S-phase entry leads to cell death in circulating T cells from HIV-infected persons

Abstract

Central memory T cells are thought to play a critical role in memory T cell homoestasis by undergoing self-renewal and by maturating into effector T cells that mediate immunity at tissue sites. Circulating T cells in S phase of the cell cycle are found at increased frequencies during HIV infection and are predominantly composed of cells with a central memory phenotype. Here, we tested the hypothesis that CD4 and CD8 S-phase T cells have different capacities to complete cell cycle and survive. S-phase T cells in peripheral blood from HIV-infected donors were identified by incubating whole blood with BrdU ex vivo. Upon in vitro cultivation, S-phase T cells were more likely to die than to complete mitotic division. Intrinsic differences were observed between CD4 and CD8 S-phase T cells during incubation. Higher frequencies of CD4+ S-phase T cell underwent apoptosis after incubation in medium alone or after TCR stimulation, and CD4+ S-phase T cells were less readily induced to proliferate after incubation with IL-2 than were CD8+ S-phase T cells. CD4+ and CD8+ S-phase T cells expressed low levels of Bcl-2, which could contribute to their heightened susceptibility to cell death. Intrinsic differences in the proliferation and survival of CD4+ and CD8+ S-phase T cells could influence the homeostatic maintenance of these T cell subsets in HIV disease.

Figures

Fig. 1

Differential requirements for proliferation of CD4 and CD8 S-phase T cells. Whole blood from a HIV-infected person was incubated with BrdU for 1 h at 37°C. PBMC were isolated and labeled with CFSE-tracking dye. Cells were incubated in medium alone (RPMI, 10% FBS, L-glutamine, and antibiotics) or in medium supplemented with rIL-2 (360 U/ml) or anti-CD3 antibody (100 ng/ml). Cells were assessed by flow cytometry for expression of CD4, CD8, BrdU, and CFSE after 3 days of incubation. For IL-2-treated cells, the numbers in parentheses represent the percentages of BrdU+ or BrdU− cells that diluted tracking dye.

Fig. 2

IL-2 induces S-phase T cell proliferation. Cells from HIV+ and HIV− donors were labeled with BrdU and then CFSE as described in Figure 1. Cells were incubated for 3 days with IL-2 (360 U/ml) before being assessed for cellular proliferation by flow cytometry. The average number of divisions among CFSElow S-phase cells, proliferation index (PI), or the average number of cell divisions among all S-phase cells, division index (DI), was determined with FlowJo software. Statistically significant differences are shown. Comparison between CD4+ and CD8+ S-phase cells within the same subject population was performed with paired Wilcoxon signed ranks test, and differences between subject categories were determined with Kruskal-Wallis (P<0.001) and Mann-Whitney tests; n = 16 for HIV+ donors, and n = 7 for healthy donors.

Fig. 3

Poor recovery and increased apoptosis among CD4 S-phase T cells after activation with anti-CD3 antibody. (A) Results from 16 HIV+ donors showing the difference in the percentage of CD4 or CD8 S-phase T cells derived from subtracting the percentage of S-phase T cells in medium alone from the percentage of S-phase T cells in cells cultured with anti-CD3 antibody. (B) A representative experiment demonstrating flow cytometric analyses of Annexin V binding among CD4 and CD8 S-phase (BrdU+) T cells after 2 days of incubation in medium alone or in medium treated with anti-CD3 antibody. The donor was HIV+ with a CD4 cell count of 847 cells/μl and a plasma HIV RNA level of 37,600 copies/ml.

Fig. 4

CD4 S-phase T cells die more rapidly than CD8+ S-phase T cells in vitro. PBMC derived from BrdU-labeled whole blood were incubated for indicated periods in medium alone or in medium with rIL-2 (360 U/ml) and assessed for the percentages of BrdU+ CD4+ and BrdU+ CD8+ T cells that bound Annexin V. The mean percentage of Annexin V+ S-phase T cells is shown after 18 h (n=10), 44 h (n=5), and 68 h (n=5) incubation. Apoptosis of CD4+ S-phase cells was significantly higher than apoptosis of CD8+ S-phase T cells at 44 h and 68 h (P<0.05; Wilcoxon signed ranks test) among cells incubated in medium alone.

Fig. 5

Low Bcl-2 expression in S-phase T cells and induction with IL-2 or anti-CD3 antibody stimulation. (A) Whole blood from a HIV-infected person was incubated 1 h with BrdU ex vivo, RBC were lysed with FACS Lyse solution, and cells were stained for surface expression of CD4 and CD8 as well as the intracellular expression of Bcl-2 and BrdU. The results shown are representative of four experiments with different HIV+ donors. (B) PBMC from ex vivo BrdU-labeled whole blood was incubated for 3 days in medium alone or in medium treated with rIL-2 or anti-CD3 antibody. Results are representative of three experiments.