Early changes in natural killer cell function indicate virologic response to interferon therapy for hepatitis C

Abstract

Background & aims: Mathematical modeling of hepatitis C virus (HCV) kinetics indicated that cellular immune responses contribute to interferon (IFN)-induced clearance of HCV. We investigated a potential role of natural killer (NK) cells in this process.

Methods: Phenotype and function of blood and liver NK cells were studied during the first 12 weeks of treatment with pegylated IFN-alfa and ribavirin, the time period used to define the early virological response.

Results: Within hours of treatment initiation, NK cells of patients that had an early virological response increased expression of activating receptors NKG2D, NKp30, and CD16 and decreased expression of NKG2C and 2B4, along with inhibitory receptors SIGLEC7 and NKG2A, resulting in NK cell activation. NK cell cytotoxicity, measured by degranulation and tumor necrosis factor-related apoptosis-inducing ligand production, peaked after 24 hours (P<.01), concomitant with an increase in alanine aminotransferase levels (P<.05), whereas IFN-γ production decreased within 6 hours and did not recover for more than 4 weeks (P<.05). NK cells from liver biopsies taken 6 hours after treatment initiation had increased numbers of cytotoxic CD16+NK cells (P<.05) and a trend toward increased production of tumor necrosis factor-related apoptosis-inducing ligand. Degranulation of peripheral blood NK cells correlated with treatment-induced, first-phase decreases in viral load (P<.05) and remained higher in early virological responders than in nonresponders for weeks.

Conclusions: IFN activates NK cells early after treatment is initiated. Their cytotoxic function, in particular, is strongly induced, which correlates to virologic response. Therefore, NK cell activation indicates responsiveness to IFN-α-based treatment and suggests the involvement of the innate immune cells in viral clearance.

Figure 1. Frequency of NK cells and their subsets during PegIFN/RBV therapy

(A) Gating strategy. (B–D) Frequency of CD56+CD3− NK cells (B) and their CD56^bright (C) and CD56^dim subsets (D) in PBMCs during PegIFN/RBV therapy. Mean ± SEM are shown (n=22 patients). h, hour; wk, week. (***) P<0.001 with repeated measures ANOVA; *P≤0.05, ** P<0.01 with paired Student t-test comparing the indicated individual time points to the 0 h time point.

Figure 2. Phenotype of peripheral blood NK cells during PegIFN/RBV therapy

(A–E) MFI of NKG2D (A), NKp30 (B), NKG2C (C), SIGLEC7 (D) and CD69 expression (E) on CD56+CD3− NK cells. (F) Frequency of CXCR3+ cells in the CD56+CD3− NK cell population. Mean ± SEM are shown (n=22 patients). h, hour; wk, week. (***) P<0.001 with repeated measures ANOVA; *P≤0.05, *** P<0.001 with paired Student t-test comparing the indicated individual time points to the 0 h time point.

Figure 3. PegIFN/RBV therapy polarizes NK cell function towards increased degranulation and TRAIL production and decreased IFN-γ production

(A–C) Changes in NK cell CD107a (A), TRAIL (B) and IFN-γ expression (C) during PegIFN/RBV therapy. Mean ± SEM of NK cell frequency and MFI are shown in 21, 20 and 22 patients, respectively. Statistical analysis: paired Student t-test comparing the indicated individual time points to 0 h. h, hour; wk, week. *P≤0.05, ** P<0.01, *** P<0.001.

Figure 4. Changes in the intrahepatic NK cell subset during the early phase of PegIFN/RBV therapy

(A) The size of the CD16+ NK cell subset, the percentage of TRAIL-expressing NK cells and the TRAIL MFI in a cross-sectional analysis of patients who were randomized to undergo liver biopsies either immediately prior to (n=19), or 6 h after (n=9) initiation of PegIFN/RBV therapy. Lines indicate the median. (B) Relative serum ALT levels during PegIFN/RBV therapy. Statistical analysis: Wilcoxon matched paired test comparing the individual time points to 0 h. Mean ± SEM are shown; h, hour; wk, week.

Figure 5. Degranulation of peripheral blood NK cells correlates to treatment response

(A–B) Correlation between the 24 h and 48 h change in the CD107a expression on either all blood NK cells (A) or their CD56^dim subset (B) and the first phase virological response (log decline in HCV titer during the first 48 h of PegIFN/RBV therapy). (C) Comparison between CD107a expression on CD56^dim NK cells of responders (filled circles, n=10) and nonresponders (open circles, n=15). Mean ± SEM are shown; h, hour; wk, week. The p-value was calculated by ANOVA.