Figure 2.

Lactate Uptake by CD4⁺ T Cells Impacts Intracellular Utilization of Central Carbon Metabolic Pathways

(A) Glucose and glutamine uptake rates for CD4⁺ T cells isolated from HC PBMCs, then activated with anti-CD3 and anti-CD28 mAbs for 24 h followed by further 48-h culture with lactate alone or in the presence of SLC5A12 Ab, or left untreated, in medium containing low glucose (5 mM) and 5% FBS (n = 3, each in duplicate).

(B) NAD⁺ and NADH intracellular levels in CD4⁺ T cells (n = 2) treated with sodium lactate (10 mM) for the indicated time points after 72-h activation and shown as NAD⁺/NADH ratio. Lactate-untreated CD4⁺ T cells (CN, dotted line) set to 1.

(C) Seahorse measurements of extracellular acidification (left) and oxygen consumption (right) rates (ECAR and OCR, respectively) by 12-h-activated CD4⁺ T cells (n = 3, technical replicates). 1 h prior to the experiment, cells were seeded in a 96-well microplate in XF Assay medium in the presence of 10 mM of glucose. Sodium lactate (10 mM) or PBS was injected during measurement. Data representative of n = 2 independent experiments.

(D and E) ¹³C tracing of [U¹³C]-lactate into pyruvate and citrate. Activated CD4⁺ T cells were incubated for 48 h with [U¹³C]-lactate in the presence or absence of SLC5A12 Ab in medium containing low glucose (5 mM) and 5% FBS (n = 2, each in duplicate). Polar metabolites were extracted, analyzed by LC-MS and peak areas of mass isotopologues normalized to cell number are represented.

(F and G) Acetyl-CoA (F) and citrate (G) intracellular levels in CD4⁺ T cells (n = 3) treated with sodium lactate (10 mM) for the indicated time points after 72-h activation. Lactate-untreated CD4⁺ T cells (CN, dotted line) set to 1.

Two tailed Student’s t test. Data expressed as mean ± SEM. ^∗p ≤ 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001.