All experiments and protocols were approved by the regional animal ethics committee of Northern Stockholm (dnr N78/15, N101/16). For T cell purification, wild type donor and recipient C57BL/6J (RRID: IMSR_JAX:000664) animals were purchased from Janvier Labs.

Sodium Chloride (Sigma, S5886) and Sodium L-Lactate (Sigma, L7022) solutions were prepared by dissolving each compound into RPMI 1640 (Thermo Fisher, 21875) at a maximum concentration of 500 mM. Solutions were serially diluted, and osmolality measurements were obtained with a micro-osmometer (Löser Messtechnik, D-1000 Berlin 38).

Spleens were harvested from 8-12 week old C57BL/6J (RRID : IMSR_JAX:000664) mice, mashed over a 40 µm cell strainer (VWR, 10199-654), and CD8+ T cells were purified by positive magnetic bead selection (Miltenyi Biotec, 130-117-044) according to manufacturer’s instructions. Purified CD8+ T cells were counted and cell diameter measured using a Moxi Z mini (Orflo, MXZ001) or a TC20 (Bio-Rad, 145-0101) automated counter. 1x10⁶ (24-well plate) or 5x10⁵ (48-well plate) CD8+ T cells were activated with Dynabeads Mouse T-Activator CD3/CD28 (Thermo Fisher, 11456D) in a 1:1 bead to T cell ratio and cultured in 2 ml (24-well plate) or 1 ml (48-well plate) RPMI 1640 (Thermo Fisher, 21875) supplemented with 10% Fetal Bovine Serum (Thermo Fisher, 10270-106), 50 µM 2-mercaptoethanol (Thermo Fisher, 21985023), 100 U/ml penicillin-streptomycin (Thermo Fisher, 15140122), and 10 U/ml recombinant human IL-2 (Sigma, 11011456001), and incubated at 37˚ C for 3 days in a humidified CO₂ incubator.

Peripheral blood mononuclear cells (PBMCs) were harvested from standard buffy coat preparations of healthy donors, aged 20 to 40 years old, obtained from the Department of Transfusion Medicine at Karolinska University Hospital and processed no later than 4 hours after collection. PBMCs were isolated by gradient centrifugation using Histopaque-1077 (Sigma, 10771) and enriched for CD8+ T cells by magnetic bead selection, according to manufacturer’s instructions (Miltenyi Biotec, 130-045-201, RRID : AB_2889920). Purified CD8+ T cells were counted and 5x10⁵ (48-well plate) or 1x10⁵ (96-well plate) naïve CD8+ T cells were activated with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher, 11132D) in a 1:1 bead to T cell ratio and cultured in 1 ml (48-well plate) or 200µl (96-well plate) RPMI 1640 (Thermo Fisher, 21875) supplemented with 10% Fetal Bovine Serum, (Thermo Fisher, 10270-106), 100 U/ml penicillin-streptomycin (Thermo Fisher, 15140122), and 30 U/ml recombinant human IL-2 (Sigma, 11011456001), and incubated at 37˚C for 4 days in a humidified CO₂ incubator. For long-term expansion after 4 days, Dynabeads were removed using a DynaMag-2 Magnet (Thermo Fisher, 12321D), washed with PBS, counted, and resuspended in fresh media supplemented with 30 U/ml IL-2 and incubated as described above. Media supplemented with cytokines was added every 2-3 days to maintain cell densities below 2x10⁶/ml. Cells were counted again at day 7 and maintained in culture under the above-described conditions until day 14.

Sodium L-Lactate (Sigma, L7022), L-Lactic Acid (Sigma, L1750) and Sodium Chloride (Sigma, S5886) were prepared as 10x concentrated solutions in complete media. Compounds were added to T cells at the point of activation (day 0) minutes before addition of CD3/CD28 Dynabeads. Sodium Chloride and/or plain media was used as control. For long-term expansion, after beads removal cells were cultured in complete media supplemented with cytokines without further addition of compounds.

After magnetic bead purification, CD8+ T cells were loaded with 5 µM CellTrace CFSE or CellTrace Violet (CTV) dyes (Thermo Fisher, C34554 and C34557, respectively) according to manufacturer’s instructions. CFSE or CTV dilution was determined by flow cytometry.

Single-cell suspensions were washed and stained with Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher, L10119) followed by staining of extracellular antigens with fluorochrome labelled antibodies. The Fixation/Permeabilization Solution Kit (BD Biosciences, 554714) was used for exposing cytoplasmic antigens. The Transcription Factor Buffer Set (BD Biosciences, 562574, RRID : AB_2869424) was used for exposing nuclear antigens. Fluorochrome-labelled antibodies against mouse antigens CD44 (clone IM7), CD8 (clone 53- 6.7), CTLA-4 (clone UC10-4F10-11), LAG-3 (clone C9B7W), and PD-1 (clone J43) were purchased from BD Biosciences, CD27 (clone LG.3A10), CD28 (clone 37.51), CD62L (clone MEL-14), and ICOS (clone C398.4A) were purchased from BioLegend, and 4-1BB (clone 17B5), CD127 (clone A7R34), CD25 (clone PC61.5), Eomes (clone Dan11mag), T-bet (clone eBio4B10), and anti-human/mouse Granzyme B (clone GB12) was purchased from Thermo Fisher. Cell counting was performed with CountBright Absolute Counting Beads (Thermo Fisher, C36950). Samples were processed in a FACS Canto II flow cytometer (BD Biosciences). Data analysis was performed with FlowJo (RRID : SCR_008520, version 10.7.2).

Mouse CD8+ T cells were activated ex vivo and cells harvested at 0, 6, 12, 24, 48, and 72 hours post-activation. For each time point, total protein was extracted using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents kit (78833, Thermo Fisher) and the cytoplasmic fraction used for western blots. Total RNA was extracted using the RNeasy Mini Kit (74104, Qiagen). For each sample, 15 µg of total protein were separated in SDS-PAGE, blotted onto a PVDF membrane and probed with antibodies against MCT1 (Santa Cruz Biotechnology Cat# sc-50325, RRID : AB_2083632), MCT4 (Santa Cruz Biotechnology Cat# sc-50329, RRID : AB_2189333), GzmB (Cell Signaling Technology Cat# 4275, RRID : AB_2114432) and PPIB (ABclonal Cat# A7713, RRID : AB_2771764) and detected using infra-red labeled secondary antibodies in an Odyssey imaging system (LICOR). Western blot data were analyzed with the Image Studio Lite software (Image Studio Lite, RRID : SCR_013715). REVERT Total protein stain (926-11010, Li-COR) was used for lane normalization. Two micrograms of total RNA were reverse transcribed using iScript cDNA synthesis kit (BioRad) in a total volume of 20 μl. Real-time RT-PCR was used for mRNA quantification (7500 Fast Real-Time PCR system, Applied Biosystems Inc., Foster City, California, USA). All primers were designed to cover exon-exon boundaries to avoid amplification of genomic DNA. Predesigned qPCR primers used to detect mouse Slc16a1 (forward: 5’-ACTTGCCAATCATAGTCAGAGC-3’, reverse: 5’-CGCAGCTTCTTTCTGTAACAC-3’), Slc16a3 (forward: 5’-GACGCTTGTTGAAGTATCGATTG-3’, reverse: 5’-GCATTATCCAGATCTACCTCACC-3’), granzyme B (GzmB) (forward: 5’-CTGCTAAAGCTGAAGAGTAAG-3’, reverse: 5’-TAGCGTGTTTGAGTATTTGC-3’), and Hprt (forward: 5’-TGACACTGGCAAAACAATGCA-3’, reverse: 5’-GGTCCTTTTCACCAGCAAGCT-3’) were purchased from IdtDNA or Sigma. All reactions were performed in 96-well MicroAmp Optical plates in duplicates. The total reaction volume was 15 µl, containing 5 µl sample cDNA, 0.4 mM of each primer forward and SYBR Green PCR Master Mix (4309155E, Applied Biosystems Ins.). All quantification reactions were controlled with a melting curve and primer efficiency was tested with standard curves, and did not differ between the primer pairs. Gene expression data was normalized to Hprt levels.

Mouse CD8+ T cells were activated for 72 hours in the presence of either 40 mM sodium lactate or plain media. At the end of activation cells were washed twice with PBS and cell pellets were snap-frozen in RLT Plus lysis buffer (Qiagen, #1053393). Total RNA was extracted with the Qiagen’s RNeasy kit according to the manufacturer’s instructions. All samples were quality checked with Agilent Tapestation RNA screen tape. To construct libraries suitable for Illumina sequencing, the Illumina TruSeq Stranded mRNA Sample preparation protocol which includes cDNA synthesis, ligation of adapters, and amplification of indexed libraries was used. The yield and quality of the amplified libraries were analyzed using Qubit by Thermo Fisher and the Agilent Tapestation. The indexed cDNA libraries were normalized and combined, and the pools were sequenced on the Nextseq 550 (Illumina NextSeq 550 System, RRID : SCR_016381) for a 50-cycle v2.5 sequencing run, generating 71 bp single-end reads (and 2*10 bp index reads). Demultiplexing was performed using bcl2fastq (bcl2fastq, RRID : SCR_015058, v2.20.0.422), generating fastq files for further downstream mapping and analysis.

Bcl files were converted and demultiplexed to fastq using the bcl2fastq v2.20.0.422 program. STAR 2.7.5b (33) was used to map the fastq files to the mouse reference genome (mm10/GRCm38) and to remove PCR duplicates. Uniquely mapped reads were then counted in annotated exons using featureCounts v1.5.1 (34). The gene annotations (Mus_musculus.GRCm38.99.gtf) and reference genome were obtained from Ensembl. The count table from featureCounts was imported into R/Bioconductor and differential gene expression was performed using the EdgeR package (35) and its general linear models pipeline. For the gene expression analysis, genes that had 1 count per million in 3 or more samples were used and normalized using TMM normalization. Genes with an FDR adjusted p value ≤ 0.01 were termed significantly regulated. Principal Component Analysis (PCA) plots were obtained by normalizing gene counts after filtering out lowly expressed genes.

For metabolite analysis, 5x10⁶ cells were harvested and washed 3 times with PBS before adding 800 µL of ice-cold extraction mixture consisting of chloroform:MeOH:H2O (1:3:1), containing 500 pg/µL of 2-Deoxy-D-glucose 6-phosphate and 70 pg/µL of myristic acid-¹³C₃ were added to each sample. Metabolites were extracted using a mixer mill set to a frequency 30 Hz for 2 min, with 1 tungsten carbide bead added to each tube. Obtained extracts were centrifuged at 14000 rpm for 10 min. The collected supernatants were divided for GC-MS and Sugar-P analysis. 300 µL of the supernatant was transferred into GC and LC-vial respectively, the supernatants were evaporated until dryness using a SpeedVac.

For derivatization, dried samples were dissolved in 20 µl of methoxylamine and incubated on a heat block at 60°C, 30 min. After overnight incubation at room temperature, 12µl of 1- Methylimidazol and 6 µl of propionic acid anhydride were added and heated at 37°C for 30 minutes. The reaction mixture was then evaporated to dryness by N2 gas. Prior to LC-MS analysis, derivatized metabolites were dissolved in 100µl of aqueous 0.1% formic acid. Quantitative analysis was performed by combined ultra-high-performance liquid chromatography electrospray ionization-triple quadrupole-tandem mass spectrometry (UHPLC-ESI-QqQMS/MS) in dynamic multiple-reaction-monitoring (MRM) mode. An Agilent 6495 UHPLC chromatograph equipped with a Waters Acquity BEH 1.7 µm, 2.1 x 100 mm column (Waters Corporation, Milford, USA) coupled to a QqQ-MS/MS (Agilent Technologies, Atlanta, GA, USA) was used. The washing solution, for the autosampler syringe and injection needle, was 90% MeOH with 1% HCOOH. The mobile phase consisted of A, 2% HCOOH and B, MeOH with 2% HCOOH. The gradient was 0% B for 1 min followed by linear gradients from 0.1 to 30% from 1 to 3 min then 30 to 40% B from 3 to 6 min, hold at 40% B from 6 to 10 min, followed by 40 to 70% B from 10 to 12.5 min, hold at 70% B from 12.5 to 15 min, and thereafter 70 to 99% B from 15 to 17.5 min. B was held at 99% for 0.5 min, and thereafter the column was re-equilibrated to 0% B. The flow rate was 0.65 mL min-1 during equilibration and 0.5 mL min-1 during the chromatographic runs. The column was heated to 40°C, and injection volumes were 1 μL. The mass spectrometer was operated in negative ESI mode with gas temperature 230°C; gas flow 12 L min-1; nebulizer pressure 20 psi; sheath gas temperature 400°C; sheath gas flow 12 L min-1; capillary voltage 4000 V (neg); nozzle voltage 500 V; iFunnel high pressure RF 150 V; iFunnel low pressure RF 60 V. Fragmentor voltage 380 V and cell acceleration voltage 5 V. Data were processed using MassHunter Qualitative Analysis and Quantitative Analysis (QqQ; Agilent MassHunter WorkStation - Qualitative Analysis for GC/MS, RRID : SCR_016657) and Excel (Microsoft, Redmond, Washington, USA) software.

The GC-MS samples were spiked with 1050 pg of each GC-MS internal standard before evaporation. Derivatization was performed according to Gullberg et al. (36). In detail, 10µL of methoxyamine (15 µg/µL in pyridine) was added to the dry sample that was shaken vigorously for 10 minutes before being left to react at room temperature. After 16 hours 10 µL of MSTFA was added, the sample was shaken and left to react for 1 hour at room temperature. 10 µL of methyl stearate (1050 pg/µL in heptane) was added before analysis. One µL of the derivatized sample was injected by an Agilent 7693 autosampler, in splitless mode into an Agilent 7890A gas chromatograph equipped with a multimode inlet (MMI) and 10 m x 0.18 mm fused silica capillary column with a chemically bonded 0.18 μm DB 5-MS UI stationary phase (J&W Scientific). The injector temperature was 260°C. The carrier gas flow rate through the column was 1 ml min-1, the column temperature was held at 70°C for 2 minutes, then increased by 40°C min-1 to 320°C and held there for 2 min. The column effluent 16 is introduced into the electron impact (EI) ion source of an Agilent 7000C QQQ mass spectrometer. The thermal AUX 2 (transfer line) and the ion source temperatures were 250°C and 230°C, respectively. Ions were generated by a 70 eV electron beam at an emission current of 35 µA and analyzed in dMRM-mode. The solvent delay was set to 2 minutes. Data were processed using MassHunter Qualitative Analysis and Quantitative Analysis (QqQ; Agilent Technologies, Atlanta, GA, USA) and Excel (Microsoft, Redmond, Washington, USA) software.

Mouse CD8+ T cells were activated with CD3/CD28 beads and 10 U/ml IL-2 in glucose-free RPMI supplemented with 2 mM glutamine, 10% FBS, 100 U/ml penicillin/streptomycin and 2-mercaptoethanol, and either i) 11 mM [U¹³C₆] glucose, ii) 11mM [U¹³C₆] glucose and 40 mM lactate, or iii) 11 mM glucose and 40 mM [U¹³C₃] sodium lactate, and isotopic labelling was performed for 24, 48 and 72 hours. Sodium chloride (40 mM) was added to i) to control for osmolality. Cells harvested at each time point were washed with PBS, and metabolic activity quenched by freezing samples in dry ice and ethanol, and stored at -80°C. Metabolites were extracted by addition of 600 μl ice-cold 1:1 (vol/vol) methanol/water (containing 1 nmol scyllo-Inositol as internal standard) to the cell pellets, samples were transferred to a chilled microcentrifuge tube containing 300 μl chloroform and 600 μl methanol (1500 μl total, in 3:1:1 vol/vol methanol/water/chloroform). Samples were sonicated in a water bath for 8 min at 4°C, and centrifuged (13000 rpm) for 10 min at 4°C. The supernatant containing the extract was transferred to a new tube for evaporation in a speed-vacuum centrifuge, resuspended in 3:3:1 (vol/vol/vol) methanol/water/chloroform (350 μl total) to phase separate polar metabolites (upper aqueous phase) from apolar metabolites (lower organic phase), and centrifuged. The aqueous phase was transferred to a new tube for evaporation in a speed-vacuum centrifuge, washed with 60 μl methanol, dried again, and derivatized by methoximation (20 μl of 20 mg/ml methoxyamine in pyridine, RT overnight) and trimethylsilylation (20 μl of N,O-bis(trimetylsilyl)trifluoroacetamide + 1% trimethylchlorosilane) (Sigma, 33148) for ≥ 1 h. GC-MS analysis was performed using an Agilent 7890B-5977A system equipped with a 30 m + 10 m × 0.25 mm DB-5MS + DG column (Agilent J&W) connected to an MS operating in electron-impact ionization (EI) mode. One microliter was injected in splitless mode at 270°C, with a helium carrier gas. The GC oven temperature was held at 70°C for 2 min and subsequently increased to 295°C at 12.5°C/min, then to 320°C at 25°C/min (held for 3 min). MassHunter Workstation (B.06.00 SP01, Agilent Technologies) was used for metabolite identification by comparison of retention times, mass spectra and responses of known amounts of authentic standards. Metabolite abundance and mass isotopologue distributions (MID) with correction of natural 13C abundance were determined by integrating the appropriate ion fragments using the GC–MS Assignment Validator and Integrator (GAVIN) (37).

Soluble Interferon-gamma (IFN-γ) was quantified in culture media 3 days after CD8+ T cell activation using the Mouse IFN-gamma Quantikine ELISA Kit (RnD Systems, MIF00).

CD8+ T cells activated for 3 days were washed and oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in a Seahorse Extracellular Flux Analyzer XF96 (Agilent). 3x10⁵ CD8+ T cells were plated onto poly-D-lysine coated wells and assayed in XF RPMI medium (Agilent) pH 7.4 supplemented with 10 mM glucose and 2 mM glutamine. A minimum of 4 technical replicates per biological replicate was used. During the assay, wells were sequentially injected with 1 µM oligomycin (Sigma), 1.5 µM FCCP (Sigma) and 100 nM rotenone (Sigma) + 1 µM antimycin A (Sigma).

Purified CD8+ T cells were activated for 12 hours, then 3x10⁵ CD8+ T cells were plated onto poly-D-lysine coated wells in order to form a homogeneous monolayer. Cells were assayed in XF RPMI medium (Agilent) pH 7.4 supplemented with 2 mM glutamine. A minimum of 4 technical replicates per each biological replicate was used. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined through a Seahorse Extracellular Flux Analyzer XF96 (Agilent). During the assay, wells were sequentially injected with 40 mM NaCl/NaLac with or without the MCT1-selective inhibitor AZD3965 (Cayman Chemical, 19912) or the LDH inhibitor GSK2837808A (Bio-Techne, 5189), or plain media alone, followed by 10 mM glucose (Sigma), 1 µM oligomycin and 50 mM 2-deoxyglucose (2-DG; Sigma D6134).

Analysis of transcription factor regulators was carried out on transcripts abundantly expressed (1^st quartile) and up-regulated with lactate exposure (n = 685) queried against positional weight matrixes of human and rodent transcription factors from Transfac and JASPER. EnrichR (38), combined with single-cell sequencing data from human T-cells (39) were used. Transcription factors detected at the transcriptional levels in human T cells and in the present experiment with a motif-enrichment FDR of <5% were deemed as transcriptional regulators.

Statistical analyses were performed with Prism 9 version 9.2.0 (GraphPad). Statistical tests and number of replicates are stated in figure legends.

All animal experiments were approved by the regional animal ethics Committee of Northern Stockholm, Sweden (dnr N78/15 and N101/16). The Stockholm Regional Ethical Review Board does not require an ethical permission for the use of non-identified healthy donor blood samples.

In metabolically perturbed tissues, glycolysis will lead to protonated lactate, i.e., lactic acid, being released into the extracellular environment. The subsequent dissociation of the lactate anion from the proton leads to a localized decrease in pH ( Figure 1A ). In the physiological setting, a reduction in pH is counteracted by ample buffering systems, such as the bicarbonate buffer (22). In order to test the effect of lactate on CD8+ T cell activation and differentiation, independently of pH reduction, we activated purified mouse CD8+ T cells ex vivo for 3 days in the presence of increasing doses of either lactic acid, or its pH-neutral salt sodium lactate, or sodium chloride as a control for osmolality ( Figure 1B and Supplementary Figure 1A ). This allowed us to distinguish between the effects of the lactate anion itself and the lactate anion in conjunction with acidification. Addition of lactic acid to culture media caused a sharp decrease in pH, while equimolar doses of sodium lactate or sodium chloride did not alter media pH ( Supplementary Figure 1B ). As reported previously (40), pH-lowering lactic acid has deleterious effects on CD8+ T cell activation, resulting in significantly reduced proliferation and cell numbers ( Figures 1C, D ). In contrast, exposure to increasing doses of pH-neutral sodium lactate, for 72 hours during TCR triggering, did not impair cell expansion and had only a minimal effect on cell proliferation ( Figures 1C–E and Supplementary Figure 1C ). A decrease in proliferation was observed at NaCl and sodium lactate concentrations above 50 mM, likely due to increased osmolality ( Figures 1C, D ). Based on this, we selected 40 mM as the highest tolerated dose of sodium lactate that could induce a significant effect without irreversibly impairing cell fitness ( Figure 1E ). Interestingly, human CD8+ T cells activated in the presence of increasing doses of sodium lactate or sodium chloride showed a higher sensitivity to the rise in osmolality, but overall, a similar response to the one of murine cells ( Supplementary Figure 1D ). Exposure to 40 mM sodium lactate during activation was well tolerated by human CD8+ T cells, with no significant impact over prolonged in vitro culture ( Supplementary Figure 1E ).

In line with our findings from a previous publication (30), addition of sodium lactate during activation elicited a dose-dependent increase in Granzyme B (GZMB) expression, beginning at 10 mM and peaking at 40-50 mM ( Figures 1F–H and Supplementary Figures 1F, G ). Granzyme B is one of the most highly expressed effector proteins in activated mouse CD8+ T cells (41) and essential to granule-mediated apoptosis of target cells (42), suggesting that lactate can alter the effector profile of CD8+ T cells.

Aiming to expand our understanding of the role of lactate during T cell activation, we compared the transcriptome of CD8+ T cells prior activation (naïve) and 72 hours after activation in the presence of media or 40 mM sodium lactate. TCR triggering caused a drastic reprogramming of the cellular transcriptome, which was further altered by the presence of lactate ( Figure 1I and Supplementary Figures 1H, I ). Specifically, lactate had a substantial effect on the expression of genes involved in CD8+ T cell differentiation and cytotoxic activity, including Granzyme B (Gzmb), interferon-gamma (IFN-γ) (Ifng), perforin 1 (Prf1) and ICOS (Icos) ( Figure 1J ). By means of flow cytometry, we could confirm that lactate also altered protein levels of a range of factors involved in CD8+ T cell differentiation, increasing the levels of CD44, CTLA-4, 4-1BB, and ICOS, and lowering the levels of the transcription factor T-bet and the lymph node homing molecule CD62L ( Figure 1K and Supplementary Figure 1I ). In addition, CD8+ T cells activated with 40 mM sodium lactate showed increased secretion of IFN-γ, a key mediator of cytotoxicity ( Figure 1L ).

In addition to altering the expression of effector molecules, further analysis of the transcriptome revealed that exposure to sodium lactate during the activation window induced a concerted upregulation of genes involved in the TCA cycle ( Figure 2A and Supplementary Figure 2A ). In contrast, expression of genes involved in glycolysis was not uniformly affected by lactate compared to media alone ( Figure 2B and Supplementary Figure 2A ).

The significant shift in metabolic gene expression led us to characterize the immediate effects of lactate exposure on CD8+ T cell metabolism. For this purpose, a short activation signal was given 12 hours before carrying out real-time metabolic analyses. During the metabolic flux monitoring, 40 mM of sodium lactate was administered to the cells ( Figure 2C ). Relative to cells treated with media or an equimolar dose of NaCl, addition of sodium lactate reduced the extracellular acidification rate (ECAR) of activated CD8+ T cells and concomitantly increased the oxygen consumption rate (OCR), used as surrogate measurements for glycolysis and oxidative respiration, respectively ( Figures 2D, E ). Despite suppressing glycolysis, addition of sodium lactate only slightly reduced the glycolytic capacity of the cells ( Figures 2D, E ). Similar effects were observed in human CD8+ T cell cultures ( Supplementary Figures 2B–D ), suggesting that exogenous lactate is taken up by metabolically active CD8+ T cells and instantly shifts down the endogenous production of lactic acid in the activated cells.

As previously described, upon TCR triggering, CD8+ T cells undergo a rapid induction of glycolytic metabolism which is followed by an increase in both intracellular and extracellular lactate levels ( Supplementary Figures 2E, F ). The lactate anion is transported across cell membranes via the proton-linked monocarboxylate transporters (MCT1, 2, 3, and 4) (43, 44). Of these, only MCT1 and MCT4 are expressed in CD8+ T cells (41, 45, 46). While neither transporter is expressed at high levels in naïve CD8+ T cells, ex vivo activation causes MCT1 mRNA and protein to peak at 12 to 24 hours post-activation, whereas MCT4 expression peaks later, between 48 and 72 hours after activation ( Supplementary Figures 2G, H ). Activation of CD8+ T cells in the presence of an MCT1-selective inhibitor (AZD3965) inhibited cell proliferation at the low nanomolar range, irrespective of the presence of lactate ( Supplementary Figure 2I ), suggesting a crucial role for monocarboxylate transport in early T cell expansion.

To confirm that the metabolic effect observed in Figures 2C–E was due to the presence of exogenous lactate, we assessed the glycolytic and oxidative rates of T cells after administration of lactate in combination with an MCT1 inhibitor ( Figure 2F ). Blocking the membrane trafficking of lactate via MCT1 mimicked the effects of sodium lactate, causing reduced ECAR and increased OCR ( Figure 2F ). This suggests that retention of endogenously generated lactate impacts T cell metabolism in a similar way as increased exogenous lactate. As shown in Figure 2F , adding exogenous sodium lactate during treatment with the MCT1 inhibitor did not further alter the T cell metabolic parameters, supporting efficient blocking of lactate transport and a dependence on an active uptake for the metabolic effects seen with lactate.

The lactate dehydrogenase enzyme (LDH) plays a crucial role in lactate metabolism as it catalyzes the interconversion between pyruvate and lactate. Activated CD8+ T cells express high levels of LDHA and lower levels of LDHB ( Supplementary Figure 2J ) (47). We hypothesized that inhibition of LDH would prevent incorporation of exogenous lactate into carbon metabolism and abrogate its effects. However, inhibition of LDH activity with the LDH inhibitor GSK2837808A (48) caused a drastic reduction of T cell expansion ( Supplementary Figure 2K ), and led to suppression of both basal glycolytic and oxidative respiration rates, in both the presence and absence of exogenous lactate ( Figure 2F ). This result highlights the central role played by LDH in the metabolism of activated CD8+ T cells, and indicates that disrupting the shuttling between pyruvate and lactate has dramatic consequences for the metabolic fitness of the cell. However, due to the deleterious effects on cell viability, inhibition of LDH did not allow us to determine if the effects of exogenous lactate on CD8+ T cell metabolism are directly dependent on the LDH-mediated conversion to pyruvate.

To understand how T cell metabolism is altered by prolonged exposure to lactate during TCR triggering, metabolite quantification by mass spectrometry and metabolic flux analysis was conducted after activation of CD8+ T cells for 72 hours in the presence of 40 mM sodium lactate ( Figure 3A ). Mass spectrometric analysis of the T cell metabolome revealed increased levels of glycolytic intermediates, including glucose-6-phosphate (G-6-P), 3- and 2-phosphoglyceric acid (3-PGA, 2-PGA) and pyruvate, in CD8+ T cells exposed to sodium lactate for 3 days ( Figures 3B, C and Supplementary Figure 3A ). Within the TCA cycle intermediates, lactate exposure caused an increase in the amount of succinate and a reduction in the downstream metabolites fumarate and malate ( Figures 3B, C and Supplementary Figure 3A ). Mitochondrial stress tests confirmed that CD8+ T cells activated in the presence of sodium lactate for 72 hours exhibit higher basal and maximal respiration as well as increased glycolytic rates and glycolytic capacity at the end of the exposure ( Figures 3D, E ). Altogether, these results demonstrate that CD8+ T cells adapt with an enhanced metabolic capacity when exposed to lactate during activation, by increasing the flux through glycolysis and the TCA cycle. The increased levels of pyruvate after lactate exposure and the shift in TCA metabolites indicate that lactate is taken up and incorporated into cellular metabolism.

To determine how the exogenously provided sodium lactate is taken up by CD8+ T cells and incorporated into carbon metabolism, we activated CD8+ T cells in the presence of 11.1 mM [U-¹³C₆]glucose alone, in combination with 40 mM sodium lactate, or 40 mM [U-¹³C₃]sodium lactate in combination with 11.1 mM glucose ( Figure 4A and Supplementary Figure 4 ). Labelling of cellular metabolites was measured by mass spectrometry at 24, 48 and 72 hours after activation ( Figures 4B–D and Supplementary Figure 4 ). Activation in the presence of [U-¹³C₆]glucose alone resulted initially (24-48h) in labelling of metabolites in the glycolytic pathway, including phosphoenolpyruvate (PEP), then pyruvate and lactate, followed by TCA cycle intermediates at 48-72 hours. Progressive labelling of ribose, originating from the pentose phosphate pathway (PPP), and the amino acids serine, glycine, alanine, glutamate, proline, and aspartate was also observed, suggesting incorporation of glucose-derived carbons through glycolysis and its branch pathways, as well as the TCA cycle. Addition of sodium lactate significantly displaced the contribution of [U-¹³C₆]glucose to the labelling of pyruvate and lactate, but not to PEP or ribose ( Figure 4C and Supplementary Figure 4 ). Glucose labelling of TCA cycle metabolites was markedly decreased by addition of lactate, as was the labelling of amino acids ( Figure 4C and Supplementary Figure 4 ). Tracing of [U-¹³C₃]sodium lactate revealed that exogenously sourced lactate enters T cells and is oxidized to pyruvate ( Figure 4D and Supplementary Figure 4 ). Labelling of metabolites downstream of pyruvate, namely alanine and citrate, occurs rapidly. Within 24 hours, labelling from lactate-derived carbons can be found in the TCA cycle metabolites succinate, fumarate and malate. In addition to alanine, lactate-derived carbons were incorporated into amino acids derived from the TCA cycle such as glutamate, proline and aspartate, but not into amino acids derived from the glycolytic pathway upstream of pyruvate, e.g., serine and glycine ( Figures 4B–D and Supplementary Figure 4 ). These findings show that exogenous lactate can be taken up, oxidized, and displace glucose as a carbon source for both the TCA cycle and amino acid synthesis in activated CD8+ T cells.

Based on the consistent differential expression of critical molecules for metabolism and effector function following lactate exposure during activation of CD8+ T cells, we sought to identify potential up-stream transcriptional regulators that could explain the observed alterations. To this aim, we utilized the 184 genes significantly differentially regulated with lactate exposure, to do a transcription-factor motif-enrichment analysis. Seventeen transcription factors had significant motif-enrichment and were readably detected transcriptionally in T cells. NRF1 was the transcription factor with the highest motif enrichment (OR 2.4 p 1e-14) but with low transcript abundance - 64 TPM (present data) and 14 TPM (single-cell). HIF-1α had a motif-enrichment (OR 2.2 p 1.5e-11) and high T cell transcript abundance - 388 TPM (present data) and 92 TPM (single-cell). The transcription factor with the highest affinity score was HIF-1α (affinity score 4.1, p = 0.002) together with c-MYC (affinity score 4.2, p = 0.002) ( Figure 5A ). The transcription factor analysis indicated that the HIF-1α transcription factor motif is enriched in the genes differentially expressed after exposure to sodium lactate. To follow up on this finding, we measured the differentiation of CD8+ T cells activated in the presence of 40 mM sodium lactate at ambient oxygen tension (21% O₂) or hypoxic conditions (1% O₂), using wild type or HIF-1α null CD8+ T cells ( Figures 5B ). Activation in hypoxic conditions severely limited CD8+ T cell proliferation but led to greater expression of the effector markers CD44, CTLA-4, 4-1BB, GZMB and ICOS, in agreement with previously published data ( Figures 5C–H ) (4, 49). Exposure to lactate led to an increase in expression of effector markers, which was further enhanced when combined with hypoxia ( Figures 5B–H ). Previous findings showed that effector differentiation was compromised in the absence of HIF-1α (4, 49, 50). Here, we observed that CD8+ T cells lacking HIF-1α failed to upregulate CTLA-4 in all of the tested culture conditions, suggesting a high level of HIF-1α-dependency ( Figure 5E ). In contrast, expression of CD44, 4-1BB, GZMB and ICOS was increased in hypoxia even in the absence of HIF-1α. The additive effect of lactate and hypoxia on the expression of 4-1BB and ICOS was lost in HIF-1α null CD8+ T cells. This HIF-dependency was not observed for GZMB and CD44. To gain insights on the role of HIF-1α in regulating the metabolism of CD8+ T cells, especially in the presence of lactate, we activated murine CD8+ T cells under mild hypoxic conditions (5% O2) and with or without sodium lactate. Interestingly, the lack of HIF1α led to increased basal and maximal oxidative respiration as well as spare oxidative capacity, independently of lactate presence and even at reduced oxygen tension ( Figures 5I–K ). This confirms the critical role played by HIF1α in triggering glycolysis. The addition of lactate leads to elevated extracellular acidification rate (ECAR) in both WT and HIF1α KO cells, suggesting saturation of glycolytic activity. This is in accordance with the increased oxygen consumption rate. Altogether, these results show that lactate and hypoxia have additive effects in driving expression of CD8+ T cell effector markers and metabolic activity and indicate that this relationship is in part dependent on HIF-1α.