IL-21 Expands HIV-1-Specific CD8⁺ T Memory Stem Cells to Suppress HIV-1 Replication In Vitro

Figure 5

More robust suppression of HIV-1 production with IL-21-generated CD8⁺ T memory stem cells. (a) Schematic diagram of the transwell suppression assay for in vitro-generated CD8⁺ T memory stem cells (T_SCMs). Naïve CD8⁺ T cells (CD3⁺CD8⁺CD45RA⁺CD45RO⁻CD62L⁺CCR7⁺CD95⁻CD122⁻) were sorted from the PBMCs of HIV-1-infected individuals, and then, the cells (/ml) were mixed with irradiated autologous antigen-presenting cells (APCs; /ml). The cell mixtures were stimulated with a peptide cocktail (each peptide at a concentration of 2 μM) with interleukin- (IL-) 21 (20 ng/ml) or IL-15 (20 ng/ml) for 7 days. The medium and cytokines were refreshed every 2 days. After 1 week, the cell mixtures were stimulated with anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) antibodies with IL-21 (20 ng/ml) or IL-15 (20 ng/ml) for 14 days. The medium and cytokines were refreshed every 2 days. The polyclonal CD8⁺ T_SCMs () were then sorted and cocultured with autologous CD8⁺ T-lymphocyte-depleted PBMCs () that were activated with PMA (500 ng/ml) and ionomycin (1 μg/ml) in the presence of IL-2 (20 ng/ml). After 1 week, the mixtures were moved to the upper chambers of a transwell system and fresh CD4⁺ T cells, isolated from healthy donors, were activated with phytohemagglutinin (0.5 μg/ml) and IL-2 (20 ng/ml) and added to the lower chamber. The concentrations of HIV-1 p24 in the supernatants were tested by ELISA. (b) The inhibitory effect of CD8⁺ T_SCMs on the replication of HIV-1. Viral replication was monitored by the continuous detection of HIV-1 p24 antigen in the supernatant of each treatment group by ELISA. The data are shown as the error of the mean of three independent experiments ().

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