Fig. 6: Lysophosphatidic acid shifts metabolism to consume fatty acids for oxidation.

A–D Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) by both naïve and effector CD8 T cells given media without LPA (RPMI+Glutamine) or treated with 1 µM LPA for 30 min, 2 h, or 4 h prior to starting the Seahorse metabolic flux assay. Assay was performed with injections of oligomycin (oligo), (4-(trifluoromethoxy) phenyl) carbonohydrazonoyl dicyanide (FCCP), antimycin A (ant), and rotenone (rot) at 18-min intervals in media supplemented with 25 mM glucose. Data are representative and show n = 6 technical replicates. E–H Capacity calculations from Seahorse metabolic flux assay showing basal respiration, maximal respiration, ATP-linked production, and proton leak. Data show n = 5 independent experiments with technical replicate error propagated into biological replicate error. Exact p-values for (E) are as follows, RPMI+Glutamine vs 4 h LPA p = 0.0149. Exact p-values for (F) are as follows, RPMI+Glutamine vs 2 h LPA p = 0.0455; RPMI+Glutamine vs 4 h LPA p = 0.0489. Exact p-values for (G) are as follows, RPMI+Glutamine vs 30 min LPA p = 0.0491. Exact p-values for panel (H) are as follows, 30 min LPA vs 4 h LPA p = 0.0129; 2 h LPA vs 4 h LPA p = 0.0347. I, J Flow cytometric analysis of BODIPY in effector CD8 T cells given media without LPA (RPMI+Glutamine) or treated with 1 µM LPA for 30 min, 2 h, or 4 h. I Shows representative histogram and (J) shows quantitative analysis of normalized geometric mean fluorescence intensity (gMFI) across n = 3 independent experiments with 3 mice per group. Exact p-values are as follows, RPMI+Glutamine vs 2 h LPA p = 0.0002; RPMI+Glutamine vs 4 h LPA p = 0.0002. K Seahorse metabolic flux analysis performed with acute injection of etomoxir to a final concentration of 1 µM. Effector CD8 T cells were cultured in normal media (RPMI+Glutamine) or 1 µM LPA for 4 h prior to starting the assay. n = 6 technical replicates. Exact p-values are as follows, at t = 60 min RPMI+Glutamine vs 4 h LPA p = 0.0049; 4 h LPA vs 4 h LPA + Etomoxir p = 0.0044, at t = 66 min RPMI+Glutamine vs 4 h LPA p = 0.0045; 4 h LPA vs 4 h LPA + Etomoxir p = 0.0042, at t = 72 min RPMI+Glutamine vs 4 h LPA p = 0.0030; 4 h LPA vs 4 h LPA + Etomoxir p = 0.0030. Statistics for (E–K) were performed using an ANOVA statistical test with a Tukey’s post-hoc analysis was performed where *p < 0.05, **p < 0.005, and ***p < 0.0005. Error bars for panels (A–H, J, K) represent standard error of the mean.