Extended interferon-alpha therapy accelerates telomere length loss in human peripheral blood T lymphocytes

Abstract

Background: Type I interferons have pleiotropic effects on host cells, including inhibiting telomerase in lymphocytes and antiviral activity. We tested the hypothesis that long-term interferon treatment would result in significant reduction in average telomere length in peripheral blood T lymphocytes.

Methods/principal findings: Using a flow cytometry-based telomere length assay on peripheral blood mononuclear cell samples from the Hepatitis-C Antiviral Long-term Treatment against Cirrhosis (HALT-C) study, we measured T cell telomere lengths at screening and at months 21 and 45 in 29 Hepatitis-C virus infected subjects. These subjects had failed to achieve a sustained virologic response following 24 weeks of pegylated-interferon-alpha plus ribavirin treatment and were subsequently randomized to either a no additional therapy group or a maintenance dose pegylated-IFNα group for an additional 3.5 years. Significant telomere loss in naïve T cells occurred in the first 21 months in the interferon-alpha group. Telomere losses were similar in both groups during the final two years. Expansion of CD8(+)CD45RA(+)CD57(+) memory T cells and an inverse correlation of alanine aminotransferase levels with naïve CD8(+) T cell telomere loss were observed in the control group but not in the interferon-alpha group. Telomere length at screening inversely correlated with Hepatitis-C viral load and body mass index.

Conclusions/significance: Sustained interferon-alpha treatment increased telomere loss in naïve T cells, and inhibited the accumulation of T cell memory expansions. The durability of this effect and consequences for immune senescence need to be defined.

Trial registration: ClinicalTrials.gov NCT00006164.

Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflicts. Financial relationships of the authors with Hoffmann-La Roche, Inc., are as follows: H.L. Bonkovsky receives research support. Authors with no financial relationships related to this project are J.M. O'Bryan, J.A. Potts, A. Mathew and A.L. Rothman.

Figures

Figure 1. Telomere length (TL) measurement using modified flowFISH.

(A) FlowFISH cytometry gating strategy used to assess TL in CD4+ and CD8+ T cells. Singlet cells in a G0/G1 (diploid DNA content) gate were further selected using a lymphocyte gate based on forward and side scatter. Flow scatter plots of CD4+ by CD8+ staining with and without flowFISH hybridization are shown. TL estimates in molecules of equivalent soluble fluorescence (MESF) in CD4+ and CD8+ populations. (B) Linear regression analysis of naïve (CD45RA+ CD57−) CD4+ (circles) and naïve CD8+ (squares) TL at screening for cHCV subjects (n = 29, left panel) and healthy, age-matched donors (n = 19, right panel) versus age at blood draw. (C) FlowFISH-determined baseline TL estimates within total CD4+ and CD8+ T lymphocytes and indicated subpopulations for all 29 HALT-C subjects at screening. Graphs are box-whisker 10–90 percentile with outliers.

Figure 2. Screening T cell telomere lengths inversely correlated with hepatitis C viral RNA levels and BMI.

(A) TL in total CD4+ (gray circles) and total CD8+ (black squares) T cells from the screening (S00) time point versus screening HCV RNA levels, (B) TL in naïve CD4+ (circles) and CD8+ (squares) CD45RA+ CD57− subsets versus screening HCV RNA levels, and (C) TL in memory CD4+ (circles) and CD8+ (squares) CD45RA− CD57− subset versus screening HCV RNA levels. (D) Baseline body-mass index (BMI, in kilograms per meter squared) from screening assessment inversely correlated with naïve phenotype CD4+ (gray circles) and CD8+ (filled squares) T cells. Correlation (r-squared) and p values are from linear regression testing with best-fit lines as shown.

Figure 3. Accelerated telomere length (TL) loss in naïve T cell subsets for the IFN group.

Individual TL trajectories for (A) all CD4+, (B) naïve CD4+ (CD45RA+ CD57−), (C) memory CD4+ (CD45RA− CD57−), (D) all CD8+, (E) naïve CD8+ (CD45RA+ CD57−), and (F) memory CD8+ (CD45RA− CD57−) T cell subsets are shown for peg-IFNα subjects (left panels) and the no-therapy, control subjects (right panels). Solid lines connect the TL of each subject at three time points plotted by age at blood draw. The dashed line on each plot derives from a linear regression based on the averaged slope and y-intercept from each individual's linear regression equation, with the average slope (± standard. deviation) in MESF per year of age shown in the upper right corner.

Figure 4. Accelerated telomere length loss (delta TL) occurs in the first 21 months.

Delta TL analysis between treatment and control groups for (A) CD4+ and (B) CD8+ T cells and their naïve and memory subsets for the S00 to M21, and M21 to M45 intervals. P values from Mann-Whitney testing. Error bars are mean ± standard error.

Figure 5. TL loss with therapy was lost with increasing age in a T cell subset-dependent manner.

Naïve CD4+ (upper left plot), memory CD4+ (lower left plot), naïve CD8+ (upper right plot), and memory CD8+ (lower right plot) T cell change in telomere length (delta TL) from screening to month 45 from subjects in both treatment groups. Each symbol is an individual subject's delta TL for that T cell subset. Filled symbols are IFNα treatment group subjects, open symbols are control group subjects. Horizontal bars are mean values.

Figure 6. Serum ALT correlates with changes in naïve T cell telomere lengths in the control group.

Average serum alanine aminotransferase (ALT) levels during the randomization phase correlated with telomere length loss (delta TL) in the no-therapy control group (right-hand panel), but not in the IFN therapy group (left-hand panel). Delta TL shown is from screening (S00) to month 45 (M45).

Figure 7. Sustained interferon therapy associated with suppression of CD8+ CD45RA+ CD57+ expansions.

CD45RA+ CD57+ subset frequency (%) within CD8+ T lymphocytes across the three time points for the peg-IFNα subjects (left panel) and the no-therapy, control subjects (right panel). Individual lines on the plots represent values from each subject across the three time points. * p<0.05; *** p = 0.0001 by Wilcoxon paired analysis (decr = decrease; incr = increase; ns = not significant).