Natural killer (NK) cells are lymphocytes that play important roles in the host defense against hepatitis C virus (HCV) infection. Killer cell immunoglobulin-like receptors (KIRs) are a group of regulatory molecules expressed on NK cells and a subset of T cells (Parham 2005). Ligands for KIRs are human leukocyte antigen (HLA) class Ⅰ molecules, and HLA-C1 is a ligand for the inhibitory receptors KIR2DL2, KIR2DL3 and the activating receptor KIR2DS2 (Robinson et al. 2003; Du et al. 2007). In 2002, the National Institutes of Health Consensus Development Conference concluded that a combination therapy of pegylated alpha interferon (PEG-IFN) with ribavirin (RBV) manages HCV infections effectively (Gebo and Bartlett 2002). Before the direct-acting antiviral agent treatment was approved, PEG-IFN and RBV were the main antiviral treatments for chronic HCV in China (Chinese Society of Hepatology et al. 2015). Patients that receive the same standard combination therapies are classified as non-responders (NR) and sustained virological responders (SVR) according to their responses to the treatment by detecting HCV RNA 24 weeks after treatment (Asselah 2012). The NR do not mount a sufficient anti-HCV response, which is defined by a consistent positive viral load during treatment, at its end, or at 24-weeks posttreatment. The SVR are defined by consistent undetectable HCV RNA levels in serum at 24-weeks posttreatment (Fried et al. 2002).
To investigate the association of the KIR frequencies with HCV infection and therapy responses in Chinese Han population, we recruited 333 patients infected with HCV- 1b and 320 healthy individuals in Hubei Province between October 2010 and August 2012. In 333 HCV patients, 98 treated by PEG-IFN and RBV were successfully tracked and divided into NR and SVR groups, which were detected with Real-Time Quantitative PCR Detection System and ELISA (Kehua Bio-Engineering Co, Shanghai, China). All subjects were negative from other disorders, such as infection with hepatitis B virus, hepatitis D virus, and HIV, which were detected by quantitative PCR and ELISA, as well as diabetes, malignant tumor, or any autoimmune diseases.
We extracted the genomic DNA from the blood of donors with a SE Blood DNA kit (Omega Bio-Tek Inc, Norcross, GA, USA), and the KIR alleles were determined by the sequence-specific PCR primers (Bunce et al. 1995; Hsu et al. 2002). The results were analyzed with a Chi square test based on P values and odds ratios (OR). The gene frequencies of KIR2DL2 and KIR2DS3 were significantly higher in HCV patients (Supplementary Table S1, Table 1) (P < 0.01), and only the frequency of KIR2DL2 was significantly higher in the SVR group (Supplementary Table S2). The gene frequency of full-length KIR2DS4 was significantly lower in HCV patients and the SVR group. HLA-C1, as a ligand, is necessary for the function of KIR2DL2 (Bashirova et al. 2006; Du et al. 2007).
Table Table S1. Frequency of KIR genes in HCV and healthy patients
Table 1. Clinical characteristics and occurrence of KIR2DL2 and HLA-C1 in HCV and healthy patients.
Table Table S2. Frequency of KIR genes in NR and SVR HCV patients
Therefore, we analyzed the association among KIR2DL2, its ligand HLA-C1, HCV-1b infection, and treatment response. As shown in Table 1, although the frequency of HLA-C1 was not different compared with healthy controls, both KIR2DL2 and HLA-C1 positive (KIR2DL2+/C1- pairs) showed a risk association with HCV-1b infection. As shown in Table 2, in contrast to the SVR group, the gene frequency of KIR2DL2 was significantly higher in the NR group, whereas the frequency of HLA-C1 was not different. The joint analysis revealed that the frequency of KIR2DL2+/C1- pairs in the NR group was significantly higher than the SVR group (P < 0.05), and the KIR2DL2+/C1- pairs showed no difference. This suggests that the patients carrying KIR2DL2/C1 genes have a high risk of being infected by HCV-1b and they are not beneficial for HCV treatment. But 186 of the 333 HCV patients had the KIR2DL2 gene, of which 180 had HLA-C1 group genes and 6 did not. Ninety-four of the 320 healthy control participants had the KIR2DL2 gene, of which 84 had the HLA-C1 gene and 10 did not. There was no statistical difference in the P values and OR values of KIR2DL2+/C1- between HCV patients and the healthy controls.
Table 2. Clinical characteristics and occurrence of KIR2DL2 and HLA-C1 in NR and SVR HCV patients.
We further performed meta-analysis to assess the association between KIR2DL2 and the response to therapy. Twelve studies were identified through databases searching (e.g., PubMed, Science Direct) for all case-control studies evaluating KIRs and HCV-1b treatment in humans (up to April 2017). Studies were further selected if they fulfilled the following criteria: (1) have a NR-SVR design, used the same therapy strategy, reported the KIR2DL2 genotype frequencies, and confirmed that the recruited HCV patients had no other disease; (2) supplied sufficient information to calculate the OR in a peer-reviewed journal. Finally, five articles covering populations in three countries (Brazil, Spain, Ireland) were eligible for meta-analysis (Carneiro et al. 2010; Vidal-Castineira et al. 2010; Dring et al. 2011; de Vasconcelos et al. 2013; Vidal-Castineira et al. 2014). A significant association of the KIR2DL2 gene with the response to therapy was detected under the fixed effects model, including 934 NRs and 732 SVRs (shown in Supplementary Table S3 and Fig. 1). The aggregated OR was 1.41 (P < 0.01, 95% CI = 1.05–1.88), and the heterogeneity was moderate (P = 0.16, I2 = 39%). The publication bias of the literature was estimated by funnel plots (shown in Supplementary Figure S1). The shapes of funnel plots appeared symmetrical for NR versus SVR in the studies. Together with our data, KIR2DL2 is not beneficial for HCV therapy in multiple populations. However, none of the above papers discussed the relationship between the KIR2DL2/C1 pair and HCV patients.
Table Table S3. Meta-analysis of the occurrence of KIR2DL2 in NR and SVR HCV patients
Figure 1. Forest plot of the meta-analysis for KIR2DL2 in NR and SVR HCV patients under the fixed effects model.
Figure Supplementary Figure S1. Funnel plot analysis to detect publication bias using Egger's linear regression test
This study displayed a significantly higher frequency of the KIR2DL2 gene among patients infected with HCV-1b compared with healthy controls in a Chinese Han population. As previously mentioned, the frequency of the KIR2DL2 gene in this Chinese Han population was 10%– 30% and is present in 40%–60% of Caucasians (Single et al. 2007). In this study, the KIR2DL2 gene frequency in Chinese Han HCV patients (55.86%) is similar to that in Caucasian patients, which also implies that KIR2DL2 is a risk factor for HCV-1b infection in the Chinese Han population.
KIR2DL2 is an inhibitory receptor on the surface of NK cells, which can transmit inhibitory signals to the cell, but all these require the involvement of HLA-C1. In the early stages of virus infection and development, the elimination of viruses in the body also depends on innate immunity, including NK cells. The killing ability of NK cells carrying KIR2DL2 may be impaired, which has also been verified in studies on KIR2DL2 and HIV (Zwolinska et al. 2016). In this study, one possibility is that the inhibition of virus infection mediated by KIR2DL2 can overcome the activation of NK cells by IFN-alpha and decrease the antiviral ability of NK cells.
In conclusion, KIR2DL2/C1 is a risk factor of HCV-1b infection and is associated with nonresponse to PEG-IFN and RBV combination therapy in Chinese Han patients. These findings might contribute to our understanding of the pathogenic mechanisms of HCV infection and to the development of more efficient therapeutic strategies for HCV that consider host genetic factors.
We thank all subjects who donated samples for this study. This work was supported by Grants from the National Major Science and Technology Project for Infectious Diseases of China (2012ZX10004503), and Major State Basic Research Development Program of China (973 Program; No. 2013CB530505).
KIR2DL2/C1 is a Risk Factor for Chronic Infection and Associated with Non-response to PEG-IFN and RBV Combination Therapy in Hepatitis C Virus Genotype 1b Patients in China
- Received Date: 27 January 2018
- Accepted Date: 14 June 2018
- Published Date: 23 July 2018
Abstract: Natural killer (NK) cells play an important role in innate immunity to hepatitis C virus (HCV), especially in response to HCV infections. Killer cell immunoglobulin-like receptors (KIRs) are the primary receptors of NK cells that regulate NK cell behavior to antivirus infection response. Our aim was to determine the association of KIR frequencies with HCV infection and therapy responses in a Chinese Han population, and we compared that of 333 patients infected with HCV-1b and 320 healthy controls, as well as 36 non-responders (NR) and 62 sustained virological responders (SVR) treated with a combination therapy of pegylated alpha interferon and ribavirin. It was found that the frequency of the KIR2DL2 gene was higher in patients infected with HCV than that in controls. Additionally, a higher frequency of the KIR2DL2 gene was observed in NRs compared with SVRs, which was also supported by the results of a meta-analysis. Moreover, in a Chinese Han population, we found that KIR2DL2 in combination with its ligand HLA-C1 is a risk factor for infection and is not useful for HCV therapy; also, the KIR2DL2 gene alone is a risk factor for the development of infection. Results suggested that KIR2DL2 downregulates the killing ability of NK cells in the clearance of HCV.