Fig 1: Cardiolipin Synthesis Supports CD8+ TM Cell Development, Metabolism, and Function(A) Schematic of CD8+ T cells activated with aCD3/aCD28 + IL-2 for 48 h and then cultured ± AD for 72 h during IL-15 TM cell development.(B) Quantification of total CL content in IL-15 TM cells cultured as in (A).(C) OCR of IL-15 TM cells cultured as in (A) at baseline and after exposure to aCD3/aCD28 coated beads, Oligo, FCCP, and Rot/Ant.(D) IL-15 TM cells were cultured as in (A) and then restimulated with aCD3/aCD28 for 20 h. Percentage of producing IFN-? cells, and IFN-? MFI are shown.(E) Schematic of the activation of CD8+ T cells isolated from Ptpmt1flox/floxErt2-Cre-/- (PTPMT1 iWT) Ptpmt1flox/floxErt2-Cre+/- (PTPMT1 i?T) mice and 4-OHT treatment.(F) CL content in Ptpmt1 iWT and Ptpmt1 i?T CD8+ T cells cultured as in (E).(G) OCR of Ptpmt1 iWT and Ptpmt1 i?T CD8+ T cells cultured as in (E) at baseline and after exposure to PMA/Ionomycin, Oligo, FCCP, and Rot/Ant.(H) Ptpmt1 iWT and Ptpmt1 i?T CD8+ T cells cultured as in (E) and stimulated with aCD3/aCD28 for 20 h. Percentage of producing IFN-? cells and IFN-? MFI are shown.(I) Schematic of WT CD8+ T cells activated with aCD3/aCD28 + IL-2 for 48 h, cultured additional 24 h in IL-2 before performing Ctrl/Ptpmt1 CRISPR. 24 h after CRISPR, equal number of CRISPR Ctrl/Ptpmt1 KO cells were transferred in congenic recipient mice and donor cell frequencies followed for 21 days before LmOVA WT infection.(J) Percentage of CD8+ CD45.2+ Tet+ CRISPR Ctrl/Ptpmt1 KO cells treated as in (I), shown as mean ± SEM.(K) Bacterial burden shown as CFU per µg of liver isolated from mice treated as in (I) 3 days post-LmOVA challenge. Dots represent individual mice.(L) Schematic of WT CD8+ T cells activated and cultured in IL-2 in the presence of BSA-conjugated 50 µM PG(18:1)2 or 50 µM PG(18:2)2 for 48 h.(M) Quantification of CL species in IL-2 TE CD8+ T cell cultured ± PG(18:2)2 for 48 h.(N) Maximal cristae width measured in IL-2 TE CD8+ T cell cultured as in (M).(O) SRC of IL-2 TE CD8+ T cells cultured as in (M) stimulated with aCD3/aCD28 coated beads.(P) Cytokine production (normalized to untreated cells) measured in IL-2 TE CD8+ T cells activated as in (M) and stimulated for 20 h with aCD3/aCD28 + IL-2.(Q) Schematic of adoptive transfer strategy of OT-I IL-2 CD8+ TE cells—pre-treated as in (L)— into congenic recipient mice. Twenty-one days post-transfer, mice were infected i.v. with 1 × 106 CFU of LmOVA ?Acta.(R) Survival of donor cells 21 days post-transfer, expressed as number of donor cells in the spleen. Dots represent individual mice.(S) Percentage of circulating donor cells 21 days post-transfer. Dot represent individual mice.(T) Number of donor cells recovered from spleens of recipient mice 5 days after infection with LmOVA ?Acta. Dots represent individual mice.Data shown as mean ± SEM of =3 independent experiments unless indicated. Statistical comparisons for 2 groups calculated by unpaired two-tailed Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001.See also Figure S4.