To investigate how natural killer (NK) cells are affected in the elimination of hepatitis C virus (HCV) by sofosbuvir/ledipasvir, two highly effective direct-acting antivirals (DAAs).

Thirteen treatment-naïve and treatment-experienced chronic hepatitis C (CHC) patients were treated with sofosbuvir/ledipasvir, and NK cells were detected at baseline, weeks 2, 4, 8 and 12 during therapy, and week post of treatment (Pt)-12 and 24 after the end of therapy by multicolor flow cytometry and compared with those from 13 healthy controls.

All patients achieved sustained virological response. There was a significant decline in CD56^bright NK cell frequencies at week 8 (P = 0.002) and week 12 (P = 0.003), which were altered to the level comparable to healthy controls at week Pt-12, but no difference was observed in the frequency of CD56^dim NK cells. Compared with healthy controls, the expression levels of NKG2A, NKp30 and CD94 on NK cells from CHC patients at baseline were higher. NKG2A, NKp30 and CD94 started to recover at week 12 and reached the levels similar to those of healthy controls at week Pt-12 or Pt-24. Before treatment, patients have higher interferon (IFN)-γ and perforin levels than healthy controls, and IFN-γ started to recover at week 8 and reached the normalized level at week Pt-12.

NK cells of CHC patients can be affected by DAAs, and phenotypes and function of NK cells recover not at early stage but mainly after the end of sofosbuvir/ledipasvir treatment.

Chronic hepatitis C virus (HCV) infection is a disease that affects about 71 million people worldwide[1]. It can lead to mortality from hepatic as well as extra-hepatic causes[2]. In the era of HCV treatment with pegylated-interferon (PEG-IFN)/ribavirin (RBV), the sustained virological response (SVR) rate is different according to HCV genotypes: Approximately 40% to 50% in patients with genotype 1 and 80% in patients with genotype 2 or 3[3-5]. However, because of their higher SVR rate (> 95%) and less toxicity[6], IFN-free direct antiviral agents (DAAs) have replaced PEG-IFN/RBV as a first-line treatment option recommended by international guidelines[7,8].

IFN-α can induce immunomodulatory effects by acting the innate and adaptive immune systems of various cells[9,10]. Since IFN-free DAAs take the virus life cycle as a target, they can inhibit NS3 protease, NS5A replication complex or NS5B polymerase activity specifically[11]. These regimens can help us clarify the interaction between HCV clearance and the innate immune response, regardless of the IFNα induced immune modulation[12]. This provides a unique opportunity to analyze whether DAAs can change natural killer (NK) cell activation when HCV replication is inhibited. Previous studies have explored the effect of IFN therapy on the NK cells. It has been shown that chronic hepatitis C (CHC) patients with SVR to IFN therapy exhibited greater levels of NK cell degranulation and enhanced NK cytotoxicity[13,14]. Combination therapy of PEG-IFN-α/RBV reversed NK subtype distribution and function in HCV-eliminated patients[15]. Until now, only few studies have reported the effect of DAAs on NK cells[16-18], in which the results and conclusions were controversial.

NK cells are enriched among lymphocytes in the blood (5%-20%), and their percentage increases further in viral hepatitis[19]. NK cells play an important role in the antiviral immune defense and undergo great changes in subsets, phenotypes and function during persistent viral infection[20]. NK cells can be divided into three subgroups according to the expression levels of CD56 and CD16, including CD56^bright NK cells, CD56^dim NK cells and CD56^neg (CD16^positive) NK cells[21]. CD56^bright NK cells, which can produce IFN-γ mainly and inhibit viral replication, are the less-mature subset that can differentiate into CD56^dim NK cells[22]. CD56^dim NK cells are cytotoxic NK cell subset expressing higher levels of killer immunoglobulin-like receptors (KIR), CD16 and perforin[21]. CD56^neg NK cells express lower perforin and exhibit lower cytotoxicity[23].

The function of NK cells is regulated by interaction of NK cell receptors (NKRs), which can be divided into activating and inhibitory NKRs, and their respective ligands[24]. Activating NKRs include NKG2C, NKG2D and the “natural cytotoxicity receptors”, for example, NKp30, NKp44 and NKp46. Inhibitory receptors comprise NKG2A and the KIR family members[25]. During viral infection, the balance shifts from inhibition to activation because the threshold value of activation receptors exceeds that of inhibition[26]. For example, the integration of all signals results in activation of blood and liver NK cells in HCV infection[27] and altered functional phenotypes with increased cytotoxicity and decreased antiviral cytokine production[27,28].

Up to now, more and more DAAs have been approved for clinical practice. In China, sofosbuvir/ledipasvir have been increasingly used among CHC patients, especially those with genotype 1 HCV infection. Sofosbuvir is an NS5B polymerase inhibitor and ledipasvir is an NS5A replication complex inhibitor. In the current study, we aimed to observe the dynamic changes of NK cell subsets, phenotypes and functional parameters during and after DAAs treatment, and to investigate the effect of DAAs (sofosbuvir/ledipasvir) treatment on innate immunity in genotype 1b HCV-infected patients.

In our study, we evaluated the innate immune effects in 13 CHC patients successfully treated with sofosbuvir/ledipasvir. All patients achieved SVR12 and SVR24, and no patients had virologic breakthrough.

The development of DAAs has opened a new era of HCV treatment[29]. IFN-free regimens for HCV infection provide a unique opportunity to study the interaction between HCV and the immune system because DAAs rapidly decrease viremia to undetectable levels regardless of the IFNα induced immune modulation[12]. Due to the direct effect of IFN-α on NK cells, the consequence of viral load decline on NK cells could not be examined precisely. Our study examined the innate immune effects of sofosbuvir/ledipasvir therapy induced HCV RNA level decline. There was no difference in the frequency of total NK cells from CHC patients at baseline and healthy controls, which is inconsistent with other two studies assuming that NK cell frequency decreases in the blood in chronic HCV infection[28,30]. We found that only CD56^bright NK cells showed a slightly decline at week 8 and week 12 during DAAs treatment and normalized at week Pt-12. There was no change in total NK cells or CD56^dim NK cells. However, another study reported that DAA therapy enhances the frequency of CD56^dim and decreases CD56^bright cells in chronically HCV-infected patients[30].

As for NK cell phenotypes and function of CHC patients, we showed that reduced HCV RNA load altered NK cell phenotype and function, including NKG2A, CD94, NKp30, NKp46, IFN-γ and perforin. Most of these phenotypes and cytokines became to change at week 12 approximately and normalized to the levels of healthy controls at week Pt-12 or Pt-24, which are different from previous studies[17,30]. Serti et al[17] demonstrated that the expression levels of NKp46 and NKG2A normalized in patients with undetectable viremia by week 8 in daclatasvir (DCV) and asunaprevir (ASV) therapy, and they assumed that the percentage of IFN-γ producing NK cells and the IFN-γ expression level were significantly lower in chronically HCV-infected patients compared with healthy controls and increased within the first 8 wk. Spaan et al[30] illustrated that NK cell phenotype is already normalized at week 12 during DCV/ASV therapy, and they assumed that DAAs treatment did not alter the frequency of NK cells producing perforin. However, we found that the MFI of perforin⁺ NK cells was higher than that of healthy controls before, during and after DAAs therapy persistently, which has not been found in previous studies. Importantly, researchers in these two studies have not demonstrated the changes of NK cells after the end of DAAs therapy.

Patients in other two studies were treated with DCV/ASV and all patients were HCV-infected non-responders to previous PEG-IFN/RBV therapy[17,30]. However, patients in our study were treatment-naïve CHC patients and treatment-experienced CHC patients who had a relapse after PEG-IFN/RBV therapy. NK cells might be affected by PEG-IFN/RBV therapy[13,14]. Combination therapy of PEG-IFN-α/RBV reversed NK subtype distribution and functions in HCV-eliminated patients[15]. However, we found that there was no difference in the changes of the NK cells during and after DAAs treatment between patients who were treatment-naïve and those who had a relapse after PEG-IFN/RBV therapy. And the effect of non-response to PEG-IFN/RBV treatment on the changes of NK cells during and after DAA treatment should not be excluded. Because it is unclear whether the response pattern to PEG-IFN/RBV treatment can affect the subsequent changes of NK cells induced by DAAs in treatment-experienced patients. Second, HCV genotype may affect dynamic changes of NK cells during DAAs therapy. In a study of Ning et al[31] in which patients with different genotypes were included, they assumed that different HCV genotypes may have an impact on their results. After treatment with sofosbuvir/ledipasvir or sofosbuvir/daclatasvir, the frequency of CD16⁺CD56⁺ NK cells gradually increased to normal levels of healthy controls at week 12. Third, different DAAs may have different impact on NK cells. Sofosbuvir is an NS5B polymerase inhibitor, ledipasvir and DCV are NS5A replication complex inhibitor and ASV is an NS3 protease inhibitor. As Ning et al[31] described, we cannot exclude the possibility that different DAA regimens induce different dynamic changes of NK cells. Fourth, the race of CHC patients may be an important factor. Whether in era of PEG-IFN/RBV or DAAs treatment, race is one of the most important factors which can affect SVR[32,33]. Therefore, we cannot rule out the distinction induced by different races between our studies and others. Additionally, we assumed that it may take a long term for NK cell recovery and NK cells cannot recover immediately after the influence from DAAs was relieved.

We confirm earlier studies on increased inhibitory NKG2A expression on NK cells in HCV infection[18,27]. NKG2A is a major and prominent inhibitory NK cell receptor and is known as lectin superfamily group A[34]. DAA-induced NKG2A level reduction might be the consequence of compensatory mechanisms exerted upon declining activating signals. Besides, CD94 is mainly expressed as a heterodimer with NKG2A, NKG2B or NKG2C protein[35]. The inhibitory signals of NK cells are mainly mediated by HLA class I-binding receptors, including KIRs and CD94/NKG2A[36]. Accordingly, there are something in common of the changing trends between NKG2A and CD94 MFI in our study.

Frequency of NKp30⁺ NK cells at baseline in our cohort was higher than those of healthy controls. During DAAs treatment, the percentage of NKp30⁺ NK cells was reduced to the level of healthy controls, which is consistent with the studies of Serti et al[17] and Spaan et al[30], but there was no difference in the MFI of NKp30.

In all changed phenotypes, NKp46 is a special one. The changing trend of NKp46 is completely different from that of other receptors during sofosbuvir/ledipasvir treatment. There was a significant decline only at week 8 and week 12. As we all know, NKp46, a member of the natural cytotoxicity receptor family, is a main activating NK-cell receptor[37]. NK cells with high expression of NKp46 were characterized by a high functional capacity (e.g., high cytotoxicity and IFN-γ production) and a high antiviral activity in vitro[38,39]. However, the percentage of IFN-γ⁺ NK cells was similar to the level of healthy controls at week Pt-12, and this level was maintained until week Pt-24.

The effect of HCV infection on NKG2D was controversial. Oliviero et al[28] reported that CHC patients have increased NKG2D expression, while Dessouki et al[15] demonstrated that the frequency of NKG2D⁺ NK cells decreased in HCV infection. In our study, the expression of NKp44, NKG2C, NKG2D and Granzyme B did not differ between NK cells from CHC patients at baseline and healthy donors in our study, and there was no change in the expression during and after the end of treatment (EOT) of DAAs, which is consistent with the published articles[19,40].

Our results showed that NK cells phenotypes and function started to change in the later period of sofosbuvir/ledipasvir treatment and reversed to the normalized level of healthy individuals mainly after EOT. What we found in our research is different from previous studies which assumed that HCV clearance induced by DAAs can mediate NK recovery rapidly. Whether dynamic changes of NK cells in DAA-treated patients are related to HCV reinfection or liver carcinogenesis after HCV elimination is a great topic in the future.