Fig. 3

TNF-α-treated Th9 cells exhibit increased antitumor efficacy in vivo. (a) Naïve CD4⁺ T cells from OT-II mice were cultured under Th9 polarizing conditions with or without the addition of TNF-α for 2 days. C57BL/6 mice (five mice/group) were injected s.c. with 2 × 10⁵ B16-OVA cells. On Day 2 after tumor challenge, Th9 or TNF-α-treated Th9 cells (1 × 10⁶) were injected i.v. into the B16-OVA tumor-bearing mice. Mice treated by PBS served as controls. Shown are the tumor growth curves. The experiments were performed twice with a total of 10 mice per group (n = 10). (b-d) C57BL/6 mice were injected s.c. with 5 × 10⁵ B16-OVA cells. OT-II Th9 cells and TNF-α-treated Th9 cells were generated in vitro as in (A). On Day 5 after tumor challenge, mice (n = 3/group) were given i.v. with Th9 or TNF-α-treated Th9 cells (3 × 10⁶) or PBS control. On Day 3 after T cell transfusion, cells were isolated from tumor-draining lymph nodes (TDLNs). (b) Flow cytometry analysis of IL-9-producing CD4⁺ T cells. Numbers in the dot plots represent the percentages of double-positive T cells. Right, summarized results of three independent experiments obtained as at left. (c, d) CD4⁺ T cells were isolated from TDLNs by MACS. qPCR examined the expression of Il9 (c), Il5 and Il13 (d) in CD4⁺ T cells. (e) C57BL/6 mice were injected i.v. with 5 × 10⁵ B16-OVA cells. OT-II Th9 and TNF-α-treated Th9 cells were generated in vitro as in (a). On Day 5 after tumor challenge, mice (n = 3/group) were given i.v. with Th9 or TNF-α-treated Th9 cells (3 × 10⁶) or PBS control. On Day 3 after T cell transfusion, CD4⁺ T cells were separated from the lung tumor tissues by MACS. qPCR assessed the expression of Il9 in CD4⁺ T cells. Data are representative of three (b) independent experiments or presented as mean ± SD of three (b-e) independent experiments. *P < 0.05; **P < 0.01