Method to Study Metabolism in Lymphoid Cells using Chemistry to Measure Puromycin Incorporation by Flow Cytometry

Summary

The protocol shows a flow cytometry-based method using chemistry to study energy metabolism in cells.

Abstract

Upon antigen stimulation, naïve T cells undergo rapid proliferation and expansion to effector T cells. Metabolism plays an important role in the generation of biomass needed for these rapidly proliferating cells and for the generation of molecules required for effector T cell differentiation and function, which influence the outcome of the adaptive immune response in infection or cancers. Naïve T cells reprogram their metabolism upon antigenic stimulation to increase the generation of ATP, which is required to support their growth, biosynthesis, and effector functions. ATP can be generated in a cell either by the mitochondrial-oxidative phosphorylation (OXPHOS) pathway or by the glycolytic pathway. Because most of the ATP generated in a dividing, growing cell is used up for the synthesis of proteins, protein synthesis has been used as a surrogate for ATP levels. Protein synthesis can be measured by the incorporation of puromycin, which mimics the 3′ adenosine of a tRNA charged with a modified tyrosine and leads to spontaneous termination of protein translation. Metabolic inhibitors like 2-deoxyglucose (2DG), which blocks the glycolytic pathway, and Oligomycin (O), which blocks complex 5 of the electron transport chain (ETC), can be used to study the dependencies of cellular ATP generation on these two pathways in conjunction with evaluation of protein synthesis in a method called SCENITH. We describe here a variation of this method that detects puromycin incorporation by flow cytometry using chemistry. This method of studying metabolism is relatively easy and can be used for evaluating rare cell populations, as well as patient samples, by flow cytometry.

Introduction

In recent years, there has been a growing appreciation of the important role of metabolism in the regulation of T cells. Cellular metabolism regulates many critical functions in lymphocytes, including clonal expansion of pathogen-specific T cells and their differentiation into effector cell subsets, which synthesize a variety of effector molecules, including inflammatory cytokines and cytolytic granzymes required to kill pathogens¹. The study of T cell metabolism is therefore important both for evaluating normal T cell function and for the development of potential therapeutics.

Naïve T cells are metabolically quiescent and rely on catabolic metabolism and oxidative phosphorylation (OXPHOS) for their energy needs^1,2. Upon T cell receptor (TCR) activation, T cells undergo rapid metabolic reprogramming driven in part by the upregulation of nutrient transporters on the surface of the cells, which increase the uptake of glucose and multiple amino acids, including glutamine, from the environment. These nutrient building blocks are then used in anabolic pathways to make macromolecules that contribute to cellular growth and support the increased energy demands associated with increased cell growth and division^1,2,3. Metabolic reprogramming in activated T cells makes the cells more dependent on aerobic glycolysis and glutaminolysis for their energy needs^1,2,3. Upon TCR stimulation, the uptake of nutrients also plays a critical role in activating downstream molecular pathways³. For example, uptake of amino acids and increased local amino acid concentration activate mTORC1 signaling⁴. Conversely, reducing the availability of intracellular amino acids diminishes the mTORC1 signaling pathway^2,3,5,6,7. Thus, metabolism and cellular function are tightly linked in an effector T cell.

The large increase in biomass required for rapidly dividing T cells requires the upregulation of protein synthesis/translation⁸. Protein synthesis can be quantified in a cell by studying the incorporation of puromycin⁹, a tyrosyl-tRNA mimic that blocks translation by incorporating, marking, and releasing elongating polypeptide chains from translating ribosomes⁹. Protein translation is a high-energy-dependent process; indeed, most of the ATP generated in dividing, growing cells is used for the synthesis of proteins. Thus, protein synthesis has been used as a surrogate measurement for cellular ATP¹⁰. To generate ATP, cells commonly rely on glycolysis and/or the mitochondrial OXPHOS pathway¹¹. Glycolysis generates two ATP molecules per glucose molecule. The OXPHOS pathway generates 36 ATPs per glucose^12,13, but glycolysis can generate ATP faster¹⁴. Thus, rapidly dividing activated T cells, like cancer cells, rely heavily on the glycolytic pathway for ATP generation.

The dependency of a cell on each metabolic pathway can be studied by the use of specific inhibitors. The glycolytic pathway inhibitor 2-Deoxyglucose (2-DG), which is a glucose analog, acts as a competitive inhibitor for the glycolytic enzyme hexokinase¹⁵. 2-DG is phosphorylated to 2DG-phosphate (2DG-P) by hexokinase. 2DG-P cannot be further metabolized by phosphohexose isomerase, which normally converts glucose-6-phosphate (G-6-P) to fructose-6-phosphate¹⁶. 2DG-P is, therefore, trapped and accumulates in the cells, leading to inhibition of glycolysis and depletion of cellular ATP, thereby blocking protein synthesis¹⁷. In a similar manner, the dependency of cells on the mitochondrial OXPHOS pathway can be studied using Oligomycin, which binds to the proton channel of ATP synthase of the electron transport chain (ETC)¹⁸, thus blocking mitochondrial ATP synthesis (Figure 1). The use of these inhibitors in conjunction with an evaluation of protein synthesis can provide a picture of the metabolic state of cells and has been used in a powerful single-cell method known as SCENITH that evaluates puromycin incorporation using flow cytometry¹⁰. However, the availability of SCENITH reagents can be limiting. Other commonly used techniques for evaluating glycolysis and OXPHOS include Seahorse extracellular flux analyses, which require larger cell numbers, which may not be experimentally feasible. In this article, we describe an alternative method for evaluating puromycin incorporation using a commercially available Click-it chemistry reagent. The ease of this method allows rapid evaluation of the metabolic state of lymphocytes, including rare cell populations and patient samples.

Protocol

Animal husbandry and experiments were performed in accordance with approved protocol G98-3 by the National Human Genome Research Institute's Animal Use and Care Committee, NIH, and approved protocol LISB 22E by the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee, NIH. Male C57Bl/6J mice aged 6-12 weeks, bred and housed in specific pathogen-free (SPF) conditions, were used for the present study. Similar results were obtained with female mice. Human whole blood was obtained from the NIH Blood Bank under NIH Clinical Center IRB-approved protocol 99-CC-0168 Collection and Distribution of Blood Components from Healthy Donors for In Vitro Research Use. Written informed consent was obtained from all subjects in accordance with the Declaration of Helsinki.

1. Mouse naïve CD4⁺ T cell isolation and culture

1. Purify naïve CD4⁺ T cells by negative selection from pooled single-cell suspensions of mouse spleen or lymph nodes from 6 to 12-week-old male mice using a magnetic cell separation system according to the manufacturer's protocol.
2. Culture cells (as described below) in IMDM media containing L-alanyl-L-glutamine dipeptide and supplemented with 10% Fetal Bovine Serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin, and 0.05 mM 2-β-mercaptoethanol.
3. Co-culture naive CD4 T cells at a ratio of 1:8 with mitomycin-C-treated T-depleted splenocytes (described below) as APCs in 48-well plates under non-polarized (1 µg/mL of anti-CD3 [clone 2C11] and 3 µg/mL of anti-CD28 [clone 37.51]), and Th0 conditions (1 µg/mL of anti-CD3, 3 µg/mL of anti-CD28, 10 µg/mL each of anti-IFNg [clone XMG1.2], anti-IL-12 [clone C17.8], and anti-IL-4 [clone 11B11]) for 72 h at 37 °C. Set up cultures using 1 x 10⁶ APCs and 0.13 x 10⁶ naïve CD4 T cells per well of a 48-well plate (Table of Materials)
4. Generation of mitomycin-C-treated T-depleted splenocytes as APCs: In brief, stain a single cell suspension of mouse spleen with FITC-conjugated CD4 and CD8 antibodies. Subsequently, label the cells magnetically with Anti-FITC Microbeads (see Table of Materials). Then, using a magnetic cell separation system according to the manufacturer's protocol, negatively select the APCs from T cells. Next, treat the APCs with 50 µg/mL Mitomycin C for 40 min at 37 °C incubator, followed by washing 3x with 1x PBS.

2. Human PBMCs isolation and culture

1. Isolate PBMCs from whole blood using lymphocyte separation media and according to the manufacturer's protocol (see Table of Materials).
2. Culture PBMCs in RPMI media supplemented with 2 mM L-glutamine, 0.01 M HEPES, 1 mM sodium pyruvate, 1% non-essential amino acids, 100 U/mL penicillin and Streptomycin, 0.05 mM 2-β-mercaptoethanol, and 5% Human Serum.
3. Culture 1 x 10⁶ PBMCs per well of a U-bottomed 96-well plate with 5 µg/mL of Phytohemagglutinin-L (PHA-L) for 18-24 h at 37 °C.

3. Metabolic assay

NOTE: It is important to keep all cells, inhibitors [Oligomycin (O), 2-Deoxyglucose (2-DG), DMSO (Co)], and O-propargyl-puromycin (OPP) reagent as close to 37 °C as possible. Aliquot out the cells, inhibitors, and OPP reagent onto U-bottomed 96 plates and leave the plates in a 37 °C incubator as much as possible. Aliquot inhibitors 2-DG and O into smaller volumes to avoid multiple freeze-thaws.

1. Make a 2 M (20x) stock solution of 2-DG and a 40 µM (40x) stock solution of O. For OPP, dilute 1 µL of OPP in 49 µL of medium so that the concentration is 400 µM-this will be the 20x stock solution.
2. Make sure all other components of the OPP protein synthesis kit (see Table of Materials) are at room temperature (RT), before starting the experiment.
3. Prepare 1x OPP reaction buffer by diluting an aliquot from the Component C bottle 1:10 with deionized water. After use, store any remaining 1x solution at 2-8 °C.
4. Seed 0.1-2 x 10⁶ cells per well of round-bottom 96-well plate for each condition (Co / DG / O / DGO) in 100 µL of complete IMDM (for mouse CD4 T cells) or complete RPMI (for human PBMCs) or any complete media of choice. Keep the plate in the incubator as much as possible.
5. Add 1x inhibitors from stock solutions (5 µL of 20x DG, Co), (2.5 µL of 40x O) and incubate for 15 min at 37 °C, 5% CO₂. The final concentrations on cells are 100 mM DG and 1 µM O. Mix the inhibitors with the cells carefully with a pipette. For ease, aliquot out inhibitors in a 96-well plate and add cells to the inhibitors and mix well by pipetting up and down a few times.
6. In the DGO sample, add 1x of DG (5 µL), mix carefully with a pipette, and incubate for 10 min at 37 °C, 5% CO₂. Then, add 1x O (2.5 µL), pipette a couple of times, and incubate for 5 min at 37 °C, 5% CO₂.
7. Without washing out the inhibitors, add 5 µL of OPP from the 20x (400 µM) stock solution to each sample so that the concentration is 20 µM in cells, pipette up and down multiple times, and incubate 30 min at 37 °C, 5% CO₂. For ease, aliquot out OPP onto a 96-well plate, add cells with inhibitors to the OPP reagent, and mix well.
   NOTE: For less metabolically active cells and cells isolated directly ex vivo, increase the incubation time after OPP addition. Incubation time with OPP needs to be standardized for each cell type.
8. After incubation with OPP, add 100-150 µL of ice-cold FACS Buffer (2% FBS and 1 mM EDTA in 1x PBS). Centrifuge at 750 x g for 2 min at 4°C. Discard the supernatant and repeat the wash step.
9. Stain cells with appropriate surface markers, anti-mouse CD4 antibody (used at 1/200 dilution) or anti-human CD4 antibody (used at 1/50 dilution), Live/Dead reagent (used at 1/600 dilution), in the presence of Fc Block (used at 1/200 dilution). Incubate for 25-30 min at 4 °C in the dark.
10. Wash the plate with 150-200 µL of FACS buffer and centrifuge at 750 x g for 2 min at 4 °C. Gently flick the plate to discard the supernatant, then repeat the wash step. At this stage, the cells are stained on the surface.
11. Resuspend the cells in 1% PFA in PBS, mix well, and incubate for 10-15 min at 4 °C. Protect from light.
12. Wash with FACS Buffer and centrifuge at 750 x g for 2 min at 4 °C, and repeat washing.
13. Resuspend the cell pellet in 200 µL of cold 1x Saponin permeabilization buffer (0.1% (wt/vol) Saponin + 3% FBS in 1x PBS). Incubate at RT, protected from light for 5-15 min. The cells are fixed and permeabilized at this time.
14. Prepare 1x OPP reaction buffer additive (Component E) by diluting the 10x solution 1:10 in deionized water. Prepare this solution fresh and use it on the same day.
15. Prepare OPP reaction cocktail according to Table 1. Use the reaction cocktail within 15 min of preparation.
16. Centrifuge the samples from step 3.13 at 750 x g for 2 min. Discard the supernatant. Wash the cells 2x with 200 µL of 1x Saponin permeabilization buffer. Spin and discard the supernatant.
17. Add 50-100 µL of OPP reaction cocktail (prepared in step 3.12) to each well and mix well. Incubate for 30 min at RT, protected from light. Then, spin plates at 750 x g or 2 min at 4 °C. Discard the supernatant.
18. Resuspend the pellet in 200 µL of 1x Saponin permeabilization buffer and centrifuge samples at 750 x g for 2 min at 4 °C. Discard the supernatant.
19. Resuspend in 50 µL of Reaction Rinse Buffer (Component F) per well and centrifuge at 750 x g for 2 min at 4°C. Resuspend stained cells in 200 µL of FACS buffer.
20. Analyze samples by flow cytometry (see Supplementary Figure 1) the same day, or store the cells in the dark at 4 °C and analyze them the next day.

4. Analysis of metabolic assay

1. Measure the incorporation of puromycin (OPP) as a surrogate for protein synthesis under the following conditions: 1) control condition (Co); 2) in cells where the glycolytic pathway is blocked by 2-DG; 3) in cells where the mitochondrial OXPHOS pathway is blocked by O; and 4) in cells where both the glycolytic and mitochondrial pathways are blocked by inhibitors (DGO), by evaluating the mean fluorescence intensity (MFI) of OPP (Figure 2).
2. Analyze the dependency of cells towards a particular pathway by looking at the MFI of OPP in each condition. Directly evaluate the MFI as a measure of protein synthesis. Calculate the glucose dependence, glycolytic capacity, fatty acid oxidation (FAO) and amino acid oxidation (AAO) capacity, and mitochondrial dependence according to¹⁰.

Results

Naïve CD4⁺ T cells were isolated from the spleen and lymph nodes of mice and differentiated to Th0 or non-polarized (NP) lineages for 72 h. Cells were harvested after 0 h, 48 h, and 72 h of culture, and OPP incorporation was measured by looking at the MFI of the OPP. Naïve CD4⁺ T cells were less synthetic than the activated T cells, as seen by the MFI of OPP (Figure 3). Naïve cells were partially dependent on glycolysis but more so on the OXPHOS pathway as evaluated by ...

Discussion

This paper provides a method to study metabolism in lymphoid cells by flow cytometry, based on SCENITH technology, but using chemical labeling of incorporated puromycin. Although we have focused on lymphocytes, this protocol can be adjusted to study metabolism in any cell type. This method is also advantageous to study metabolism in rare populations of cells and patient samples, where other techniques, such as Seahorse, require larger numbers of cells.

A critical step in the protocol is to mak...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Mercaptoethanol	Thermo Fisher	21985-023
Alexa Fluor 488 anti-Puromycin Antibody	Biolegend	381506	Dilution used: 1/100
Alexa Fluor 647 anti-human CD4 Antibody	Biolegend	357422	Dilution used: 1/50
BD Horizon BUV737 Rat Anti-Mouse CD4	BD Biosciences	612761	Dilution used: 1/200
Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kit	Thermo Fisher	C10456
HEPES (1 M)	Thermo Fisher	15630080
Human Serum	Millipore Sigma	H4522
IMDM, GlutaMAX Supplement, 500 ml	Thermo Fisher	31980030
InVivoMAb anti-mouse CD28, Clone: 37.51	BioXcell	BE0015-1
InVivoMAb anti-mouse CD3ε, Clone: 145-2C11	BioXcell	BE0001-1
InVivoMAb anti-mouse IFNγ, Clone: XMG1.2	BioXcell	BE0055
InVivoMAb anti-mouse IL-12 p40, Clone: C17.8	BioXcell	BE0051
InVivoMAb anti-mouse IL-4, Clone: 11B11	BioXcell	BE0045
L-Glutamine (200 mM)	Thermo Fisher	25030081
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation	Thermo Fisher	L34966	Dilution used: 1/600
LSM lymphocyte separation medium	MP Biomedicals	50494X
MEM Non-Essential Amino Acids Solution (100X)	Thermo Fisher	11140050
Mitomycin C from Streptomyces caespitosus	Sigma	M0503-10X2MG
Naive CD4 Isolation Kit, mouse	Miltenyi Biotec	130-104-453
Paraformaldehyde 16% Aqueous Solution EM Grade	Electron Microscopy Sciences	15710
PBS (1X), pH 7.4, 500 mL	Quality Biologicals	114-058-101
Penicillin-Streptomycin (10,000 U/mL). 100mL	Thermo Fisher	15140122
Phytohemagglutinin-L (PHA-L)	Millipore Sigma	11249738001
Premium Grade Fetal Bovine Serum (FBS)	Avantor	97068-085
RPMI 1640 Medium, no glutamine	Thermo Fisher	21870076
Saponin	Thermo Fisher	A18820.22
Sodium Pyruvate (100 mM)	Thermo Fisher	11360070