Decreased IFN-γ production in AMPK KO T cells is cell-intrinsic. (A-C) WT or AMPK KO T cells were transplanted individually into irradiated B6D2F1 recipients, recovered on day 7, stimulated for 6 hours with fresh B6D2F1 splenocytes in the presence of monensin, and analyzed for intracellular cytokine production. Representative flow plots are shown in panel A, with the percentage of IFN-γ⁺ cells (B) and the median fluorescence intensity (MFI) of cells falling within the IFN-γ⁺ gate shown in panel C, respectively. (D-E) WT (CD90.1/2) and AMPK KO (CD90.2) T cells were transplanted separately or in a 1:1 combination (mixed) into irradiated B6D2F1 recipients and intracellular IFN-γ detected as outlined in panels A-C. Representative flow plots for individual and mixed samples are shown in panel D, whereas panel E represents the percentage of IFN-γ⁺ cells in CD4 vs CD8 T cells from multiple samples. (F-H) T cells were recovered from a separate cohort of B6D2F1 recipients on day 21 and analyzed by flow cytometry for CD4 vs CD8 expression (F), with CD4 percentages plotted for individual mice (G). CD4 and CD8 percentages were then multiplied by the total number of lymphocytes recovered to calculate the total number of CD4⁺ (left) and CD8⁺ (right) T cells on day 21 (H). n = 7 to 8 recipients per group for panels A-E, and n = 12 to 14 recipients per group for panels F-H. ∗P < .05; ∗∗∗∗P < .0001.