Carbon source availability drives nutrient utilization in CD8+ T cells

**Background:** CD8+ T cells are critical for adaptive immunity against pathogens and tumors. Glucose has traditionally been considered the primary fuel for T cell metabolism and effector function. However, recent in vivo studies using 13C-labeled metabolite infusion have revealed that glucose is used less prominently for TCA cycle metabolism in CD8+ effector T cells in vivo compared to in vitro, suggesting that other undefined substrates in immune microenvironments may influence glucose utilization. The authors hypothesized that environmental nutrient availability, particularly physiologic carbon sources (PCSs) found at >100 μM in mouse serum, may shape T cell metabolic phenotypes and function.

**Methods:** Naive CD8+ T cells from C57BL/6 mice were activated with anti-CD3/CD28 antibodies and cultured in standard IMDM or VIM (Van Andel Institute-modified Iscove's medium) with or without PCSs (acetate, β-hydroxybutyrate, citrate, lactate, pyruvate) at mouse serum concentrations. 13C-based stable isotope labeling (SIL) with [U-13C]-glucose and other 13C-labeled substrates was used to track metabolite utilization via LC-MS and GC-MS. Metabolic flux analysis (MFA) was performed using the INCA software package. RNA sequencing assessed transcriptional changes. T cell function was evaluated by flow cytometry for effector molecules (IFN-γ, TNF-α, granzyme B), proliferation (CellTrace Violet, Ki67), and viability. Bioenergetics were measured using a Seahorse XF96 Extracellular Flux Analyzer. For in vivo studies, Thy1.1+ OT-I CD8+ T cells transduced with control or Ldha-targeting shRNA were adoptively transferred into naive Thy1.2+ hosts, followed by LmOVA infection. Antigen-specific T cell expansion and cytokine production were assessed 7 days post infection.

**Key Results:** The presence of PCSs dramatically reduced glucose contribution to the TCA cycle. In VIM with PCSs, 13C-glucose contribution to citrate synthesis decreased from 62.6% ± 2.2% to 7.0% ± 3.0% after 2 h of labeling. This shift occurred independent of transcriptional changes in metabolic gene expression. Lactate, acetate, and β-hydroxybutyrate directly fueled the TCA cycle, with lactate contributing equally with glucose to the intracellular pyruvate pool. CD8+ T cells responding to Listeria infection preferentially consumed lactate over glucose as a TCA cycle substrate ex vivo, with 13C-lactate contributing almost 4-fold more than 13C-glucose to intracellular acetyl-CoA production (measured as M+2 enrichment in acetyl-carnitine). Exogenous lactate (2 mM) promoted a 15-20% increase in basal ATP production and 30% increase in maximal ATP production from OXPHOS. PCSs enhanced CD8+ T cell effector function, boosting IFN-γ, TNF-α, and granzyme B production, and improved viability under glucose deprivation. Silencing Ldha via shRNA reduced lactate-to-pyruvate conversion, OXPHOS-dependent ATP production from lactate, and significantly impaired antigen-specific CD8+ T cell expansion in vivo (reduced percentage and number of OVA-specific CD8+ effector T cells in spleen at 7 dpi). Ldha knockdown also reduced overall NAD+ abundance by ~50% and decreased lactate- and glucose-dependent lipid synthesis.

**Clinical Implications:** This study demonstrates that environmental nutrient availability, particularly the presence of physiologic carbon sources like lactate, profoundly shapes CD8+ T cell metabolism and function. Lactate, traditionally viewed as a metabolic waste product, is identified as a major bioenergetic and biosynthetic fuel for CD8+ effector T cells at physiologic concentrations (1-3 mM), while high concentrations (>5 mM) suppress T cell function. These findings explain discrepancies between in vivo and in vitro T cell metabolism and suggest that T cell function in diverse immune microenvironments (e.g., tumors, inflamed tissues) may be shaped by local nutrient availability. The data support using physiologic culture media (e.g., VIM or HPLM) for metabolic studies and suggest that dietary or metabolic interventions targeting nutrient availability could modulate T cell responses to improve protective immunity.