Article Title: Glycolysis Fuels Phosphoinositide 3-Kinase Signaling to Bolster T Cell Immunity
Figure Lengend Snippet: Reduced glycolytic ATP production accounts for defective PI3K/Akt/Foxo1 signaling in LDHA-deficient T cells following antigen stimulation ( A ) A schematic depicting how naïve CD8 + T cells were treated for assessment of kinase signaling and PIP3 generation. ( B ) Naïve CD8 + T cells were isolated from Ldha fl/fl (wild-type, WT) and CD4 Cre Ldha fl/fl (knockout, KO) mice, and stimulated with 5 μg/ml anti-CD3, 2 μg/ml anti-CD28, and 100 U/ml IL-2 for 3 days. Day-3 activated T cells and freshly isolated naïve WT and KO CD8 + T cells were left untreated, or labeled with biotinylated anti-CD3 and anti-CD28 and re-stimulated by streptavidin crosslinking. Cell lysates were prepared and immunoblotted for p-Akt (T308), Akt, p-Foxo1 (T24), p-Foxo1 (S256), Foxo1, LDHA, p-Zap70 (T319), Zap70, p-LAT (T191), LAT, p-AMPKα (T172), AMPKα, and β-Actin. Normalized expression of p-Akt (T308) to Akt, p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1, p-Zap70 (T319) to Zap70, p-LAT (T191) to LAT, and p-AMPKα (T172) to AMPKα were marked. ( C ) Day-3 activated WT and KO CD8 + T cells were collected, rested in RPMI medium for 3 hours, and incubated in the absence or presence of 10 mM ATP and/or 250 U/ml Streptolysin O (SLO). T cells were subsequently re-stimulated with biotinylated anti-CD3 and anti-CD28 through streptavidin crosslinking, and immunoblotted for p-Akt (T308), Akt, p-Foxo1 (T24), p-Foxo1 (S256), Foxo1, LDHA, and β-Actin. Normalized expression of p-Akt (T308) to Akt, p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1 were marked. ( D ) Representative immunofluorescent images of PIP3 and its quantification in WT and KO CD8 + T cells after receiving the indicated treatments as described in ( C ). Unpaired t tests for the measurements between the two groups ( D ): **p
Article Snippet: Antibodies for in vitro T cell culture: Monoclonal anti-mouse CD28-biotin (Clone 37.51; Cat. # 13-0281-81), monoclonal anti-mouse and CD3ε-biotin (Clone 145-2C11; Functional Grade; Cat. # 36-0031-85) were purchased from Invitrogen.
Techniques: Isolation, Knock-Out, Mouse Assay, Labeling, Expressing, Incubation