Flow-cytometry was used to detect γ-H2AX, a marker of DNA double-strand breaks, to investigate the relation between peripheral CD8+ T-lymphocyte telomere length and downstream signalling (A). Subjects with CD8 + CD45RO+ T lymphocytes with shortened telomeres had an increased proportion of circulating γ-H2AX + CD8+ T lymphocytes (p = 0.006, Rs = −0.23, B). The proportion of circulating γ-H2AX + CD8+ T lymphocytes in HCV-RNA+ patients with severe fibrosis (median 2.8% (1.5–4.6)) was higher than in healthy controls (1.8% (0.98–2.4)), HCV-RNA-negative HCV-exposed subjects (2.0% (1.1–3.5)) and HCV-RNA-positive patients with mild fibrosis (1.4% (0.8–2.4), Kruskal Wallis p = 0.0023, C).
Fig. 1 γ-H2AX (Ser-139) expression on circulating CD8+ T lymphocytes in healthy controls and HCV-infected subjects. (A) Gating strategy for γ-H2AX; cells within the live lymphocyte gate by scatter characteristics (left panel) and positive staining (more ...)
To study the independent association between clinical and demographic factors with CD8+ T-lymphocyte γ-H2AX expression in HCV-RNA+ subjects (n = 109), a multiple linear regression model was constructed (Supplementary Fig. 1
and ). By univariate analysis, increased fibrosis stage (p
<0.001) and a low ALT (p
= 0.059) were associated with increased γ-H2AX expression. On multivariate analysis, increased fibrosis stage (p
<0.001) and low ALT (p
= 0.025) were independently associated with increased γ-H2AX expression ().
Clinical and demographic factors associated with γ-H2AX expression on CD8+ T lymphocytes from HCV-RNA+ subjects (n = 109) by multiple linear regression.
There was increased γ-H2AX expression on CD8+ T lymphocytes in subjects with past exposure to CMV (Supplementary Fig. 1G–I
), which is probably explained by a higher proportion of CD8+ T lymphocytes with an advanced cell surface phenotype, defined by CD27 and CD57 expression and described previously in CMV 
(Supplementary Fig. 1H
). There was no increase in γ-H2AX expression when the analysis was restricted to the CD27- subset, controlling for this increase in highly differentiated cells (Supplementary Fig. 1I
γ-H2AX expression was associated with evidence of downstream DNA damage response (DDR) signalling, as γ-H2AX + CD8+ T lymphocytes had increased phosphorylation of p53 at serine 15 (D). There was no difference in Ki67 expression between unfractionated CD8+ T lymphocytes (0.7%; 0.57–1.15) and γ-H2AX + CD8+ T lymphocytes (1.3%; 0.45–1.75, p = 0.24, E and F), indicating γ-H2AX expression was unrelated to DNA replication.
γ-H2AX was present in a higher proportion of CD8+ T lymphocytes expressing the mature phenotypes CD27-CD57- (3.1% (1.4–5.6)) or CD27-CD57+ (3.2% (1.7–6.2)) than the less mature CD27 + CD57- subset (0.8% (0.4–1.6), p <0.0001, G). Consistent with this observation, CD8 + CD27- T lymphocytes had shorter telomeres (117 (111 – 134)) than CD8 + CD27+ T lymphocytes (146 (139–161), p = 0.0003, H). Immunoblotting revealed increased γ-H2AX expression in CD8 + CD27- T lymphocytes compared to CD8+ CD27+ T lymphocytes (I).
Without stimulation, a higher proportion of γ-H2AX + CD8+ T lymphocytes expressed IFN-γ (3.9% (2.48–13.5)) compared with unfractionated CD8+ T lymphocytes (0.4% (0.08–1.2), p = 0.002, A). Following overnight stimulation (with anti-CD3 and anti-CD28), a higher proportion of γ-H2AX + CD8+ T lymphocytes expressed IFN-γ (20.0% (7.8–26.9) compared with 9.6% (4.9–18.1), p = 0.02) than unfractionated CD8+ T lymphocytes (C). However, the proportion of unstimulated γ-H2AX + CD8+ T cells expressing IL-2 was lower than unfractionated CD8+ T lymphocytes (1.8% (0–3.1) compared with 3.1% (2–5.3), p = 0.03, D).
Fig. 2 Cytokine expression pattern of peripheral CD8+ T lymphocytes from 10 subjects with chronic HCV infection. Unfractionated CD8+ lymphocytes or γ-H2AX + CD8+ subsets were analysed for expression of (A and C) IFN-γ, and (B (more ...)
We studied co-expression of γ-H2AX and markers of activation and known inhibitory receptors. γ-H2AX + CD8+ T cells were CD38 positive, but CD69 negative (Supplementary Fig. 2
). Further, these cells did not express the inhibitory receptors PD-1 or Tim3 (Supplementary Fig. 2
). γ-H2AX + CD8+ T cells were negative for CD161 
in 15 further patients with viraemic HCV infection (data not shown). To study potential antigen-specificity, circulating γ-H2AX + CD8+ T lymphocytes derived from three HLA-2 positive patients with positive CMV serology and current HCV infection (HCV-RNA+ in serum) showed no specificity for either HCV-NS3 or CMV-pp65, although unfractionated CD8+ T lymphocytes responded to both pentamers (Supplementary Fig. 3
Induction of pSTAT1 expression was used as a measure of the response to in vitro
IFN-α (A and B). CD8+ T lymphocytes from healthy controls responded with an EC50 of 99 IU/ml. Unfractionated CD8+ T lymphocytes from HCV-infected subjects had an increased EC50 when compared to healthy controls (308 IU/ml), which was still higher in γ-H2AX + CD8+ T lymphocytes (781 IU/ml) (A–C). The maximal response of unfractionated CD8+ T lymphocytes from HCV infected patients was similar to that in healthy controls (p
= 0.29, D) but the response of γ-H2AX+ T lymphocytes was impaired by comparison (p
<0.0001). These defects could not be explained by total STAT1 content (Supplementary Fig. 4A and B
), surface expression of IFN-AR components (E-G), failure to phosphorylate Tyk2 or Jak1 (Supplementary Fig. 4C and D
) or IFN-α induced downregulation of IFN-AR from the cell surface of γ-H2AX + CD8+ T lymphocytes (H).
Fig. 3 CD8+ γ-H2AX+ lymphocytes fail to phosphorylate STAT1 after incubation with IFN-α unrelated to IFN-α receptor function. Circulating T lymphocytes were incubated with variable concentrations of IFN-α2b before staining for (more ...)
A higher proportion of intrahepatic CD8+ T lymphocytes expressed γ-H2AX (2.15%, (1.28–3.65)) compared to circulating CD8+ T lymphocytes simultaneously isolated (0.25% (0.1–1.43), p = 0.03, A and B). Intrahepatic γ-H2AX + CD8+ T lymphocytes had a similar phenotype to circulating T lymphocytes with low expression of CD27 (C). Intrahepatic γ-H2AX + CD8+ T lymphocytes also failed to phosphorylate STAT1 in response to IFN-α2b (D).
Fig. 4 CD8+ γ-H2AX+ T lymphocytes are more frequent within the liver of HCV-infected subjects. (A) Example plots of CD8 and γ-H2AX expression on peripheral (left panel) and intrahepatic (right panel) lymphocytes from an HCV-RNA+ subject. (B) (more ...)
To investigate whether the defect in Jak/Stat signalling was confined to the IFN-α pathway, the response of γ-H2AX + CD8+ T lymphocytes to IL-6 was investigated, as this also signals through STAT1. The proportion of unfractionated CD8+ T lymphocytes that expressed pSTAT1 after exposure to 300 IU/ml IL-6 was higher than in γ-H2AX + CD8+ T lymphocytes (16.4% (12.3–28.4 and 3.9% (1.9–5.6), p = 0.002, E and G) suggesting that defective STAT1 phosphorylation was not confined to IFN-α signalling.
IL-2-induced STAT5 phosphorylation was investigated to determine if there was an overall failure of Stat activation in γ-H2AX + CD8+ T-lymphocytes. After exposure to 10 IU/ml IL-2 the proportion of unfractionated CD8+ T lymphocytes that expressed pSTAT5 was higher than in γ-H2AX + CD8+ T lymphocytes (36.2% (23.6–39.5) and 2.7% (1.8–6.8), p = 0.0039, F and H) suggesting a more widespread failure of Jak/Stat signalling in γ-H2AX + CD8+ T-lymphocytes.