our protocol did not allow us to perform early liver biopsies nor we were permitted to recall patients for venepuncture before the second week on therapy, our data are compatible with those of Ahlenstiel [13], since the proportion of CD69+ cells was upregulated in all patients, early after initiation of treatment. However, while this activated NK phenotype persisted until treatment discontinuation in the subjects who achieved SVR, it was only transient and limited to week 2 on therapy in NR patients, suggesting that an early and sustained NK-cell
activation is of paramount importance for a successful treatment outcome. These findings are further corroborated by evidence that CD69 expression is statistically and consistently higher in RVR patients compared to the remainder. Interestingly, the frequency of CD56dim/CD16 NK cells, a reportedly activated NK subpopulation [28], was increased in the first 12 weeks of treatment in the SVR group. The role of CD56dim/CD16 NK cells is still poorly defined, although previous studies suggested that these cells represent the only subset able to degranulate when exposed to tumour targets, whereas CD56dim/CD16+ cells would be involved in antibody-dependent cytotoxicity [29]. Further studies actually showed that CD56dim/ CD16 NK cells are mature cytotoxic CD16+ cells that have downregulated CD16 following exposure to targets (16 and our own data not shown). In line with these findings, a progressive proportional increase of these cells in SVR patients would suggest CD16 downmodulation as a result of contact with, and subsequent successful killing of HCV-infected hepatocytes in patients eventually developing SVR. Progressive reduction of Siglec-7 expression to lower than baseline values appeared to be associated with antiviral treatment in all patient groups during and after discontinuation of treatment akin to Ahlenstiel’s study [13]. However, the observation that levels of NK Siglec-7 expression over time were statistically lower than baseline in SVR patients only, suggests that reduced expression of this inhibitory receptor [30,31] would contribute to maintain a stronger and sustained NK-cell activated status in this group. One important issue was to determine whether IL28B polymorphism was associated with specific NK profiles in this study. Indeed, it is possible that members of the IFNk family can modulate NK activity and that IL28B polymorphism is a marker for this interaction. Interestingly, it has recently been reported that the unfavourable rs12979860 IL28B TT homozygosis was associated with increased expression of the NKG2A inhibitory receptor and reduced expression of TRAIL on CD56dim NK cells [32], providing evidence in support of IL28B polymorphism regulating innate immune responses in HCV infection. As extensively reported in recent studies, reviewed in [33], IL28B CC homozygosis was significantly associated with SVR also in our series of patients, but no correlation was seen between NK-cell phenotype and function and IL28B polymorphism with respect to response to treatment. These findings are in agreement with those of a recent study, in which analysis of the same IL28B SNP was shown to be associated with the favourable CC SNP in early virological responders [13]. The difference in NK-cell activation between responders and non-reponders was maintained even when the analysis was restricted to CC homozygotes, suggesting that early NK activation during IFNa therapy is independent of IL28B polymorphism. This interpretation is further supported by our data confirming that IL28B polymorphism is associated with treatment outcome independently from the patients’ NK dynamic profile. REL patients exhibited a peculiar phenotypic pattern characterised by a consistently increased expression of NKp30 from baseline to week 12 on treatment and a transient (until week 4) increase of NKp46, which prompted us to analyse this small group separately. We have no simple explanation for such findings, but it is possible that NKp30, which correlates with cytotoxicity [34], plays a specific role in the relapse process, since upregulation is already present at baseline. Interestingly, lower frequencies of NKp30+ and NKp46+ NK cells have been recently
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Please cite this article in press as: Oliviero B et al. Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection. J Hepatol (2013), http://dx.doi.org/10.1016/j.jhep.2013.03.003
Research Article associated with the ability to clear HCV following acute infection [35], suggesting that NCR upregulation may reflect incomplete NK-cell activation in individuals who do not clear infection either spontaneously or following successful antiviral treatment. In summary, we have identified specific NK-cell phenotypic and functional profiles that may help distinguish patients according to treatment outcome. Early NK-cell activation and robust cytolytic function appear to be fundamentally important in the rapid IFNa-induced elimination of HCV-infected hepatocytes. Even though the standard of care for chronic HCV infection is rapidly changing, we submit that NK dynamic profiling could still provide valuable information on dual therapy outcome in this clinical setting.
Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Acknowledgements We thank Dr. E. Tagliabue, Molecular Targeting Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, for providing the BT474 HER2-positive breast cancer cell line.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2013.03. 003.
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Please cite this article in press as: Oliviero B et al. Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection. J Hepatol (2013), http://dx.doi.org/10.1016/j.jhep.2013.03.003
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Please cite this article in press as: Oliviero B et al. Natural killer cell dynamic profile is associated with treatment outcome in patients with chronic HCV infection. J Hepatol (2013), http://dx.doi.org/10.1016/j.jhep.2013.03.003