Staphylococcal Enterotoxin C2 Mutant–Directed Fatty Acid and Mitochondrial Energy Metabolic Programs Regulate CD8+ T Cell Activation

Xuanhe Fu, Mingkai Xu, Huiwen Zhang, Yongqiang Li, Yansheng Li and Chenggang Zhang
J Immunol October 15, 2020, 205 (8) 2066-2076; DOI: https://doi.org/10.4049/jimmunol.2000538

Key Points

  • ST-4 induces CD8 T cell activation and increases TCRVβ 8.2 and 8.3 subgroup expression.

  • ST-4 promotes CD8 fatty acid metabolic program via mTOR/PPARγ/SREBP signal pathway.

  • ST-4 promotes CD8 mitochondrial energy metabolic program via p38 signal pathway.

Abstract

CD8+ T cells can switch between fatty acid catabolism and mitochondrial energy metabolism to sustain expansion and their cytotoxic functions. ST-4 is a TCR-enhanced mutant derived from superantigen staphylococcal enterotoxin C2 (SEC2), which can hyperactivate CD4+ T cells without MHC class II molecules. However, whether ST-4/SEC2 can enhance metabolic reprogramming in CD8+ T cells remains poorly understood. In this study, we found that ST-4, but not SEC2, could induce proliferation of purified CD8+ T cell from BALB/c mice in Vβ8.2- and -8.3–specific manners. Results of gas chromatography–mass spectroscopy analysis showed that fatty acid contents in CD8+ T cells were increased after ST-4 stimulation. Flow cytometry and Seahorse analyses showed that ST-4 significantly promoted mitochondrial energy metabolism in CD8+ T cells. We also observed significantly upregulated levels of gene transcripts for fatty acid uptake and synthesis, and significantly increased protein expression levels of fatty acid and mitochondrial metabolic markers of mTOR/PPARγ/SREBP1 and p38-MAPK signaling pathways in ST-4–activated CD8+ T cells. However, blocking mTOR, PPARγ, SREBP1, or p38-MAPK signals with specific inhibitors could significantly relieve the enhanced fatty acid catabolism and mitochondrial capacity induced by ST-4. In addition, blocking these signals inhibited ST-4–stimulated CD8+ T cell proliferation and effector functions. Taken together, our findings demonstrate that ST-4 enhanced fatty acid and mitochondria metabolic reprogramming through mTOR/PPARγ/SREBP and p38-MAPK signaling pathways, which may be important regulatory mechanisms of CD8+ T cell activation. Understanding the effects of ST-4–induced regulatory metabolic networks on CD8+ T cells provide important mechanistic insights to superantigen-based tumor therapy.

Introduction

Staphylococcal enterotoxins are potent bacterial superantigens secreted by Gram-positive Staphylococcus aureus (1). Staphylococcal enterotoxins can cross-bind to the antigenic groove of MHC class II (MHC II) molecules and the specific Vβ-chain of the TCR to form a trimeric complex, which stimulates CD4+ and CD8+ T cell proliferation and release of massive amounts of proinflammatory cytokines like IL-2, TNF-α, and IFN-γ (2, 3). During this process, activated CD8+ T cells can trigger antitumor activity through the superantigen dependent cytotoxic effect and cytotoxic molecule production (4).

In the tumor microenvironment (TME), activated CD8+ T cells use distinct metabolic programs to maintain expansion and functions, which is extremely important for successful tumor immunotherapy. The modest energetic and biosynthetic needs of CD8+ T cells are primarily met through the acquisition and metabolism of pyruvate derived from glucose, fatty acid oxidation, or mitochondrial oxidative phosphorylation to generate ATP (5). However, in nutrient-depleted settings, such as the TME, a lack of glucose and oxygen is the main constraint of CD8+ T cell function, which leads to CD8+ T cells anergy or apoptosis (6). Recent advances in cancer immunotherapy have revealed that modulation or reprogramming of altered energy metabolism in CD8+ T cells through mechanistic target of rapamycin (mTOR) and p38-MAPK signaling pathways might supply a potential strategy to achieve superior antitumor efficacy (7, 8).

SEC2 has been used as a supplementary therapeutic drug against tumors in China for many years (9). To enhance the antitumor activity of SEC2, our laboratory constructed an SEC2 mutant, named ST-4, that exhibits enhanced immunocyte stimulation and antitumor activity, even in an MHC II–independent manner (10, 11). However, mechanisms of SEC2/ST-4–enhanced metabolic reprogramming on CD8+ T cells have not been investigated.

At present, little is known about staphylococcal enterotoxin–induced CD8+ T cell activation and metabolic regulatory mechanisms. Our previous studies showed that SEC2/ST-4 could trigger T cell activation via TCR and CD28 engagement and drive rapid proliferation through PI3K and mTOR signaling pathways (12). As a key regulator of T cell function, mTOR integrates intracellular and extracellular signals to coordinate shifting metabolic states with cell differentiation, cytokine production, and proliferation (13–15). In CD4+ and CD8+ T cells, mTOR activates peroxisome proliferator-activated receptors (PPARs) and mediates fatty acid uptake for energy production (8, 16). This process involves the participation of several proteins, such as the CD36 receptor for lipoproteins, plasma membrane-associated fatty acid–binding proteins (Fabps), and low-density lipoprotein receptors (Ldlr) (17). In addition, mTOR positively regulates SREBPs, which promotes the expression of lipid synthesis genes, including acetyl-CoA carboxylase α (ACACA), fatty acid synthase (Fasn), stearoyl-CoA desaturase (SCD), and elongation of very long chain fatty acid–like 1 (Elovl1), as well as hydroxymethylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol synthesis (18–22). Upon cellular uptake and synthesis, fatty acids are activated by cytosolic acyl-CoA synthases. Next, carnitine palmitoyltransferase 1 (CPT1) facilitates the entry of fatty acids into mitochondria for the β-oxidation spiral (23, 24). Mitochondria are required for T cell activation partly through the generation of reactive oxygen species (ROS) (25). However, mitochondrial metabolism in T cells is repressed in the TME (26). Recent study has established that additional costimulatory signals can induce mitochondrial biogenesis and enhanced T cell respiratory capacity through the p38-MAPK signaling pathway (27).

To better understand SEC2/ST-4–induced CD8+ T cell activation and the metabolic reprogramming mechanism, we investigated roles of mTOR and p38-MAPK signaling pathways in SEC2/ST-4–induced metabolic activation of CD8+ T cells. In this study, we used the mTOR inhibitor rapamycin, PPARγ inhibitor GW9662, and SREBP1 inhibitor fatostatin to investigate the fatty acid metabolic mechanism of SEC2/ST-4–induced CD8+ T cell activation. Furthermore, we used the p38-MAPK inhibitor SB203580 to analyze mitochondrial function in CD8+ T cells activated by SEC2/ST-4.

In this work, we identified that only MHC II–independent mutant ST-4, but not its closely similar homolog wild-type SEC2, could induce specific Vβ8.2 and -8.3 CD8+ T cell activation. Furthermore, mTOR/PPARγ/SREBP and p38-MAPK signaling pathways are essential for activation of the fatty acid and mitochondrial energy metabolic program in ST-4–induced CD8+ T cell activation. Our study provides important insights into fatty acid and mitochondrial energy metabolism processes, which permit rapid proliferation and activation of CD8+ T cells after ST-4 stimulation.

Materials and Methods

Animals

Female wild-type BALB/c mice (4–6 wk old) were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). All mice were housed under pathogen-free conditions with feed and water supplied ad libitum. All experiments involving mice were approved by the institutional animal care committee.

Isolation of CD8+ T cells

Spleens from BALB/c mice were minced through a nylon screen (200 mesh) into medium to obtain splenocytes. CD8+ T cells were isolated from splenocytes by negative selection using labeled magnetic beads following the manufacturer’s instructions (Miltenyi Biotec). Purified CD8+ T cells were >97% pure as assessed by cytometry.

Reagents and Abs

Recombinant prototype SEC2 and its mutant ST-4 were expressed in Escherichia coli BL21 (DE3) strains containing each vector, respectively, and purified as previously described (10, 28). RPMI 1640 and FBS were purchased from Thermo Fisher Scientific (HyClone, Waltham, MA). Rapamycin, GW9662, fatostatin, and SB203580 were purchased from Selleck (Houston, TX). Monoclonal CD3-FITC/APC (catalog nos. 100204 and 100235), CD36-PE (catalog no. 102605), TCR Vβ8.1, 8.2-PE (catalog no. 140103), and TCR Vβ8.3-PE (catalog no. 156303) Abs were purchased from BioLegend (San Diego, CA). CFSE Cell Proliferation Assay and Tracking Kit was purchased from Beyotime Biotechnology (Haimen, Jiangsu, China). BODIPY-labeled palmitate (BODIPY FLC16; Invitrogen) was used in conjunction with flow cytometry for uptake experiment. An ELISA kit for mouse IL-2, TNF-α, and IFN-γ were purchased from Abcam (Cambridge, U.K.). SYBR Premix Ex Taq Kit, Prime Script RT Master Kit, and the RNA-extracting reagent RNAiso Plus were purchased from Takara Biotechnology (Dalian, China). Radioimmunoprecipitation assay lysis buffer was purchased from Beyotime Biotechnology. Abs against phospho-PPARγ (ab59256), phospho-SREBP1 (ab28481), T-bet (ab53174), CPT1 (ab234111), phospho-p38 (ab195049), phospho–activating transcription factor 2 (ATF2; ab32019), mitofusin 2 (ab124773), β-actin (ab8227), and goat anti-rabbit IgG (conjugated to HRP; ab6721) were purchased from Abcam.

CFSE proliferation assay

Mice splenocytes/CD8+ T cells were obtained and labeled with CFSE as previously described (11, 12). The cells were stimulated with SEC2/ST-4 as indicated. For the metabolic mechanism analysis, labeled CD8+ T cells were resuspended in RPMI 1640 medium supplemented with 10% FBS at a final concentration of 5 × 106 cells/ml. Then, labeled cells were incubated with each inhibitor at final concentrations of 0.1 μM rapamycin, 50 μM GW9662, 10 μM fatostatin, and 10 μM SB203580, respectively. All CD8+ T cells were incubated in 48-well, flat-bottom plates at a density of 3 × 106 cells per well in 0.5 ml medium at 37°C in a humidified atmosphere containing 5% CO2 for 30 min and then stimulated with SEC2/ST-4 at a final concentration of 10,000 ng/ml for 72 h. Untreated cells served as a negative control. After incubation, cell division analysis was performed using a BD Biosciences LSRFortessa, and data were analyzed with FlowJo software (Tree Star, Ashland, OR). Percent divided is the percentage of the cells of the original sample that were divided. Histograms represent the mean ± SD (error bars) of three independent experiments. Statistical significance (*p < 0.05) was calculated by two-way ANOVA.

Flow cytometry analysis

Freshly isolated CD8+ T cells (5 × 106 cells/ml) were incubated and stimulated as indicated above. Cell surface markers were determined by staining with fluorochrome-conjugated mAbs. The Ab panel consisted of APC-conjugated anti-mouse CD3 for T lymphocytes; PE-conjugated anti-mouse TCR Vβ8.1, -8.2, and -8.3 for the specific TCR Vβ analysis; and FITC-conjugated anti-mouse CD3 for T lymphocytes, PE-conjugated anti-mouse CD36 for the receptor for lipoproteins analysis. Cells stimulated in 48-well plates were centrifuged and washed twice with PBS. Then, cells (1 × 106 cells per tube) were resuspended in 50 μl PBS containing moderate concentrations of Abs (according to the manufacturer’s instructions) and incubated for 30 min at 4°C in the dark. Flow cytometry was performed using a BD LSRFortessa, and data were analyzed with FlowJo software. Determination of mitochondrial mass, membrane potential, and cellular ROS were performed using MitoTracker Green, MitoTracker Deep Red, and Cell ROX Green reagents, respectively (all from Life Technologies), and cells were incubated with these dyes in final concentrations of 0.25, 0.25, and 0.625 μM, respectively, at 37°C in a 5% CO2 humidified incubator for 30 min before being detected with flow cytometry. Histograms represented the mean ± SD (error bars) of three independent experiments. Statistical significance (*p < 0.05) was calculated by two-way ANOVA.

Metabolic profiling

CD8+ T cells cultured in 24-well, flat-bottom plates at a density 1 × 107 cells per well in 1 ml medium were stimulated with SEC2/ST-4 at concentration of 10,000 ng/ml for 72 h. After incubation, cells were centrifuged and washed twice with PBS. Then, metabolic profiles of all samples were analyzed with gas chromatography–mass spectrometry (GC-MS) method as described in previous studies (29). Briefly, GC-MS analysis was performed with a 7890A GC/5975C mass spectrometry (MS) system (Agilent Technologies) fitting a fused silica capillary column (HP-5 MS, 30 m × 0.25 mm inner diameter, 0.25 μm film thickness; Agilent J&W Scientific, Folsom, CA). Each sample in 1 μl was injected in splitless mode into an inlet held at 275°C. The temperature rise programs had an initial temperature of 50°C for 2 min, which was raised 10°C/min to 200°C and then maintained for 2 min, raised to 250°C at 5°C/min and then maintained for 6 min, and raised to 280°C at 10°C/min and then maintained isothermally for 5 min. The column flow rate was 1 ml/min, with helium as the carrier gas. The MS was run in electron ionization mode (70 eV). The transfer line, electron ionization source, and quadrupole were maintained at 250, 230, and 150°C, respectively. Chromatographic and spectral analysis was performed using ChemStation and MassHunter (Agilent Technologies).

Seahorse analysis

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured with an XFe96 extracellular flux analyzer (Agilent Technologies). Cultured CD8+ T cells were seeded at a density of 400,000 cells per well in XFe96 plate. Oligomycin, FCCP, rotenone, and antimycin A, four pharmaceutical modulators of mitochondrial oxidative phosphorylation, which were supplied with an XFe Cell Mito Stress Test Kit (Seahorse Bioscience), were added sequentially as described in previous studies (30).

Cytokine assay

Freshly isolated CD8+ T cells (5 × 106 cells/ml) were incubated and stimulated as indicated above for 72 h. Cell culture supernatants were collected, and the concentrations of IL-2, TNF-α, and IFN-γ within the supernatants were determined using the ELISA kits following the instructions of the kit. Absorbance values were measured with a microplate reader at a measurement wavelength of 450 nm and a reference wavelength of 620 nm.

Quantitative real-time PCR

After treatment with SEC2/ST-4 at the concentration of 10,000 ng/ml and inhibitor as indicated above, CD8+ T cells were harvested and total RNA was extracted from 5 × 106 cells using the RNA-extracting reagent RNAiso Plus, and 0.5 μg of total RNA was reverse transcribed using a PrimeScript RT Master Kit according to the manufacturer’s instructions. Resulting cDNA was used for quantitative real-time PCR (qRT-PCR) analysis with an SYBR Premix Ex Taq Kit and ABI Prism 7000 (Applied Biosystems, Norwalk, CT). PCR conditions were described in previous study (12). Specific primers for mouse TCR Vβ elements were synthesized according to sequence designs previously described (31), as well as the primers for ACACA, Fasn, Elovl1, Scd1, Insig1, HMGCR, Ldlr, Lrp8, Fabp5, perforin, and granzyme B (Table I) (32–40). Relative transcription levels were determined using the 2−ΔΔCt analysis method (11).

Western blotting

After treatment with SEC2/ST-4 at the concentration of 10,000 ng/ml and inhibitor as indicated above, a total of 1 × 107 CD8+ T cells were collected and lysed in radioimmunoprecipitation assay lysis buffer at 4°C for 10 min to extract cellular protein. Samples containing equal amounts of total protein (8 μg) were mixed with 5× Laemmli buffer, boiled, and separated on a 12% SDS-PAGE gel. Samples were then transferred onto polyvinylidene fluoride (PVDF) membranes (MilliporeSigma, Billerica, MA). After blocking with 5% skimmed milk, primary Ab incubation was done overnight at 4°C in an appropriate dilution. After washing, HRP-conjugated secondary Ab incubation was done at room temperature for 60 min. Detection was performed using an ECL method.

Statistical analysis

All values are given as mean ± SD. Data were analyzed using a two-way ANOVA method. The follow-up least significant difference test was used for post hoc comparison with assess differences between groups. Differences with p values < 0.05 considered to be statistically significant.

Results

Analysis of SEC2/ST-4–induced proliferation and changes in TCR Vβ gene expression in murine CD8+ T cells

Both SEC2 and its mutant ST-4 significantly induced splenocyte proliferation at a concentration of 10,000 ng/ml. Furthermore, the stimulatory activity of ST-4 was significantly higher than that of SEC2 (p < 0.05) (Fig. 1A), in accordance with our previous results (11). To investigate whether ST-4 could also induce purified CD8+ T cell proliferation, murine CD8+ T cells were stimulated with SEC2/ST-4. As shown in Fig. 1B and 1C, ST-4, but not SEC2, could significantly induce CD8+ T cell proliferation in time- and dose-dependent manners (Table I).

FIGURE 1.
FIGURE 1.

Analysis of SEC2/ST-4–induced splenocyte/CD8+ T cell proliferation. (A) CFSE-labeled splenocytes were cultured in 48-well, flat-bottom plates at a density of 1.5 × 106 cells per well. Then, cells were subsequently stimulated with SEC2 or ST-4 at a final concentration of 10,000 ng/ml. Untreated CFSE-labeled splenocytes were served as a negative control. After 72 h, cells were harvested and analyzed by flow cytometry. Percent divided is the percentage of the cells of the original sample that divided. Histograms, representing the mean ± SD (error bars) of three independent experiments. (B) CFSE-labeled CD8+ T cells were stimulated with 10,000 ng/ml SEC2/ST-4 for 24, 48, or 72 h. Untreated CFSE-labeled CD8+ T cells were served as a negative control. After incubation for the indicated time, cells were harvested and analyzed by flow cytometry. (C) CFSE-labeled CD8+ T cells were stimulated for 72 h with 500, 1000, 5000, 10,000, or 50,000 ng/ml SEC2/ST-4. Untreated CFSE-labeled CD8+ T cells were served as a negative control. After incubation for 72 h, cells were harvested and analyzed by flow cytometry. (D) CD8+ T cells were stimulated with 10,000 ng/ml SEC2/ST-4. Untreated CD8+ T cells were served as a negative control. After 72 h, total RNA was collected, and transcription levels of Vβ were identified by qRT-PCR, as described in Quantitative real-time PCR. (E) CD8+ T cells were stimulated with 10,000 ng/ml SEC2/ST-4. Untreated CD8+ T cells were served as a negative control. After 72 h, cells were collected and stained with APC-conjugated anti-CD3, PE-conjugated anti-TCR Vβ8.1/8.2, or PE-conjugated anti-TCR Vβ8.3 Ab before analysis by flow cytometry. Dot plots of total events are shown with frequencies of cells in each quadrant. Each value indicates mean ± SD of results obtained from three independent experiments. *p < 0.05 by two-way ANOVA.

View this table:
Table I. Sequences for qRT-PCR primers

TCR Vβ repertoires are important indicators to characterize the properties and classification of staphylococcal enterotoxin superantigens. In our previous study, ST-4 induced significant expression of Vβ8.2 and -8.3 in CD4+ T cells (11). In this study, we investigated relative expression of TCR Vβ-chains stimulated by SEC2 and ST-4 in purified CD8+ T cells. As shown in Fig. 1D and 1E, SEC2 did not elicit changes in amplification levels of any TCR Vβs. However, Vβ8.2 and -8.3 CD8+ T cells were significantly amplified after stimulation by ST-4 compared with SEC2 (p < 0.05). These results strongly suggest that ST-4 can also trigger CD8+ T cell activation via enhanced TCR signaling, similar to CD4+ T cells (11).

Importance of fatty acid metabolism for ST-4–induced CD8+ T cell proliferation

Cellular lipid metabolism has a critical effect on T cell activation and cell fate decisions. To elucidate the role of fatty acid metabolism in SEC2/ST-4–induced CD8+ T cell proliferation, we first evaluated amounts of lipid metabolites in SEC2/ST-4 stimulated CD8+ T cells using GC-MS analysis. As shown in Fig. 2A, after stimulation for 72 h, the ST-4–treated group exhibited significantly higher amounts of most kinds of lipid metabolites compared with SEC2. Moreover, we examined whether ST-4 could stimulate CD8+ T cells to acquire fatty acids from the external environment using fluorescently labeled palmitate (BODIPY FLC16) (41). The results showed that ST-4, but not SEC2, could stimulate CD8+ T cells to acquire palmitate in a time-dependent manner (Fig. 2B). In addition, we used the mTOR inhibitor rapamycin, PPARγ inhibitor GW9662, and SREBP1 inhibitor fatostatin to investigate the role of fatty acid metabolism in ST-4–induced CD8+ T cell proliferation. As expected, fatty acid uptake (FLC16 intensity) of CD8+ T cells was almost completely blocked by the three inhibitors (Fig. 2C). Moreover, ST-4–induced CD8+ T cell proliferation was significantly inhibited by rapamycin, GW9662, and fatostatin (Fig. 2D). These results indicate that fatty acid metabolism played an important role in ST-4–induced CD8+ T cell proliferation.

FIGURE 2.
FIGURE 2.

Fatty acid metabolism is required for ST-4–induced CD8+ T cell proliferation. (A) CD8+ T cells were collected 72 h after SEC2/ST-4 stimulation. Untreated CD8+ T cells served as a negative control. Log2 values for each metabolite represent the average of duplicates. (B) Representative plots of BODIPY FLC16 in CD8+ T cells collected at the indicated time after SEC2/ST-4 stimulation. Untreated CD8+ T cells served as a negative control. After incubation for the indicated time, cells were harvested and analyzed by flow cytometry. (C) Representative plots of BODIPY FLC16 in CD8+ T cells collected 72 h after SEC2/ST-4 stimulation in the presence of rapamycin, GW9662, or fatostatin. Untreated CD8+ T cells served as a negative control. (D) CFSE-labeled CD8+ T cells were preincubated with rapamycin, GW9662, or fatostatin for 30 min at the indicated concentrations. Cells were subsequently stimulated with 10,000 ng/ml SEC2/ST-4 for 72 h before analysis by flow cytometry. Untreated CFSE-labeled CD8+ T cells served as a negative control. Histograms, representing the mean ± SD (error bars) of three independent experiments. *p < 0.05 by two-way ANOVA.

ST-4–induced mTOR activation controls fatty acid metabolism in CD8+ T cells

To better understand the involvement of mTOR signaling in fatty acid uptake and biosynthesis programs in CD8+ T cells activated by SEC2/ST-4, we assessed the transcription levels of genes associated with these metabolic pathways after pharmacological inhibition of mTOR. Results of qRT-PCR analysis showed that ST-4, but not SEC2, could upregulate transcription levels of fatty acid uptake program-related genes including Ldlr, Lrp8, and Fabp5. The percentage of CD36-positive events was also increased by ST-4 stimulation in flow cytometry analysis (Fig. 3A, 3B). Similarly, transcription levels of genes associated with fatty acid biosynthesis, including ACC1, Fasn, Elovl1, Scd1, Insig1, and HMGCR, were also increased after ST-4 stimulation of CD8+ T cells (Fig. 3C). However, upregulation of those genes induced by ST-4 was significantly inhibited by rapamycin in CD8+ T cells (Fig. 3A–C). A previous study suggested that mTOR signaling could activate PPARγ and SREBP pathways and control fatty acid metabolism in murine CD4+ T cells (16). Accordingly, we evaluated mTOR/PPARγ/SREBP pathways in SEC2/ST-4–stimulated CD8+ T cells. As shown in Fig. 3D, Western blotting results indicated that ST-4, but not SEC2, could significantly activate PPARγ and SREBP in CD8+ T cells (p < 0.05). Meanwhile, rapamycin almost completely blocked upregulation of PPARγ and SREBP induced by ST-4 in CD8+ T cells. Notably, we found that ST-4 could significantly enhance protein expression of CPT1, which is needed for fatty acid oxidation in CD8+ T cells. The upregulation of ST-4–stimulated CPT1 was also repressed by rapamycin (Fig. 3D). Taken together, these results indicate that ST-4–induced mTOR activation controlled the fatty acid metabolism program in CD8+ T cells through PPARγ and SREBP pathways.

FIGURE 3.
FIGURE 3.

ST-4–induced mTOR activation controls fatty acid metabolism of CD8+ T cells. (A) qRT-PCR analyses for relative expression of genes encoding enzymes in the fatty acid uptake program in SEC2/ST-4–stimulated CD8+ T cells in the presence of 0.1 μM rapamycin. Heatmap represents log2 values of relative mRNA transcription levels (see color scale). The log2 value of each gene in control cells was set to 0. Untreated CD8+ T cells served as a negative control. (B) CD8+ T cells were preincubated with 0.1 μM rapamycin for 30 min and subsequently stimulated with 10,000 ng/ml SEC2/ST-4 for 72 h. Untreated CD8+ T cells served as a negative control. Next, cells were stained with FITC-conjugated anti-CD3 and PE-conjugated anti-CD36 Abs before analysis by flow cytometry. Dot plots of total events are shown with frequencies of cells in each quadrant. (C) qRT-PCR analyses for relative expression of genes encoding enzymes in the fatty acid synthesis program of SEC2/ST-4–stimulated CD8+ T cells in the presence of 0.1 μM rapamycin. Untreated CD8+ T cells served as a negative control. (D) CD8+ T cells were preincubated with 0.1 μM rapamycin for 30 min and subsequently stimulated with 10,000 ng/ml SEC2/ST-4 for 72 h. Untreated CD8+ T cells served as a negative control. Protein levels of phospho-PPARγ, phospho-SREBP1, T-bet, and CPT1 were detected by Western blot analysis. Each value indicates mean ± SD of results obtained from three independent experiments. *p < 0.05 by two-way ANOVA.

ST-4–induced PPARγ and SREBP fatty acid metabolic signaling controls CD8+ T cell activation

To investigate roles of PPARγ and SREBP signaling in SEC2/ST-4–stimulated fatty acid metabolism of CD8+ T cells, we assessed the expression of genes encoding enzymes involved in fatty acid uptake and biosynthesis programs after pharmacological inhibition of PPARγ and SREBP. As shown in Fig. 4A and 4B, upregulation of fatty acid uptake and biosynthesis genes induced by ST-4 was significantly inhibited by GW9662 and fatostatin in CD8+ T cells (p < 0.05). Meanwhile, the percentage of CD36-positive events induced by ST-4 was decreased by inhibition with either GW9662 or fatostatin (Fig. 3B).

FIGURE 4.
FIGURE 4.

ST-4–induced PPARγ and SREBP signaling controlled fatty acid uptake, synthesis, and effector function of CD8+ T cells. (A) qRT-PCR analyses for relative expression of genes encoding enzymes in the fatty acid uptake program in SEC2/ST-4–stimulated CD8+ T cells in the presence of 50 μM GW9662. Untreated CD8+ T cells served as a negative control. Heatmap represents log2 values of relative mRNA transcription levels (see color scale). The log2 value of each gene in control cells was set to 0. (B) qRT-PCR analyses for relative expression of genes encoding enzymes in the fatty acid synthesis program in SEC2/ST-4–stimulated CD8+ T cells in the presence of 10 μM fatostatin. Untreated CD8+ T cells served as a negative control. (C) CD8+ T cells were preincubated with rapamycin, GW9662, or fatostatin for 30 min at the indicated concentration. Cells were subsequently stimulated with 10,000 ng/ml SEC2/ST-4 for 72 h. Untreated CD8+ T cells served as a negative control. Total cellular RNA was extracted and reverse transcribed. Transcription levels of perforin and granzyme B were detected by qRT-PCR, as described in Materials and Methods. Each value indicates mean ± SD of results obtained from three independent experiments. (D) ST-4 drives fatty acid metabolic programs and CD8+ T cell activation. *p < 0.05 by two-way ANOVA.

Activation of CD8+ T cells is accompanied by elevated production of tumoricidal cytokines, such as IL-2, perforin, granzymes, TNF-α, and IFN-γ (42). As one of the T-box transcription factors, T-bet is important for expression of cytotoxic molecules in CD8+ T cells (43). As shown in Fig. 3D, ST-4, but not SEC2, could significantly induce T-bet protein expression. At a final concentration of 0.1 μM, rapamycin almost completely blocked upregulation of T-bet induced by ST-4 in CD8+ T cells. These data strongly suggest that ST-4 could induce secretion of cytotoxic molecules via mTOR-mediated fatty acid metabolism signaling pathways in CD8+ T cells. Subsequently, our results showed that ST-4, but not SEC2, could stimulate CD8+ T cells to produce IL-2 (53.39 ± 3.88 pg/ml), TNF-α (848.10 ± 25.40 pg/ml), and IFN-γ (486.08 ± 24.28 pg/ml) and upregulated mRNA transcription of perforin and granzyme. Rapamycin (0.1 μM), GW9662 (50 μM), or fatostatin (10 μM) could completely inhibit ST-4–induced secretion of IL-2, TNF-α, and IFN-γ (not detected in culture medium) and significantly downregulated mRNA transcription of perforin and granzyme B (Fig. 4C). These results indicate that ST-4 could enhance mTOR/PPARγ/SREBP fatty acid metabolism signaling pathways to control CD8+ T cell activation (Fig. 4D).

Importance of mitochondrial metabolism for ST-4–induced CD8+ T cell proliferation

Mitochondrial metabolism is important for cellular longevity and avoidance of senescence (44). In CD8+ T cells, increased mitochondrial activity results in enhanced cytotoxicity to tumor cells, which is important for tumor immunotherapy (26). In addition, 4-1BB activation through stimulation of p38-MAPK was shown to require increased T cell respiratory capacity, which could be used upon full T cell activation (27). To investigate whether p38-MAPK–related mitochondrial activation is involved in SEC2/ST-4–induced CD8+ T cell proliferation, we examined the effect of p38-MAPK inhibition on SEC2/ST-4 stimulation. As shown in Fig. 5A and 5B, ST-4, but not SEC2, could significantly increase the percentage of events positive for mitochondrial membrane potential, mitochondrial mass, and mitochondrial ROS (p < 0.05). However, percentages of mitochondrial activation-positive events induced by ST-4 were significantly decreased by treatment with SB203580 (p < 0.05). Meanwhile, ST-4–stimulated CD8+ T cell proliferation was significantly inhibited by SB203580 (Fig. 5C, p < 0.05). These results indicate that ST-4 could stimulate mitochondrial activation through p38-MAPK signaling. Thus, ST-4–induced mitochondrial metabolism plays an important role in CD8+ T cell proliferation.

FIGURE 5.
FIGURE 5.

ST-4–induced p38-MAPK activation controls mitochondrial capacity and effector function of CD8+ T cells. (A) CD8+ T cells were preincubated with 10 μM SB203580 for 30 min and subsequently stimulated with 10,000 ng/ml SEC2/ST-4 for 72 h. Untreated CD8+ T cells served as a negative control. Next, cells were stained with MitoTracker Green and MitoTracker Deep Red reagents before analysis by flow cytometry. Dot plots of total events are shown with frequencies of cells in each quadrant. (B) CD8+ T cells were preincubated with 10 μM SB203580 for 30 min. Cells were subsequently stimulated with 10,000 ng/ml SEC2/ST-4 for 72 h and then stained with Cell ROX Green reagent before analysis by flow cytometry. Untreated CD8+ T cells served as a negative control. (C) CFSE-labeled CD8+ T cells were preincubated with 10 μM SB203580 for 30 min and subsequently stimulated with 10,000 ng/ml SEC2/ST-4 for 72 h before analysis by flow cytometry. Untreated CFSE-labeled CD8+ T cells served as a negative control. (D) CD8+ T cells were preincubated with 10 μM SB203580 for 30 min and subsequently stimulated with 10,000 ng/ml SEC2/ST-4 for 72 h. Protein levels of phospho–p38-MAPK, phospho-ATF2, mitofusin, and T-bet were detected by Western blot analysis. Untreated CD8+ T cells served as a negative control. (E and F) OCR and ECAR of CD8+ T cells activated by SEC2/ST-4 for 72 h with or without 10 μM SB203580 under basal conditions (time point 0) and in response to sequential treatment with oligomycin, FCCP, and rotenone–antimycin A. Untreated CD8+ T cells served as a negative control. (G) CD8+ T cells were preincubated with 10 μM SB203580 for 30 min and subsequently stimulated with 10,000 ng/ml SEC2/ST-4 for 72 h. Untreated CD8+ T cells served as a negative control. Total cellular RNA was extracted and reverse transcribed. Transcription levels of perforin and granzyme B were detected by qRT-PCR, as described in Materials and Methods. Each value indicates mean ± SD of results obtained from three independent experiments. (H) ST-4 drives mitochondrial energy metabolic programs and activation of CD8+ T cells. *p < 0.05 by two-way ANOVA.

Inhibited p38-MAPK signaling reduced SEC2/ST-4–induced mitochondrial energy metabolism and CD8+ T cell function

p38-MAPK can activate ATF2 to promote mitochondrial fusion and energy metabolism (27, 45, 46). Thus, we next examined whether ST-4–induced p38-MAPK signaling was the dominant pathway for mitochondrial energy metabolism. As shown in Fig. 5D, ST-4, but not SEC2, could activate p38-MAPK, resulting in increased phosphorylation of ATF2 and expression of mitofusin 2. Meanwhile, SB203580 completely blocked upregulation of phospho–p38-MAPK, phospho-ATF2, and mitofusin 2 induced by ST-4 in CD8+ T cells.

Analysis of cellular energy metabolism revealed that the ST-4–treated group had significantly higher OCR (an indicator of mitochondrial respiration), ATP turnover, and ECAR (a consequence of lactic acid production) compared with control CD8+ T cells in the basal state. In contrast, SEC2 did not promote mitochondrial energy metabolism or the rate of acid efflux in CD8+ T cells (Fig. 5E, 5F). Levels of ST-4–induced OCR, ATP turnover, and ECAR were significantly decreased in the presence of SB203580 (Fig. 5E, 5F). Additionally, blocking p38-MAPK signaling could completely inhibit ST-4–induced T-bet expression and cytokine secretion (IL-2, TNF-α, and IFN-γ) and significantly downregulated mRNA transcription of perforin and granzyme B (Fig. 5D, 5G). Our results indicate that ST-4 could regulate mitochondrial energy metabolism and improve CD8+ T cell function through the p38-MAPK signaling pathway (Fig. 5H).

Discussion

We identified ST-4 as a positive regulator of CD8+ T cell fitness with regard to metabolism and function. ST-4 triggered mTOR/PPARγ/SREBP and p38-MAPK signals and, in turn, promoted fatty acid and mitochondrial energy metabolism in CD8+ T cells. By treating CD8+ T cells with rapamycin, GW9662, or fatostatin, we obtained evidence that fatty acid uptake and biosynthesis are required to support the proliferation and function of ST-4–activated CD8+ T cells. Furthermore, by applying the p38-MAPK inhibitor SB203580, we demonstrated the important role of p38-MAPK signaling in regulation of ST-4–induced CD8+ T cell respiratory capacity and function. Our findings provide critical mechanistic insights into ST-4–directed fatty acid and mitochondrial energy metabolism, which control CD8+ T cell proliferation and cytotoxic function.

By binding to both MHC complex and specific TCR Vβ segments, staphylococcal enterotoxins can stimulate CD4+ Th cells and CD8+ cytotoxic T cell activation with excessive release of multiple inflammatory cytokines and chemokines. Emerging evidence suggests that T cells dramatically alter their metabolic activity during TCR Vβ-mediated activation (47, 48). Our previous results show that the SEC2-TCR–enhanced mutant ST-4 could upregulate both Vβ8.2 and -8.3 subgroups of purified CD4+ T cells (11), indicating that ST-4 might also enhance CD8+ T cell immune response through similar Vβ-specific selection. Indeed, our results show that ST-4 could stimulate purified CD8+ T cell proliferation in time- and dose-dependent manners and increased expression of both TCR Vβ8.2 and -8.3 subgroups. In contrast, SEC2 lost almost all of its superantigen activities in purified CD8+ T cells. As such, these results suggest that ST-4 has more powerful potential than SEC2 with regard to TCR affinity-mediated CD8+ T cell activation and metabolic programming.

Upon entering the TME, CD8+ T cells undergo metabolic reprograming from glycolysis to fatty acid oxidation (16). As a serine and threonine kinase, mTOR plays key roles in the promotion of cell growth, proliferation, and metabolism by detecting the nutritional status of intracellular and extracellular environments (49). Recently, Angela et al. (16) revealed that mTOR/PPARγ signaling is needed for fatty acid uptake by activated CD4+ T cells. Our previous study showed that SEC2/ST-4 can induce T cell activation via the PI3K/mTOR pathway (12). However, it is unclear whether SEC2/ST-4 could promote fatty acid uptake in CD8+ T cells via the mTOR pathway. Thus far, little is known about the mTOR-mediated fatty acid uptake program in CD8+ T cells. In this study, we demonstrated that ST-4–activated CD8+ T cells acquired external fatty acids by upregulating the mTOR-mediated PPARγ pathway. Blocking of either mTOR or PPARγ inhibited the ability of cytotoxic CD8+ T cells to produce effector cytokines and decreased the expression of genes associated with fatty acid uptake. Membrane uptake of long chain fatty acids is the first step of cellular fatty acid use, and CD36 mediates long chain fatty acid transport in key tissues (50). Previous studies found that CD36 expression was not increased, but rather decreased, after antigenic stimulation of CD4+ T cells (16). However, in our study, we found that ST-4 could upregulate CD36 expression through the mTOR/PPARγ signaling pathway. In addition, the long chain fatty acids heneicosanoic acid and eicosapntemacnioc acid were the top-ranked metabolites increased by changes in lipid metabolism after ST-4 stimulation, which could partly be attributed to ST-4–induced upregulation of CD36 expression. These results suggest that ST-4 can promote fatty acid uptake and expression of lipid transport receptors via mTOR/PPARγ signaling. Uptake and accumulation of lipids from the extracellular environment play a key role in sustaining the proliferation and function of CD8+ T cells.

mTOR-mediated regulation of fatty acid synthesis has been associated with SREBP localization and enrichment on the gene promoter loci of lipogenic enzymes, such as ACACA, following activation of CD8+ T cells (21). ACACA produces malonyl-CoA, which is used by FASN to produce palmitate. De novo synthesis of fatty acids can be achieved by elongations with elongases, such as ELOVL1, or desaturation by desaturases, such as SCD (20). Consistent with these findings, our results using specific inhibitors of mTOR and SREBP1 revealed that ST-4 could significantly promote expression of mRNA transcripts for fatty acid synthesis enzymes and induce rapid proliferation and cytotoxic effects of CD8+ T cells through the mTOR/SREBP pathway (Fig. 4D). Although fatty acid synthesis is a strictly anabolic process that supports cell proliferation, de novo synthesis of fatty acids can also be used as an energy source via a catabolic process. In this study, we found that ST-4 could significantly upregulate the expression of CPT1, the key rate-limiting enzyme of fatty acid β-oxidation via mTOR signaling. This result implies that CD8+ T cells can use fatty acid β-oxidation as an energy source to maintain effector activity after ST-4 stimulation. This conclusion is further supported by recent research, suggesting that mTOR can promote CPT1 activity and enhance lipid oxidation to meet the energy demands of hypoxic lymphocytes (51).

Activated by TCR signaling in most cells, p38-MAPK participates in a signaling cascade controlling cellular responses to cytokines and stress (52). However, the TME can repress mitochondrial sufficiency during T cell activation. A recent study found that 4-1BB pretreatment was sufficient to enhance T cell respiratory capacity through activation of p38-MAPK in cancer immunotherapy (27). However, it is unclear whether staphylococcal enterotoxins could also promote mitochondrial energy metabolic via p38-MAPK signaling. Thus far, studies have primarily focused on staphylococcal enterotoxin–induced inflammation, such as vasodilation and vascular leak through p38-MAPK signaling (53). This study shows that ST-4 could significantly promote the expression phospho–p38-MAPK, phospho-ATF2, and mitofusin 2. In addition, ST-4 could significantly enhance mitochondrial mass, ROS generators, and mitochondrial respiratory capacity. Using the p38-MAPK inhibitor SB203580, we revealed that ST-4–stimulated upregulation of mitochondrial capacity, proliferation, and effector function of CD8+ T cells relied on activation of p38-MAPK signaling. These results emphasize the role of ST-4–induced activation of p38-MAPK in the positive regulation of mitochondrial capacity of CD8+ T cells. The resulting substantial increases of CD8+ T cell respiratory capacity and energy could be used upon CD8+ T cell activation.

In conclusion, our findings elucidated that ST-4 enhances the proliferation and effector function of CD8+ T cells by activating fatty acid and mitochondrial energy metabolic pathways to satisfy increased demands for biomass and energy. The results of this study, to our knowledge, provide new insights for understanding the mechanism of ST-4 in antitumor immunotherapy.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac) for editing the English text of a draft of this manuscript.

Footnotes

  • This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences grant (XDA12020225), the Liaoning Revitalization Talents Program (XLYC1807226), the Shenyang High-Level Innovative Talents Program (RC190060), and the Science and Technology Plan Projects of Shenyang City (Z17-7-013).

  • Abbreviations used in this article:

    ACACA
    acetyl-CoA carboxylase α
    ATF2
    activating transcription factor 2
    CPT1
    carnitine palmitoyltransferase 1
    ECAR
    extracellular acidification rate
    Elovl1
    elongation of very long chain fatty acid–like 1
    Fabp
    fatty acid–binding protein
    Fasn
    fatty acid synthase
    GC-MS
    gas chromatography–mass spectrometry
    HMGCR
    hydroxymethylglutaryl-CoA reductase
    Ldlr
    low-density lipoprotein receptor
    MHC II
    MHC class II
    MS
    mass spectrometry
    mTOR
    mechanistic target of rapamycin
    OCR
    oxygen consumption rate
    PPAR
    peroxisome proliferator-activated receptor
    qRT-PCR
    quantitative real-time PCR
    ROS
    reactive oxygen species
    SCD
    stearoyl-CoA desaturase
    TME
    tumor microenvironment.

  • Received May 11, 2020.
  • Accepted August 10, 2020.

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