Fig 2: Transcriptomics data suggest that the loss of FFAR4 suppresses immune reactions. A transcriptomics approach was used to identify differentially expressed genes in the colons of fl/fl and GKO mice with DSS-induced colitis. A volcano plot shows the distribution of upregulated (red) and downregulated (green) genes (a). GO enrichment analysis of upregulated (red) and downregulated (green) genes (b). Heatmap showing genes in the most significant cluster (negative regulation of immune response-related genes) (c). It is noteworthy that the Treg cell marker Cd25 is among the clusters.

Fig 3: Tregs in the Tumor Microenvironment Exhibit a Suppressive Phenotype with Mitochondrial Alterations and Type I IFN Signaling(A) Gene expression levels in Tregs isolated from human breast cancer relative to Tregs from healthy donor PBMC generated from previously published data (Plitas et al., 2016).(B) Mice were injected with E.G7-OVA, and Tregs were isolated from the tumor or spleen on D14. Mean (±SEM) gene expression of type I IFN-related genes from intratumoral Tregs, normalized to Tregs isolated from the spleen (n = 5), is shown. Results represent two independent experiments.(C) Representative histograms and mean (±SEM) quantification of Treg lipid uptake in vivo following labeled C16 administration. Results are representative of 5 mice from one experiment.(D) Representative histograms and mean (±SEM) quantification of Mitotracker deep red staining. Results represent two independent experiments.(E) Representative histograms and mean (±SEM) quantification of CD25 and ICOS from Tregs isolated from the spleen or tumor. Results represent two independent experiments.(F) Representative flow plots and mean (±SEM) quantification of IL-10 expression from Tregs isolated from the spleen or tumor. Results represent two independent experiments. Gating controls are depicted in red.(G) Representative confocal photomicrographs of mitochondria and DNA of Tregs isolated from the tumor or spleen and quantification of mitochondrial sphericity. Scale bar, 2 µm. Results represent one experiment.(H) Mice were injected with E.G7-OVA, and serum, spleens, and tumors were excised on D14. The total lipid content of serum or interstitial fluid of the spleen or tumor was quantified by MS. Results are representative of 8 mice from one experiment.(I) Mean (±SEM) quantification of Fabp5 gene expression and protein expression in Tregs following overnight culture in lipid or glucose depleted media (n = 4).(J) Mean (±SEM) quantification of labeled C16 uptake in vitro in Tregs following overnight lipid starvation (n = 4) or from nTregs ex vivo from the spleen or tumor of mice (n = 5) as per (F). Results represent two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. P values were calculated using a two-way ANOVA with Bonferroni correction (A and B) or two-tailed, unpaired t test (C, D, E, G, H, I, and J).

Fig 4: Flotillin expression is required for activation of mouse primary CD4 T cells. a Western blot of splenocyte lysate from WT and flotillin2 KO mice probed with antibodies against flotillin1 and flotillin2. b–e Representative flow cytometry histograms of CD4 T cells isolated from the spleen of WT or flotillin2 KO mice, activated on anti-CD3 and anti-CD28 surfaces for 20 and 36 h and stained with antibodies against CD69 (b) or CD25 (d). Mean fluorescent intensity (MFI) at the surface of 10,000 cells of WT and flotillin2 KO CD4 T cells stained with antibodies against CD69 (c) or CD25 (e). Data obtained from three mice for each condition. Error bars indicate mean (±SEM). *p < 0.01; **p < 0.001; ***p < 0.0001; ANOVA test

Fig 5: FABP5 Inhibition of Fully Differentiated Tregs Increases Suppression and Expression of the Regulatory Cytokine IL-10Treg suppression assays were performed following acute FABP5 blockade.(A) Quantification of the mean suppression (±SEM) of responder effector cells from in vitro-differentiated Tregs following overnight BMS309403 treatment (n = 4). Results represent five independent experiments.(B) Quantification of mean suppression (±SEM) from human Tregs following BMS309403 treatment (n = 4). Results represent the combined results from 4 independent donors.(C) Quantification of mean suppression (±SEM) from ex vivo Tregs following 30 min BMS309403 treatment (n = 4). Results represent two independent experiments.(D) Representative histograms and mean (±SEM) quantification of CD25 and ICOS expression on Tregs following acute FABP5 blockade (n = 4). Results represent six independent experiments.(E) Representative histograms and mean (±SEM) quantification of CD25 and ICOS expression on Tregs following lentiviral knockdown of Fabp5. Results represent two independent experiments.(F) Mean (±SEM) expression of Il10 gene expression measured by qPCR (n = 4).(G) Mean (±SEM) protein expression of IL-10 in Tregs after overnight BMS309403 treatment (n = 5). Results represent two independent experiments. Gating controls are depicted in red.(H) Mean quantification (±SEM) of suppression from in vitro-generated Tregs following overnight BMS309403 treatment in the presence or absence of anti-IL-10 antibody (aIL10) (n = 4). Results represent four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. P values were calculated using a two-way ANOVA with Bonferroni correction (A, B, C, and H) or a two-tailed, unpaired t test (D, E, F, and G).