Measuring Mitochondrial Function of Naïve and Effector CD8 T Cells

CD8 T cell bioenergetics can be interrogated using the Mito Stress Test. This methodology can be used to study acute and chronic metabolic programming. This protocol describes approaches to examine the relationships between T cell receptor biology and bioenergetic analysis.

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. Lymphocyte biology has been explored using bioenergetic analysis and has now become a critically import tool in the field. Thus, we sought to optimize a bioenergetic analysis assay that can be adapted with pretreatments and acute injection for receptor stimulations. Here, we evaluated CD8 T cell ex vivo metabolism using the Cell Mito Stress Test to assess rates of oxygen consumption and extracellular acidification in naïve and effector CD8 T cells. Antigen-specific effector CD8 T cells were derived via ex vivo stimulation, and naïve CD8 T cells harvested from splenocytes and isolated with magnetic bead column separation.

Pretreatments are performed in microplates and we detail how to prepare sensor cartridges. We show how injection ports can loaded with drugs to indirectly measure metabolic capacities and with metabolic modulators, this protocol can be used to study specific enzyme activity. T-cell receptor stimulations can be studied in real time with acute injection and stimulation with anti-CD3/CD28 using the injection ports. Instrument analyzers are used for measurements and data collection and data visualization is done with software programs to interpret cellular metabolism. This strategy produces an extensive amount of data on immune cell biology and mitochondrial bioenergetics allowing researchers to customize the protocol in numerous ways to explore CD8 T cell metabolism.

The fate and functionality of immune cells are significantly impacted by metabolism, oxidative consumption, and anaerobic respiration^1,2,3,4. Recently, there has been growing interest in targeting metabolic modulation as a strategy to re-program or revigorate CD8 T cell fate and effector function and improve viral clearance or enhance endogenous anti-tumor immunity^5,6,7,8,9. Notab....

Mice were kept in a pathogen-free environment and maintained according to the Institutional Animal Care and Use Committee standards and regulations.

1. Generation and expansion of CD8 T cells via antigen-specific stimulation

1. On the first day, harvest splenocytes derived from OT-I mice; then, prepare and activate them in vitro with the SIINFEKL (4) peptide.
   1. Clean the hood space, prepare reagents, and gather the mouse medium. Warm the mouse media in a water or bead incubator.
      1. In a 6-well plate, put 7 mL of mouse medium into one well and then another 3 mL of mouse medium into a di....

The glycolytic and oxidative metabolic capacities can be measured using a mitochondrial functional assay which evaluates capacities by targeting components of the electron transport chain at particular time points (Figure 2A). Different injection schemes can be loaded onto the sensor cartridge ports to modify the traditional assay and assess acute TCR stimulation (Figure 2B,C). Cell number and drug concentration for various cell types should be .......

In this article, we outline a protocol to assess mitochondrial function of naïve and effector CD8 T cells. We detail and compare methods to prepare both antigen-specific and polyclonal CD8 T cells using OT-I and C57BL/6 mice. Our results demonstrate that there are similar trends in metabolism despite the method of activation and pretreatment in CD8 T cells. The data reveal that antigen-specific activation leads to more metabolically active OT-I CD8 T cells compared to their C57BL/6 wild-type counterparts stimulated .......

The authors have no competing interests to disclose.

The Hertz Foundation, the Amy Davis Foundation, the Moore Family Foundation, and the Heidi Horner Foundation have provided invaluable support, for which we are grateful. This work was also supported in part by NIH grants to RMT (AI052157, AI136534), while JAT was supported by the Hertz Graduate Fellowship.