Liver cirrhosis in HIV/HCV-coinfected individuals is related to NK cell dysfunction and exhaustion, but not to an impaired NK cell modulation by CD4+ T-cells

Abstract

Introduction: HIV worsens HCV-related liver disease by accelerating fibrosis progression; however, progression rates are extremely variable among HIV/HCV-coinfected individuals. NK cells are associated with modulation of liver fibrosis and are profoundly altered during HCV and HIV infections. CD4⁺ T-cells modulate NK cell function, and are also affected by HIV infection. Here, we aim to characterize the association of hepatic fibrosis with both the phenotype and function of peripheral NK cells and their regulation by CD4⁺ T-cells, in HIV/HCV-coinfected individuals.

Methods: Thirty-four HIV/HCV-coinfected individuals with minimal (n = 16) and advanced (n = 18) fibrosis (METAVIR F0/F1 and F4 scores respectively) and 20 healthy volunteers were enrolled. PBMC were obtained from peripheral blood samples and NK and CD4⁺ T-cells were isolated and analysed. NK cell phenotype (CD25, CD69, Nkp46, NKG2D, PD-1), degranulation (CD107a) and IFN-γ and TNF-α production, as well as CD4⁺ T-cell activation (CD69, CD25 and CD38) were measured by flow cytometry. CD4⁺ T-cell conditioned medium (CM) derived from F0/F1 or F4 individuals was assessed for IL-2 levels by ELISA. Modulation of NK cell functionality by these CMs was also analysed.

Results: When comparing to NK cells from individuals with minimal fibrosis, degranulation and cytokine secretion by NK cells from subjects with F4 scores was significantly impaired, while PD-1 expression was augmented. On the one hand, neither the expression of activation markers nor IL-2 secretion was distinctly induced in CD4⁺ T-cells from subjects with F0/F1 or F4 METAVIR scores. Finally, NK cell degranulation and cytokine secretion were not differentially modulated by CD4⁺ T-cell CM, whether CD4⁺ T-cells derived from subjects with minimal or advanced fibrosis.

Conclusions: Low levels of NK and CD4⁺ T-cells in HIV/HCV-coinfected individuals with advanced liver fibrosis have been previously described. Here, we show that advanced liver fibrosis in coinfected individuals is associated to a defective function of NK cells and an increased expression of the exhaustion/senescence marker PD-1. This NK signature could not be attributed to changes in the ability of CD4⁺ T-cells to modulate NK cell function.

Keywords: HIV; CD4-positive T-lymphocytes; Immunology; NK cells; hepatitis C virus; liver fibrosis.

Figure 1. NK cell phenotype and functionality in HIV/HCV‐coinfected individuals with minimal or advanced liver fibrosis

(A) CD107a externalization in PBMC from control and HIV/HCV‐coinfected individuals with METAVIR F0/F1 or F4 scores incubated with cRPMI (basal) or K562 cells (+K562). (B) Correlation between frequency of NK cells and degranulation capacity in F4 individuals. (C and D) For IFN‐γ and TNF‐α expression, NK cells were pretreated with conditioned medium (CM) from CD4+ T lymphocytes or cRPMI, and subsequently exposed to K562 cells. (E) PBMCs were stained for CD25, CD69, NKp46, NKG2D and PD‐1 markers. Individual NK cell frequencies, median and 25th and 75th percentiles are indicated. Statistical comparisons were performed using Kruskal‐Wallis followed by Dunn′s multiple‐comparison tests (A and D) and Mann‐Whitney test (E). NK, natural killer.

Figure 2. Activation of CD4+ T‐cells from HIV/HCV‐coinfected individuals with minimal or advanced liver fibrosis

(A) CD69, CD25 and CD38 expression were measured on CD4+ T‐cells from HIV/HCV‐coinfected subjects with METAVIR F0/F1 or F4 scores after 48 hours of vehicle (basal) or anti‐CD3/CD28 stimulation (bead‐to‐cell ratio of 1:1). (B) The same markers were determined after anti‐CD3/CD28 stimulation (1:5 or 1:35 ratios; 24 hours). Individual CD4+ T‐cell frequencies, median and 25th and 75th percentiles are indicated. Statistical comparisons were performed using Wilcoxon matched paired test (A) and Mann‐Whitney test (B).

Figure 3. Ex vivo effect of CD4+ T‐cells from HIV/HCV‐coinfected individuals with minimal or advanced liver fibrosis on NK cells

(A) IL‐2 levels in CM. (B) Conditioned medium (CM) from activated CD4+ T‐cells from HIV/HCV‐coinfected subjects with METAVIR F0/F1 or F4 scores were collected, and subsequently used for pretreatment of NK cells from one healthy individual. Finally, NK cells were coincubated with K562 cells. (C) CD107a, IFN‐γ and TNF‐α expression on NK cells. Individual CD4+ T‐cell frequencies, median and 25th and 75th percentiles are indicated. Statistical comparisons were performed using Mann‐Whitney test. NK, natural killer.