Fig. 3: CD8⁺ T cells from mice with a constitutive activation of PI3Kγ lipid kinase displays enhanced PIP3 formation, Akt phosphorylation and cellular metabolism toward effector functions.

a, b Immunocytochemistry representation (b) of PIP3⁺ (green) expression in CD8⁺ (red) T cells isolated from the spleen of PI3Kγ^+/+ and PI3Kγ^CX/CX mice and plated onto anti-CD3 and anti-CD28 coated wells. Nuclei are stained by DAPI and visualized in blue. b Quantitative analysis of the % of PIP3⁺ CD8⁺ T cells was performed in n = 3 PI3Kγ^+/+ and n = 4 PI3Kγ^CX/CX mice. Images were acquired by confocal microscopy at 40X magnification (scale bar = 20 µm). c–f Western blot analysis of Akt phosphorylation in Thr308 (c) and Ser473 (e) in CD8⁺ T cells isolated from the spleens of PI3Kγ^+/+ and PI3Kγ^CX/CX mice. Representative blot (c, e) and quantitative analysis of phosphorylated Akt at Thr308 (d) and Ser473 (f) as fold changes over β-actin levels, in n = 6 PI3Kγ^+/+ and PI3Kγ^CX/CX mice in d, and n = 8 PI3Kγ^+/+ and PI3Kγ^CX/CX mice in (f). Metabolic-flux analysis by the Seahorse XF Analyzer was used to measure the mitochondrial respiration and the aerobic glycolysis by the oxygen-consumption rate (OCR) (g–j) and extracellular acidification rate (ECAR) (k–m) under basal conditions and after drug-induced mitochondrial inhibition in CD8⁺ T cells purified from the spleen of PI3Kγ^CX/CX and PI3Kγ^+/+ mice. For g–m, n = 9 PI3Kγ^+/+ and n = 8 PI3Kγ^CX/CX animals were analyzed and measured in triplicates. All data are expressed as mean ± SEM. Statistical analysis was performed by unpaired Mann–Whitney test in b; two-sided unpaired T-test in d, f, h, i, j, l, m. N represents biologically independent samples. Source data are provided as a Source Data file.