Name: Measuring Mitochondrial Function of Naïve and Effector CD8 T Cells (Video) | JoVE Journal
Uploaded: 2025-03-28T12:30:00.000+00:00
Duration: 6 min 7 s
Description: Learn to measure mitochondrial function in naïve and effector CD8 T cells using the Mito Stress Test. This protocol enables real-time bioenergetic analysis of T cell receptor signaling and metabolic flexibility.

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5 minutes

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.

The authors have no competing interests to disclose.

In this article, we outline a protocol to assess mitochondrial function of na&#239;ve 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 fate and functionality of immune cells are significantly impacted by metabolism, oxidative consumption, and anaerobic respiration1,2,3,4. 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 immunity5,6,7,8,9. Notably, antigen receptor signaling through the T cell receptor (TCR) is a key requirement for CD8 T cell differentiation resulting in downstream signaling and activation10,11,12 (Figure 1). Prolonged exposure to immunological insults causes persistent antigen-specific stimulation on the TCR eventually leading to chronically inflamed states, T cell fatigue, a remodeling of the immune microenvironment, and immune escape11,13,14,15,16,17,18,19.
The metabolism of exhausted CD8 T cells is fundamentally distinct from that of functional effector CD8 T cells2,3,14,15,18,20. T cell differentiation, interferon &#947; (IFN&#947;) secretion, and recall capacity are, in part, determined by mitochondrial function and &#946;-oxidation break-down products. IFN&#947;+ CD8 T cells are critical components of both anti-tumor and anti-viral immune responses21,22,23. Specific metabolic flux via glycolysis and the electron transport chain is important for CD8 T cell activation, cytokine secretion, and memory responses4,11,13,15,18,24,25,26,27,28. Optimal responses, including T cell activation and effector differentiation, require a coordinated and specific mitochondrial response, while mitochondrial defects and excessive reactive oxygen species (ROS) characterize exhausted or dysfunctional T cells9,29. Recently, persistent TCR stimulation of CD8 T cells in vitro promotes CD8 T cell exhaustive differentiation in part by inducing oxidative stress and reprogramming oxidative metabolism and metabolic capacities required for T cell proliferation1,2,13,20,24,29. Altogether, metabolic control axes are critical components in directing CD8 T cell differentiation and their progression to effector, memory, or exhausted/dysfunctional phenotypes.
Metabolic compounds also direct immune cell responses by functioning as autocrine or paracrine signaling molecules9,30,31,32,33,34,35. Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive and inflammatory lipids that signal via G-protein coupled receptors (GPCRs) to modulate lymphocyte egress and cytotoxicity by CD8 T cells36. LPA signaling via GPCR LPA receptors on CD8 T cells reprograms metabolism to increase lipolysis, fatty acid oxidation, and proton leak9. Altogether, the bioenergetics and metabolism of CD8 T cells are largely driven by substrate availability, environmental cues, and energetic requirements.
Methodologies to interrogate CD8 T cell metabolism have become ever more important. The Cell Mito Stress Test provides a comprehensive evaluation of bioenergetics and is now recognized as a hallmark technique in the field of immunometabolism and CD8 T cell energetics9,37. Adherent cells were historically used for the Mito Stress Test assay38; however, there is increasing interest in applying this protocol for cells grown in suspension and specifically using immune cells for the Cell Mito Stress Test assay. Here, we present a detailed protocol to measure the metabolic activity of CD8 T cells based on our recent publication9. We provide a detailed explanation of the expansion of CD8 T cells, na&#239;ve CD8 T cell isolation, assay preparation, and treatment with protocols for both pretreatments and acute injections in the Cell Mito Stress Test assay. Importantly, we compare and contrast multiple methods for TCR stimulation and CD8 T cell activation, including polyclonal and antigen-specific TCR stimulation.
This protocol details antigen-specific stimulation using OT-I transgenic mice (a classical transgenic mouse model) for which all mouse T cells express the same V&#945;2 and V&#946;5 genes39. The OT-I mouse CD8 T cells all harbor the same TCR that is specific against ovalbumin octapeptide (OVA257-264 also written as the amino acid sequence SIINFEKL or N4 a widely studied epitope that, upon presentation by major histocompatibility complex (MHC) class I, activates cytotoxic CD8 T cells39 (Figure 1A). Overall, the OT-I transgenic mouse model is widely used by immunologists to study TCR signaling and antigen-specific T cell effector function. As opposed to monoclonal activation with the OT-I mouse model, polyclonal CD8 T cells may be generated with anti-CD3/CD28 antibodies against TCR CD3 subunits and CD28 co-stimulatory molecule40 (Figure 1B). Anti-CD3/CD28 antibodies bypass the antigen-specific component of TCR signaling to activate a polyclonal population of T cells40. Ultimately, the results described in this report compare multiple methods for using the Cell Mito Stress Test to quantify dynamic metabolic flux in CD8 T cells.

<table><tbody><tr><td>Antimycin A</td><td>Sigma-Aldrich</td><td>A8674</td><td></td></tr><tr><td>Anti-CD28</td><td>Biolegend</td><td>102116</td><td></td></tr><tr><td>Anti-CD3/CD28 Dynabeads</td><td>ThermoFisher</td><td>11456D</td><td></td></tr><tr><td>Biotinylated anti-CD3</td><td>Biolegend</td><td>317320</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>108321-42-2</td><td></td></tr><tr><td>CD8a+ T cell isolation kit</td><td>Miltenyi Biotec</td><td>130-104-075</td><td></td></tr><tr><td>Cell Strainers (100 &amp;micro;m)</td><td>CELL TREAT</td><td>229485</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>E8008</td><td></td></tr><tr><td>Ficoll</td><td>Sigma-Aldrich</td><td>26873-85-8</td><td>density gradient medium&amp;nbsp;</td></tr><tr><td>FCCP ((4-(trifluoromethoxy) phenyl) carbonohydrazonoyl dicyanide)</td><td>Sigma-Aldrich</td><td>C2920</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G-6152</td><td></td></tr><tr><td>Glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>Positive selection columns</td></tr><tr><td>Magnetic cell separation column</td><td>Miltenyi Biotec</td><td>130-042-301</td><td></td></tr><tr><td>Microplate</td><td>Agilent</td><td>102601-100</td><td></td></tr><tr><td>Oligomycin</td><td>Sigma-Aldrich</td><td>75351</td><td></td></tr><tr><td>Pyruvate</td><td>Sigma-Aldrich</td><td>113-24-6</td><td></td></tr><tr><td>Recobinant IL-2</td><td>PeproTech</td><td>200-02</td><td></td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>Seahorse media</td><td>Agilent</td><td>103576-100</td><td></td></tr><tr><td>Sensor cartridge</td><td>Agilent</td><td>102601-100</td><td></td></tr><tr><td>Streptavidin</td><td>Sigma-Aldrich</td><td>A9275</td><td></td></tr><tr><td>Sterile 6 well plate</td><td>CELL TREAT</td><td>230601</td><td></td></tr><tr><td>Sterile 24 well plate</td><td>CELL TREAT</td><td>229524</td><td></td></tr><tr><td>XF Calibrant</td><td>Agilent</td><td>102601-100</td><td></td></tr></tbody></table>

measuring mitochondrial function of na&#239;ve and effector cd8 t cells

Measuring Mitochondrial Function of Na&#239;ve and Effector CD8 T Cells

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 na&#239;ve and effector CD8 T cells. Antigen-specific effector CD8 T cells were derived via ex vivo stimulation, and na&#239;ve 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 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 ...

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. ...

measuring-mitochondrial-function-of-nave-and-effector-cd8-t-cells

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Learn to measure mitochondrial function in naïve and effector CD8 T cells using the Mito Stress Test. This protocol enables real-time bioenergetic analysis of T cell receptor signaling and metabolic flexibility.

Measuring Mitochondrial Function of Naïve and Effector CD8 T Cells

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