NK cells and their dysfunction have been implicated in all stages of HCV infection [15
]. Recently, aberrant expansion of functionally impaired CD56-negative NK cells has been demonstrated to be a harbinger of anti-HCV treatment failure [16
]. NK cells are known to become rapidly activated by cytokine stimulation. In order to test the premise that the nature and function of NK cells are associated with the antiviral effects of IFN-α, we used the well-characterized, longitudinal Virahep-C cohort [5
]. We found that functionally impaired CD56-negative NK cells circulated at higher levels in chronically infected HCV patients (compared to normals), correlated with poor early viral kinetics following initiation of antiviral therapy, and were highest in those resistant to treatment (Supplemental Figure 2
The dominant “default” feature of NK class I receptor-mediated function is inhibition [17
]. Although it is accepted that the balance of activating and inhibitory signals control NK cell activity, the relative importance of these signals in mediating antiviral efficacy to IFN in vivo
had not been defined. We compared the expression of these markers as a predictor of early virologic response, i.e., within 28 days following initiation of combination therapy, and found pre-treatment differences in multiple inhibitory and activating receptors. Moreover, by univariate analysis, we found pre-treatment levels of stimulatory receptors NKRp44, TRAIL, CD161 were positively associated with SVR, whereas the expression levels of inhibitory receptors NKG2A, CD158e (KIR3DL1) and CD158b (KIR2DL2/DL3) were negatively correlated with SVR. By multivariate modeling, the expression of two inhibitory receptors (NKG2A and CD158e) was predictive of treatment failure. The robustness of the model ROC indicates that it provides useful discriminative clinical information.
A recent study found an association between the IL-28B SNP and an inhibitory KIR gene, KIR2DL3 [18
]. In the current report, we found that expression levels of another inhibitory receptor NKG2A were higher in patients homozygous for the poor response T allele of the IL-28B (IFN-λ 3) SNP. Little is known about the interaction between type I and type III IFNs; they utilize unique signaling receptors but share common downstream signaling [19
]. We examined potential mechanisms whereby NKG2A expression on NK cells might contribute to the differential response of IFN-α -based therapy. We provide experimental evidence that the surface expression of NKG2A demarcate significant intracellular gene expression differences. Using the Virahep-C cohort, Taylor and colleagues had previously demonstrated blunted transcription of ISGs in whole PBMC of poor-response patients [20
]. The importance of examining the cell of origin with regards to ISG expression was underscored recently by the finding that ISG up-regulation was more pronounced in hepatocytes in nonresponders to IFN therapy, but in Kupffer cells in responders [21
]. Reduced expression of IFN-α receptor on NKG2A-positive NK cells compared to their NKG2A negative counterparts suggests one mechanism contributing to the differential response of IFN-α -based therapy.
Higher serum and hepatic levels of interferon-γ–inducible protein-10 (IP-10), a CXC chemokine (CXCL10), have been associated with nonresponse to HCV therapy [22
]. However, the fold induction in IP-10 has been associated with early viral kinetics in patients who are IFN-responders [24
], as well as SVR [25
]. Our data indicate that NK cells are a source of IP-10 following stimulation with IFN-α. Indeed, NK cells depleted of NKG2A-positive NK cells produced significantly greater IP-10. Prior studies have demonstrated that antiviral effector cells, including CTLs and NK, express CXCR3 and are responsive to IP-10 in chemotaxis [26
]; further work is warranted to examine the role of NKG2A expression on chemotaxis within the hepatic compartment.
We found statistically higher pretreatment levels TRAIL on total NK cells in patients who responded to treatment as compared to nonresponders. Recent reports demonstrated that 4 hrs after IFN-α treatment, TRAIL (TNFSF10
) mRNA is markedly upregulated in liver biopsies from HCV patients, and on circulating NK cells correlated inversely with serum HCV-RNA levels [13
]. Moreover, in the latter study, HCV recovered patients had superior up-regulation of TRAIL after in vitro
IFN-α stimulation than non-responder patients. In keeping with these results, we found that PEG IFN-α upregulated expression of TRAIL on NK cells, but that NKG2A expression attenuated this effect.
Taken together, our data indicate that NKG2A-positive cells are increased in frequency in patients with chronic HCV infection who are more likely to fail treatment and demonstrate impaired response to exogenous PEG IFN-α. As a single variable, when NKG2A expression on lymphocytes was greater than 8.0%, the sensitivity and specificity for futility (lack of SVR) was 71.7% and 75.6%, respectively; the ROC curve (c-statistic of 0.80) is shown in supplemental Figure 3
. Because PEG-IFN-α and ribavirin is likely to remain the backbone of antiviral therapy for the foreseeable future, our results may affect how clinicians use direct antiviral agents such as protease or polymerase inhibitors. For example, for patients with NK receptor expression associated with poor early viral kinetics, response to IFN-α therapy is slower and thus are, in theory, more likely to develop resistance to direct antiviral agents [12
]. Furthermore, IL-28B genotype has now been incorporated into treatment algorithms to predict patients' responses to anti-HCV therapy; however, because of low negative predictive value for SVR, IL-28B genotype cannot be used to determine whether to withhold treatment in an individual patient [12
]. Our results indicate that a simple model incorporating inhibitory NK receptor expression levels can accurately discern likelihood of SVR across different IL-28B genotypes. These results have implications for improving prediction of treatment response and development of new approaches that target NK inhibitory pathways.