Fig. 2. Feedback regulation of T-cell glycolysis by pH establishes an acidic extracellular milieu at the steady-state.

a Time course of extracellular acidification rate (ECAR) was measured by Seahorse in B6 T-cells (Mean ± SD, n = 7 biological samples.). Injection of activating antibody (or vehicle for control) at 20 min evoked an increase in ECAR, due to the activation of T-cell glycolysis. b Proton production rate (PPR) measured by Seahorse in B6 or OT-II T-cells is reduced under acidic conditions. In paired experiments on B6 or OT-II T-cells, oxygen consumption rate (OCR), measured by Seahorse, is increased under acidic conditions (two-tailed, unpaired t-test, mean ± SD, n = 8 biological samples. PPR (B6, p = 1.35E-11; OT-II, p = 1.62E-11), OCR (B6, p = 2.22E-5; OT-II, p = 1.14E-5). Asterisks (***) represent p < 0.0001). c Glucose consumption and lactate production as a function of pHe in OT-I T-cells, expressed as mean ± SD; n = 3 biological samples. Significance tested by one-way ANOVA with multiple comparisons p < 0.001. d Schematic diagram of feedback loop between lactic acid production by glycolysis, and its inhibitory feedback by extracellular pH. e Time course of pHe measured in 60 µl volumes of 5 mM HEPES-buffered media containing no cells, nonactivated T-cells or activated (CD3-coated plates, then incubated in media containing 2 µg/ml CD28) T-cells at the densities indicated. n = 3 biological replicates. Data shown as mean ± S.E.M. Activated T-cells acidify the restricted extracellular volume towards pH 6.3 within several hours. f Schematic representation of mathematical model used to simulate the relationship between extracellular pH and lactate for a system featuring glycolytic lactic acid production and feedback inhibition by extracellular pH, as determined from panel e (i.e. linear inhibition towards zero production at pH 6.3), for a LN paracortex of intracellular volume fraction v_i, and fluid turnover (perfusion) of τ. (g) Results of simulation for extracellular pH (upper panel) and lactate (lower panel). Black line shows the combination of v_i and τ that simulates experimentally observed data for pHe (6.3; Fig. 1) and lactate (9.4 mM; Fig. S5). h Replotting of the best-fit curves from panel g. Red dashed line shows solution of this mathematical problem using the literature value for τ of 20 min. This indicates that ~70% of the paracortical zone is occupied by T-cells, engaged in lactic acid production, the source of low pHe measured in LNs.