Generalized Liver- and Blood-Derived CD8+ T-Cell Impairment in Response to Cytokines in Chronic Hepatitis C Virus Infection

Abstract

Generalized CD8+ T-cell impairment in chronic hepatitis C virus (HCV) infection and the contribution of liver-infiltrating CD8+ T-cells to the immunopathogenesis of this infection remain poorly understood. It is hypothesized that this impairment is partially due to reduced CD8+ T-cell activity in response to cytokines such as IL-7, particularly within the liver. To investigate this, the phenotype and cytokine responsiveness of blood- and liver-derived CD8+ T-cells from healthy controls and individuals with HCV infection were compared. In blood, IL-7 receptor α (CD127) expression on bulk CD8+ T-cells in HCV infection was no different than controls yet was lower on central memory T-cells, and there were fewer naïve cells. IL-7-induced signalling through phosphorylated STAT5 was lower in HCV infection than in controls, and differed between CD8+ T-cell subsets. Production of Bcl-2 following IL-7 stimulation was also lower in HCV infection and inversely related to the degree of liver fibrosis. In liver-derived CD8+ T-cells, STAT5 activation could not be increased with cytokine stimulation and basal Bcl-2 levels of liver-derived CD8+ T-cells were lower than blood-derived counterparts in HCV infection. Therefore, generalized CD8+ T-cell impairment in HCV infection is characterized, in part, by impaired IL-7-mediated signalling and survival, independent of CD127 expression. This impairment is more pronounced in the liver and may be associated with an increased potential for apoptosis. This generalized CD8+ T-cell impairment represents an important immune dysfunction in chronic HCV infection that may alter patient health.

Fig 1. HCV⁺ individuals have fewer blood-derived naïve CD8⁺ T-cells and a lower expression of CD127 on central memory cells than HCV^- controls.

CD8⁺ T-cell subset distribution was determined by CD45RA and CCR7 staining in (A) controls (n = 10) and (B) HCV infection (n = 12). (C) Subset distribution data for controls and HCV-infected individuals are graphically represented as means (T_N p = 0.006). (D) The expression of CD127 was measured on blood-derived bulk CD8⁺T-cells (control n = 30, HCV⁺ n = 50) and their subsets (control n = 10, HCV⁺ n = 12) and presented as (E) percentage (T_CM p = 0.02, unpaired Student’s t-test) and (F) mean fluorescence intensity of CD127 expressing cells (error bars represent ±S.D.).

Fig 2. IL-7-induced signaling of blood-derived CD8⁺ T-cells is impaired in HCV infection.

(A) Phosphorylation of STAT5 was measured as mean fluorescence intensity (MFI) as shown in a representative histogram. (B) The expression of pSTAT5 was significantly increased by increasing concentrations of IL-7 (0.01–10 ng/ml) in blood-derived CD8⁺ T-cells from controls (p < 0.001, n = 10) or chronically infected HCV⁺ individuals (p = 0.003, n = 9) is summarized, as assessed by ANOVA, yet responses of the latter group were significantly less pronounced than controls (t: p = 0.005, non-linear regression analysis). (C) The expression of pSTAT5 in CD8⁺ T-cell subsets were distinguished by CD45RA and CCR7 expression, with significance in T_CM and T_N subsets (t: p<0.0001 for each subset, non-linear regression, control n = 7, HCV⁺ n = 5). Error bars in the graphs represent ± S.D.

Fig 3. IL-7-induced proliferation is not impaired while production of Bcl-2 is reduced in blood-derived CD8⁺ T-cells from HCV⁺ individuals.

Cell proliferation in response to IL-7 (10ng/ml) and/or PHA (0.2mg/ml) was measured as CFSE dilution of CD8⁺ T-cells from (A) HCV^- (n = 8) and (B) HCV⁺ individuals (n = 8), with markers indicating the proportion (%) of CFSE^low (dividing) cells. (C) Proliferation of isolated CD8⁺ T-cells induced by IL-7 + suboptimal PHA was significantly increased in both groups (control p = 0.0001 and HCV⁺ p<0.0001, unpaired Student’s t-test), yet there was no difference between these groups (n.s. = not significant). (D) Bcl-2 expression of blood-derived CD8⁺ T-cells was measured. (E) Bcl-2 expression (MFI) of unstimulated CD8⁺ T-cells is summarized (control n = 4, HCV⁺ n = 5) and (F) Bcl-2 production in response to IL-7 after 48 hours is summarized as MFI (t p = 0.0006, non-linear regression, control n = 8, HCV⁺ n = 9). (G) The IL-7-induced expression of Bcl-2 in blood-derived CD8⁺ T-cells from HCV⁺ individuals with low fibrosis (F0-F2) was compared to that of high fibrosis (F3-F4). Values are expressed relative to medium alone (* p = 0.02, unpaired Student’s t-test, error bars represent ±S.D.).

Fig 4. The proportion of CD8⁺ T _CM cells is increased, while T_N and T_EMRA cells are decreased in the liver in HCV infection and CD127 expression does not differ between IH- and blood-derived CD8⁺ T-cells.

Representative dot plots of subset distribution are shown for (A) Blood-derived and individually matched (B) IH-CD8⁺ T-cell subsets, as analysed by flow cytometry which distinguished between subsets on the basis of CD45RA and CCR7 expression. (C) The means of these observations are summarized in a bar graph (* T_N p = 0.03, T_CM p = 0.01, T_EMRA p = 0.03, unpaired Student’s t-test, n = 3, error bars represent ± S.D.). Membrane CD127 expression on bulk and CD8⁺ T-cells subsets did not differ between locations, as measured by (D) percentage expression nor (E) intensity of expression (MFI).