INTRODUCTION
On a global scale, hepatocellular carcinoma is one of the common malignant tumors. The most recognized surgical treatment of hepatocellular carcinoma primarily includes surgical resection and liver transplant.[1] However, most patients with hepatocellular carcinoma cannot be cured due to multifocal diseases, portal hypertension or liver dysfunction.[2,3]
It was reported in the literature that γδ T cells,[4] as a unique subpopulation of T cells, can play an essential scientific and clinical role in T cell anti-tumor immunity.[5–8] Compared to αβ T cells, it is antigen-free, independent of antigen-presenting cells, and is unrestricted by major histocompatibility complex (MHC).[9]
In 1994, Boom et al.[10] reported that a group of heat-resistant polypeptide antigens derived from Mycobacterium tuberculosis H37Ra strain can positively activate γδ T cells. Another study confirmed this phenomenon[11,12] and the authors named the antigen Mtb-HAg. Furthermore, some pyrophosphate-containing phosphorylated antigens (HDMAPP) of the pyrophosphate family also can stimulate the proliferation of γδ T cells.[13] Additionally, studies have shown that zoledronate (ZOL) can also efficiently stimulate γδ T cells in vitro, and the expanded γδ T cells still have an obvious killing effect on tumor cells.[14,15] However, comparing the ratio of HDMAPP, Mtb-HAg, and ZOL stimulation to the proliferation of γδ T cells in human peripheral blood and whether the activity of killing tumor cells after amplification is different has not been reported so far.
Extracellular signal-regulated kinase (ERK) 1/2 is an essential member of the ERK family. When activated by signals, such as the external environment or cytokines, they can transmit signals to the nucleus and regulate biological behaviors, such as cell proliferation, differentiation, and apoptosis.[16]PD98059 is a specific blocker of the ERK1/2 signaling pathway, and it has been widely used in studying the role of the ERK1/2 signaling pathway in regulating cell activity and function. Many in vitro and in vivo studies[17] discovered that PD98059 inhibited the proliferation of lymphocytes after inhibiting the ERK1/2 signaling pathway.
As one of the neutral killer (NK) cell–activating receptors, NKG2D can be expressed in NK cells, CD8+ (cytotoxic) T cells, and the surface of γδ T cells membrane;[18,19] its ligand, can activate T cells and NK cells, kill tumor cells using NKG2D ligand expression, and play the role of tumor immune regulation. In this study, NKG2D blockers blocked effector cells from identifying target cells, and PD98059 blocked intracellular signaling pathways.
Some companies are developing γδ T cell therapy to fight cancer, including TC Biopharm (TCB), Gadeta, etc. In April 2019, TCB announced that it had launched a phase I clinical study (NCT03790072) of TCB002 to treat acute myeloid leukemia (AML). TCB002 is an allogeneic cell therapy composed of activated and expanded γδ T cells from healthy donors. This study aims to obtain safety data for the use of γδ T allografts and promote the development of chimeric antigen receptor T-cell products. Professor Yan Xu’s team from Jinan University in China published their work in the journal, Cellular & Molecular Immunology, clinical trial data of γδ T cells in patients with advanced liver cancer or lung cancer.[20] In terms of safety, in phase I clinical trial involving 132 patients (414 times of cell reinfusion have been completed), allogeneic γδ T cells reinfusion did not have serious side effects. In one case, some patients had transient mild clinical reactions. In terms of curative effect, among 132 patients, 8 patients with liver cancer and 10 patients with lung cancer received cell reinfusion ≥5 times, and the survival time was significantly extended. These results preliminarily verified allograft T cells’ efficacy and provided an essential basis for new anti-cancer immunotherapy.
MATERIALS AND METHODS
Materials
Reagents
On December 24, 2018, it was approved by the ethics committee of Bengbu Medical College. RPMI 1640, containing L-glutamine and phenol red (Gibco company); Human lymphocyte separation fluid (Biosharp company); IL-2 recombinant human protein TL-104 (Tongli Haiyuan Company); PHA (Dayou Company); HDMAPP (Shanghai Binzhi Biotechnology Co., Ltd.); Mtb-HAg: Prepared by the Experimental Center of Infection and Immunology of Anhui Province, according to the literature’s method;[21] ZOL (TCI company); The staining buffer was prepared by the Experimental Center of Infectious and Immunological Sciences of Anhui Province by Bengbu Medical College; CD8FITC (Tianjin Xieke Biotechnology Co., Ltd.); CD3PE (Tianjin Xieke Biotechnology Co., Ltd.); CD3APC (1:20) (BioLegend company); CD3VB (Meitian company); TCRγδFITC (Invitrogen company); γδ1VB (Meitian company); γδ2PE (BioLegend company); CFSE (MCE company); PI (BioFroxx company); PD98059 (Apexbio company); (NKG2D) Monoclonal Antibody (1D11) (eBioscience company); γδTPE (eBioscience company); Anti-PE MicroBeads (Meitian company); 4% Paraformaldehyde (PFA) (Biomiky company).
Instruments
Flow cytometry (DxP AthenaTM, Cytek, and FACSCalibur, USA, BD); Live Cell Workstation (Axio Observer Z1, ZEISS, Germany); MACS Magnetic Cell Separator and Reagent (Germany Meitian Company).
Methods
Heparin anticoagulant venous blood separation of peripheral blood mononuclear cells
After signing the informed consent form for voluntary blood donation, 10 ml of peripheral venous blood from healthy volunteers was obtained from the heparin anticoagulant tube, and placed in an equal amount of serum-free 1640 to mix. 10 ml of human lymphocyte separation solution was added to a centrifuge tube and it was centrifuged at 1550 rpm for 15 minutes at 20°C. When the upper layer of plasma was sucked into the white membrane, using a papillon pipette at 0.5 cm, the Pasteur pipette was gently inserted into the tunica layer, and the leucocytic cells were aspirated along the circumference of the tube wall and transferred to another 15-ml centrifuge tube, after which 15 ml of the white membrane layer was taken, and 15 ml of phosphate buffer saline (PBS) was added and suspended. Centrifugation was conducted thrice at 1800 rpm, 1600 rpm, and 1400 rpm for 10 minutes each, at 4°C. Then, 2-ml RPMI-1640 medium containing 5% FBS and 5% autologous serum was added, resuspended, and counted.
Proliferation test of γδ T cells
The cell density was adjusted to 2 × 106 cells/ml using the above RPMI-1640 medium. A 24-well plate was taken, and 1 ml of 2 × 106 cells/ml cell suspension was added to each well. HDMAPP (2 × 10-9 mol/ml), Mtb-HAg (279-ng/μl), Zol (5-μM), PHA (10-μg/ml), and IL-2 (1000 IU/ml) were added to each well, and the final volume per well was 1 ml, while the final cell density was 1–2 × 106 cells/ml. The cell density was adjusted to 2–5 × 105 cells/ml every 1–3 days depending on cell growth, and IL-2 was added to a final concentration of 1000-IU/ml.
Determination of PBMC ratio by flow cytometry
Here, 3–5 × 105 cells were taken, resuspended in 2 ml of staining buffer, rotated at 1500 rpm, centrifuged for five minutes, and repeated. In the control group, only the cell suspension was added. The experimental tube was supplemented with CD8FITC 3-μl, CD3PE 3-μl, CD3APC (1:20) 1-μl, CD3VB 3-μl, and then TCRγδ-FITC 1.5-μl, CD3APC (1:20) 2-μl, Vδ1VB 3-μl, Vδ2PE (1:10) 2-μl. Then the cell suspension was added, the light ice bath was avoided for 20 minutes at 4°C, rotated at 1500 rpm, and centrifuged for five minutes. The supernatant was discarded, the pellet in was resuspended in 2-μl PBS, spun at 1500 rpm, centrifuged for five minutes, and repeated. The supernatant was discarded, 0.2–0.5-μl 4% PFA was added to each tube, and the proportion of γδ T cells was detected using flow cytometry.
Culture of HepG2 cells
The cell cryotube was placed in a constant temperature water tank to dissolve. 5-ml cell culture medium and the liver cancer cell suspension was added to a 10-ml centrifuge tube at a speed of 1000 rpm and centrifuged for five minutes. The supernatant was discarded, resuspended in 3–5 ml complete medium, transferred to a cell culture flask, and cultured overnight in a cell culture incubator for one day. The culture solution was aspirated, rinsed with PBS on the second day, and the cells were digested using trypsin. The cell suspension was resuspended, transferred to a 10-ml centrifuge tube, and centrifuged at 1000 rpm for five minutes; the supernatant was discarded, and then 3–6 ml of complete medium was added and pipetted into a single cell suspension. The liver cancer cell suspension was pipetted into a new cell culture flask, supplemented with a complete medium, and cultured in an incubator.
Hepatoma cell HepG2 passage
When the reconstituted liver cancer cells were grown for 1–3 days, they were passaged as appropriate. The method is the same as above.
Proportion of γδ T cell expansion after 12–14 days
Each group of γδ T cells was resuspended into a 50-ml centrifuge tube. The method is the same as determination of PBMC ratio by flow cytometry.
Flow cytometry to detect the killing effect of γδ T cells stimulated by PBMC on HepG2
Marking of target cells HepG2
Digest HepG2. HepG2 cells in exponential growth period were washed twice using PBS, 1 ml of trypsin digest was added to digest the cells for 2–3 minutes, and 2 ml of complete medium was added for neutralization and digestion. Also, 10-μl count 1 × 106 was taken, rotated at 1000 rpm, and centrifuged for five minutes. Resuspend HepG2 was taken in 5-ml dulbecco’s modified eagle medium (DMEM), spun at 1000 rpm, centrifuged for five minutes, and repeated. It was then resuspend in 1-ml DMEM. The target cells were labeled using carboxyfluorescine diacetate succinimidyl ester (CFSE) at a final concentration of 5 μmol/L, placed in a cell culture incubator for 10 minutes and suspended once every 5 minutes, after which it was washed thrice using 5–10 volumes of complete medium, centrifuged at 1300 rpm for 4 minutes at 4°C, and suspended in 1 ml of complete medium, incubated in cell culture incubator for 2 hours, then washed using 5–10 volumes of complete medium. The temperature was 1300 rpm, centrifuged for 5 minutes, suspended in 1 ml of complete medium, and placed in ice for later use.
Preparation of effector cells γδ T cells
The amplified γδ T of each group was resuspended, and 20 ml of cell suspension was counted using a cytometer.
Cytotoxicity experiment
Each group of γδ T cells was uniformly mixed with HepG2 and placed in a U-shaped 96-well plate, and the reaction volume was set to 200 μl. Three to four parallel duplicate wells were set; the effective target ratio (effector cells:target cells) were 0:1, 10:1, and 40:1. 10,000 labeled target cells were added to each well, and then effector cells were added and centrifuged at 120 g for two minutes and incubated for four hours in a cell culture incubator. After centrifugation at 1000 rpm for four minutes at 4°C, the supernatant was discarded, and each well was resuspended in 200 ml of staining buffer and centrifuged twice. 125 μl staining buffer was added to each well and resuspended. The cell suspension was aspirated into a flow tube and 2.5 mμl propidium iodide (PI) was added to each flow tube. The reaction was performed for five minutes on ice, followed by analysis using flow cytometry.
Magnetic beads sorting γδ T cells
The number of γδ T cells was determined. The cell suspension was centrifuged at 300 g for 10 minutes. The supernatant was discarded, and 107 total cells were resuspended in 100-l buffer, after which 10 μl of gdTPE was added, mixed and incubated in the dark for 10 minutes at -4°C. 1–2 ml of buffer was added per 107 cells and centrifuged at 300 g for 10 minutes to remove unbound primary antibody. The supernatant was discarded and the cells were resuspended in 8 μl of buffer per 107 cells. Also, 20-μl Anti-Phycoerythrin (PE) MicroBeads were added per 107 cells. The cells were mixed and incubated in the refrigerator for 15 minutes at 2–8°C. The cells were washed using 1–2 ml of buffer per 107 cells and centrifuged at 300 g for 10 minutes. The supernatant was discarded, and 108 cells were resuspended in 500 μl of buffer. The column was placed in the magnetic field of a suitable MACS separator. Rinsing was done using an appropriate amount of buffer and 3-ml LS column was prepared. The cell suspension was placed on the column. The unlabeled cells were obtained and the column was washed using the appropriate amount of buffer. When the column was empty, the buffer was added thrice. Three LS(3ml) columns are needed here. The column was removed from the separator and placed on a suitable collection tube. Onto the column, 5 ml of buffer was pipetted. The magnetically labeled cells were flushed immediately.
Dynamic observation of PBMC using Mtb-HAg and ZOL stimulated amplification and sorting to kill HepG2 cells
Marking of target cells
A polylysine 2-ml coating was spread overnight on a confocal dish. HepG2 was digested. HepG2 was resuspended in 5 ml of complete medium, spun at 1000 rpm, centrifuged for five minutes, and repeated. Also, 105 cells were plated in a confocal dish overnight in a cell culture incubator. The culture solution was aspirated and washed thrice in 2 ml of DMEM. 1 ml of DMEM was added, then 0.5 ul of CFSE was added, incubated in a cell culture incubator for 10 minutes, and the 2-ml complete culture solution was washed thrice. 1 ml of complete culture solution was added, washed in the cell incubator for two hours, and washed twice. Then 0.5 ml of serum 1640 culture solution was added.
Effector cell preparation
During the preparation, 2 × 106 effector cells were taken according to the effective target ratio (effector cells:target cells = 20:1). They were resuspended in a centrifuge tube, rotated at 1000 rpm, and centrifuged for five minutes. 5 ml of serum-free 1640 medium was used for resuspension, spun at 1000 rpm, centrifuged for five minutes, and repeated once more. After this, 0.2 ml of 1640 serum-free medium was added to light suspension. Also, 1 μl 4’,6-diamidino-2-phenylindole (DAPI) (1 mg/ml) was added and incubated for 1 hour in a cell culture incubator. 5-ml serum-free medium was washed over time, and 0.5 ml of 1640 medium containing fetal bovine serum was added and resuspended.
Co-cultivation
Effector cells were added to the co-focusing dish containing the target cells and incubated for 1 hour in a cell culture incubator.
Live Cell Workstation
The living cell workstation was observed, and 10 ml of propidium iodide (PI) was added. One picture was taken at a total of three positions and shoot for three hours in a row.
Flow cytometry to detect the killing mechanism of HepG2 by γδ T cells stimulated by HDMAPP, Mtb-HAg, and ZOL in PBMC
Marking of target cells
Same as the above-mentioned method for marking target cell HepG2.
Preparation of effector cells
The effector cells were collected 2 × 106, resuspended in a centrifuge tube, centrifuged for 5 minutes at 1000 rpm, and 0.5 ml of cell culture medium was added. The effector cells were divided into three groups: without the blocker group, NKG2D blocker group, and PD98059 blocker group.
Cytotoxicity experiment
After mixing γδ T with HepG2, it was placed in a U-shaped 96-well plate, and the reaction volume was set to 200 μl. The effective target ratio was 0:1, 10:1, 40:1. 8,000 labeled target cells were added to each well, and then effector cells were added and centrifuged at 120 g for two minutes. The remaining methods were the same as the cytotoxicity experiment mentioned above.
Establishment of tumor model in nude mice
In the first batch of tumor models, the effective target ratio was 10:1, that is, 107 effector cells and 106 target cells were taken. Three nude mice were injected with target cells under the right forelimb, and right hindlimb as the control group, and two nude mice were taken as the experimental group. One nude mouse was injected with target cells and γδ T cells (amplified by ZOL stimulation) under the right forelimb, and another nude mouse was injected with target cells and γδ T cells (amplified by ZOL stimulation) under the right forelimb and at the right hindlimb, at the same time.
In the second batch of tumor models, the effective target ratio was 40:1, that is, 107 effector cells and 2.5 × 105 target cells were taken. Four nude mice were taken. Only target cells were injected into the armpit of the right forelimb of each nude mouse. The amplified γδ T cells were stimulated by ZOL and Mtb-Hag at the left hindlimb and right hindlimb, respectively.
Preparation of target cells and effector cells
At this time, HepG2 was selected as in the laboratory to construct lentivirus using luciferase, near-infrared fluorescent protein, and puromycin resistance gene. HepG2 hepatoma cell line was infected, and the cell line stably expressing luciferase and near-infrared fluorescent protein gene was screened, which is called mHepG2 cell line. The two have the same tumor tissue structure, which is characterized by the accumulation and growth of tumor cells, irregular cell morphology, abundant cytoplasm, different nuclear size and morphology, uneven staining, and a more abnormal nuclear division.
Establishment of tumor model
The cells were resuspended and sucked into the syringe. The 150-μl cell suspension was subcutaneously inoculated at the fixed position of the nude mice under sterile conditions. On the second day, the state of the mice was observed. The tumor length was measured, short diameter every 3–4 days, volume = long diameter *(Short diameter) 2 *1/2. Then, tumor growth curve and scatter diagram were drawn. The tumor was weighed and photographed when its volume was close to 1000 mm3; intraperitoneal injection of luciferase substrate (30 mg/ml, 5 μl/g, plus 200 ml PBS and mixed well), photos using small animal imager were taken, and white light and bioluminescence were observed.
Cell therapy
When the tumor volume of the first tumor model was close to 400–500 m3, 200 ml of PBS was injected around the tumor tissue in the control group, and 200-μl of γδ T cells (amplified by ZOL stimulation) was injected around the tumor tissues in the experimental group, namely, 107 cells, four times, twice a week for two consecutive weeks.
The tumor model was photographed using a small animal imager
Each nude mouse was weighed, measured, fixed in a position, and photographed at an equal distance from the tumor site. Each nude mouse was observed after injection of luciferase substrate for 10–15 minutes, during which pentobarbital sodium (0.01 ml/g) was injected. Each nude mouse was placed on a tray and photos using a small animal imager were taken (white light: QQ-MWL-0.175 Sec-160.00mm-ExOEmO-2; bioluminescence (injection of luciferase substrate): LHG-LUM-5.000 Min-160.00mm-BinX4Y4).
The first batch of tumor models was killed, stripped, fixed in a position, and photographed at an equal distance by a small animal imager.
Statistical analysis
CellQuest Pro software was used to analyze statistics for streaming results. SPSS 22.0 statistical analysis software was used; the data were expressed using ± s (mean ± standard deviation); the statistical method was analyzed using t test and variance analysis, and the data were considered statistically significant when P < 0.05.
RESULTS
Heparin anticoagulated venous blood was isolated using density gradient centrifugation to obtain peripheral blood mononuclear cells
γδ T cells accounted for about 1–10% of T cells. The ratio of γδ T cells in TB cells in flow cytometry was (4.64 ± 0.37)%. Figure 1 indicates that γδ T cells accounted for 6.7% of T cells in the sample. The ratio of Vδ1 cells in γδ T cells was (20.65 ± 5.58)%, and the ratio of Vδ2 cells in γδ T cells was (77.26 ± 11.92)% in PBMCs. Figure 1 1 shows that Vδ1 cells accounted for 14.6% of γδ T cells in the sample. Vd2 cells accounted for approximately 76.8% of γδ T cells.
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Figure 1: Proportion of γδ T cells in T cells and the proportion of subpopulations in γδ T cells in isolated PBMC
Peripheral blood mononuclear cells stimulated and expanded in vitro
The culture protocol was divided into five groups: the control group, which was separately added with IL-2 and PHA, and the experimental group, supplemented with HDMAPP, ZOL, and Mtb-HAg. The first picture shows the morphological features of PBMC after separation. The rest is a picture of 100 times γδ T under 12–14 days. The experimental group’s large cell mass indicates that γδ T cells are effectively amplified in vitro, and the control group PHA was round. The activated cells were activated ab T cells, and the IL-2 group cells were scattered [Figure 2].
Figure 2: Microscopic features of PBMC and γδ T cells in each group after amplification (×100)
Proportion of γδ T cells in T cells after expansion and culture in vitro by flow cytometry
When the cells were cultured in vitro on the 12th day, the ratio of γδ T cells in each group was measured using flow cytometry [Figures 3 and 4], the IL-2 group was close to PBMC, and the PHA group was slightly higher. In the three experimental groups, γδ T cells indicated a large amount of amplification (**P < 0.01), and the ratio of γδ T amplified by HDMAPP and ZOL stimulation was significantly higher than that of the Mtb-HAg group (*P < 0.05).
Figure 3: Proportion of γδ T cells in T cells Compared with fresh isolated PBMC ((4.64 ± 0.37) % of total T cells): ** P < 0.01
Figure 4: Proportion of γδ T cells in T cells
Cytotoxicity test
When the cells of each group were cultured for 12 days, the tumor cell killing experiments were conducted according to the effective target ratio, and the killing ratio was determined using flow cytometry [Figures 5 and 6]. According to the experimental results, the efficiency of γδ T cells killing tumor cells in each experimental group also increased with increase in the effective target ratio (*P < 0.05). Among them, when the ratio of the effective target was 10:1, the killing rate of γδ T cells stimulated by ZOL in the experimental group was significantly higher than that in the HDMAPP and Mtb-HAg groups (*P < 0.05), and stimulated by the HDMAPP and Mtb-Hag groups. The killing rate of γδ T cells was significantly higher than that of the PHA group (*P < 0.05), but the γδ T cell killing rate of the HDMAPP and Mtb-HAg groups did not exhibit any difference. When the target ratio was 40:1, the killing rate of γδ T cells stimulated using ZOL and Mtb-HAg in the experimental group was significantly higher than that in the HDMAPP group (*P < 0.05). And the γδ T cell killing rate of all experimental groups also indicated a difference from the PHA group (*P < 0.05). At this time, the γδ T cell’s killing rate of the ZOL group and the Mtb-HAg group did not show a difference.
Figure 5: Killing rate of γδ T cells in different E:T ratios Note - E: γδ T cells; T: HepG2 cells. (a). Killing rate of each treatment group at different effective target ratios. (b). Comparison of the killing rate of the ZOL group with the target ratio of 10:1 and other treatment groups. (c). Comparison of the killing rate of the Mtb-HAg group and the ZOL group with a target ratio of 40:1 and other treatment groups * P < 0.05. △ P < 0.05. ▲ P < 0.05. & P < 0.05
Figure 6: Killing rate of γδ T cells at different E:T ratios
Live-cell workstation dynamic observation of killing effect
The γδ T cells of the Mtb-HAg group and the ZOL group with higher purity were obtained through magnetic bead sorting, and the target cells were co-actuated for three consecutive hours to obtain a dynamic killing video. When CFSE marks target cells, it can easily penetrate the cell membrane, covalently binding to intracellular proteins in living cells, and release green fluorescence after hydrolysis. The PI added to the apoptosis before photographing can pass through the damaged cell membrane to stain the nucleus and emit red fluorescence after inserting the double-stranded DNA. Through the video, it can be observed that under the action of effector cells, a series of atrophy, necrosis, and exocytosis of tumor cells appear from green to red, which visually confirms the killing effect of γδ T cells on target cells and makes them become visual [Figure 7]. A picture was taken every 20 minutes. The image was fusiform, the CESE mark was green, and the larger volume was the target cell HepG2. DAPI is labeled blue, and the smaller cells were effector γδ T cells. The PI labeled as red was a dead cell.
Figure 7: View of ZOL-stimulated activated γδ T cells killing tumor cells
Killing block experiment
To further investigate the mechanism by which γδ T kills HepG2, γδ T cells were treated using NKG2D blocker and PD98059 to observe block killing [Figures 8-11]. NKG2D blocker and PD98059 can reduce the killing effect to some extent under the same target ratio, and PD98059 plays a major role (*P < 0.05). When PD98059 was used, and the target ratio was 40:1, the killing effect was observed in each experimental group. Among them, in the HDMAPP group, when the target ratio was 40:1, the NKG2D blocker showed a significant blocking effect (*P < 0.05). In the ZOL group, when the effect ratio was 10:1, the effector cells were significantly blocked after treatment with PD98059 (*P < 0.05), to further illustrate the major blocking effect of PD98059. It can also be said that the intracellular ERK1/2 signaling pathway plays an essential role when effector cells kill target cells.
Figure 8: Changes in killing rate of activated γδ T cells in each experimental group after application of blocking agents at different effective target ratios. Note - (a). The kill rate at different target ratios after using two blockers. (b). Comparison of kill rates after application of two blockers at an Mtb-HAg and ZOL group target ratio of 40:1. * P < 0.05. # P < 0.05. ▲ P < 0.05. &P < 0.05
Figure 9: Killing rate of untreated groups of γδ T cells at different effective target ratios
Figure 10: Killing rate of γδ T cells treated with NKG2D blocker at different target ratios
Figure 11: Killing rate of γδ T cells treated with PD98059 at different target ratios
Establishment of tumor model
The first batch of nude mice were divided into the control and experimental group. In the control group, only target cells were injected into the armpit of the right hindlimb and the right forelimb of nude mice [Figure 12]. In the experimental group, effector and target cells were injected into the armpit of the right hindlimb and the right forelimb of nude mice at the same time. When the tumor volume reached 400–500 mm3 (day 30), it was found using cell therapy twice a week for two consecutive weeks. At first, there was little difference in tumor volume between the control and experimental groups. After treatment, there was a difference in the tumor growth rate between the control and the experimental groups [Figure 13]; the killing effect of γδ T cells is reflected in vivo. In the second batch of nude mice, only target cells were injected into the armpit of the right forelimb, and effector cells and target cells were injected into the left and right hindlimbs simultaneously; the effective target ratio was increased to 40:1. After regular measurement of volume and small animal imager, it was found that only the tumor under the armpit of the right forelimb grew [Figure 14], which further proved the killing effect of γδ T cells in vivo, thereby providing a scientific basis for the clinical treatment of tumors.
Figure 12: Nude mouse tumor models
Figure 13: Nude mouse tumor growth curve Compared with untreated group: * P < 0.05
Figure 14: Fluorescence imaging before and after treatment of nude mouse tumor model
DISCUSSION
In China, hepatocellular carcinoma is one of the many malignant tumors.[22] Although traditional methods such as surgical resection of the liver, radiotherapy, chemotherapy, and interventional therapy has specific effects, the problems of low cure rate and a high rate of postoperative recurrence have been incompletely solved. Recently, tumor cell immunotherapy has been widely recognized in the clinical community.[23]
The reason why γδ T cells were selected in this study is that γδ T cells can recognize antigens without the stimulation of specific antigens and are unrestricted by MHC when they exert the activity of killing tumor cells. Recently, many scientific studies and clinical applications have shown that γδ T cell immunotherapy has a good clinical application potential.[24]
After the in vitro amplification of γδ T cells, the effectiveness of the three antigens in stimulating the expansion of γδ T was also demonstrated by group comparison. Other studies have not reported a comparison of the proportion of γδ T cells amplified by these three stimulators. Simultaneously, a subpopulation of γδ T cells was determined using flow cytometry, and it was also discovered that Vγ9Vδ2T cells were more than γδ1 T cells in the cell ratio.
By studying the killing of tumor cells by γδ T cells, we found that there are few killings of liver cancer cells by γδ T cells amplified by different stimulators. In this experiment, the efficiency of killing HepG2 in each group of γδ T cells was compared by setting different target ratios, such as 0:1, 1:1, 10:1, 20:1, and 40:1. It can be seen that HDMAPP and the amplification efficiency of ZOL stimulating agent were higher, and the antagonizing power of γδ T cells amplified by Mtb-HAg and ZOL was higher.
To further study the path through which γδ T cells may be killed, NKG2D blocker and PD98059 were used in this experiment. It was discovered that the use of both blockers’ did not indicate the advantage of using only one blocker. Therefore, only the killing effect in blocking agents alone was repeated in the later stage. The killing path was established in the reverse direction through the blocking experiment, and the main blocking effect of PD98059 was also confirmed. Thus, the experiments using two blockers demonstrated that γδ T cells involved NKG2D activation and intracellular ERK1/2 signaling pathway in killing HepG2 cells, and compared the effects of NKG2D blocker and PD98059 on killing rate, making mechanism discussion more comprehensive.
Magnetic beads sorted the γδ T cells of the Mtb-HAg group and ZOL group, and the purity and proportion of γδ T cells were further improved. The killing effect was observed by living-cell workstation. At first, the effector and target cells were simultaneously placed in a confocal dish to observe the killing effect. The two cells were not highly recognizable. Later, they were adjusted to pre-lay the tumor cells. After overnight growth, they were placed in effector cells to observe the killing. At this time, the target cells were observed. It has a distinct fusiform shape, larger than the effector cells, and has good recognition. The use of live-cell workstations makes the entire killing process visual and persuasive, becoming a new method and means of demonstrating the killing effect.
Finally, the tumor formation experiment in nude mice was used to observe whether the γδT-cells stimulated and expanded in vitro play a killing role in vivo. For the first batch of nude mice tumor models, the effect target ratio of the experimental group was 10:1. It was found that the tumor volume of the experimental group did not exhibit any difference from the control group. When cell therapy was conducted, the growth rate of the tumor gradually showed statistical significance, which indicated that the tumor cell immune experiment should improve the effect target ratio and explains the significance, and value of cell therapy in tumor therapy. Therefore, in the second batch of nude mice tumor models, when the effective target ratio reached 40:1, it was intuitively proved by the monitoring and measurement of tumor volume and small animal imager that γδ T cells have a stronger killing effect on target cells.
CONCLUSION
The above experimental results show that the ratio of γδ T cells in the peripheral blood of healthy people can be effectively induced in vitro, and the activity of killing tumor cells is also enhanced after stimulation and activation, and has potential for cancer cell immunotherapy.
Based on decades of basic and clinical research results, the clinical use of DC-based cell vaccines has shown the lack of other cellular vaccines.[25] However, studies have suggested that activated γδ T cells may be a valuable and useful cellular vaccine for patients with cancer.[26] In future studies, the activity of γδ T cells in killing tumor cells will be further enhanced, making them play a more significant role in cellular immunotherapy.
Financial support and sponsorship
National Natural Science Foundation of China (81472656) Key research and development program of Anhui Province (201904a0702019).
Conflicts of interest
There are no conflicts of interest.
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