Carbon source availability drives nutrient utilization in CD8+ T cells

Ketolysis is a metabolic driver of CD8+ T cell effector function through histone acetylation

“…For mouse T cell isolation, naïve CD8 + T cells were purified from spleen and peripheral lymph nodes by negative selection (StemCell Technologies, Vancouver, BC) as previously described ( 2 ). Cells were cultured in Iscove’s Modified Delbecco’s Medium (IMDM) or Van Andel Institute-modified Iscove’s Medium (VIM) ( 3 ) supplemented with 10% dialyzed FBS (Wisent, St. Bruno, QC), penicillin-streptomycin (Invitrogen), and 2-ME (Sigma-Aldrich, St. Louis, MO). Unless stated otherwise, CD8 + T effector (Teff) cells were generated by activation in standard IMDM containing 25 mM glucose and 6 mM L-glutamine.…”

“…For physiologic culture conditions, Seahorse experiments, and 13 C tracing experiments, CD8 + Teff cells were culture in medium containing 5 mM glucose and 0.5 mM L-glutamine, respectively. Physiologic carbon sources (PCS) were added to culture medium (IMDM or VIM) as indicated at the following concentrations: acetate (400 μM), βOHB (850 μM), citrate (215 μM), lactate (3 mM), and pyruvate (150 μM) ( 3 ). In vitro -activated CD8 + Teff cells were generated by stimulating naïve CD8 + T cells (1 x 10 6 cells/mL) with plate-bound anti-CD3ε (clone 2C11) and anti-CD28 (clone 37.51) antibodies (eBioscience, San Diego, CA) for 3 days.…”

“…SIL experiments with in vitro -activated T cells using liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometry (MS) were conducted as previously described ( 2, 3 ). In brief, naïve CD8 + T cells were activated as above washed, in IMDM or VIM containing 10% dialyzed FBS, and re-cultured (2.5 × 10 6 cells/well in 24-well plates) for indicated times in medium containing 13 C-labeled metabolites (Cambridge Isotope Laboratories) at the following concentrations: [U- 13 C 6 ]glucose, 5 mM; [U- 13 C 2 ]acetate, 400 μM; [U- 13 C 4 ]AcAc, 2 mM; [U- 13 C 3 ]alanine, 200 μM; [U- 13 C 4 ]πOHB, 0.85 or 2 mM; [ 13 C 6 ]citrate, 215 μM; [ 13 C 3 ]lactate, 3 mM; [ 13 C 3 ]pyruvate, 150 μM.…”

“…1e ). We recently reported that the presence of physiologic carbon sources (PCS) enhances CD8 + T cell effector function, including IFN-γ and granzyme production ( 32 ). Removing βOHB from PCS-supplemented cell culture medium reduced IFN-γ production by CD8 + T cells, while addition of βOHB alone was sufficient to increase IFN-γ levels, suggesting that βOHB was the active metabolite in PCS acting to enhance IFN-γ production ( Fig.…”

“…To further characterize the metabolic substrates contributing to acetyl-CoA production, we cultured activated CD8 + T cells in the presence of competitive uniformly 13 C-labeled fuels at their physiologic concentrations (as in Fig. 2b ) ( 32 ). Strikingly, over 50% of the intracellular acetyl-CoA (M+2) pool was derived from [U- 13 C 4 ]βOHB, compared to less than 10% from [U- 13 C 6 ]-glucose, in CD8 + T cells ( Fig.…”