Increased degranulation of natural killer cells during acute HCV correlates with the magnitude of virus-specific T cell responses

Abstract

Background & aims: Natural killer (NK) cells provide early defense against viral infections by killing infected cells and producing cytokines that inhibit viral replication. NK cells also interact with dendritic cells (DCs) and this reciprocal interaction regulates both innate and adaptive immunity. Genetic studies have suggested that NK cell activity is a determinant of HCV infectious outcome but a functional correlation has not been established. We hypothesized that increased NK cell activity during acute HCV infection correlates with spontaneous viral clearance.

Methods: We used multiparametric flow cytometry to monitor longitudinally the phenotype and the activity of NK cells in a cohort of intravenous drug users following HCV exposure. Three groups were studied: acute HCV with chronic evolution (n = 13), acute resolving HCV (n = 11), and exposed un-infected individuals (n = 10). We examined the expression of several NK cell-activating and -inhibiting receptors, IFN-γ production and CD107a degranulation upon stimulation, and the kinetics of NK cell responses relative to T cell responses.

Results: We observed decreased expression of the inhibitory NKG2A receptor in NK cells following spontaneous HCV clearance. In addition, we observed increased NK cell degranulation during acute HCV irrespective of infectious outcome. NK cell peak responses preceded or coincided with peak T cell responses. Furthermore, NK cell degranulation correlated with the magnitude of HCV-specific T cells.

Conclusions: Our results demonstrate that NK cells are activated during acute HCV regardless of infection outcome and may play an indirect role through induction and priming of T cell responses.

Figure 1. No change in frequency of CD56^dimCD16⁺ NK cells following HCV-exposure

A) Strategy for gating on the two NK cell subsets by flow cytometry: viable lymphocytes CD3⁻CD56^dimCD16⁺ and CD3⁻CD56^brightCD16⁻. Frequency of B) CD3⁻CD56^dimCD16⁺ NK cells and C) CD3⁻CD56^brightCD16⁻ NK cells was determined ex-vivo in patients with HCV chronic evolution (•), HCV spontaneous resolution (Δ), exposed un-infected (■) and healthy donors (○). The acute phase of HCV infection is represented by the shaded area. Mean is represented by a horizontal bar. *p < 0.05; **p < 0.01; ***p < 0.001. 2-way ANOVA (repeated measures) or 1-way ANOVA (comparison with healthy donors).

Figure 2. Acute HCV infection is associated with increased NK cell degranulation regardless of infection outcome

Degranulation was measured by CD107a surface staining in patients with HCV chronic evolution (•), HCV spontaneous resolution (Δ), exposed un-infected (■) and healthy donors (○). PBMCs were co-incubated with or without K562 target cells. Background expression of CD107a was subtracted from expression with target cells. A) Frequency of CD107a⁺IFN-γ⁻ cells gated on CD56^brightCD16⁻ NK cells and B) CD56^dimCD16⁺ NK cells. C) Longitudinal changes in frequency of CD107a⁺IFN-γ⁻ cells represented for each individual patient in the HCV chronics group (•) gated on CD56^brightCD16⁻ NK cells and D) CD56^dimCD16⁺ NK cells. For each individual patient, different infection time points are joined by a line. E) Longitudinal changes in frequency of CD107a⁺IFN-γ⁻ cells in individual patients in the spontaneous resolvers group (Δ) gated on CD56^brightCD16⁻ NK cells and F) CD56^dimCD16⁺ NK cells. For each individual patient, different infection time points are joined by a line. The acute phase of HCV infection is represented by the shaded area. Mean is represented by a horizontal bar. *p < 0.05; **p < 0.01; ***p < 0.001. 2-way ANOVA (repeated measures) or 1-way ANOVA (comparison with healthy donors).

Figure 3. HCV infection is associated with decreased IFN-γ regardless of infectious outcome

Cytokine production was measured by intracellular IFN-γ in patients with HCV chronic evolution (•), HCV spontaneous resolution (Δ), exposed un-infected (■) and healthy donors (○). PBMCs were co-incubated with or without K562 target cells. Background expression of IFN-γ was subtracted from expression with target cells. A) Frequency of CD107a-IFN-γ⁺ cells gated on CD56^brightCD16⁻ NK cells and B) CD56^dimCD16⁺ NK cells. C) Frequency of CD107a⁺IFN-γ⁺ cells gated on CD56^brightCD16⁻ NK cells and D) CD56^dimCD16⁺ NK cells. The acute phase of HCV infection is represented by the shaded area. Mean is represented by a horizontal bar. *p < 0.05; **p < 0.01; ***p < 0.001. 2-way ANOVA (repeated measures) or 1-way ANOVA (comparison with healthy donors).

Figure 4. Expression of activation markers, activating receptors and inhibitory receptors by NK cells

Expression of phenotypic markers by NK cells was determined ex-vivo in patients with HCV chronic evolution (•), HCV spontaneous resolution (Δ), exposed un-infected (■) and healthy donors (○). A) Frequency of CD69⁺ cells gated on CD56^brightCD16⁻ NK cells and B) CD56^dimCD16⁺ NK cells. C) Frequency of NKG2A⁺ cells gated on CD56^brightCD16⁻ D) Frequency of CD161⁺ cells gated on CD56^dimCD16⁺ NK cells. The acute phase of HCV infection is represented by the shaded area. Mean is represented by a horizontal bar. *p < 0.05; **p < 0.01; ***p < 0.001. 2-way ANOVA (repeated measures) or 1-way ANOVA (comparison with healthy donors).

Figure 5. NK cell peak precedes or coincides with peak HCV-specific T cell responses

NK cell activity was measured by degranulation of the CD56^dimCD16⁺CD107a⁺IFN-γ⁻NK cell population or IFN-γ production by the CD56^brightCD16⁻CD107a⁻IFN-γ⁺ NK cell population and T cell responses measured by IFN-γ secretion in response to overlapping HCV peptides spanning the immunodominant HCV core and NS3 proteins [27-30] using the ELISPOT assay were measured longitudinally in a subset of HCV acutely infected patients with either spontaneous resolution (n=6, Panel A) or chronic evolution (n=6, Panel B). Plasma HCV viral load was measured by an in-house real time PCR assay (Sensitivity 1000 IU/ml) and represented as grey area graph. For patients R3-R6, HCV-RNA was below the sensitivity of our assay. Patients R3 and R6 were HCV RNA+ by qualitative PCR (sensitivity 50 IU/ml) at acute HCV diagnosis and negative afterwards. Patient R4 was never HCV-RNA+, acute HCV was diagnosed based on a positive anti-HCV antibody test following a previous negative test within a 16 week interval. Patient R5 was HCV RNA+ by qualitative PCR (sensitivity 50 IU/ml) at acute HCV diagnosis and the first time point tested then negative afterwards. Patient C5 tested HCV RNA+ by qualitative PCR (sensitivity 50 IU/ml) at all time points studied but samples were not available for viral load quantification.

Figure 5. NK cell peak precedes or coincides with peak HCV-specific T cell responses

NK cell activity was measured by degranulation of the CD56^dimCD16⁺CD107a⁺IFN-γ⁻NK cell population or IFN-γ production by the CD56^brightCD16⁻CD107a⁻IFN-γ⁺ NK cell population and T cell responses measured by IFN-γ secretion in response to overlapping HCV peptides spanning the immunodominant HCV core and NS3 proteins [27-30] using the ELISPOT assay were measured longitudinally in a subset of HCV acutely infected patients with either spontaneous resolution (n=6, Panel A) or chronic evolution (n=6, Panel B). Plasma HCV viral load was measured by an in-house real time PCR assay (Sensitivity 1000 IU/ml) and represented as grey area graph. For patients R3-R6, HCV-RNA was below the sensitivity of our assay. Patients R3 and R6 were HCV RNA+ by qualitative PCR (sensitivity 50 IU/ml) at acute HCV diagnosis and negative afterwards. Patient R4 was never HCV-RNA+, acute HCV was diagnosed based on a positive anti-HCV antibody test following a previous negative test within a 16 week interval. Patient R5 was HCV RNA+ by qualitative PCR (sensitivity 50 IU/ml) at acute HCV diagnosis and the first time point tested then negative afterwards. Patient C5 tested HCV RNA+ by qualitative PCR (sensitivity 50 IU/ml) at all time points studied but samples were not available for viral load quantification.

Figure 6. NK cell degranulation correlates positively but IFN-γ correlates negatively with HCV-specific T cell adaptive immune response

NK cell degranulation and IFN-γ production were determined in patients with HCV chronic evolution and HCV spontaneous resolution during the acute phase of HCV as shown in Figures 2 and 3. The magnitude of the HCV-specific T cell response approximately 1 month later in these same patients was determined by IFN-γ secretion in response to overlapping HCV peptides spanning the entire HCV polyprotein using the ELISPOT assay. A) Correlation between HCV-specific T cell response and frequency of CD107a⁺IFN-γ⁻ cells gated on CD56^brightCD16⁺ NK cells, B) frequency of CD107a⁺IFN-γ⁻ cells gated on CD56^dimCD16⁻ NK cells, C) frequency of CD107a⁻IFN-γ⁺ cells gated on CD56^brightCD16⁺ NK cells, D) frequency of CD107a⁻IFN-γ⁺ cells gated on CD56^dimCD16⁻ NK cells, E) frequency of CD107a⁺IFN-γ⁺ cells gated on CD56^brightCD16⁺ NK cells, F) frequency of CD107a⁺IFN-γ⁺ cells gated on CD56^dimCD16⁻ NK cells. Correlation determined with Spearman test.