Extended Data Fig. 6: T cells exposed to pEtn have an altered intracellular lipid phenotype.
From: Tumour interstitial fluid-enriched phosphoethanolamine suppresses T cell function
A) PCA analysis of lipidomics performed in 4I-O and S6B B) Heatmap showing Log2FC over mean for each lipid measured in lipidomics described in Fig. 4i-o). C-E) Lipidomics analysis of activated OT-I T cells cultured in either RPMI or RPMI supplemented with pEtn. C) Ratio of phosphatidylethanolamine (PE) to Phosphatidylcholine (PC) (p-value p = 0.0142). Boxes range from 25th to 75th percentiles, while the whiskers extend to the minimum and maximum value for each dataset. Fold change of D) PE species and E) PC species in pEtn treated OT-I T F-H) Cells treated with F) pEtn, G) Choline, or H) pCholine at TIFM-levels for 3 or 5 days and analyzed via lipidomics as described in Fig. 4i-o). Each volcano plot shows Log2 fold change of treated cells over RPMI-treated controls on the x axis, and Log10 p-value on the y axis. Red dots indicate DAG species, tan dots are PE species, blue dots are PC species, and purple dots are sphingomyelins. I) DAG levels in OT-I T cells that were cultured as described in 4I-O). Normalized DAG MFI is quantified on the left from six independent experiments (n = 18 mice/group, RPMI vs. pEtn p = 0.0037, RPMI vs. Chol p = 0.8314, RPMI vs. pChol p = 0.9923) and statistical analysis for all indicated comparisons in was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. J) RNA expression of choline and choline-like transporters in PD1+Tim3+ tumor-infiltrating CD8 + T cells. Data retrieved from previous report46. ND = transcripts not detected. K-M) Representative flow plots for quantification shown in Fig. 4 Panels Q-S). N) Western blot for Pcyt2 and β-Actin on Day 7 WT and Pcyt2 KO CD8 T cells from Fig. 4p-s. (n = 4 mice/group from 3 independent experiments) Statistical significance for indicated comparison in ED6C) quantified using a two-tailed paired t-test (p = 0.0098). Proteins were probed across multiple independent blots that were loaded with the same original proteins. Source numerical data are available in source data.
