Fig. 1. Activated characteristics of CCR9⁺ T cells in spleen and 4T1 tumors.

Spleens and tumors were harvested from 4T1 tumor–bearing mice when tumor volume was about 500 mm³ and analyzed for surface CCR9 expression in splenic lymphocytes and TILs. Spleen cells from normal BALB/c mice were used as controls. (A) Representative flow cytometry plots demonstrating the CCR9⁺ cells in CD4⁺, CD8⁺, CD49b⁺, and CD19⁺ cells in CD45⁺ cells in the spleens and tumors. (B) Frequencies of CD4⁺ T cells, CD8⁺ T cells, B cells, and NK cells in CD45⁺ cells in spleens and 4T1 tumors. (C) Frequencies of CCR9⁺ cells in CD4⁺ T cells, CD8⁺ T cells, B cells, and NK cells in the spleens and 4T1 tumors. (D) Frequencies of CCR9⁺ cells, CCR9⁺CD4⁺ T cells, CCR9⁺CD8⁺ T cells, CCR9⁺ B cells, and CCR9⁺ NK cells in CD45⁺ cells in the spleens and 4T1 tumors. (E and F) Representative flow cytometry plots (E) for gating strategy to define frequencies (F) of CD62L⁻CD44^hi cells in CCR9⁺CD8⁺, CCR9⁺CD4⁺ (CCR9⁺CD3⁺CD8⁻) and CCR9⁻CD8⁺, and CCR9⁻CD4⁺ (CCR9⁻CD3⁺CD8⁻) T cells isolated from the spleens harvested from normal BALB/c mice. (G and H) CCR9⁻CD3⁺ T cells were purified from the spleen of normal BALB/c mice and activated for 5 days by plating 2 × 10⁵ cells in plates coated with CD3 and CD28 antibodies. Representative flow cytometry plots showing CCR9 expression on CD3⁺ T cells (G) and frequencies of CCR9⁺ cells in CD3⁺ T cells (n = 4 per group) (H) at 0, 72, 96, and 120 hours after activation. (I and J) Representative flow cytometry plots (I) and frequencies (J) CD62L⁻CD44^hi cells in CCR9⁺CD8⁺ and CCR9⁻CD8⁺ T cells in the spleens and 4T1 tumors when tumor volumes were about 500 mm³ (n = 3 to 4 per group). (K and L) CCR9⁺CD8⁺ and CCR9⁻CD8⁺ T cells were prepared from the spleens (SP) of normal BALB/C mice and the spleens and tumors of 4T1 tumor–bearing BALB/c mice (tumor volumes were about 500 mm³) and analyzed for PD-1 expression by flow cytometry. (K) Representative flow cytometry profiles showing PD-1 expression in gated CCR9⁺CD8⁺ and CCR9⁻CD8⁺ T cells. (L) Frequencies of PD-1⁺ cells in CCR9⁺CD8⁺ and CCR9⁻CD8⁺ T cells (n = 3 to 4 per group). Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P< 0.001; ****P < 0.0001.