Figure 6.

Eomes confers increased mitochondrial respiratory capacities of CD4⁺ T cells and enhanced their survival abilities under stress conditions. (A) Number and volume of mitochondria were determined in Eomes-TKO, Eomes⁻ and Eomes⁺ CD4⁺ T cells by TOMM20 staining and confocal fluorescence microcopy after anti-CD3 and anti-CD28 mAbs stimulation during 48 h. (B) Mitochondrial cristae architecture was imaged using transmission electron microscopy (TEM) in Eomes-TKO and Eomes⁺ CD4⁺ T cells. Scale bars represent 2 µm (left) and 200 nm (right). (C) Mitochondrial membrane potential (ΔΨm) of Eomes-TKO, Eomes⁻, and Eomes⁺ CD4⁺ T cells stimulated in vitro for 48 h using MitoFM DeepRed and TMRE stainings (n = 4 mice/group). (D) OCR was assessed in Eomes-TKO, Eomes⁻, and Eomes⁺ cells stimulated in vitro for 48 h (left). Maximal OCR and SRC were also determined (right) (each point represents an experiment replicate from a pool of three or six mice/group for Eomes-TKO and Eomes-GFP, respectively). (E) ATP synthase expression was determined using confocal fluorescence microcopy. (F and G) (F) ΔΨm and (G) mitochondrial dependency were assessed in 2D2-Eomes⁻ and 2D2-Eomes⁺ CD4⁺ T cells purified from the brain of immunized mice (n = 6 and 8 mice for F and G, respectively). (H and I) Eomes-TKO and Eomes-TWT CD4⁺ T cells were stimulated during 48 h with anti-CD3 and anti-CD28 mAbs in presence of either (H) antimycin A and rotenone or (I) sulfasalazine, etoposide, and idarubicin (n = 4 mice/group). Data are a pool of two (E), three (A), or representative of at least two independent experiments. Error bars = SEM; P values in F and G (Mann–Whitney U test), other P values (two-way ANOVA with Bonferroni correction)—****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. See also Fig. S4.