Figure 4.

Cardiolipin Synthesis Supports CD8⁺ T_M Cell Development, Metabolism, and Function

(A) Schematic of CD8⁺ T cells activated with αCD3/αCD28 + IL-2 for 48 h and then cultured ± AD for 72 h during IL-15 T_M cell development.

(B) Quantification of total CL content in IL-15 T_M cells cultured as in (A).

(C) OCR of IL-15 T_M cells cultured as in (A) at baseline and after exposure to αCD3/αCD28 coated beads, Oligo, FCCP, and Rot/Ant.

(D) IL-15 T_M cells were cultured as in (A) and then restimulated with αCD3/αCD28 for 20 h. Percentage of producing IFN-γ cells, and IFN-γ MFI are shown.

(E) Schematic of the activation of CD8⁺ T cells isolated from Ptpmt1^flox/floxErt2-Cre^−/− (PTPMT1 iWT) Ptpmt1^flox/floxErt2-Cre^+/− (PTPMT1 iΔT) mice and 4-OHT treatment.

(F) CL content in Ptpmt1 iWT and Ptpmt1 iΔT CD8⁺ T cells cultured as in (E).

(G) OCR of Ptpmt1 iWT and Ptpmt1 iΔT 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 iΔT CD8⁺ T cells cultured as in (E) and stimulated with αCD3/αCD28 for 20 h. Percentage of producing IFN-γ cells and IFN-γ MFI are shown.

(I) Schematic of WT CD8⁺ T cells activated with αCD3/αCD28 + 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 T_E CD8⁺ T cell cultured ± PG(18:2)2 for 48 h.

(N) Maximal cristae width measured in IL-2 T_E CD8⁺ T cell cultured as in (M).

(O) SRC of IL-2 T_E CD8⁺ T cells cultured as in (M) stimulated with αCD3/αCD28 coated beads.

(P) Cytokine production (normalized to untreated cells) measured in IL-2 T_E CD8⁺ T cells activated as in (M) and stimulated for 20 h with αCD3/αCD28 + IL-2.

(Q) Schematic of adoptive transfer strategy of OT-I IL-2 CD8⁺ T_E 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 ΔActa.

(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 ΔActa. 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.