This study shows that IFN-α-induced modulation of STAT1 phosphorylation underlies the in vivo
polarization of NK cells towards increased cytotoxicity and decreased IFN-γ production. This result is consistent with the observation that LCMV-induced IFN-α secretion in mice has been shown to increase STAT1 expression in NK cells resulting in preferential STAT1 over STAT4 phosphorylation (6
). It also extends the findings by Miyagi et al. on preferential STAT1 phosphorylation in HCV-infected patients (11
) because we show that IFN-α exposure in vivo
results in increased pSTAT1 levels and that it correlates to increased TRAIL production and degranulation and decreased IFN-γ production (). The clinical relevance of IFN-α signaling in NK cells is suggested by our observation that NK cell responsiveness and refractoriness correlate with the first phase virological response to IFN-α-based therapy ().
This analysis of NK cells is relevant for current research on biomarkers predicting interferon responsiveness and treatment outcome. Advantages of using NK cells as biomarkers of IFN responsiveness are that they are readily accessible from the peripheral blood and that both in vivo
and in vitro
NK cell responsiveness can easily be assessed in a short, standardized flow cytometry-based assay by checking pSTAT1 levels. How does our system compare to other biomarkers of IFN-responsiveness? A well-established biomarker for interferon responsiveness is the intrahepatic expression of interferon-stimulated genes (ISGs). Typically, ISGs are most highly expressed pretreatment in HCV-infected patients who respond poorly to IFN-α-based therapy (17
). As potential explanation it has been proposed that high baseline activation of the endogenous interferon system does not allow a further increase of ISGs during interferon-based therapy, possibly because the ISG response has already reached maximal levels and/or inhibitory autocrine feedback mechanisms have been induced (18
). In contrast to these ISG data, we did not find any evidence that pretreatment pSTAT1 levels or in vitro
inducibility differed among HCV-infected patients (data not shown). Thus, pSTAT1 induction is an independent measure for IFN responsiveness and may complement ISG analysis.
How does the NK cell response correlate to the treatment response? Because all patients in our study achieved an early virological response to PegIFN/RBV therapy at week 12 we were not able to assess NK cell responses in the context of the ultimate treatment outcome. On the other hand, we believe that late time points of PegIFN/RBV therapy are less relevant for our study because NK cells exhibited their greatest response within the first days of therapy in parallel to the first phase virological response (–). Our data clearly indicate that near-maximal NK cell activation can be reached within hours of the first injection of PegIFN because the response to additional in vitro
stimulation with IFN-α was significantly reduced at later treatment time points (). Here, we made the interesting observation that NK cells from patients with a weak first phase decline in HCV titer, who displayed lower levels of in vivo
pSTAT1 induction than patients with a strong first phase decline in HCV titer, nevertheless retained responsiveness to in vitro
stimulation with IFN-α. Thus, both patient groups differed in their in vivo
responsiveness to IFN-based therapy, but not in their overall response to IFN-α (). These results suggest that NK cell responsiveness depends to a certain extent on the environment. One explanation is that in vivo
levels and pharmacokinetics of IFN differ among patients. Another possible explanation is that certain factors, such as suppressive cytokines, interfere with the responsiveness of NK cells to PegIFN therapy in vivo
, and that these are overcome once NK cells are stimulated with high doses of IFN-α in vitro
. However, removal of inhibitory factors can be excluded, because the in vitro
NK cell stimulation was performed in whole blood. A third possibility is that genetic determinants, such as IL28B SNP at rs12979860
) and KIR/HLA compound genotype (19
) cannot completely be ruled out due to the small size of the analyzed patient cohort (, ). However, if rs12979860
SNPs play a role, it would be an indirect rather than direct effect on NK cells because NK cells retain their responsiveness to in vitro
stimulation with IFN-α () and because they do not respond directly to type III IFN including IL28B (20
). Thus our study opens the interesting possibility that in vivo
responsiveness to IFN-α-based therapy may be improved.
Another relevant result of this study was the observed refractoriness of NK cells to in vitro IFN-α stimulation, which occurred in all patients within the first week of IFN-α-based therapy and was maintained for the entire study (). NK cells were not only refractory to in vitro IFN-α stimulation but exhibited refractoriness in vivo as shown in the patients who consented to a blood draw prior to and 6h after the week 12 PegIFN injection and did not exhibit an increase in vivo pSTAT1 levels during this period (). This refractoriness to STAT1 phosphorylation striking because STAT1 levels continued to increase while pSTAT1 levels declined in NK cells. There are at least three possible explanations:
First, the half-life time of STAT1 is longer than that of pSTAT1, because STAT1 has been shown to persist for many days in response to IFNs whereas pSTAT1 levels decrease by SHP1, SHP2 and SOCS1-dependent negative regulation and tyrosine phosphatase-mediated dephosphorylation. Second, the accumulated unphosphorylated STAT1 itself is able to induce the expression of a subset of ISGs such as 2’5’ OAS, Mx1 and STAT1 creating a pSTAT1-independent positive feed-back loop (21
). Third, STAT1 can also be induced independently from signaling via the IFN-alpha/beta receptor in NK cells as suggested by the observation that NK cells from STAT1-deficient mice show a greater level of impairment in cytotoxicity and ability to reject transplanted tumors than NK cells from mice that lack IFN receptors (22
Collectively, the results suggest that continued exposure to high levels of IFN-α may reign in the NK cell response to prevent collateral damage. Similar mechanisms may be operative in acute HCV infection, which is known to induce high levels of type I IFN-induced genes without evidence of significant liver injury throughout the incubation phase of 1–2 months (23
). Thus IFN-α-induced NK cell refractoriness may contribute to the often observed, but in its mechanisms not yet understood, clinically asymptomatic nature of acute HCV infection.