Figure 6. Ldha regulates T cell metabolism and effector cell expansion in vivo.

(A) Immunoblot of Ldha and actin protein levels in whole cell lysates from activated CD8⁺ T cells expressing a control (Ctrl) or Ldha-targeting (shLdha) shRNA.

(B) Relative abundance of intracellular [U-¹³C]-lactate-derived lactate (Lac) and pyruvate (Pyr) in activated CD8⁺ T cells expressing a control (Ctrl) or Ldha-targeting (shLdha) shRNA. T cells were cultured in VIM containing 5 mM glucose and 2 mM [U-¹³C]-lactate for 4 h (mean ± SEM, n = 3/group).

(C) Basal (left) and maximal (right) ATP production rates from OXPHOS for CD8⁺ T cells expressing a control (Ctrl) or Ldha-targeting (shLdha) shRNA cultured as in (B) (mean ± SD, n = 10–24/group).

(D) Relative abundance of intracellular [U-¹³C]-glucose-derived lactate (Lac) in control (Ctrl) or shLdha-expressing CD8⁺ T cells cultured with 5 mM [U-¹³C]-glucose and 2 mM lactate for 4 h (mean ± SEM, n = 3/group).

(E) Flow cytometry plots showing abundance of OVA-specific Thy1.1⁺ OT-I T cells in the spleen of LmOVA-infected mice 7 dpi. Right: percentage and total number of Thy1.1⁺ CD8⁺ OT-I T cells (mean ± SEM, n = 5 mice/shRNA).

(F) Flow cytometry plots showing the percentage of IFN-γ⁺ control (Ctrl) or shLdha-expressing Thy1.1⁺ CD8⁺ OT-I T cells in the spleen of LmOVA-infected mice 7 dpi. Right: percentage and total number of IFN-γ⁺ Thy1.1⁺ CD8⁺ T cells (mean ± SEM, n = 5 mice/shRNA).

(G and H) MIDs for indicated intracellular metabolites in activated control (Ctrl) or shLdha-expressing CD8⁺ T cells. Cells were cultured for 4 h in VIM containing (G) 2 mM [U-¹³C]-lactate and unlabeled 5 mM glucose or (H) 5 mM [U-¹³C]-glucose and unlabeled 2 mM lactate (mean ± SEM, n = 3/group).

(I) [U-¹³C]-lactate and [U-¹³C]-glucose labeling into palmitate (in VIM for 24 h) in activated CD8⁺ T cells expressing a control (Ctrl) or Ldha-targeting (shLdha) shRNA (mean ± SEM, n = 3/group).

(J) Heatmap depicting relative abundance of intracellular metabolites in activated control (Ctrl) or shLdha-expressing CD8⁺ T cells following 4 h culture in VIM. Shown are the top 45 significant metabolites (p < 0.05) based on differential metabolite levels between groups (n = 6/group).

(K) NAD⁺:NADH ratio for control (Ctrl) or shLdha-expressing CD8⁺ T cells cultured for 2 h in VIM containing 0 or 2 mM sodium lactate (n = 2–4/group).

(L) NAD⁺ abundance in activated CD8⁺ T cells expressing a control (Ctrl) or Ldha-targeting (shLdha) shRNA following 4 h culture in VIM (mean ± SEM, n = 6/group).