Unraveling the Impact of Fatty Acids on T-Cell Functionality

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).