Figure 3. Glycolysis Modulates the Ca²⁺-NFAT1 Signaling Pathway in CD4+ T Cells.

(A) Naive CD4+ T cells from Nur-77-eGFP mice were left unstimulated or stimulated with anti-CD3/anti-CD28 mAbs for 5 hr in the indicated conditions and GFP fluorescence was measured by flow cytometry. Glc.: glucose; 2-DG: 2-deoxy-D-glucose.

(B and C) Intracellular Ca²⁺ levels were measured in Fluo-4- and Fura-Red-labeled TH1 CD4+ T cells cultured in 10 mM glucose or 0.1 mM glucose before and after activation with anti-CD3 crosslinking antibodies (B) or ionomycin (C). The ratio of Fluo-4 and Fura-Red fluorescence was measured using flow cytometry.

(D) Intracellular Ca²⁺ levels were measured as above in naive CD4⁺ T cells isolated from wild-type (Wt) or GLUT-1-knockout (Glut1-KO) mice.

(E) T_H1 CD4⁺ T cells were stimulated with anti-CD3/anti-CD28 in the indicated conditions and amounts of phospho-PLCγ1 were measured by flow cytometry.

(F) T_H1 cells were stimulated with ionomycin in medium containing 10 mM or 0.1 mM glucose for 10 min and the cytoplasmic versus nuclear distribution of NFAT1 and NFAT2 was determined by Amnis Imagestream. Representative histograms and images show the similarity profiles of NFAT1 (yellow, left) or NFAT2 (red, right) with DAPI staining to measure nuclear localization. The percentage of T cells with nuclear NFAT1 or NFAT2 is shown.

(G) Heat map shows normalized expression of select genes associated with T cell anergy (Safford et al., 2005) in T_H1 cells stimulated for 5 hr with anti-CD3/anti-CD28 mAbs in glucose-sufficient (10 mM) or glucose-deficient (0.1 mM) conditions.

Data shown are representative of two (D and E) and three (A–C, F) independent experiments or cumulative of three (G) independent experiments (n = 2 mice/group).