Figure 5.

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).