Recovery of natural killer cells is mainly in post-treatment period in chronic hepatitis C patients treated with sofosbuvir plus ledipasvir

Abstract

Aim: 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).

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Direct-acting antivirals therapy; Hepatitis C virus; Natural killer cells; Natural killer subsets.

Figure 1

Serum hepatitis C virus RNA levels and liver inflammation decrease rapidly with sofosbuvir and ledipasvir therapy. A: Serum hepatitis C virus RNA levels of patients who all responded to therapy (n = 13). A response to sofosbuvir and ledipasvir therapy was defined as undetectable viremia at end of treatment (week 24); (B) Serum alanine aminotransferase. (C) Serum aspartate aminotransferase. HCV: Hepatitis C virus; Lloq: Lower limit of quantitation; Tnd: Target not detected; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase.

Figure 2

Flow cytometry results. A: Isolation of human peripheral blood natural killer (NK) cells and subsets; B: The expression of NKp46, NKp30, NKG2D, CD94, NKG2C and NKG2A during and after the end of direct-acting antivirals (DAAs) treatment; C: The expression of Granzyme B, IFN-γ and perforin during and after the end of DAAs treatment. NK: Natural killer; IFN: Interferon; DAAs: Direct-acting antivirals.

Figure 3

Effect of sofosbuvir and ledipasvir therapy on the frequencies of natural killer cell subsets from chronic hepatitis C patients. A: Frequencies of CD56⁺ natural killer (NK) cells in peripheral blood mononuclear cells (PBMCs); B: Frequencies of CD56^bright NK cells in PBMCs; C: Frequencies of CD56^dim NK cells in PBMCs. ^aP < 0.05 and ^bP ≤ 0.01 and ^cP ≤ 0.001, different time points of CHC patients vs healthy controls; ^dP < 0.05 and ^eP ≤ 0.01 and ^fP ≤ 0.001, different time points of CHC patients (2 wk, 4 wk, 8 wk, 12 wk, Pt-12 wk, Pt-24 wk vs 0 wk). NK: Natural killer; CHC: Chronic hepatitis C; Ctrl: Healthy controls; Pt: Post of treatment; PBMCs: Peripheral blood mononuclear cells.

Figure 4

Sofosbuvir and ledipasvir therapy modulates the expression of NKG2A and CD94 on natural killer cells. Flow cytometric analyses of inhibitory receptor NKG2A and CD94 on natural killer (NK) cells. The graphs display the frequencies and mean fluorescence intensity (MFI) of NKG2A (A-B) and CD94 (C-D) on NK cells from chronic hepatitis C (CHC) patients during and after direct-acting antivirals therapy and healthy controls (n = 13). ^aP < 0.05 and ^bP ≤ 0.01 and ^cP ≤ 0.001, different time points of CHC patients vs healthy controls; ^dP < 0.05 and ^eP ≤ 0.01 and ^fP ≤ 0.001, different time points of CHC patients (2 wk, 4 wk, 8 wk, 12 wk, Pt-12 wk, Pt-24 wk vs 0 wk). NK: Natural killer; PBMCs: Peripheral blood mononuclear cells; CHC: Chronic hepatitis C; Ctrl: Healthy controls; Pt: Post of treatment; MFI: Mean fluorescence intensity.

Figure 5

Sofosbuvir and ledipasvir therapy modulates the expression of NKp30 and NKp46 on natural killer cells. Flow cytometric analyses of activating receptors NKp30 and NKp46 on natural killer (NK) cells. The graphs display the frequencies and mean fluorescence intensity (MFI) of NKp30 (A-B) and NKp46 (C-D) on NK cells from chronic hepatitis C (CHC) patients during and after Direct-acting antivirals therapy and healthy controls (n = 13). ^aP < 0.05 and ^bP ≤ 0.01 and ^cP ≤ 0.001, different time points of CHC patients vs healthy controls; ^dP < 0.05 and ^eP ≤ 0.01 and ^fP ≤ 0.001, different time points of CHC patients (2 wk, 4 wk, 8 wk, 12 wk, Pt-12 wk, Pt-24 wk vs 0 wk). NK: Natural killer; PBMCs: Peripheral blood mononuclear cells; CHC: Chronic hepatitis C; Ctrl: Healthy controls; Pt: Post of treatment; MFI: Mean fluorescence intensity.

Figure 6

Sofosbuvir and ledipasvir therapy modulates the expression of natural killer cell-related cytokine interferon-γ, perforin and Granzyme B. Flow cytometric analyses of interferon (IFN)-γ, perforin and Granzyme B expression in natural killer (NK) cells. The graphs display the frequencies and mean fluorescence intensity (MFI) of IFN-γ (A-B), perforin (C-D) and Granzyme B (E-F) in NK cells from CHC patients during and after direct-acting antivirals therapy and healthy controls (n = 13). ^aP < 0.05 and ^bP ≤ 0.01 and ^cP ≤ 0.001, different time points of CHC patients vs healthy controls; ^dP < 0.05 and ^eP ≤ 0.01 and ^fP ≤ 0.001, different time points of CHC patients (2 wk, 4 wk, 8 wk, 12 wk, Pt-12 wk, Pt-24 wk vs 0 wk). IFN: Interferon; NK: Natural killer; CHC: Chronic hepatitis C; Ctrl: Healthy controls; Pt: Post of treatment; MFI: Mean fluorescence intensity.

Figure 7

Frequency of CD56⁺ natural killer cells, phenotypes and cytokine expression of natural killer cells between treatment-naïve and treatment-experienced chronic hepatitis C patients. Flow cytometric analyses of the frequency of CD56⁺ natural killer (NK) cells (A), the expression of NKG2A (B), NKG2D (C), NKG2C (D), CD94 (E), NKp30 (F), NKp46 (G), IFN-γ (H), perforin (I) and Granzyme B (J) expression of NK cells and the changing trend between treatment-naïve (n = 7) and treatment-experienced (n = 6) chronic hepatitis C patients during and after direct-acting antivirals therapy. Pt: Post of treatment; IFN: Interferon; NK: Natural killer; PBMCs: Peripheral blood mononuclear cells; CHC: Chronic hepatitis C.