Fig. 7: Lysophosphatidic acid receptor 5 modulates metabolic adaptability and efficiency in effector CD8 T cells. | Nature Communications

Fig. 7: Lysophosphatidic acid receptor 5 modulates metabolic adaptability and efficiency in effector CD8 T cells.

From: Lysophosphatidic acid modulates CD8 T cell immunosurveillance and metabolism to impair anti-tumor immunity

Fig. 7

A, B Seahorse metabolic flux assay performed on effector CD8 T cells from (A) an OT-I mouse or Lpar5−/− OT-I mouse in the absence of LPA treatment and (B) given normal media without LPA (RPMI+Glutamine), treated with 1 µM LPA for 4 h prior to starting the assay, or co-treatment of 1 µM LPA and the LPA receptor antagonist (TC LPA5 4 at 1 µM) for 4 h prior to starting the assay. Data are representative and show n = 6 technical replicates. C–G Capacity calculations from Seahorse metabolic flux assay showing (C) ratio of maximal respiratory capacity / basal respiratory capacity where exact p-values are as follows, OT-I vs Lpar5−/− OT-I p = 0.0026; Lpar5−/− OT-I vs OT-I 4 h LPA p = 0.0015; Lpar5−/− OT-I vs OT-I 4 h LPA + TC LPA5 4 p = 0.0002, D basal respiration where exact p-values are as follows, OT-I vs Lpar5−/− OT-I + 4 h LPA p = 0.048; OT-I vs OT-I 4 h LPA p = 0.0052; Lpar5−/− OT-I vs Lpar5−/− OT-I + 4 h LPA p = 0.0010; Lpar5−/− OT-I vs OT-I 4 h LPA p = 0.0010, (E) maximal respiration where exact p-values are as follows, OT-I vs Lpar5−/− OT-I p = 0.0052; OT-I vs Lpar5−/− OT-I + 4 h LPA p = 0.07; OT-I vs OT-I 4 h LPA p = 0.0055; Lpar5−/− OT-I + 4 h LPA vs OT-I 4 h LPA p = 0.0049; Lpar5−/− OT-I + 4 h LPA vs OT-I + 4 h LPA + TC LPA5 4 p = 0.0042; OT-I 4 h LPA vs OT-I 4 h LPA + TC LPA5 4 p = 0.0049, (F) ATP-linked production where exact p-values are as follows, OT-I vs Lpar5−/− OT-I 4 h LPA p = 0.0001; Lpar5−/− OT-I vs Lpar5−/− OT-I + 4 h LPA p = 0.0041; Lpar5−/− OT-I + 4 h LPA vs OT-I + 4 h LPA p = 0.0050; Lpar5−/− OT-I + 4 h LPA vs OT-I + 4 h LPA + TC LPA5 4 p = 0.0050, and (G) proton leak where exact p-values are as follows, OT-I vs Lpar5−/− OT-I p = 0.0080; OT-I vs OT-I + 4 h LPA p = 0.0050; Lpar5−/− OT-I vs OT-I + 4 h LPA p < 0.0001; Lpar5−/− OT-I + 4 h LPA vs OT-I + 4 h LPA p = 0.0001; OT-I + 4 h LPA vs OT-I + 4 h LPA + TC LPA5 4 p < 0.0001. Data show n = 3 independent experiments with technical replicate error propagated into biological replicate error. H, I Seahorse metabolic flux assay on effector CD8 T cells from an Lpar5−/− OT-I mouse 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 assay. Data are representative and show n = 6 technical replicates. J–M Capacity calculations from Seahorse metabolic flux assay showing basal respiration, maximal respiration, ATP-linked production, and proton leak. Data show n = 3 independent experiments with technical replicate error propagated into biological replicate error. Exact p-values for (J) are as follows, RPMI+Glutamine vs 4 h LPA p = 0.0328. Exact p-values for (L) are as follows, RPMI+Glutamine vs 30 min LPA p = 0.0495; RPMI+Glutamine vs 2 h p = 0.0485; RMPI+Glutamine vs 4 h LPA p = 0.0215. Statistics for this entire figure were performed using an ANOVA statistical test with a Tukey’s post-hoc analysis was performed where *p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001. Error bars for panels (A-M) represent standard error of the mean.

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