(A) Naive T cells were activated with anti-CD3/28 for 1, 2, or 3 days and were CFSE labeled and cultured with or without oligomycin. Proliferation was measured by CFSE dilution at 24 and 72 hr post-CFSE labeling.
(B) Naive T cells were activated with anti-CD3/28 for 3 days. Cells were then CFSE labeled and cultured with mitochondrial inhibitors. Proliferation was measured by CFSE dilution at 24, 48, and 72 hr post-CFSE labeling (left), and OCR and ECAR were measured at 72 hr after culturing with or without mitochondrial inhibitors (O, oligomycin; R/A, rotenone and antimycin-A) (right).
(C) Naive T cells were activated with anti-CD3/28 in glucose medium for 3 days, followed by CFSE labeling. The CFSE-labeled activated cells were then cultured in medium supplemented either with glucose (+Glc), glucose plus 1 μM oligomycin (Glc+Oligo), without glucose −Glc), or with galactose (+Gal), or galactose plus 4 nM oligomycin (Gal+Oligo). CFSE dilution was measured at 24 and 72 hr post-CFSE labeling.
(D) Live-cell (7-AAD−) numbers of activated CD4+ T cells were determined at 72 hr and normalized to the number of live cells cultured in glucose medium at 0 hr.
(E and F) T cells were activated with anti-CD3/28 in glucose medium for 3 days. OCR and ECAR were measured 1 hr after these cells were switched into media with glucose (+Glc) or without glucose (−Glc) or with galactose (+Gal).
Data are representative of at least three independent experiments (A), (B, left), and (C) or of two independent experiments and depict mean ± SEM (error bars) of quadruplicates (B, right), (D), (E), and (F); *p = 0.0001. See also .