Figure 6.

Effect on Th17-cell generation and effector T-cell proliferation by CD8⁺CD122⁺ T cells in vitro. (A) Effect on Th17-cell generation by coculturing CD8⁺CD122⁺ T cells with effector CD4⁺CD25⁻ T cells. Naïve CD4⁺CD25⁻ T cells from Ly5.1 C57BL/6 mice were stimulated under Th17 conditions. Different ratios of effector cells to CD8⁺CD122⁺ T cells either from naïve Ly5.2 wild type C57BL/6 (Ly5.2 WT) or from Ly5.2 IL-15 transgenic C57BL/6 mice (Ly5.2 IL-15-Tg) were cocultured with CD4⁺CD25⁻ T cells for 4 days. The percentage of TM7 cells was detected by intracelluar staining for IL-17A by flow cytometry. (B) Effect on Th17-cell generation by naive CD8⁺CD122⁺ T cells in vitro. Instead of coculture with CD4⁺CD25⁻ T cells for 4 days, the CD8⁺CD122⁺ T cells from Ly5.2 IL-15-Tg mice were provided on day 3 and continued with the following procedures to generate and detect Th17 cells (CD4⁺CD25⁻/naïve CD8⁺CD122⁺). Selected groups were cultured with CD8⁺CD122⁺ T cells for 4 days along with 10 μg/mL anti-IL-10 (CD4⁺CD25⁻+activated CD8⁺CD122⁺+anti-IL-10). The percentage of Th17 cells was detected by intracellular staining for IL-17A by flow cytometery. Data shown are from single experiments representative of three experiments performed. (C) Effect of CD8⁺CD122⁺ T cells on IL-17 secretion by CD4⁺CD25⁻ T cells. Naïve CD4⁺CD25⁻ T cells from Ly5.1 C57BL/6 mice were stimulated under Th17 conditions, CD8⁺CD122⁺/CD8⁺CD122⁻ T cells either from naïve Ly5.2 wild type C57BL/6 (WT) or from Ly5.2 IL-15 transgenic mice (IL-15 Tg) were cocultured with CD4⁺CD25⁻ T cells for 4 days at a 1:1 ratio. Supernatants were collected and IL-17 was detected by ELISA. In selected groups, 10 μg/mL of anti-IL-10 was added at the initiation of the incubation. *p < 0.05 compared to effector cells alone group analyzed by Student’s t-test. (D) Effect of CD8⁺CD122⁺ T cells on the proliferation of CD4⁺CD25⁻ T-cell proliferation upon anti-CD3 stimulation. Different ratios of effector cells to potential regulatory cells (CD8⁺ CD122⁺ or CD8⁺ CD122⁻ T cells) were cocultured with naïve CD4⁺CD25⁻ T cells upon plate bound anti-CD3 stimulation for 72 h, cell proliferation was measured in the final 6 h by ³H-TdR incorporation. (C, D) Data are shown as mean ± SD of three samples from a single experiment representative of three independent experiments performed. *p < 0.05 compared to effector cells alone group (Student’s t-test).