To meet the demand for energy and biomass, T lymphocytes (T cells) activated to proliferation and clonal expansion, require uptake and metabolism of
glucose (Gluc) and the
amino acid (AA)
glutamine (Gln). Whereas exogenous Gln is converted to
glutamate (Glu) by
glutaminase (GLS), Gln is also synthesized from the endogenous pool of AA through Glu and activity of
glutamine synthase (GS). Most of this knowledge comes from studies on cell cultures under ambient
oxygen conditions (normoxia, 21% O2). However, in vivo,
antigen induced T-cell activation often occurs under moderately hypoxic (1-4% O2) conditions and at various levels of exogenous nutrients. Here, CD4+ T cells were stimulated for 72 h with
antibodies targeting the CD3 and CD28 markers at normoxia and
hypoxia (1% O2). This was done in the presence and absence of the GLS and
GS inhibitors, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl
sulfide (BPTES) and
methionine sulfoximine (MSO) and at various combinations of exogenous Gluc, Gln and
pyruvate (Pyr) for the last 12 h of stimulation. We found that T-cell proliferation, viability and levels of endogenous AA were significantly influenced by the availability of exogenous Gln, Gluc and Pyr as well as inhibition of GLS and GS. Moreover, inhibition of GLS and GS and levels of
oxygen differentially influenced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Finally, BPTES-dependent down-regulation of ECAR was associated with reduced
hexokinase (HK) activity at both normoxia and
hypoxia. Our results demonstrate that Gln availability and metabolism is rate-limiting for CD4+ T-cell activity.