Figure 7.

Blockade of FoxO1 impairs the immune regulatory effects of GSK

(A and B) CD4 T cells were isolated from C57BL/6 mice and stimulated by anti-CD3/28 in the presence of GSK and/or HS or not for 3 days (A) Western blot analysis was employed to assess the expression levels of Glut1, HK2, LDHA, and FoxO1 in CD4 T cells from C57BL/6 mice (n = 3). (B) Quantitative analysis of Glut1, HK2, LDHA, and FoxO1 expression in CD4 T cells from C57BL/6 mice (n = 3). (C, D) CD4 T cells were isolated from peripheral blood of RA patients and stimulated by anti-CD3/28 in the presence of GSK and/or HS for 3 days.

(C) Western blot analysis was employed to assess the expression levels of Glut1, HK2, LDHA, and FoxO1 in CD4 T cells from RA patients (n = 3).

(D) Quantitative analysis of Glut1, HK2, LDHA, and FoxO1 expression in CD4 T cells from RA patients (n = 3).

(E–J) Naive CD4 T cells from C57BL/6 mice were polarized into Th17 cells or Treg cells in the presence of different combinations of HS, GSK, 2-DG and iFoxO1 for 3 days. For Treg stability assay, naive CD4 T cells were initially cultured for 3 days under Treg polarization condition. Then, Treg cells were harvested, washed and cultured for an additional 2 days under Th17 condition in the presence of HS, GSK, 2-DG or iFoxO1. (E, H) The proportion of Th17 cells within CD4 T cells following distinct stimulation (n = 3). (F, I) The proportion of Treg cells within CD4 T cells following distinct stimulation (n = 3). (G, J) The proportion of IL-17A producing cells within total Foxp3⁺ Treg cells following distinct stimulation (n = 3). Statistical significance was calculated by unpaired Student’s t test and data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.