Research ArticleExperimental Studies

Cytotoxicity of Human Hepatic Intrasinusoidal Gamma/Delta T Cells Depends on Phospho-antigen and NK Receptor Signaling

YOORHA KANG, MINA HAN, MINSONG KIM, HYUN JU HWANG, BYUNG CHAN AHN, EUNYOUNG TAK, GI-WON SONG, SHIN HWANG, KYUNG-NAM KOH, DONG-HWAN JUNG and NAYOUNG KIM
Anticancer Research January 2023, 43 (1) 63-73; DOI: https://doi.org/10.21873/anticanres.16135

Abstract

Background/Aim: We previously showed that human hepatic intrasinusoidal (HI) natural killer (NK) T cells selectively eliminate hepatocellular carcinoma (HCC) cell lines. In this study, we investigated the underlying mechanisms on how HI γδ T cells, expanded with zoledronate, exhibit a superior cytotoxic effect on HI NK-resistant Huh7 HCC cells. Materials and Methods: γδ T cells were obtained from living liver transplant donors or from peripheral blood mononuclear cells (PBMC) of healthy volunteers and were expanded in the presence of IL-2, IL-15, and zoledronate for 2 weeks. Cytotoxicity was measured using the lactate dehydrogenase (LDH) assay in vitro and by flow cytometry using carboxyfluorescein succinimidyl ester (CFSE) in vivo. Results: The cytotoxicity of expanded HI γδ T cells against Huh7 cells was associated with a higher pyrophosphate expression in Huh7 cells compared to SNU398 cells. In contrast, the cytotoxicity of HI γδ T cells against SNU398 cells depended on NKG2D. HI γδ T cells expressed less PD-1 than PB γδ T cells. The cytotoxicity of HI γδ T cells against Du145 and PC3 prostate cancer cells was also associated with pyrophosphate expression in these cells, as well as NKG2D and DNAM-1. Conclusion: The expression levels of phospho-antigen in tumor cells determined the cytotoxicity of HI γδ T cells, although the NK activating receptors, death ligands, and immune checkpoint molecules also contribute to their cytotoxicity. γδ T cells are attractive candidates for cancer immune cell therapy.

Key Words:

γδ T cells are cytotoxic lymphoid cells with T cell receptor (TCR) γ and δ chains and constitute a minor subset of lymphoid cells in mice and humans. Unlike conventional αβ T cells, γδ T cells are derived from CD4-CD8- double-negative cells and develop into functional γδ T cells depending on the intensity of TCR signaling (1). γδ T cells act at the interface of the innate and adaptive immune systems to play an important role in stress surveillance and homeostasis (2). γδ T cells share many common effector functions with αβ T cells, but there is one distinct difference between them, γδ T cells can recognize a wide range of antigens in the absence of major histocompatibility complex (MHC) proteins (3).

Human γδ T cells are grouped by a δ chain rather than a γ chain and are classified into Vδ2 and non-Vδ2 γδ T cells. Vδ2 T cells are typically paired with a Vγ9 chain and constitute most of the peripheral blood (PB) γδ T cells (4, 5), whereas Vδ1 γδ T cells are predominant in solid tissues and mucosal epithelial. Human Vγ9Vδ2 T cells, which constitute the majority of circulating γδ T cells, require cell–to–cell interactions to induce their reactivity. Their proliferation and activation occur via direct phospho-antigen recognition (6). Through the mevalonate pathway, aminobisphosphonates (NBPs), such as zoledronate, lead to accumulation of isopentenyl pyrophosphates (IPP) by inhibiting farnesyl-pyrophosphate synthase. IPP is then recognized by a γδ T cells, and this induces their proliferation (7). The γδ T cells expanded and activated in response to IPP exert anti-tumor, anti-microbial, and anti-viral responses (8-10). In particular, among the PB mononuclear cells (MC), Vγ9Vδ2 T cells exert strong cytotoxic effects against various cancers, including neuroblastoma and liver and lung cancers (11, 12).

The cytotoxicity of γδ T cells relies on the engagement of T cell receptors (TCR) and NK cell receptors (NKRs), such as natural killer group 2D (NKG2D) (13) and DNAX accessory molecule-1 (DNAM-1) (14). Notably, NKG2D, expressed on most γδ T cells, contributes to recognition and killing of tumor cells. The binding of NKG2D-NKG2D ligands triggers γδ T cells to lyse tumor cells (15). DNAM-1 is involved in the γδ T cell-mediated killing of tumor cells. The interaction between DNAM-1 and its cognate ligands, such as CD112 (Nectin-2) and CD155 (poliovirus receptor; PVR), is required to lyse tumor cells, as it is known that blocking with anti-DNAM-1 does not affect the cytotoxic effect of γδ T cells on the Burkitt’s lymphoma cell line, Daudi, which has no ligand expression (14). In addition to NKRs, various receptors have been identified as playing an important role in the effector function of γδ T cells. For example, upon γδ TCR activation, the expression of Fas ligand (FasL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is increased, thereby promoting the killing of Fas+ and TRAIL receptor+ tumor cells (16). γδ T cells are also known to express immune checkpoint receptors, such as programmed cell death protein (PD)-1 and B and T lymphocyte attenuator (BTLA) (17).

Lymphocytes within the liver differ from those in peripheral blood; innate lymphoid cells [including NK cells, invariant NK T (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and γδ T cells] are enriched in the liver (18). In particular, hepatic γδ T cells represent up to 6.6% of the T lymphocytes in the liver and only 0.9% in the peripheral blood (19). Intrahepatic γδ T cells display tissue-resident features (20). Secretion of cytokines, such as IFN- γ, TNF-α, interleukin-2, and interleukin-4, by hepatic γδ T cells are increased in malignancy, and they exhibit strong cytotoxic effects on target cell K562 and Daudi cell lines, suggesting the potential antitumor role of liver-resident γδ T cells in controlling HCC (19).

We previously showed that hepatic intrasinusoidal (HI) NK cells have distinct features, such as the strong and selective cytotoxic effects of CD56bright NK cells on HCC cell lines (21, 22). Specifically, HI NK cells failed to kill Huh7 cells, but they successfully killed SNU398 cells (22). In this study, we aimed to characterize the HI γδ T cells and to investigate the mechanisms underlying the cytotoxicity of HI γδ T cells against solid tumor cells, such as HCC and prostate cancer cells.

Materials and Methods

Human HI and PB γδ T cell expansion. Right lobe grafts from healthy living donors were washed with 1 l of histidine–tryptophan–ketoglutarate solution, and liver perfusate was collected. All participants provided written informed consent, and all procedures were approved by the Institutional Review Board (IRB) of the Asan Medical Center, Seoul, Republic of Korea (Approval No. 2019-1665). The participants were healthy donors for liver transplant surgery (21 males aged 51.2±14.4 years and 6 females aged 50.2±12.7 years). HI mononuclear cells (MC) were isolated using the Ficoll–Paque density gradient method (GE Healthcare Life Sciences, Waukesha, WI, USA). Isolated cells suspended in 90% fetal bovine serum (FBS; Welgene, Gyeongsan-si, Gyeongsangbuk-do, Republic of Korea) and 10% DMSO (Sigma Aldrich, St. Louis, MO, USA) were frozen in liquid nitrogen until use.

Peripheral blood mononuclear cells (PBMC) from healthy adult volunteers for hematopoietic cell transplantation were delivered to us after αβ T cell depletion (23) and were further isolated using a Ficoll–Paque density gradient method (GE Healthcare Life Sciences). The isolated cells were suspended in 90% FBS (Welgene) and 10% DMSO (Sigma Aldrich) and frozen in liquid nitrogen until use. All participants provided written informed consent, and all procedures were approved by the IRB, Asan Medical Center, Seoul, Republic of Korea (Approval No. 2018-0445). The participants were five females aged 36.2±9.4 years and two males aged 37±15.6 years. All the studies were performed ethically in accordance with the Declaration of Helsinki.

HI MC or PBMC were cultured at 2×106 cells per well in RPMI 1640 medium (WelGene) supplemented with 10% (v/v) heat-inactivated FBS (WelGene), 100 U/ml penicillin, 100 μg/ml streptomycin (Cellgro, Manassas, VA, USA), 5 mM sodium pyruvate (Pan Biotech, Aidenbach, Germany), and 55 μM 2-mercaptoethanol (Thermo Scientific, Waltham, MA, USA). HI MC or PBMC were cultured with 100 U/ml recombinant human IL-2 (rhIL-2) (PeproTech, Rocky Hill, NH, USA), 1 μM zoledronate (Selleckchem, Houston, TX, USA), and 50 ng/ml recombinant human IL-15 (rhIL-15) (PeproTech) for 2 weeks. The culture was treated with zoledronate once on day 0 and then treated with RhL-2 and rhIL-15 at the same concentration every 2 days.

Tumor cell culture. Human tumor cell lines, Huh7, PC3, Du145, and LNCaP, were purchased from ATCC (Manassas, VA, USA), and SNU398 cells were purchased from Korean Cell Line Bank (Seoul, Republic of Korea). Huh7, PC3, and Du145 cells were cultured in DMEM medium (WelGene) supplemented with 10% (v/v) heat-inactivated FBS (WelGene), 100 U/ml penicillin, and 100 μg/mL streptomycin (Cellgro). SNU398 and LNCaP cells were cultured in RPMI 1640 medium (WelGene) supplemented with 10% (v/v) heat-inactivated FBS (WelGene), 100 U/ml penicillin, 100 μg/ml streptomycin (Cellgro), and 5 mM sodium pyruvate (Pan Biotech).

Lactate dehydrogenase (LDH) assay. Target cells were seeded in round-bottom 96-well plates at 7,000 cells/well. HI γδ T cells were seeded at 140,000 cells/well. The effector to target (E: T) ratio was 20:1. The HI γδ T cells were incubated with monoclonal antibodies (mAb) against human NKG2D (Clone 1D11), TRAIL (RIK-2), FasL (NOK-1), or DNAM-1 (11A8) (all from BioLegend, San Diego, CA, USA) at 10 μg/ml for 30 min at 4°C, following 5 min incubation with human FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). Negative control was precoated with matched isotype control, mouse IgG1, Embedded Image (Biolegend). The plates were incubated at 37°C in a humidified atmosphere of 5% CO2 for 6 h. The assay was performed using a CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Optical density at 490 nm was measured using a Sunrise ELISA reader (Tecan, Mannedorf, Switzerland). Specific lysis was calculated as percent cytotoxicity =100× Experimental LDH Release (OD450) Maximum LDH Release (OD450), as the manufacturer’s instructions.

Pyrophosphate assay. A pyrophosphate assay was conducted to quantify pyrophosphates, such as isopentenyl pyrophosphate (IPP) which is known to be a phospho-antigen to γδ T cells. One million tumor cells were washed with cold PBS and lysed in RIPA buffer (Biosesang, Seongnam, Republic of Korea) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) for 30 min on ice. The lysates were collected by centrifugation for 5 min at 14,000 rpm and 4°C. A pyrophosphate assay kit (Abcam, Cambridge, UK) was performed according to the manufacturer’s instructions. Fluorescence was measured using a VICTOR X3 2030 multilabel reader at Ex/Em 316/456 nm (PerkinElmer, Waltham, MA, USA).

Degranulation assay. For the degranulation assay, HI γδ T cells as effector cells (E) and SNU398 or Huh7 cells as target cells (T) were co-incubated at a ratio of 1:2 (E:T) at 37°C with 5% CO2 for 16 h. The positive control was stimulated at 37°C with 50 ng/ml phorbol 12-myristate 13-acetate (PMA; Calbiochem, San Diego, CA, USA) and 1 μg/ml ionomycin (Sigma Aldrich) for 15 h. Monensin (eBioscience, San Diego, CA, USA) was treated at 2 μM in the presence of fluorescence-conjugated anti-CD107a (Clone H4A3; Biolegend, San Diego, CA, USA) for 15 h. After washing, the cells were stained for cell surface markers for 30 min at 4°C. Flow cytometric data were acquired using Cytoflex (Beckman Coulter, Brea, CA, USA) and analyzed with FlowJo software (TreeStar, Ashland, OR, USA).

In vivo experiments. On the day of tumor cell implantation, Huh7 and SNU398 cells were harvested and labeled using a 1.25 μM carboxyfluorescein succinimidyl ester (CSFE) using a CellTrace™ CFSE Cell Proliferation kit (Invitrogen, Waltham, MA, USA) in PBS at 37°C for 6 min. Total 16 female NOD-scid IL2Rgnull (NSG) mice, aged 6-8 weeks, were purchased from JA BIO (Suwon, Republic of Korea) and were injected intraperitoneally (i.p.) with 5×106 tumor cells 30 min prior to injection with HI γδ T cells. The experimental schemes and groups were illustrated in Figure 1B. After 48 h, peritoneal lavages were collected by washing the peritoneal cavity with 10 ml of sterile PBS, as previously described (24). The animal experiment was approved by Institutional Animal Care and Use Committee (IACUC), Asan Medical Center, Seoul, Republic of Korea (Approval No. 2022-13-162).

Figure 1.
Figure 1.

Zoledronate-expanded HI γδ T cells effectively eradicated HCC cells. (A) A lactate dehydrogenase assay was performed with zoledronate-expanded HI γδ T cells on Huh7 and SNU398, hepatocellular carcinoma cell lines. The effector to target (E:T) ratio was 20:1. N=11 for HI γδ T cells and N=3 for PBMC γδ T cells. Data are expressed as mean±SEM. The Student’s t-test was conducted. (B) HI γδ T cells degranulate more in response to Huh7 cells compared to SNU398 cells. A CD107a degranulation assay was conducted. Numeric values show % of CD107a+ cells. Representative flow cytometric plots are shown. Blue lines, isotype control; red lines, CD107a. (C) A schema of the in vivo experiment is shown in the upper panel. CFSE-labeled HCC cells were injected intraperitoneally (i.p.). into NSG mice at 5×106 cells. After 30 min, the same numbers of HI γδ T cells were injected i.p., and peritoneal lavage was collected 48 h later and analyzed by flow cytometry. Means±SEM are shown. A Student’s t-test was performed. N=4/group (D) Pyrophosphate antigen expression was assessed in Huh7 and SNU398 cells (means±SEM). A Student’s t-test was performed. N=4/group. *p<0.05. HI, Hepatic sinusoidal; HCC, hepatocellular carcinoma; PBMC, peripheral blood mononuclear cells.

Flow cytometry. Cells were incubated with the conjugated antibodies and washed twice with 1x PBS containing 2% FBS (WelGene). The following antibodies were used for surface staining; human CD3 (Clone SK7 or UCHT1), CD16 (4G7), Vδ1 (TS8.2), Vδ2 (B6), NKp46 (9E2), NKG2D (1D11), TRAIL (RIK-2), CD178 (NOK-1), BTLA (MIH26), CD226 (11A8), PD-1 (EH12.2H7), CD8 (HIT8a), and TIGIT (A15153G) mAb. Du145, PC3, and LNCaP human prostate cancer cell lines were incubated with human CD112 (TX31), CD155 (SKIL4), HLA-E (3D12), DR4 (DJR1), DR5 (DJR2-4), MICA/B (GD4), PD-L1 (29E.2A3), Fas (DX2), CD40 (5C3), HLA-ABC (G46-2.6), and HVEM (94081) mAb at 4°C for 30 min. These Ab and matched isotype controls were purchased from BD Biosciences (San Diego, CA, USA), BioLegend, or eBioscience. An FcR blocking reagent (Miltenyi Biotec) was used prior to staining. Flow cytometry data were acquired using Cytoflex (Beckman Coulter) and analyzed using FlowJo software (TreeStar).

Statistical analyses. All data are presented as the mean±SEM. p-Values for the degranulation assay, LDH assay, in vivo experiment, and pyrophosphate assay were calculated using the one-way analysis of variance (ANOVA), and the degranulation assay with blocking antibodies and expression of cell surface markers were analyzed using the two-way ANOVA. Both ANOVAs were performed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA). *p<0.05, **p<0.01, and ***p<0.001.

Results

HI γδ T cells efficiently eliminate NK-resistant huh7 cells expressing more phospho-antigen. HI and PB γδ T cells were expanded in the presence of IL-2, IL-15, and zoledronate, and cytotoxicity was measured using the LDH assay. As expected, HI γδ T cells were robustly expanded. HI γδ T cells showed comparable cytotoxicity against both NK-resistant Huh7 and NK-susceptible SNU398 HCC cells (22). The cytotoxic effect of HI γδ T cells was stronger than that of PB γδ T cells (Figure 1A). The potent cytotoxicity of HI γδ T cells against Huh7 cells was confirmed using the CD107a degranulation assay (Figure 1B). Both Vδ1 and Vδ2 subsets of HI γδ T cells expressed a greater amount of CD107a in response to Huh7 than to SNU398 cells.

To validate the anti-tumor activity of HI γδ T cells in vivo, NSG mice were injected with CFSE-labeled Huh7 or SNU398 cells intraperitoneally and with HI γδ T cells 30 min later. Peritoneal lavage was collected after 48 h and analyzed using flow cytometry. The ratio of CFSE+/CFSE− cells was calculated, to compare CFSE-labeled tumor cells between no-additional-cell-injected group where only CFSE-labeled tumor cells were injected, and HI-γδ-T-cell-injected group, where HI γδ T cells were injected following the injection of CFSE-labeled tumor cells, as shown in the lower panel of Figure 1C. HCC cells were effectively eradicated by HI γδ T cells in vivo (Figure 1C).

γδ T cells are known to share many receptors with NK cells, and hence we hypothesized that the cytotoxicity of γδ T cells against Huh7 cells was related to γδ TCR and its ligands; in this case, Vδ2 and phospho-antigen. We conducted a pyrophosphate assay to quantify pyrophosphates, which include phospho-antigen. Pyrophosphate is a source of phosphoryl groups for protein phosphorylation (25), and it forms intermediate molecules in the mevalonate pathway, notably mevalonate pyrophosphate and IPP (26). Huh7 cells expressed significantly higher amounts of pyrophosphate than SNU398 cells (Figure 1D). These results suggest that increased pyrophosphate expression on tumor cells may correlate with the anti-tumor function of HI γδ T cells.

HI γδ T cells express less PD-1. The phospho-antigen expression does not fully explain the cytotoxic effects of HI γδ T cells on SNU398 cells or the higher cytotoxicity of HI γδ T cells compared to that of PB γδ T cells. Therefore, we investigated the expression of activating receptors, death ligands, and immune checkpoint molecules of HI and PB γδ T cells expanded with zoledronate. The gating strategies and representative flow cytometric plots are shown in Figure 1B. The percentages of Vδ2 T cells were over 90% on day 14 of culture, and the Vδ2 expression levels based on the mean fluorescence indices (MFI) were also comparable (Figure 2A). None of the activating receptors was expressed at a higher level in the HI γδ T cells than in the PB γδ T cells after 14 days in culture (Figure 2B-C). The percentages of HI Vδ2 T cells expressing FasL were significantly lower than those of than in HI Vδ2 T cells and PB γδ T cells, and PD-1 expression levels by MFI were lower in HI γδ T cells than in PB γδ T cells (Figure 2C). The expression of BTLA was significantly higher in HI Vδ2 T cells than in HI Vδ1 T cells (Figure 2B-C). TIGIT was expressed significantly higher in HI Vδ1 T cells than in HI Vδ2 T cells and PB γδ T cells, even though Vδ1 T cells were less than 5% of the total cells expanded by zoledronate. In summary, the results show that lower PD-1 expression may elicit greater cytotoxic effects from HI γδ T cells when compared to PB γδ T cells.

Figure 2.
Figure 2.

HI γδ T cells and PB γδ T cells expressed activating receptors, inhibitory receptors, and death ligands differentially. (A) Percentages of Vδ1 and Vδ2 γδ T cells in HI and PB (left), and MFI of γδ TCR (right) were assessed after culture for 14 days in the presence of zoledronate, IL-2, and IL-15 (means±SEM). N=4 for LP; n=7 for PB. (B) Percentages of indicated cell surface receptor expressing γδ TCR are displayed on lower left panels (means±SEM). In HI γδ T cells, N=11 for BTLA and PD-1 expression and N=3 for NKG2D, NKp46, CD16, DNAM-1, TRAIL, Fas-L, and TIGIT expression. In PB γδ T cells, N=5. One-way ANOVA with Tukey’s multiple comparison was performed. (C) MFI of indicated cell surface receptors on γδ T cells are displayed on lower right panels (means±SEM). In HI γδ T cells, N=11 for BTLA and PD-1 expression and N=3 for NKG2D, NKp46, CD16, DNAM-1, TRAIL, Fas-L, and TIGIT expression. In PB γδ T cells, N=5. One-way ANOVA with Tukey’s multiple comparison was performed. *p<0.05, **p<0.01 and ***p<0.001. HI, Hepatic sinusoidal; MFI, mean fluorescence index; PB, peripheral blood; PBMC, peripheral blood mononuclear cells.

Cytotoxicity of HI γδ T cells against snu398 cells relies on NKG2D. As discussed above, the expression levels of activating receptors and death ligands were not related to the cytotoxicity of HI γδ T cells expanded with zoledronate, and particularly not related to that of SNU398 cells. It should be mentioned that SNU398 cells express more Fas and NKG2D ligands, MICA/B, while Huh7 cells express more TRAIL receptors, DR4 and DR5 (22). CD112 and CD155. We, thus, performed a cytotoxicity assay with blocking antibodies. The cytotoxicity of HI γδ T cells against SNU398 cells was found to be dependent on NKG2D signaling, and blocking NKG2D significantly reduced the cytotoxicity of HI γδ T cells (Figure 3A). Taken together, these results suggest that the expression of phospho-antigen and ligands of activating receptors on tumor cells can determine the cytotoxicity of HI γδ T cells.

Figure 3.
Figure 3.

NKG2D contributes to cytotoxicity of HI γδ T cells against SNU398 cells. LDH assay was performed with blocking anti-NKG2D, anti-DNAM-1, anti-FasL, and anti-TRAIL mAbs. The effector to target (E:T) ratio was 20:1. N=11 for isotype control, n=6 for anti-NKG2D and anti-DNAM-1, and n=5 for anti-FasL and anti-TRAIL groups. Means±SEM are displayed. One-way ANOVA with Tukey’s multiple comparison was performed. *p<0.05. α-DNAM-1, anti-DNAM-1 mAb-treated; α-Fas-L, anti-Fas-L mAb-treated; α-NKG2D, anti-NKG2D mAb-treated; α-TRAIL, anti-TRAIL mAb-treated; Isotype; isotype control; LDH, lactate dehydrogenase.

Phospho-antigen expression and NK receptor signaling are associated with cytotoxicity of HI γδ T cells against prostate cancer cells. To expand our findings, we evaluated the quantities of pyrophosphate in prostate cancer cell lines, Du145, PC3, and LNCaP, and the cytotoxicity of HI γδ T cells expanded with zoledronate. Du156 cells expressed pyrophosphate significantly higher than PC3 and LNCaP cells. Nonetheless, HI γδ T cells killed PC3 cells as well as Du145 cells, while they failed to kill LNCap cells (Figure 4A). In line with the results, the expression of most of the cell surface receptors was lower in LNCaP cells than in PC3 and Du145 cells, as per percentages and MFI (Figure 4B) with exception of DR4 and MICA/B. The cytotoxicity assay with blocking antibodies showed that the cytotoxicity of HI γδ T cells against Du145 also depended on NKG2D and that of PC3 depended on DNAM-1 and NKG2D (Figure 4C). DNAM-1 ligands, CD155 and CD112, were expressed highly in both Du145 and PC3 cells (Figure 4B). In addition, the cytotoxicity of HI γδ T cells against LNCaP depended on the Fas pathway, although the expression of Fas on LNCaP cells was relatively low (Figure 4C). The cytotoxicity of HI γδ T cells against Du145 prostate cancer cells appeared to be dependent on phosphor-antigen as well as NKG2D signaling.

Figure 4.
Figure 4.

Cytotoxicity of HI γδ T cells correlates to pyrophosphate antigen expression in prostate cancer cell lines and NKG2D signaling. (A) Pyrophosphate expression on human prostate cancer cell lines (left) and cytotoxicity of HI γδ T cells (right) are shown (means±SEM). N=4 for pyrophosphate assay and N=9 for LDH assay. (B) The percentages of the cells expressing activating ligands, inhibitory ligands and death receptors in prostate cancer cell lines are shown in left panel. MFI of each molecule are shown on the right. The results of two representative independent experiments are shown. (C) LDH assay was performed with anti-NKG2D, anti-DNAM-1, anti-FasL, and anti-TRAIL mAbs (means±SEM). The effector to target (E:T) ratio was 20:1. Mean±SEM, One-way ANOVA with Tukey’s multiple comparison was performed. N=3-8. *p<0.05. α-DNAM-1, anti-DNAM-1 mAb-treated; α-Fas-L, anti-Fas-L mAb-treated; α-NKG2D, anti-NKG2D mAb-treated; α-TRAIL, anti-TRAIL mAb-treated; Isotype; isotype control; MFI, mean fluorescence index; LDH, lactate dehydrogenase.

Discussion

In this study, we showed that compared to PB γδ T cells, HI γδ T cells that were expanded with zoledronate had potent cytotoxicity against HI NK-resistant Huh7 HCC cells. The results of the pyrophosphate assay of HCC cells and prostate cancer cells showed that the strong cytotoxic effects of γδ T cells were related to the expression of phospho-antigen. Increased pyrophosphate expression may intensify TCR signaling. It should be mentioned that the pyrophosphate assay detects not only IPP but also broad ranges of pyrophosphates, thus the results were not exclusively applied for IPP. Further study using analytical biochemical tools, such as LC-MS/MS, may enable measuring the precise amount of IPP. Furthermore, our preliminary microarray results showed that Huh7 cells greatly expressed genes involved in angiogenesis, cell migration, extracellular matrix, and inflammatory response (data not shown), implying that Huh7 cells are more metastatic than SNU398 cells. PC5 and Du145 cells are metastatic, but LNCaP cells are not (27). HI γδ T cells were less efficient to kill LNCaP cells. Therefore, HI γδ T cells might be more potent killers of metastatic cancer cells, and the expression levels of phospho-antigen could be used as predictive biomarkers for γδ T cell therapy.

A deregulated mevalonate/cholesterol pathway is found in certain cancers that require greater amounts of cholesterol for tumor development (26). The mevalonate pathway is up-regulated in several cancers, such as leukemia, lymphoma, multiple myeloma, as well as breast, hepatic, pancreatic, esophageal, and prostate cancers. One possible way to inhibit this is by using statins; however, NBPs, such as zoledronate, may have a dual effect on cancer as an inhibitor of farnesyl pyrophosphate synthase in the mevalonate pathway and as an activator of γδ T cells (28). Zoledronate increases the recognition of HCC and colorectal carcinoma cells by Vδ2 T cells (29). Tumor recognition by Vδ2 T cells is also improved by treating human breast tumors and their metastasis to bones with NBP (30, 31). Therefore, NBP treatment can be considered for improving γδ T cell therapy, particularly for tumors with a low phospho-antigen expression. It has been reported that repeated and combined administration of NBPs and human Vγ9Vδ2 T cells can effectively control the growth of PC3 prostate cancer cells and neuroblastoma cells in mice (11, 32). In addition, post-transplantation therapy with zoledronate and IL-2 is well-tolerated in pediatric patients with neuroblastoma (23), suggesting that more clinical trials are required after careful screening.

NKG2D is an alternative modality for killing SNU398 HCC cells and Du145 prostate cancer cells. Human HCC cells express not only NKG2D ligands, including MHC class I-chain-related antigens (MIC) A/B (33) and UL16-binding proteins (ULBPs) (34), but also DNAM-1 ligands (35), TRAIL receptor DR4 and DR5 (36), NKp46 ligand (37), and Fas (38), suggesting that γδ T cells may have additional benefits upon HCC regression in humans. NKG2D was expressed in nearly 100% of γδ T cells in this study, and MICA/B expression was higher in SNU398 cells than that of Huh7 (22), that may explain the higher dependence of HI γδ T cells on NKG2D to kill SNU398 cells. Furthermore, HI γδ T cells express less PD-1, which might permit greater cytotoxicity, when compared with PB γδ T cells. In a recent report, combining anti-PD-1 Ab with a STAT3 inhibitor failed to prevent HCC growth in mice (39). Along with a study where STAT3 are not involved in homeostasis of peripheral γδ T cells (40), this study might provide background information for incompetent combinational therapy of PD-1 blockade and STAT3 inhibition.

HI γδ T cells also effectively eliminate PC3 and Du145 metastatic prostate cancer cells, but not LNCap cells which are less metastatic. Du145 cells expressed phosphor-antigen higher than PC3 cells, and the comparable cytotoxicity against PC3 cells were mediated by DNAM-1 and NKG2D signaling. Considering that both Du145 and PC3 cells expressed DNAM-1 ligands strongly and that the cytotoxicity against Du145 cells was slightly decreased by DNAM-1 blocking, DNAM-1 signaling might play a role in the cytotoxicity of HI γδ T cells against Du145 cells as well. LNCaP prostate cancer cells express phospho-antigen and most of the activating ligands at lower levels, except DR4 and MICA/B, than PC3 and Du145 cells. HI γδ T cells thus showed only limited cytotoxicity against LNCaP cells, depending on FasL, rather than TRAIL or NKG2D. This study aimed to explore the possibility of ex vivo expanded HI γδ T cell therapy for cancer treatment, thus the natural functions of HI γδ T cells without expansion with cytokines and zoledrinic acid would expand our knowledge on γδ T immunology.

One major obstacle slowing the advance of γδ T cells use in cancer therapy is their low population levels (0.5%-5.0% of all T-lymphocytes). Considering that liver perfusate acquired from liver transplantation is an abundant source of HI T cells (41) and that HI γδ T cells were found to have potent cytotoxicity against various tumor cells in this study, HI γδ T cells could be an attractive candidate for use in immune cell therapy, particularly for metastatic cancer. Importantly, allogeneic γδ T cells are not alloreactive, and do not cause graft versus host diseases (GVHD) as they are not MHC-restricted (42), suggesting potential safety in clinics. Allogeneic γδ T cells have been proved safe in phase I clinical trials (12, 43). Nonetheless, further basic and translational studies are required.

Conclusion

In conclusion, the expression levels of pyrophosphate in tumor cells determined the cytotoxicity of HI γδ T cells, although the NK activating receptors, death ligands, and immune checkpoint molecules may modify the cytotoxicity of HI γδ T cells. These results suggest that γδ T cells are an attractive candidate for cancer immune cell therapy and that pre-screening of phospho-antigen expression in tumor cells could improve outcomes.

Acknowledgements

The Authors thank the LT staff and pediatric HCT staff for assisting the work.

Footnotes

  • Authors’ Contributions

    YK performed the experiments, analyzed the data, and wrote the manuscript. MK, MH, HJH and HCA performed the experiments and analyzed the data. ET and GWS provided materials. SH conceptualized the research and provided materials. KNK and DHJ conceptualized the research, provided materials, and obtained the grants. NK conceptualized and designed the research, analyzed the data, and wrote the manuscript.

  • Conflicts of Interest

    The Authors declare no conflicts of interest.

  • Funding

    This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (Grant No. 2019R1A2C1009175 to D.-J. Jung and Grant No. 2018R1C1B6008852 to K. N. Koh).

  • Received November 8, 2022.
  • Revision received November 22, 2022.
  • Accepted November 29, 2022.

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Cytotoxicity of Human Hepatic Intrasinusoidal Gamma/Delta T Cells Depends on Phospho-antigen and NK Receptor Signaling
YOORHA KANG, MINA HAN, MINSONG KIM, HYUN JU HWANG, BYUNG CHAN AHN, EUNYOUNG TAK, GI-WON SONG, SHIN HWANG, KYUNG-NAM KOH, DONG-HWAN JUNG, NAYOUNG KIM
Anticancer Research Jan 2023, 43 (1) 63-73; DOI: 10.21873/anticanres.16135
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