Hepa1-6 cells (American Type Culture Collection (ATCC)) and Huh-7 cells (ATCC) were cultured in buffered Dulbecco’s modified eagle medium (DMEM, C11995500, Gibco, China). CTLL-2 cells (359785, BeNa Culture Collection (BNCC), China) and CD8${}^{+}$ T cells were cultured in vitro using RPMI-1640 medium (C11875500, Gibco, China). All media were supplemented with 10% fetal bovine serum (FBS; Biological Industries, USA) and 1% penicillin/streptomycin (P1400, Solarbio, China). CTLL-2 cells were activated with 10 $\mu$g/mL anti-CD3 antibody (kx10-3A; Beijing KEXIN Biological, China) and 2.5 $\mu$g/mL anti-CD28 antibody (kx10-28A; Beijing KEXIN Biological, China) in medium containing 100 U/mL IL-2 (PeproTech, Rocky Hill, NJ, USA) for 2 days. All cell types were maintained at 37 °C in an atmosphere of 5% CO${}_2$.

4-Phenylbutyric acid (4-PBA) (P21005) was obtained from Sigma-Aldrich (Germany). Glutaminase C-IN-1/Compound 968 (HY-12682) and Telaglenastat/CB-839 (HY-12248) were purchased from MedChemExpress (MCE) (USA). DMSO (D8371) was obtained from Solarbio (China).

The primary antibodies included anti-Glutaminase (M01272-3, Boster Biological Technology, 1:1000, China), anti-GLS2 (A05334-1, Boster Biological Technology, 1:800, China), SLC1A5 (AF6610, Affinity Biosciences, 1:1000, USA), GRP78 (AF5366, Affinity Biosciences, 1:1000, USA), Phospho-PERK/Thr982 (DF7576, Affinity Biosciences, 1:1000, USA), PERK (ab229912, abcam, 1:1000, UK), PD-1 (ab214421, abcam, 1:1000, UK), TIM-3/D3M9R (83882, Cell Signaling Technology, 1:1000, USA), and anti-GAPDH (BM1623, Boster Biological Technology, 1:5000, China). The Goat anti-rabbit IgG secondary antibody (BA1054, 1:10,000) was obtained from Boster Biological Technology (China).

Co-cultures were performed in 6-well plates coated with poly-2-hydroxyethyl methacrylate (poly-Hema) (TCS011006, JET, China). In 6-well plates, 1.5 $\times$ 10${}^6$ CTLs (CTLL-2 and CD8${}^{+}$ T cells) were seeded per well as monocultures, and 1.5 $\times$ 10${}^6$ CTLs and 1 $\times$ 10${}^6$ hepatoma cells (Hepa 1–6 and Huh-7 cells) per well were seeded for co-cultures. These plates were incubated for 2 days at 37 °C in a humidified 5% CO${}_2$ atmosphere. Results of co-culture were compared with those of the control group (activated CTLs) and negative control (NC) group (respective monoculture).

Normal human peripheral blood was obtained from healthy volunteer donors with informed consent. Peripheral blood mononuclear cells (PBMCs) were obtained from these samples by centrifugation (800$\times$g, 20 min). Cell sorting was performed using a MofloXDP sorting flow cytometer (Beckman Coulter, Brea, CA, USA).

Total RNA was extracted from CD8${}^{+}$ T cells, and total RNA-Seq libraries were obtained by a three-step method: (1) RNA library construction and on-board sequencing, sequencing data were aligned to the reference genome of the project species to obtain comprehensive transcript information and perform gene expression quantification; (2) the transcriptome sequencing project was completed on the Illumina sequencing platform (San Diego, CA, USA), the IlluminaPE library (approximately 300 bp) was constructed for sequencing; and (3) the obtained sequencing data were analyzed by bioinformatics after performing quality control.

Cell culture supernatants were collected from each group and were then centrifuged for 20 min at 1000$\times$g at 2–8 °C. The concentrations of Granzyme B (ELMO003, Boxbio, China) and Perforin (E-EL-M0890c, Elabscience, China) in the supernatants were determined using ELISA kits according to the manufacturer’s instructions.

The Gln content (EGLN003, BioAssay Systems, USA) in the supernatant (ELx800${}^{\text{TM}}$, Kodak, USA) was determined colorimetrically, measured at 565 nm.

Cells in each group were digested to prepare 1 $\times$ 10${}^6$ cells/mL suspensions and centrifuged. After discarding the supernatant, the cells were fixed with 3% glutaraldehyde fixative for 4 hours and 1% osmic acid for 2 hours. After dehydration with a graded ethanol series and acetone, the samples were immersed in embedding medium for 1 hour. The cells were embedded in epoxy resin. Resin blocks were sectioned using an ultramicrotome (Leica UC-6 ultramicrotome, Germany) for double staining. Images were collected using TEM (H-7650, Hitachi, Tokyo, Japan) followed by photography (Gatan Image Analysis System, California, USA). The ultrastructural changes of the ER in each group of cells were observed by TEM.

Cells were harvested and lysed with protein sample loading buffer (1$\times$). Lysates were incubated in boiling water for 10 mins. Total proteins were separated on a polyacrylamide gel, electrotransferred to polyvinylidene difluoride (PVDF) membranes, and blocked for 1 hour at 37 °C with 5% skim milk in 1$\times$ TBST (T1082, Solarbio, China). The membranes were then incubated with the primary antibodies at 4 °C overnight. After being washed three times in 1$\times$ TBST, the membranes were incubated with secondary antibody for 2 hours at room temperature and washed again. Proteins were detected using the ChemiDoc MP Imaging System (Bio-Rad, California, USA) and form pictures. Pictures were analyzed using Image Lab (Bio-Rad, USA) and Image J (National Institutes of Health, USA) software.

The data was represented as the Mean $\pm$ Standard Deviation (SD) in this study. All analyses were performed with SPSS 21.0 software (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8.0.2 (GraphPad Software, Inc., San Diego, CA, USA). Analyses between two groups were performed using a two-tailed Student’s t test. One-way analysis of variance (ANOVA) was used for multiple group comparisons, and an LSD-t test was used for pairwise comparisons between multiple groups. Statistical significance was defined as p 0.05.

We previously found that a large number of CTLs can infiltrate liver cancer tissues. To clarify whether hepatoma cells can regulate the function of CTLs, we co-cultured CTLL-2 (CTLs) cells with Hepa1-6 (hepatoma) cells. Compared with cells in other groups, CTLL-2 cells co-cultured with Hepa1-6 cells expressed high levels of PD-1 and TIM-3 and secreted low levels of Granzyme B and perforin (Fig. 1). High expression of immune checkpoint-related proteins and low secretion of effector molecules suggested that Hepa1-6 cells can induce a functional downregulation of CTLL-2 cells.

Fig. 1.

Detection of cytotoxic T lymphocyte (CTL) functions. Hepatoma cells and CTLs were co-cultured. (A) Western blots were used to detect the protein expression levels of PD-1 and TIM-3. The levels of (B) Granzyme B and (C) perforin in the cell culture supernatants were assessed by enzyme-linked immunosorbent assays (ELISAs). *p 0.05; **p 0.01; ***p 0.001.

To investigate the functional changes of CTLs, we performed RNA-Seq on CD8${}^{+}$ T cells co-cultured with Huh-7 cells. We observed an upregulation of genes involved in ER-related reactions, such as ER to Golgi transport vesicles, and amino acid stimulation responses, such as the cellular response to amino acids (Fig. 2A). The expression of genes involved in cellular metabolism, such as “the regulation of the lipid metabolic process”, and “cellular secretion”, such as “positive regulation of secretion by cells”, was downregulated (Fig. 2B). These results indicate that CD8${}^{+}$ T cells undergo alterations associated with cell metabolism, ER-related reactions, and cell secretion after being co-cultured with hepatoma cells.

Fig. 2.

Detection of endoplasmic reticulum (ER) stress and its effects on cytotoxic T lymphocyte (CTL) functions. RNA-seq was used to detect differentially expressed genes in hepatocarcinoma-infiltrating CTLs. (A) Gene ontology (GO) Enrichment Plot of upregulated genes. (B) GO Enrichment Plot of downregulated genes. (C) The protein expression levels of GRP78, PERK, and P-PERK in CTLs were detected by western blots. (D) Morphological changes of the ER were observed by transmission electron microscopy. (E) Protein levels of PD-1 and TIM-3 in CTLs cultured with 4-PBA were analyzed using western blots. The secreted levels of (F) Granzyme B and (G) perforin in CTLs cultured with 4-PBA were assessed by enzyme-linked immunosorbent assays (ELISAs). *p 0.05; **p 0.01; ***p 0.001.

Next, we determined if hepatoma cells have any regulatory effects on the ER of CTLL-2 cells. The status of the ER in different groups was analyzed using western blots by detecting the expression of ER stress-related proteins. In contrast to the other groups, GRP78 and P-PERK protein expression levels were significantly upregulated in CTLL-2 cells co-cultured with Hepa1-6 cells (Fig. 2C). Secondly, the substructure of the ER was observed using TEM. The substructure of the ER in the control and NC groups were arranged in order and continuously. The substructure of the ER in the co-culture group was severely damaged, showing gross expansion and degranulation (Fig. 2D). These results show that hepatoma cells can induce CTLs to undergo ER stress.

Based on the role of ER stress in cellular function, we investigated whether ER stress was involved in the hepatoma cell-induced dysfunction of CTLL-2 cells. 4-PBA, an inhibitor of ER stress, was added to the co-culture system. The results showed that after 4-PBA intervention, the expression levels of PD-1 and TIM-3 were downregulated (Fig. 2E), while the secretion of Granzyme B (Fig. 2F) and perforin (Fig. 2G) were increased. Taken together, these data suggest that ER stress is involved in the hepatoma cell-mediated inhibition of CTL functions.

The RNA-Seq results (Fig. 2A) showed that genes related to the amino acid stimulation response were upregulated in CTLs after co-culture with hepatoma cells (Fig. 2A), while Gln is essential for cell metabolic and cellular proliferation. Therefore, we hypothesized that Gln is involved in the process by which hepatoma cells inhibit CTL functions. To examine this, we measured the Gln concentration in each group. The data indicate that cells in the co-culture group consumed more Gln (Fig. 3A). Next, we cultured CTLL-2 cells using Gln-free medium. The results show that CTLs in the group lacking Gln had increased expression levels of PD-1 and TIM-3 (Fig. 3B) and decreased secretion of Granzyme B and perforin (Fig. 3C,D). These data demonstrate that CTL functions are impaired following Gln deprivation. Finally, we examined the state of the ER. When cultured without Gln, there were elevated protein expression levels of GRP78 and P-PERK (Fig. 3E), as well as swollen ER (Fig. 3F). This suggests that ER stress has occurred. However, when 4-PBA was added to the medium when Gln was deprived, the expression levels of GRP78 and P-PERK were not significantly different from those of the control group. Additionally, no significant ER swelling was observed with these conditions. Overall, hepatoma cells compete with CTLs for Gln availability. Gln deficiency inhibited the function of CTLs and induced ER stress.

Fig. 3.

Effect of glutamine (Gln) deprivation on cytotoxic T lymphocytes (CTLs) co-cultured with hepatoma cells. (A) The Gln content in the supernatant of each culture group was detected by colorimetry. Activated CTLs were cultured in Gln-deprived RPMI 1640 medium, and (B) PD-1 and TIM-3 protein expression was detected by western blots. (C) Granzyme B and (D) perforin secretion was detected by enzyme-linked immunosorbent assays (ELISAs). (E) 4-PBA was added to Gln-deprived RPMI 1640 medium, and western blots were used to assess GRP78, PERK, and P-PERK protein expression levels. (F) Transmission electron microcopy was used to observe the morphology of the ER in these cells. *p 0.05; **p 0.01; ***p 0.001.

To investigate how Gln is utilized by CTLs, we detected the expression of glutaminase and glutamine transporters (SLC1A5) in cells. The expression levels of GLS2 and SLC1A5 proteins were significantly reduced in CTLL-2 cells co-cultured with Hepa1-6 cells (Fig. 4A). Next, CTLs deprived of Gln also showed low GLS2 and SLC1A5 expression levels (Fig. 4B). Additionally, there was no significant difference in GLS1 expression. Unlike CTLs, GLS1 protein expression levels were significantly reduced in Hepa1-6 cells in the absence of Gln (Fig. 4B). Thus, we speculated that CTLs mainly use GLS2 to utilize Gln. GLS inhibitors (CB-839 and Compound 968) were added to the medium to further investigate the role of glutaminase. From the western blot results, we set the effective concentration of CB-839 to 4 $\mu$M (Fig. 5A) and the concentration of Compound 968 to 40 $\mu$M (Fig. 5B). CB-839 treatment had no effect on PD-1, TIM-3, GRP78, and P-PERK expression levels and ER stress activation in CTLs compared with controls. However, CTLs treated with Compound 968 had increased protein levels of PD-1 and TIM-3, as well as decreased secretion of Granzyme B and perforin and an enhanced ER stress response (Fig. 5C–G). Overall, these results indicate that GLS2 deficiency inhibits the function of CTLs and induces an ER stress response.

Fig. 4.

Glutamine (Gln) utilization in cytotoxic T lymphocytes (CTLs) and hepatoma cells. (A) Western blots were used to detect the protein expression levels of GLS1, GLS2, and SLC1A5 in the different groups. (B) After Gln deprivation, protein expression levels of GLS1, GLS2, and SLC1A5 in both CTLs and hepatoma cells were assessed by western blots. *p 0.05; **p 0.01; ***p 0.001.

Fig. 5.

Regulation of cytotoxic T lymphocytes (CTLs) by various glutaminases. (A) CTLs were cultured using RPMI 1640 medium containing different concentrations of CB-839, and the protein expression levels of GRP78, PERK, and P-PERK were detected by western blots. (B) CTLs were cultured using RPMI 1640 containing different concentrations of Compound 968, and the protein levels of GRP78, PERK, and P-PERK were detected by western blots. CTLs were cultured using RPMI 1640 containing 4 $\mu$M of CB-839 or 40 $\mu$M of Compound 968. (C) The protein expression levels of PD-1 and TIM-3 were detected by western blots. Secretion of (D) Granzyme B and (E) perforin were measured by enzyme-linked immunosorbent assays (ELISAs). (F) Protein expression of GRP78, PERK, and P-PERK was assessed by western blots. (G) Transmission electron microscopy was performed to observe the morphology of the ER in these cells. *p 0.05; **p 0.01; ${}^{\text{ns}}$p $\mathrm{>}$ 0.05.