Figure 4.

SV.IL12 in Combination with Anti-OX40 Promotes Metabolic Reprogramming of T Cells

Tumor-bearing mice were left untreated or treated with SV.IL12 and/or anti-OX40. T cells were isolated from spleens on day 7 or otherwise indicated. (A) Selected gene set enrichment analysis (GSEA) of oxidative phosphorylation and glycolysis pathways based on DEGs in control versus SV+anti-OX40. (B) Mitochondrial respiration was assessed by measuring the median values of oxygen consumption rates (OCRs) in T cells of indicated groups using an extracellular flux analyzer. Oligomycin, FCCP, antimycin A, and rotenone were injected as indicated to identify energetic mitochondrial phenotypes. (C and D) MitoTracker Green (C) and MitoTracker Deep Red FM (D) staining of T cells from indicated groups using flow cytometry. (E) Western blot of c-Myc protein expression in T cells of control or mice treated with anti-OX40, SV.IL12, or SV.IL12+anti-OX40. GAPDH (bottom) is the loading control. (F) Baseline extracellular acidification rates (ECARs) in T cells of indicated groups derived from the CT26.Fluc and MyC-CaP.Fluc tumor models. (G) Energy profile (OCRs versus ECARs) of T cells from naive or CT26.Fluc-bearing mice treated with SV.IL12+anti-OX40 on days 7, 14, and 40. Error bars indicate SEM. Results are representatives of at least two independent experiments (B–G).