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

Figure 3

Interleukin-21 efficiently induces the production of epitope-specific CD8⁺ T memory stem cells. (a) Schematic diagram of the generation of HIV-1 antigen-specific 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; ovalbumin [OVA]_257–264 peptide alone served as the negative control peptide, abbreviated as NP) 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. (b) Flow cytometric analysis of the frequencies of antigen-specific CD8⁺ T_SCMs that were expanded from HIV-1-infected individuals in the presence of IL-15 (top panel) or IL-21 (bottom panel). Dot plots show the identification of HIV-1 antigen-specific CD95⁺CD122⁺ T_SCMs (based on a tetramer⁺CD8⁺CD45RA⁺CD45RO⁻CD62L⁺CCR7⁺ phenotype for T_SCMs stimulated with specific peptides or a CD8⁺CD45RA⁺CD45RO⁻CD62L⁺CCR7⁺ phenotype for T_SCMs stimulated with NP). The numbers in the graphs show the percentages of CD8⁺ T_SCMs after each treatment. The Kruskal–Wallis test with Dunn’s multiple comparisons was performed to assess statistical significance. The data are shown as the error of the mean (SEM) from five independent experiments. (c) Cytokine secretion profiles of HIV-1-specific CD8⁺ T_SCMs generated with IL-15 (top panel) or IL-21 (bottom panel). The CD8⁺ T_SCMs sorted by flow cytometry were cocultured with irradiated autologous APCs. Cell mixtures were incubated with NP, SL9, IL9, or TL9 (2 μM) and purified anti-CD28 antibody (1 μg/ml) and recombinant IL-2 (20 ng/ml) for 6 hours at 37°C, in the presence of brefeldin A (2 μg/ml) for the final 2 hours of incubation. The numbers in the graphs show the percentages of IFN-γ⁺ cells among tetramer⁺CD8⁺ T_SCMs after each treatment. The Kruskal–Wallis test with Dunn’s multiple comparisons was performed to assess statistical significance. The data are shown as the of five independent experiments. .

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