Figure 1.

De Novo Cardiolipin Synthesis Is a Hallmark of CD8⁺ T Cells with a High-Reserve Respiratory Capacity

(A) OCR of mouse WT CD8⁺ T cells activated with αCD3 only or αCD3/αCD28 and differentiated in IL-15 T_M cells for 6 days. OCR analysis at baseline and after exposure to αCD3/αCD28 coated beads, Oligomycin (Oligo), FCCP, and Rotenone/Antimycin (Rot/Ant).

(B) Lipids extracted from cells activated and differentiated as in (A). Data show Log2FC and p value calculated with ANOVA test.

(C) Total CL content in cells treated and analyzed as in (B).

(D) OCR of mouse WT CD8⁺ T cells activated with αCD3/αCD28 + IL-2 and cultured in complete medium until day 3, then cultured for 24 h in either complete medium (10mM glucose) or glucose restricted medium (1mM glucose) for 24 h. Normalized OCR at baseline and after (Oligo) FCCP and Rot/Ant injections.

(E) Lipids extracted from cells activated and differentiated as in (F). Data show Log2FC and p value calculated with ANOVA test.

(F) Total CL content in cells treated and analyzed as in (F).

(G) Immunoblot analysis of cells activated and differentiated as in (A).

(H) Immunoblot analysis of cells treated as in (D).

(I) MitoTracker Green staining in cells activated and differentiated as in (A).

(J) MitoTracker Green staining in cells activated and differentiated as in (D).

(K) SRC of CD8⁺ T cells activated as in (D) and cultured ± AD during 20 h of glucose restriction.

(L) Survival of CD8⁺ T cells activated as in (D) and cultured ± AD during 20 h of glucose restriction.

(M) OCR of mouse WT CD8⁺ T cells activated with αCD3/αCD28 + IL-2 and cultured in complete medium until day 3, then for additional three days in IL-2 (IL-2 T_E) or IL-15 (IL-15 T_M).

(N) Lipids extracted from WT CD8⁺ T cells differentiated as in (M). Data show Log2FC and p value calculated with ANOVA test.

(O) Total CL content from IL-2 T_E and IL-15 T_M CD8⁺ T cells cultured as in (M).

(P) Lipids extracted from T_EM (CD44^hiCD62L^lo) and T_CM (CD44^hiCD62L^hi) cells isolated ex vivo from C57BL/6 mice. Data show Log2FC and p value calculated with ANOVA test from 9 animals per group.

(Q) Total CL content from T_EM and T_CM cells isolated as in (P). Dots represent individual mice.

(R) CD8⁺ T cells were activated with αCD3/αCD28 + IL-2 for 3 days and then differentiated into IL-15 T_M cells in presence of ¹³C-glucose. At indicated time points lipids were extracted and ¹³C-glucose derived carbons traced into CL 72:8.

(S) CD8⁺ T cells were activated with αCD3/αCD28 + IL-2 for 3 days and then differentiated into IL-15 T_M cells for 72 h in the presence of ¹³C-glucose or ¹³C-linoleic acid ± CL synthesis inhibitor (AD). ¹³C-glucose and ¹³C-linoleic acid derived carbons traced into CL 72:8.

(T) CD8⁺ T cells were differentiated as in (S). Also shown is the percentage of isotopologue distribution of ¹³C-glucose and ¹³C-linoleic acid-derived carbons traced into CL 72:8.

Data shown as mean ± SEM of ≥3 independent experiments unless indicated. Statistical comparisons for two groups calculated by unpaired two-tailed Student’s t test or ANOVA test, where indicated. Comparison among multiple groups (in [R] and [S]) calculated by One-Way ANOVA and correcting for multiple comparison using Tukey test. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

See also Figure S1.