Testing the Specificity of Compounds Designed to Inhibit CPT1A in T Cells

Abstract

In response to antigen and costimulation, T cells undergo a series of metabolic transitions that fulfill the biosynthetic demands of clonal expansion, differentiation, and effector function. Following antigen clearance, the oxidation of long-chain fatty acids (LCFAO) has been implicated in the transition from effector to central memory T cells. However, studies demonstrating a role for LCFAO in memory T-cell development have largely relied on the use of etomoxir (ETO), a small molecule inhibitor of the long-chain fatty acid transporter CPT1A. Understanding how the depletion of nutrients including LCFA that might occur in tumor microenvironments affects T-cell proliferation, differentiation, and function has important implications for tumor immunotherapy. Here, we combine the analysis of posttranscriptional gene silencing with extracellular flux assays to determine if etomoxir exerts nonspecific effects on oxidative metabolism. The off-target effects of ETO that we describe highlight the challenges of using pharmacologic inhibitors in loss-of-function approaches in T cells.

1. Introduction

Following antigen stimulation, T cells follow an ordered differentiation program involving activation, proliferation, and differentiation. T-cell activation induces a series of metabolic transitions that support the entire proliferation and differentiation processes. A metabolic shift to glycolysis in activated T cells supports energy generation, macromolecular biosynthesis, and redox homeostasis. Since it was widely accepted that etomoxir inhibits CPT1A, the rate-limiting enzyme in LCFAO, long-chain fatty acids have been implicated in the formation of memory T cells following antigen clearance [1–3].

Concerns about the specificity of etomoxir have been raised in recent studies on immune cells. We showed that etomoxir concentrations at commonly used concentrations elicit nonspecific effects on oxidative metabolism, culminating in ROS production, mitochondrial matrix swelling, and GSH depletion in primary human T cells undergoing high rates of clonal expansion [4]. Complementary studies provided genetic evidence that ablating CPT1A in T cells had minimal consequence on either memory T-cell formation or regulatory T-cell development [5]. In studies on macrophage metabolism, etomoxir treatment limited the polarization of macrophages toward an M2 lineage [6]. In contrast to T cells, there was no phenotype in macrophages lacking either CPT1a [7] or CPT2 [8] despite their inability to oxidize LCFA. In isolated mitochondrial preparations, etomoxir concentrations above 10 μM inhibited the mitochondrial adenine nucleotide transporter as well as complex 1 of the electron transport chain [9]. Here, we developed an assay to test the specificity of etomoxir in cells genetically engineered to lack CPT1A expression. T cells expressing lentiviral shRNA against CPT1A were seeded on matrix-coated XF “Seahorse” cell culture dishes. Their metabolic parameters, including rates of oxygen consumption and glycolysis, were assessed in response to varying doses of etomoxir treatment.

2. Materials

2.1. Cell Culture

1. Primary human T cells.

2. T-cell growth medium: RPMI 1640 supplemented with 10% FBS, 10 mM HEPES, 2 mM l-glutamine, 100 U/ml penicillin G, and 100 μg/ml streptomycin.

3. 4.5 μm Dynabeads containing immobilized antihuman CD3 and antihuman CD28.

4. Etomoxir (sterile) diluted in QH₂O and sterile filtered with a 2 μM filter.

5. Lentiviral supernatants with shRNA targeting CPT1A or scramble shRNA against the human β-actin gene as described previously in [10].

2.2. XF Flux Analyzer

1. XF96 analyzer with WAVE software.

2. XF96 cell culture microplate and XF sensor cartridge.

3. 1.36 mg/ml Cell-Tak (Corning).

4. 0.1 M sodium bicarbonate, pH 8.0 (sterile filtered).

5. 1 N NaOH.

6. Etomoxir.

7. XF assay medium: Non-buffered RPMI 1640 containing 5.5 mM glucose, 2 mM l-glutamine, and 5 mM HEPES. Adjust the pH to 7.4 with 0.1 N NaOH.

8. Sterile filter assay medium with a 0.2 μM filter following pH adjustment.

2.3. Immunoblotting

1. RIPA-2 lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS).

2. Protease inhibitor (Mini Complete, Roche).

3. Phosphatase inhibitor (PhosSTOP, Roche).

4. PDVF membrane.

5. Running buffer (0.1% SDS, Tris/Glycine solution).

6. Transfer buffer (Tris/Glycine).

7. Tris-buffered saline (TBS).

8. TBS containing 0.1% Tween-20 (TBS-T).

9. Blocking solution: 5% nonfat dry milk in TBS-T.

10. 7.5% separating gel (solutions used: 1.5 M Tris–HCl, pH 8.8, 10% SDS, 30% acrylamide bis 37.5:1, 10% ammonium persulfate, TEMED). See Note 1 for details.

11. 4% stacking gel (solutions used: 0.5 M Tris–HCl, pH 6.8, 10% SDS, 30% acrylamide bis 37.5:1, 10% ammonium persulfate, TEMED). See Note 2 for details.

12. Sample buffer (solutions used: 0.5 M Tris-HCl, pH 6.8, 50% glycerol in QH₂O, 10% SDS, 2-mercaptoethanol, pinch of bromophenol blue). See Note 3 for details.

3. Methods

3.1. T-Cell Activation

1. Day 0. Activate 6e6 primary human T cells with anti-CD3/anti-CD28 Dynabeads at a ratio of 3 beads to 1 cell.

2. Day 1. Activated T cells are infected with either scramble or shRNA lentivirus against CPT1A.

3. Day 3. Determine infection efficiencies by flow cytometry. Count and refeed T cells at a concentration of 0.8–1.0 × 10⁶ cells/ml.

4. Day 5. Lyse 1.0 × 10⁶ scramble vs. shRNA CPT1A cells in RIPA-2 containing protease and phosphatase inhibitors.

3.2. XF Flux Analysis (See Fig. 1)

Fig. 1.

The specificity of etomoxir for CPT1A is lost at concentrations above 5 μM. Primary human T cells expressing either control or shRNA against CPT1A were restimulated with Dynabeads and expanded for 5 days. Oxygen consumption rates (OCR) in control (a) vs. shRNA cells (c) are shown. OCR was measured under basal conditions and after the introduction of etomoxir (dotted lines). The corresponding glycolytic rates (ECAR) in control (b) and shRNA cells (d) are shown. (Figure adapted from R. S. O’Connor et al., The CPT1a inhibitor, etomoxir induces severe oxidative stress at commonly used concentrations. Sci Rep 8, 6289 (2018))

1. Day 0 (7 days prior to assay). Prepare the extracellular matrix adhesive (Cell-Tak) by combining 3 ml sodium bicarbonate with 50 μl of Cell-Tak solution and 20 μl NaOH.

2. Coat all wells of the XF cell microplate with 12 μl Cell-Tak.

3. Incubate the microplate overnight at 37 °C.

4. Rinse three times in sterile QH₂O, air-dry, and then store at 4 °C until use.

5. Day 6 (1 day prior to assay). Add 200 μl of the calibrant solution to hydrate the XF sensor cartridge. Leave overnight at room temperature.

6. Day 7 (day of assay). Transfer the hydrated XF sensor cartridge to a CO₂-free, 37 °C incubator for 5–6 h (see Note 4).

7. Prepare etomoxir in assay medium at a concentration of 40 vs. 400 μM. Store in a 37 °C water bath until use.

8. Design the experiment using the WAVE software; include triplicate measurements to ensure steady-state measurement.

9. Centrifuge 2.7 × 10⁶ T cells per condition at 1200 × g for 5 min.

10. Aspirate, and wash with 1× PBS.

11. Resuspend the cell pellets in 1.575 ml of XF assay medium prepared in Subheading 2.2 step 7.

12. Mix and seed T cells at 175 μl/well for a total of 8 technical replicates.

13. Centrifuge the XF microplate at 1000 × g for 5 min and transfer to a CO₂-free, 37 °C incubator for 30 min.

14. Add 25 μl of etomoxir to injection port A of the sensor cartridge.

15. Insert the hydrated XF sensor cartridge to calibrate the instrument.

16. Following instrument calibration, replace the calibration plate with the cell culture microplate.

17. Run the XF assay to measure cellular oxygen consumption (OCR) and extracellular acidification (ECAR) under basal conditions and in response to either 5 μM or 50 μM etomoxir (see Note 5).

3.3. Immunoblotting

1. Apply the protein/sample buffer mixture to the SDS-PAGE gel matrix.

2. Perform electrophoresis for 1.5 h at 150 V.

3. Transfer the proteins from the gel to the PDVF membrane using a current of 250 mA for 110 min.

4. Incubate the membrane in 5% nonfat dry milk in TBS containing 0.1% Tween-20 (TBS-T) for 1 h.

5. With a razor blade, cut the membranes into two segments: 250–60 kDa, 60–30 kDa.

6. Probe the 250–60 kDa membrane overnight at 4 °C with a 1:500 dilution of anti-CPT1A antibody (Cell Signaling Technology) in 0.5% nonfat milk in TBS-T.

7. After a series of three washes (1× quick, 1 × 10 min, 3 × 5 min) in TBS-T, incubate the membrane with an HRP-conjugated goat anti-rabbit IgG (cell signaling), diluted 1:10,000 in the blocking solution.

8. Wash in TBS-T as follows: 1× quick, 1 × 10 min, 3 × 5 min washes.

9. Perform a final rinse in TBS.

10. Probe the 60–30 kDa segment with a 1:2000 dilution of anti-beta actin antibody (Cell Signaling Technology) in 0.5% nonfat milk in TBS-T.

11. Wash as described above, and incubate the membrane with an HRP-conjugated sheep anti-mouse IgG (Amersham), diluted 1:5000 in the blocking solution.

12. Mix equal volumes of the West Femto SuperSignal Chemiluminescent reagent A and B prior to use. Add to the blot for 5 min.

13. Wrap on saran wrap, expose the ECL Hyperfilm for 1 min in a cassette, and develop as necessary.

4. Notes

1. Separating gel preparation.

7.5% separating gel	Volume
QH2O	38.8 ml
1.5 M Tris–HCl, pH 8.8	20.0 ml
10% SDS	800 μl
30% acrylamide/bis solution	20 ml
Vacuum 5 min
10% ammonium persulfate	400 μl
TEMED	40 μl
Total	80 ml

2. Stacking gel preparation.

4% stacking gel	Volume
QH2O	12.2 ml
0.5 M Tris–HCl, pH 6.8	5.0 ml
10% SDS	200 μl
30% acrylamide/bis solution	2.66 ml
Vacuum 5 min
10% ammonium persulfate	200 μl
TEMED	20 μl
Total	20 ml

3. Sample buffer.

5x sample buffer	Volume
QH2O	175 μl
0.5 M Tris–HCl, pH 6.8	125 μl
50% glycerol in QH2O	500 μl
10% SDS	200 μl
2-Mercaptoethanol	50 μl
Bromophenol blue	Pinch
Final volume	1 ml

4. Previous protocols recommended overnight hydration of the XF sensor cartridge in a CO₂-free, 37 °C incubator. As this extended duration results in dehydration, it is preferable to add 200 μl of the calibration solution and leave overnight on the bench at room temperature. On the morning of the assay, transfer to a CO₂-free, 37 °C incubator for 5 h.

5. The inherent metabolic flexibility of T cells ensures they can use fuel sources interchangeably. Inhibiting long-chain fatty acid oxidation will promote a corresponding shift to glycolysis (increase in ECAR measurements). In cells expressing shRNA against CPT1A, there should be no further increase in ECAR at doses specific to the CPT1A target.