Fig. 6.

NNK promotes immunosuppression through IDO1-mediated Trp metabolism. a A/J mice were treated with NNK for 90 days and sacrificed, and CD45⁺ cells were isolated from lung tissues and analyzed by flow cytometry. b–e CD8⁺ T cells were harvested from C57 mice, cultured alone or in the presence of siIDO1-transfected LLC cells in Trp-free or Trp normal medium. The cells were treated with NNK at 25 μM for 72 h. Cell division was evaluated by cytometric analysis of 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (b, c), and cell death was assessed by propidium iodide (PI) and flow cytometry assay (d, e). f, g The death rate of siIDO1-transfected LLC-OVA cells cultured alone or cocultured with OT-1 CD8⁺ T cells in Trp-free or Trp normal medium in the absence or presence of NNK at 25 μM for 48 h. h, i CD4⁺ T cells were harvested from C57 mice and cultured in the absence or presence of siIDO1-transfected LLC cells in Trp-free or Trp normal medium. The cells were treated with NNK at 25 μM for 72 h. The percentage of Tregs in CD4⁺ cells was assayed by flow cytometry. j, k A/J mice were treated with NNK and/or IDO1 inhibitor 1-methyl-L-tryptophan (1-MT) for 90 days and sacrificed, and CD8⁺ T cells (j) and Tregs (k) in lung tissues were analyzed by flow cytometry. l, m A/J mice were treated with NNK and/or α-BGT for 90 days and sacrificed, and CD8⁺ T cells (l) and Tregs (m) in lung tissue were analyzed by flow cytometry. P values, Student’s t test. Error bars, sd