Fig. 3: Prostaglandin E₂ regulates mitochondrial content of intestinal LP and IEL-isolated CD8⁺ T cells.

a Mitotracker green in CD8⁺CD69⁺CD103^+/− cells isolated from the tissues of unchallenged mice. Lines in the dot plot show mean values and the data show n = 3 biological replicates over two independent experiments. b Volcano plot shows the differential distribution of metabolites isolated from the interstitial fluid of tissues. Dashed lines indicate the fold change filter of FC > 2 and FC < −2 and the p value filter of p = 0.05. Plot shows cumulative data of four independent experiments. Statistics were performed using Student’s t test between metabolites identified in the small intestine and metabolites identified in all the other tissues. No FDR correction was applied. c Relative abundance of PGE₂ in interstitial fluid isolated from the indicated tissues. Graph shows mean values ± SEM of cumulative data of four independent experiments. d Mitotracker green and TMRM in CD8⁺ T cells activated in (IL-15/TGF-β)-T cell-polarizing conditions for 5 days and treated for 24 h with 10 μM PGE₂. Lines in the dot plot show mean values and the data show n = 3 biological replicates over three independent experiments. Statistics were performed using two-tailed Student’s t test. e Mitotracker green and TMRM staining in CD8⁺ T cells activated in (IL-15/TGF-β)-T cell-polarizing conditions for 5 days and treated for 24 h with 100 nM of different prostaglandins. Lines in the dot plot show mean values and the data show n = 3 biological replicates over two independent experiments. Statistics were performed using one-way ANOVA and Dunnet’s multiple comparison correction. f UMAP of the single cell RNA and ADT sequencing data showing expression across different clusters of Ptger4. g Schematic of the Listeria monocytogenes expressing OVA (LmOVA) infection model used in Figs. 3–5. OT-I donor cells were CD45.2⁺, whereas recipient mice were CD45.1⁺. CD90.1 and CD90.2 were used to additionally separate control guide- vs target gene guides-treated donor cells. LmOVA was administered by oral gavage. Image created with Biorender.com. h–i Distribution of Ctrl vs Ptger4-deleted CD8⁺ T cells within the population of CD45.2⁺ cells upon LmOVA challenge in LP (h) and IEL fraction (i). Lines in the dot plot show pairing within single mice, and the dot plots show cumulative data of n = 15 biological replicates over three independent experiments. j Mitotracker green in Ctrl vs Ptger4-deleted CD69⁺CD103⁺ cells isolated from LP of mice orally challenged with LmOVA. Lines in the dot plot show pairing within single mice, and dot plots show cumulative data of n = 15 biological replicates over three independent experiments. k Distribution of Ctrl vs Ptger4-deleted CD8⁺ T cells gated in the population of CD45.2⁺ cells between CD69⁻CD103⁻, CD69⁺ and CD69⁺CD103⁺ populations, upon LmOVA challenge in LP. Lines in the dot plot show mean values; dot plots show cumulative data of n = 15 biological replicates over three independent experiments. Statistics were performed using two-way ANOVA and Sidak’s multiple comparison correction. a, c Statistics were performed using one-way ANOVA and Tukey’s multiple comparison correction. h, i Statistics were performed using two-tailed paired t test. Source data are provided as a Source Data file.