The Common Cold Project › Measures by Study › Biological Pathways › Telomere Length (PCS3)

Lymphocyte Telomere Length (PCS3, subsample)

Funding for measurement of lymphocyte telomere length was not included in the original PCS3 grant (NIAID R01 AI066367), but instead was obtained through a supplement awarded 3 years into the course of the study (NCCAM RC1AT005799; see Supporting Grants). Accordingly, blood samples for examination of telomere length were collected beginning with Trial 4 and continued through to the end of Trial 13. A final subsample of 152 participants provided blood samples with sufficient genetic material to assess telomere length for at least one of the 4 cell populations of interest (see below).

Telomeres are non-coding regions of DNA that form protective caps on the ends of chromosomes. After each cell division, telomeres decrease in length. In hematopoetic cells, activity of the enzyme telomerase initially compensates for cell division-mediated telomere loss by adding subunit repeats to the shortened telomeres. With repeated cell divisions, however, shortening becomes permanent and continues until the telomeres reach a critical length, which is associated with disrupted cell function and eventual cell death. The rate of progression to cell senescence and death can be affected by factors that influence telomerase activity, and differs among lymphocyte subsets. Of interest to those who study infectious diseases caused by viruses is the comparatively rapid loss of telomere length in cytolytic, CD8+ T cells relative to helper T cells (CD4+) and B cells (CD19+). After repeated replication cycles, CD8+ cells lose the capacity to express CD28, a costimulatory molecule important for antiviral function. Loss of CD28 expression is associated with reduced telomerase activity, an increased synthesis of pro-inflammatory cytokines, and a poor antibody response to vaccines.

Sample Collection

Whole blood for telomere assay was collected from a random 1/3 of the sample on each of 3 days: during the baseline screening visit (6-8 weeks before quarantine); 3-5 days before quarantine; or on the baseline day of quarantine. 15-ml samples were collected by standard venipuncture into 3 green-top (heparinized) collection tubes. Blood was drawn from only a subsample of participants at a time because we did not have resources available to conduct timely cell separations and preservation for all participants in a single day.

Lymphocyte Sorting

Lymphocytes sorting was performed in whole blood using flow cytometry (2 ml aliquot each for antibody labeling and cell counts). Peripheral blood mononuclear cells (PBMCs) and serum were separated following a protocol of Ficoll-Paque™ PLUS (Cat# 17-1440-03, Amersham Biosciences, Pittsburgh, PA)

Lymphocyte subpopulations were separated from whole blood using the RoboSep™ automated cell separator (STEMCELL™ Technologies). CD4+ cells were isolated using the EasySep® Human Whole Blood CD4 Positive Selection Kit (18082); and CD19+ cells using the EasySep® Human Whole Blood CD19 Positive Selection Kit (18084). CD8+ cells were separated using the RosetteSep® Human CD8+ T Cell Enrichment Cocktail (15063); and CD28+/CD28- cells using the EasySep® Human PE Positive Selection Kit (18551).

Assay

Mean telomere length was measured in peripheral blood mononuclear cells (PBMCs), CD4+ cells, CD19+ cells, and two subpopulations of CD8+ cells (CD8+CD28+ and CD8+CD28-) using a real-time quantitative polymerase chain reaction (qPCR) assay that determines the relative ratio of telomere repeat copy number to single-copy gene copy number (T/S ratio) in experimental samples as compared with a reference DNA sample following a published protocol¹.

All samples were run in duplicate, with replicate values being averaged to obtain the final T/S ratio values for use in analyses. Final T/S ratio values for a given cell type were computed only for those participants with complete data for both replicates. Participants missing one or both replicate values for a given cell type were assigned a missing final T/S ratio value for that cell.

Reference

¹ O’Callaghan, N.J., Dhillon, V.S., Thomas, P., & Fenech, M. (2008). A quantitative real-time PCR method for absolute telomere length. BioTechniques, 44, 807-809.