BACKGROUND: Declining telomere length (TL) is associated with T cell senescence. While TL in naive and memory T cells declines with increasing age, there is limited data on TL dynamics in virus-specific memory CD4+ T cells in healthy adults. We combined BrdU-labeling of virus-stimulated T cells followed with flow cytometry-fluorescent in situ hybridization for TL determination. We analyzed TL in T cells specific for several virus infections: non-recurring acute (vaccinia virus, VACV), recurring-acute (influenza A virus, IAV), and reactivating viruses (varicella-zoster virus, VZV, and cytomegalovirus, CMV) in 10 healthy subjects. Additionally, five subjects provided multiple blood samples separated by up to 10 years. RESULTS: VACV- and CMV-specific T cells had longer average TL than IAV-specific CD4+ T cells. Although most virus-specific cells were CD45RA-, we observed a minor population of BrdU+ CD45RA+ T cells characterized by long telomeres. Longitudinal analysis demonstrated a slow decline in average TL in virus-specific T cells. However, in one subject, VZV reactivation led to an increase in average TL in VZV-specific memory T cells, suggesting a conversion of longer TL cells from the naive T cell repertoire. CONCLUSIONS: TLs in memory CD4+ T cells in otherwise healthy adults are heterogeneous and follow distinct virus-specific kinetics. These findings suggests that the distribution of TL and the creation and maintenance of long TL memory T cells could be important for the persistence of long-lived T cell memory.

Telomere length has been shown to be a critical determinant of T cell replicative capacity and in vivo persistence in humans. We evaluated telomere lengths in virus-specific T cells to understand how they may both shape and be changed by the maintenance of memory T cells during a subsequent virus re-infection or reactivation. We used longitudinal peripheral blood samples from healthy donors and samples from a long-term HCV clinical interferon therapy trial to test our hypotheses. To assess T cell telomere lengths, I developed novel modifications to the flow cytometry fluorescence in situ hybridization (flowFISH) assay. These flowFISH modifications were necessary to enable quantification of telomere length in activated, proliferating T cells. Adoption of a fixation-permeabilization protocol with RNA nuclease treatment prior to telomere probe hybridization were required to produce telomere length estimates that were consistent with a conventional telomere restriction fragment length Southern blot assay. We hypothesized that exposure to a non-recurring, acute virus infection would produce memory T cells with longer telomeres than those specific for recurring or reactivating virus infections. We used two acute viruses, vaccinia virus (VACV) and influenza A virus (IAV) and two latent-reactivating herpesviruses, cytomegalovirus (CMV) and varicella zoster virus (VZV) for these studies. Combining a proliferation assay with flowFISH, I found telomeres in VACV-specific CD4 + T cells were longer than those specific for the recurring exposure IAV; data which support my hypothesis. Counter to my hypothesis, CMV-specific CD4 + T cells had longer telomeres than IAV-specific CD4 + T cells. We assessed virus-specific CD4 + T cell telomere length in five donors over a period of 8-10 years which allowed us to develop a linear model of average virus-specific telomere length changes. These studies also found evidence of long telomere, virus-specific CD45RA + T cell populations whose depletion may precede an increased susceptibility to latent virus reactivation. I tested the hypothesis that type I interferon therapy would accelerate T cell telomere loss using PBMC samples from a cohort of chronic hepatitis C virus patients who either did or did not receive an extended course of treatment with interferon-alpha. Accelerated telomere losses occurred in naïve T cells in the interferon therapy group and were concentrated in the first half of 48 months of interferon therapy. Steady accumulation of CD57 + memory T cells in the control group, but not the therapy group, suggested that interferon also accelerated memory turnover. Based on our data, I present proposed models of memory T cell maintenance and impacts of T cell telomere length loss as we age.

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-IFNalpha group for an additional 3.5 years. Significant telomere loss in naive 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 naive 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 naive 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.

BACKGROUND: Vaccinia viruses have been used as a model for viral disease and as a protective live vaccine. METHODOLOGY AND PRINCIPAL FINDINGS: We investigated the immunogenicity of an attenuated strain of vaccinia virus engineered to inactivate the N1L gene (vGK5). Using the intranasal route, this recombinant virus was 2 logs less virulent compared to the wildtype VACV-WR. Infection by the intranasal, intraperitoneal, and tail scarification routes resulted in the robust induction of cytolytic virus-specific CD8 T cells in the spleens and the lungs. VACV-specific antibodies were also detected in the sera of mice infected 3-5 months prior with the attenuated vGK5 virus. Finally, mice immunized with vGK5 were significantly protected when challenged with a lethal dose of VACV-WR. CONCLUSIONS: These results indicate that the attenuated vGK5 virus protects against subsequent infection and suggest that the N1L protein limits the strength of the early antiviral CD8 T cell response following respiratory infection.