Figure 1

Activated CD8 ⁺ T cells release glutamate putatively by a vesicular mechanism. (A, B) Supernatant glutamate concentrations rise exponentially with time (A; bead-to-cell ratio of 1:1, medium containing 2 mM glutamine; n = 3 mice, experiments performed in triplicates) and depend on the bead-to-cell ratio as a measure of stimulation intensity (B; stimulation for 72 hours, medium containing 2 mM glutamine; n = 3 mice, experiments performed in triplicates) during polyclonal stimulation of WT CD8⁺ T cells with anti-CD3/28 beads in vitro. (C) Glutamate liberation by WT CD8⁺ T cells depends on extracellular glutamine (Gln) and is not mediated by a known non-vesicular release mechanism (that is, system X_c-glutamate-cystine exchanger blocked by L-AAA (2.5 mM), volume-sensitive anion channels blocked by DCPIB (5 μM), excitatory amino acid transporters blocked by TBOA (500 μM), connexin hemi-channels blocked by carbenoxolone (10 μM), or P₂× receptors blocked by PPADS (100 μM); stimulation for 72 hours at a bead-to-cell ratio of 1:1, medium containing either 2 or 0 mM glutamine; n = 3 mice, experiments performed in triplicates). (D) Consistent with a vesicular mechanism of glutamate liberation, activated WT CD8⁺ T cells upregulate expression levels of glutaminase, vesicular proton ATPase (H⁺ ATPase), and vesicular glutamate transporters (VGluT 1 to 3) as revealed by quantitative RT-PCR (n = 3 mice, experiments performed in triplicates).