Replicative history of CD57⁺ and CD57⁻ memory and HIV-specific CD8⁺ T cells.

PBMCs were collected from healthy subjects, and 1 million CD57⁺ and CD57⁻CD45RO⁺CD8⁺ T cells and CD45RA⁺CD62L⁺ naive CD8⁺ T cells were sorted by flow cytometry. Sorted populations were used for telomere measurements using telomere-specific peptide nucleic acid (PNA) followed by flow cytometry. Data were gate-based such that DNA content was consistent between naive and memory CD8⁺ T cells. Histograms of telomere channel fluorescence are depicted (A). The solid line corresponds to CD57⁺memory CD8⁺ T cells (mean fluorescent intensity, 10.1), the dotted line depicts CD57⁻ memory CD8⁺ T cells (MFI 14.2), and the dashed line represents naive CD8⁺ T cells (MFI 18.3); MFI of the negative control was 7.6. This experiment was repeated on PBMCs from a total of 3 additional healthy volunteers to ensure reproducibility. Telomere length trends were identical in all 3 subjects. PBMCs then were collected from HIV^−1-12 and HIV⁺subjects13-16 and sorted by flow cytometry for CD57⁺CD8⁺ T cells (○) and CD57⁻CD45RO⁺CD8⁺ T cells (●) (B). HIV-specific CD57⁺ (○, bold numbers) or, when possible, CD57⁻ (●, bold numbers) CD8⁺T cells were additionally sorted from the 4 HIV⁺individuals based on production of IFN-γ following stimulation with HIV peptides13-16 (panel B, left). At least 10 000 sorted cells of interest were then assayed for TREC using quantitative real-time PCR. Ten is the lower limit of detection for the assay. CD57⁻ memory CD8⁺ T cells have undergone statistically significantly fewer rounds of proliferation compared with CD57⁺CD8⁺ T cells (B, right panel). Vertical bars represent 25th to 75th percentiles.