Protein Transport

One day before the T-cell activation assays, human CTL were resuspended in Aim-V media containing 2% human serum without the addition of extra cytokines. Human CTL were counted and resuspended at a concentration of 10⁶ per ml, to which the following were added: anti-CD107a antibody (1:100), GolgiPlug Protein transport inhibitor (Brefeldin A, BD Bioscience, 1:1000). The growth media was removed from the culture plate containing CHO cells, and 200 μl of the human CTL mixture was added into each well of the 96-well plate. Technical triplicates were set for the experiments. The plate was incubated at 37 °C, 5% CO₂ for 3 h. Surface and intracellular staining were done according to the manufacturer’s protocol (BD Cytofix/Cytoperm, BD Perm/Wash, BD Biosciences)

Single-cell suspensions were stained with antibodies against surface molecules (Supplementary Table 2) at RT for 30 min. For intracellular staining, cells were stimulated with cell stimulation cocktail (plus protein transport inhibitors) containing PMA, ionomycin, brefeldin A and monensin (1:500 dilution, eBioscience Cat#00-4975-93) at 37^oC in RPMI media containing 10% heat-inactivated FBS (R&D systems, Cat#S11150H) and 0.05 mM 2-mercaptoethanol (ThermoFisher Scientific, Cat#21985023) for 4 hr prior to staining with Zombie NIR (BioLegend, Cat#423105). Next, cells were stained with antibodies against surface proteins followed by fixation and permeabilization and staining with antibodies against intracellular proteins. Samples were analyzed by a BD LSRFortessa SORP equipped to detect 17 or 15 fluorescent parameters. Compensation and data analysis were carried out using FACSDiva and FlowJo software. Gating strategy is shown in Supplementary Figs. 8 and 9.

To explore the effect of fatty acids on T-cell exhaustion, isolated T cells were stained with APC-Cy7-conjugated anti-CD4, PerCP-Cy5.5-conjugated anti-CD8, PE-conjugated anti-TIM3, or BV421-conjugated anti-TIM3 (Biolegend, San Diego, CA, USA) for 30 mins at 4°C. To investigate the effects of fatty acids on T-cell proliferation, isolated T cells were marked with Cell Trace™ Violet (Thermo Fisher Scientific, Waltham, MA, USA) for 20 mins at 37°C, conforming to manufacturer’s instructions, washed with complete medium, and cultured in the presence of anti-CD3/CD28-coated Dynabeads. After incubation for 5 d, dead cells were excluded by staining with 7-aminoactinomycin D. To investigate the effects of fatty acids on CD4+ and CD8+ T-cell function, isolated T cells were activated with anti-CD3/CD28-coated Dynabeads and pre-incubated with PE-conjugated anti-CD107a for 24 h. The protein-transport inhibitor (GolgiStop; 1 μl/mL, BD Biosciences) was added to the culture for the final 6 h. After that, cells were stained with LIVE/DEAD fixable dead cell stain reagent (Invitrogen, Carlsbad, CA, USA), APC-Cy7-conjugated anti-CD4 and PerCP-Cy5.5-conjugated anti-CD8 (Biolegend, San Diego, CA, USA). Subsequently, for intracellular staining, cells were incubated with Fixation/Permeabilization working solution (eBioscience, CA, USA) for 30 mins in the dark, followed by incubation with BV421-conjugated anti-interferon (IFN)-γ and APC-conjugated anti-IL-2 for 30 mins at 4°C. To investigate the effects of fatty acid on CD4+ and CD8+ T cell activation, isolated T cells were activated with anti-CD3/CD28-coated Dynabeads and stained with Percp-Cy5.5-conjugated anti-CD8, APC-Cy7-conjugated anti-CD4, BV786-conjugated anti-CD69, BV421-conjugated anti-CD25 and BV510-conjugated anti-HLA-DR (Biolegend, San Diego, CA, USA). To explore the effects of fatty acids on mitochondrial mass and ROS, T cells co-incubated with fatty acids for 24 h were resuspended in warmed 37°C staining solution containing MitoTracker® Green FM (50nM; Thermo Fisher Scientific) and MitoSOX™ Red Mitochondrial Superoxide Indicator (5μM; Thermo Fisher Scientific) for 30 mins, washed the cells with PBS, and then stained with the LIVE/DEAD™ Fixable Aqua Dead Cell Stain kit. To investigate the effect of mito-TEMPO on T cell exhaustion, function and mitochondrial ROS, T cells were co-incubated with 200μM mito-TEMPO and 500μM eicosenoate and pre-incubated with PE-conjugated anti-CD107a for 24 h. Then the cells were stained with MitoSOX™ Red Mitochondrial Superoxide Indicator, PE-conjugated anti-TIM3, BV421-conjugated anti-PD-1 and BV421-conjugated anti-interferon (IFN)-γ. Cells were examined using a flow cytometer (BD LSR II; BD Biosciences, San Jose, CA, USA), and data were analyzed using FlowJo software (Ashland, OR, USA).