Although activated cells might be more likely to undergo proliferation, it has been reported that CD8+HLA-DR+ T cells are at an arrested phase of cell cycle and unable to divide [22-24]. In order to determine the cell cycle position of CD8+HLA-DR− naive, CD8+HLA-DR− memory, and CD8+HLA-DR+ cells and test the accuracy of those statements, simultaneous staining with 7-amino-actinomycin D (7-AAD) (DNA) and pyronin Y (RNA) was performed (Fig. 5). For studies depicted in Figure 5 and 6, magnetic bead separation, instead of FACS sorting, was used to obtain populations of CD8+HLA-DR− naive, CD8+HLA-DR− memory, and CD8+HLA-DR+ cells. This approach was necessary due to the fact that FACS-sorted cells are bound with fluorescent-conjugated antibodies that interfere with the detection of the DNA- and RNA-binding fluorescent dyes (7-AAD and pyronin Y, respectively). The strategy used to purify CD8+HLA-DR− naive, CD8+HLA-DR− memory, and CD8+HLA-DR+ subsets by magnetic beads and the purity of those populations are depicted in Supporting Information Fig. 3. Freshly isolated CD8+HLA-DR− naive cells exhibited a cell cycle profile typical of that seen in quiescent cells with the majority of cells in G0 (average: 99.4% in control, n = 5; 99.8% in HIV+ <50, n = 6; and 99.6% in HIV+ ≥50, n = 8) and <0.1% of cells in S+G2/M of the cell cycle. The CD8+HLA-DR− memory cells were virtually identical to the CD8+HLA-DR− naive cells. Although the majority of CD8+HLA-DR+ cells were also found in G0 (average: 94.5% in control; 97.9% in HIV+ <50; and 95.8% in HIV+ ≥50), these cells also contained a substantial fraction of cells in early G1 (average: 4.6% in control; 1.8% in HIV+ <50; and 3.1% in HIV+ ≥50) and in S+G2/M (average: 0.9% in control; 0.3% in HIV+ <50; and 0.9% in HIV+ ≥50). Overall, there were no discernible differences in the cell cycle profiles of CD8+HLA-DR− naive, CD8+HLA-DR− memory, and CD8+HLA-DR+ cells when comparing the control, HIV+ <50, and HIV+ ≥50 groups. Of note, if anything the ratio of G1:S was lower in the CD8+HLA-DR+ cells from the HIV+ ≥50 group (median: 3.0, range: 1.8–5.3) than in the other two populations of subjects (control group, median: 5.8, range: 3.0–7.8; and HIV+ <50 group, median: 5.5, range: 2.5–9.5). Therefore, it is unlikely that cell cycle arrest was induced at this stage of the cell cycle in the CD8+HLA-DR+ cells as a consequence of HIV-1 infection.
Figure 5. The peripheral pool of CD8+HLA-DR+ cells contains more cycling cells than that of CD8+HLA-DR− naive and memory cells. Cell cycle analysis was performed by flow cytometry using simultaneous staining with 7-AAD (DNA) and pyronin Y (PY, RNA). Definitions of G0, early G1, late G1, and S+G2/M phases of the cell cycle are described in Materials and methods and the diagram on the right helps identify positions of G0, early G1, late G1, and S+G2/M of the cell cycle. The values in the plots indicate the percentages of cells in the regions corresponding to G0, early G1, late G1, and S+G2/M. Representative patterns from two different donors are shown for control (n = 5), HIV+ <50 (n = 6), and HIV+ ≥ 50 (n = 8) groups.
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Figure 6. The proliferative capacity and death rates of CD8+HLA-DR− naive, CD8+HLA-DR− memory, and CD8+HLA-DR+ cells. DNA and RNA content were determined for cells freshly isolated by microbeads (Day 0) and for cells harvested following 1–3 days of culture with medium alone or in the presence of anti-CD3+CD28. The regions corresponding to G0, early G1, late G1, and S+G2/M are the same as those in Fig. 5. The percentages of dead cells were determined by flow cytometry with Live/Dead cell staining dye. Plots shown are from an HIV-infected individual with HIV-RNA <50 copies/mL representative of three such individuals. Gating strategy used to analyze the cell cycle status is shown in Supporting Information Fig. 8.
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