Figure 2.

GTP cyclohydrolase 1 (GCH1) increases regulatory T cells (Tregs) infiltration, reduces apoptosis, and increases the programmed cell death-1 (PD-1)⁺ fraction. Tumors were harvested from mice with orthotopic injection of a total of 3×10⁵ 4T1 cells expressing a control empty vector (Vec) or GCH1 (n=6 each). (A) Tumor volumes. (B) Representative dot plot of Tregs analyzed by flow cytometry and the corresponding quantitative results. (C) Representative dot plot of Tregs apoptosis analyzed by flow cytometry and the corresponding quantitative results. (D) Representative histograms of PD-1 on Tregs analyzed by flow cytometry and the corresponding quantitative results. (E, F, G, and H) Quantitative results of CD8⁺ T cell infiltration (E), apoptosis (F), PD-1 expression (G), and interferon-γ (IFN-γ) production (H) analyzed by flow cytometry. (I) Volumes of tumors from mice with orthotopic injection of a total of 3×10⁵ 4T1 cells expressing Vec or GCH1. The mice were treated with isotype IgG or an anti-CD25 antibody after the tumors were palpable. (J and K) Representative dot plot or histograms and the corresponding quantitative results of apoptosis (J) and PD-1 expression (K) of Tregs cocultured with BT549 cells expressing Vec or GCH1. The data are presented as the mean±SD (A, B, C, D, E, F, G, H, I, J, and K). Two-way analysis of variance (ANOVA) test for A and I; two-tailed unpaired Student’s t-test for B, C, D, E, F, G, H, J, and K; n=3 independent experiments (J and K). *P<0.05; **P<0.01; ***P<0.001; n.s., not significant. FOXP3, forkhead box protein P3; iso, isotype; SSC, side scatter.