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

Figure 4

IL-21-generated CD8⁺ T memory stem cells suppress HIV-1 production. (a) Schematic diagram of the suppression assay for in vitro-generated CD8⁺ 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. After 14 days, polyclonal CD8⁺ T_SCMs () were then sorted by flow cytometry. Autologous CD8⁺ T-lymphocyte-depleted PBMCs () that were activated with PMA (500 ng/ml) and ionomycin (1 μg/ml) plus with IL-2 (20 ng/ml) for 14 days and then cocultured with CD8⁺ T_SCMs. After 4 days of coculture, the HIV-1 production was tested by qRT-PCR. (b) HIV-1 replication was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). After various treatments for 4 days, HIV-1 viral production was determined by measuring cell-associated HIV-1 RNA by qRT-PCR (10³ copies/ cells). The data are shown as the error of the mean (SEM) of four independent experiments (). The Kruskal–Wallis test with Dunn’s multiple comparisons was performed to assess statistical significance. . (c) The antiviral capacity of CD8⁺ T_SCMs was dose-dependent. Purified CD8⁺ T_SCMs (effector cells, abbreviated as E) were cocultured with autologous CD8⁺ T-lymphocyte-depleted PBMCs (target cells, abbreviated as T). After treatment for 3 days, HIV-1 production was determined by relative quantification of cell-associated HIV-1 RNA normalized to a CD4 gene level. The data are shown as the of four independent experiments (). The slopes were compared using a Wilcoxon rank test. , .

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