Research ArticleExperimental Studies

Characteristics of Human Peripheral Blood γδ T Cells Expanded With Zoledronate

MINSONG KIM, HYORI KIM, MINA HAN, HYUN JU HWANG, HYERY KIM, HO JOON IM, NAYOUNG KIM and KYUNG-NAM KOH
Anticancer Research December 2021, 41 (12) 6031-6038; DOI: https://doi.org/10.21873/anticanres.15422

Abstract

Background/Aim: This study aimed to investigate the characteristics of human peripheral blood γδ T cells, which were expanded ex vivo in the presence of zoledronate (ZOL). Materials and Methods: Human peripheral blood cells were cultured with IL-2 and IL-15 in the presence or absence of ZOL, which was added as a phospho-antigen, and their phenotypes were assessed by flow cytometry. Expanded γδ T cells were transduced with CD19 CAR vector, and the cytotoxicity was evaluated in vitro and in vivo by flow cytometry. Results: Ex vivo expansion did not hamper the expression of activating receptors. Interestingly, ZOL promoted the expression of CD226 (DNAM-1), TRAIL, and FAS-L in the Vδ1 subset of γδ T cells. Expanded γδ T cells containing CD19 CAR+ γδ T cells removed B cell lymphoma cells effectively in vivo. Conclusion: γδ T cells could be a promising immunotherapeutic for cancer.

Key Words:

γδ T cells are a subset of T cells, which account for 1-5% of peripheral blood mononuclear cells (PBMCs), and function as a first line of defense to eradicate tumors and infections. Human γδ T cells are divided into subsets based on their chain usage, Vδ1 and Vδ2 (1). The majority (50-75%) of γδ T cells in the human blood express the Vγ9Vδ2 T cell receptor (TCR) (2-4), whereas Vδ1 γδ T cells are mainly found in tissues, such as the skin and intestine. Vδ2 γδ T cells respond to phospho-antigens accumulated in cancer cells. Zoledronate is a commonly used source of phosphor-antigen for Vδ2 γδ T cells, which expand well in the presence of zoledronate and interleukin (IL)-2 (5). Zoledronate is administered in the clinic for the treatment of osteoporosis. Vδ1 γδ T cells do not respond to phospho-antigens but recognize major histocompatibility complex (MHC) class I chain-related molecules A (MICA) and B (MICB) through the receptor natural killer group 2 member D (NKG2D) (6). Natural killer (NK) cells and γδ T cells have some activating receptors in common, such as NKG2D, CD16, and CD226 (DNAM-1). Commonly known ligands of CD226 include CD112 (nectin-2) and CD155 (poliovirus receptor; PVR). Unlike αβ T cells, which regulate proliferation through the immune checkpoint receptor cytotoxic T lymphocyte antigen 4 (CTLA-4), B and T lymphocyte attenuator (BTLA) plays an essential role in regulating the proliferation of γδ T cells (7, 8). γδ T cells express other activating and inhibitory receptors, including NKp30, NKp44, and PD-1 (9). Importantly, allogeneic γδ T cells are not alloreactive, and do not cause graft versus host diseases (GVHD) as they are not MHC-restricted (10). GVHD are caused by donor immune cells that attack host tissues after allogeneic hematopoietic cell transplantation.

Since chimeric antigen receptor (CAR)-T cell therapy has been shown to be effective for patients with B cell malignancies (11, 12), therapeutic regimens have been revolutionized to benefit patients with B cell leukemia and lymphoma. CAR is composed of an extracellular domain, including a single-chain variable fragment (scFv) of antibody (Ab), a transmembrane domain, and a signaling domain, such as CD3, 4-1-BB (CD137, TNFRSF9), or CD28 (13-15). The most commonly used CAR structures are the so-called second-generation CARs, containing two signaling domains: CD3 and 4-1-BB or CD28. The structure of CAR contains the variable regions of immunoglobulins, which is not MHC-restricted. Nonetheless, only autologous T cells are currently used clinically for CAR-T cell therapy, due to the potential risk of GVHD by endogenous TCR. Thus, γδ T cells are attractive candidates for adoptive T cell therapy, which would not cause GVHD.

In this study, we characterized the expression of activating and inhibitory receptors on ex vivo expanded human peripheral blood Vδ1 and Vδ2 γδ T cells to evaluate the effect of zoledronate (ZOL) for the first time and showed the potential action of CD19 CAR-γδ T cells against B cell lymphoma cell lines in vitro and in vivo.

Materials and Methods

Expansion of human γδ T cells. PBMC or αβ T cell-depleted PBMCs from healthy adult volunteers were isolated by density gradient centrifugation using Ficoll-Paque™ Plus (GE Healthcare, Milwaukee, WI, USA). αβ T cell-depleted PBMCs were isolated using CliniMACS (Miltenyi Biotec, Bergisch Gladbach, Germany), as previously described (16). All participants provided written informed consent, and all procedures were approved by the Institutional Review Board (IRB), Asan Medical Center, Seoul, Republic of Korea (IRB Approval No. 2018-0445). The study was performed ethically, in accordance with the Declaration of Helsinki. PBMCs were cryopreserved in heat-inactivated fetal bovine serum (FBS) (Welgene, Gyeongsangbuk-do, Republic of Korea) containing 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) and stored in a liquid nitrogen tank until use.

PBMCs were seeded into a 6-well plate at a density of 3×106 cells/ml in RPMI-1640 (Welgene) supplemented with 10% inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 mM sodium pyruvate (Sigma-Aldrich), and 0.55 mM 2-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA, USA), which we refer to as complete RPMI. γδ T cells were cultured in complete RPMI complemented with 100 U/ml recombinant human interleukin (IL)-2 (rhIL-2) (PeproTech, Rocky Hill, NH, USA) and 50 ng/ml recombinant human IL-15 (rhIL-15) (PeproTech) in the presence or absence of 1 μM ZOL (Selleckchem, Houston, TX, USA) for 2 weeks. Every two days, half the medium was replaced and refreshed with the same concentrations of rhIL-2 and rhIL-15.

Cell culture. Two human diffuse large B-cell lymphoma (DCBLC) cell lines, OCI-Ly7 (OCI) and SU-DHL6 (SU), and a human embryonic kidney cell line, HEK293T, were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). OCI-Ly7, SU-DHL6, and HEK293T (293T) cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning, Manassas, VA, USA) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (Corning). All cells were cultured in a fully humidified incubator with 5% CO2 at 37°.

Flow cytometry. Flow cytometry was performed using the following antibodies conjugated with various fluorochromes: anti-human CD314 (NKG2D) (Clone 1D11; BD Biosciences, Franklin Lakes, NJ, USA), CD226 (DNAM-1) (11A8; BioLegend, San Diego, CA, USA), CD16 (4G7; BD Biosciences), CD253 (TRAIL) (RIK-2; BioLegend), CD178 (FAS-L) (NOK-1; BioLegend), CD279 (PD-1) (EH12.2H7; BioLegend), and CD272 (BTLA) (MIH26; BioLegend). For detection of the CD19 CAR receptor, transduced cells were stained with AlexaFluor-647-conjugated Myc-Tag mouse monoclonal antibody (mAb) (clone 9B11, Cell Signaling Technology, Beverly, MA, USA) or a matched AlexaFluor-647-conjugated mouse IgG2a, κ isotype control (Clone MOPC-173, BioLegend). Before antibody treatment, cells were treated with human FcR blocking reagent (Miltenyi Biotec) on ice for 5 min. Cells were stained with the above antibodies at 4° for 30 min, and then washed with PBS. Flow cytometry was performed using a CytoFLEX (Beckman Coulter Life Sciences, Brea, CA, USA). The data were analyzed with FlowJo v10 software (Treestar, Inc., Ashland, OR, USA).

Production of CAR-γδ T cells. A CD19 CAR-expressing vector (FMC63-CD8 hinge-BBz-pCL20C-MND) was provided by Dr. Byoung Y. Ryu (St. Jude Hospital, Memphis, TN, USA). The pCAG vector backbone was derived from pCAGGS made by Dr. Jun-ichi Miyazaki (17). The CD19 CAR construct contained the FMC63 scFv, the hinge and transmembrane domain of CD8, the cytoplasmic domain of 4-1BB, and the CD3ζ signaling domain. The CD19 CAR vector was tagged with a MYC gene. Plasmid DNA was transformed by heat shock into competent E.coli Stbl3 prepared using a 0.1 M solution of CaCl2, following standard protocols. The CAR vector was isolated using a Plasmid DNA Midi prep kit (Qiagen, Venlo, Netherlands).

To produce lentivirus particles, CAR-expressing vector was transfected into 293T cells with packaging plasmid vectors, pCAG-KGP1-1R, pCAG4-RTR2, and pCAG-VSVG, at a ratio of 6:3:1:1 using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). Virus-containing medium was harvested 48 and 72 h after transfection and filtered through a 0.45 μM filter (Millipore, Burlington, MA, USA). Viral supernatant was used to transfect expanded γδ T cells pre-treated with 8 μg/ml polybrene (Millipore).

Cytotoxicity assay. Cytotoxicity was analyzed using a 7-amino-actinomycin D (7-AAD) and Annexin V (AV) assay as described previously with minor modifications (18). Target cells, OCI or SU, were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) using a Cell Trace Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. On day 2 post-transduction, transduced γδ T cells as effector cells were harvested and resuspended in complete RPMI media. γδ T cells were co-cultured with CFSE-labeled target cells at a ratio of 2:1 (effector:target). After overnight incubation, cells were washed with Annexin V binding buffer (BD Biosciences) and incubated at 37°C for 30 min. Cells were treated with human FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 5 min and stained with 7-AAD Viability Staining Solution (BioLegend) and AV-Pacific Blue antibody (BioLegend) for 15 min on ice. The CFSE-positive target cells were evaluated for early and late apoptosis by flow cytometry using a CytoFLEX (Beckman Coulter Life Sciences). Data analysis was performed using FlowJo v10 (Treestar, Inc., Ashland, OR, USA).

In vivo experiment. For in vivo evaluation, SU cells were labeled with CFSE. On day 0, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (JABio, Suwon, Republic of Korea) were injected intraperitoneally with 5×106 SU cells. After 30 min, mice were injected intraperitoneally with 5×106 γδ T cells transduced with CD19-CAR. On day 2, 10 ml 1×PBS (Biosesang, Gyeonggi-do, Republic of Korea) was injected into the peritoneal cavity. Peritoneal lavage was then harvested from mice, and the number of CFSE+ cells was measured by flow cytometry (19). The data were analyzed by FlowJo software. All mouse experimental procedures were approved by the Institutional Animal Care and Use Committee at Asan Medical Center, Seoul, Korea (Approval No. 2020-12-251).

Statistical analysis. GraphPad Prism v6 (GraphPad Software Inc., San Diego, CA, USA) was used to generate all the graphs and perform statistical analyses. p values were determined by two-way analysis of variance or the paired two-tailed t-test. p<0.05 was considered statistically significant and noted as follows: ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. All data are depicted as mean±standard error of the mean (SEM).

Results

Characterization of expanded γδ T cells. We compared the expression of the activating receptors NKG2D, CD16, and CD226, the death ligands TRAIL and FAS-L, and the inhibitory receptors PD-1 and BTLA, before and after expansion (Figure 1). We particularly aimed to assess the effect of ZOL compared with that following cytokine stimulation alone (a combination of IL-2 and IL-15). In Vδ1 γδ T cells, only CD226+ cells were significantly increased after 14 days’ expansion with IL-2 and IL-15 (IL-2+IL-15), but CD226+, TRAIL+, and FAS-L+ cells were all increased by the addition of ZOL (IL-2+ZOL+IL-15). The percentage of NKG2D+ cells was sustained, whereas the mean fluorescence indices (MFI) of NKG2D+ cells were much higher in expanded Vδ1 and Vδ2 γδ T cells. In Vδ2 γδ T cells, the percentage of FAS-L+ cells was higher after culture with ZOL. The expression of PD-1 and BTLA was not significantly changed by expansion.

Figure 1.
Figure 1.

The phenotypes of γδ T cells expanded in the presence or absence of zoledronate (ZOL). The expression of selected receptors, namely NKG2D, CD16, CD116, TRAIL, FAS-L, PD-1, and BTLA, was assessed by flow cytometry before and after culture. (A) The gating strategies. (B) Representative histograms of the cell surface receptors on γδ T cells before culture. (C) Representative histograms of the cell surface receptors on γδ T cells after culture. (D) The percentages of the receptor-expressing Vδ1 γδ T cells out of total Vδ1 γδ T cells before (Day 0) and after culture (Day 14). (E) The mean fluorescence indices (MFI) of the receptors on Vδ1 γδ T cells. (F) The percentages of the receptor-expressing Vδ2 γδ T cells out of total Vδ2 γδ T cells. (G) The MFI of the receptors on Vδ2 γδ T cells. All values are presented as mean±SEM. n=4-5. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

To understand the differences between Vδ1 and Vδ2 γδ T cells and the possible mechanisms of cytotoxicity, the receptor expression shown in Figure 1 was also evaluated as shown in Figure 2. Before culture, CD226 was more expressed in Vδ2 γδ T cells than Vδ1 γδ T cells. However, the expression of CD226 was comparable in both subsets after culture. After culture, the expression of NKG2D was lower in Vδ2 γδ T cells compared with that in Vδ1 γδ T cells. The expression of NKG2D appeared to be reduced in Vδ2 γδ T cells after culture with ZOL compared to untreated cells, although the difference was not statistically significant. The percentage of CD16+ cells increased in expanded Vδ2 γδ T cells compared with that of Vδ1 γδ T cells. The percentage of TRAIL+ cells was less in expanded Vδ2 γδ T cells than in expanded Vδ1 γδ T cells. The expression of PD-1 was reduced in expanded Vδ2 γδ T cells in the absence of ZOL compared with that in expanded Vδ1 γδ T cells.

Figure 2.
Figure 2.

Comparison of the phenotypes of Vδ1 and Vδ2 γδ T cells. The expression of selected receptors, NKG2D, CD16, CD116, TRAIL, FAS-L, PD-1, and BTLA, was assessed by flow cytometry before and after culture. The gating strategies and representative flow cytometric analysis are as described in Figure 1. (A) The percentages of the receptor-expressing Vδ1 and Vδ2 γδ T cells before culture. (B) The MFI of the receptors on Vδ1 and Vδ2 γδ T cells before culture. (C) The percentages of activating receptor (NKG2D, CD16, and CD226)-expressing Vδ1 and Vδ2 γδ T cells after culture. (D) The MFI of activating receptors (NKG2D, CD16, and CD226) on Vδ1 and Vδ2 γδ T cells after culture. (E) The percentages of death ligand (TRAIL and FAS-L)-expressing Vδ1 and Vδ2 γδ T cells after culture. (F) The MFI of death ligands (TRAIL and FAS-L) on Vδ1 and Vδ2 γδ T cells after culture. (G) The percentages of immune checkpoint receptor (PD-1 and BTLA)-expressing Vδ1 and Vδ2 γδ T cells after culture. (H) The MFI of immune checkpoint receptors (PD-1 and BTLA) on Vδ1 and Vδ2 γδ T cells after culture. All values are presented as mean±SEM. n=4-5. *p<0.05, **p<0.01, ***p<0.001.

Anti-tumor effect of CD19 CAR-γδ T cells in vitro and in vivo. We evaluated the anti-tumor effect of CD19 CAR-γδ T cells against human B cell lymphoma cell lines in vitro. As seen in Supplementary Figure 1, the proliferation rate was very low in the absence of ZOL. Thus, the experiment was performed with γδ T cells expanded with IL-2, IL-15, and ZOL. On culture day 10 and 11, expanded γδ T cells were transduced with lentiviral particles expressing CD19 CAR and on day 13 were co-incubated with CFSE-labeled OCI and SU target cells overnight (Figure 3A and B). CFSE-labeled target cells was reduced by approximately 30-37% following co-incubation for 4 h (Figure 3C). The death of target cells was also measured using AV and 7-AAD assays (Figure 3D). Early apoptotic target cells, defined as AV+ 7-AAD−, were significantly increased in the presence of CD19 CAR-γδ T cells. Late apoptotic target cells were also slightly increased.

Figure 3.
Figure 3.
Figure 3.

Evaluation of CAR-γδ T cells in vitro and in vivo. (A) In vitro Experimental scheme. (B) The gating strategies to identify CFSE-labelled B cell lymphoma cells (upper panels) and dying cells with the AV/7-AAD cell death assay (lower panels). (C) The ratio of CFSE+ cells. OCI-Ly7 (left) and SU-DHL6 (SU) (right) were used as target cells. A control group was transduced with empty lentiviral particles (n=5). (D) The ratio of AV+ 7-AAD− early apoptotic cells (left) and AV+ 7-AAD+ late apoptotic cells (right). OCI-Ly7 cells were used as target cells. A control group was transduced with empty lentiviral particles n=7. All values are presented as mean±SEM. *p<0.05. (E) In vivo Experimental scheme. (F) The gating strategies. (G) The percentages of CFSE+ SU target cells in the peritoneal lavage 48 h after the injection of target cells and γδ T cells n=3. All values are presented as mean±SEM. i.p.: Intraperitoneal injection.

The efficacy of CD19 CAR-γδ T cells was next assessed in vivo in peritoneal lavage. CFSE-labeled SU target cells were injected intraperitoneally, and CAR19 CAR-γδ T cells were injected intraperitoneally 30 min later. CD19 CAR-γδ T cells were prepared as described above and injected two days after the second transduction. Peritoneal lavage was collected 48 h later (Figure 3E). The ratio of CFSE-labeled target cells was dramatically reduced in 48 h, although only a very small portion of γδ T cells actually expressed CD19 CAR (Supplementary Figure 2). As seen in Figure 3F-G, approximately 90% of tumor cells were eliminated by adopted γδ T cells. The results suggest that CAR-γδ T cells could very effectively remove B cell lymphoma.

Discussion

This study shows the characteristics of γδ T cells expanded by ZOL and the anti-tumor potential of CAR-γδ T cells. First, the phenotypes of expanded Vδ1 and Vδ2 γδ T cells and the effect of ZOL on these cells were characterized. Prominently, the expression levels (by MFI) of NKG2D were increased more than two-fold after culture regardless of the presence of ZOL, implying effective cytotoxicity of ex vivo expanded γδ T cells against tumor cells expressing MICA/B or ULBPs. Further, the percentage of CD226+ Vδ1 γδ T cells was significantly upregulated in culture. In multiple myeloma, increased expression of NKG2D and CD226 enhances γδ T cell-mediated lysis (20). In particular, Vδ1 γδ T cells exert cytotoxicity against multiple primary myeloma cells though NKG2D and CD226 (21). In this study, ZOL unexpectedly stimulated Vδ1 γδ T cells to upregulate the expression of some activating receptors and death ligands, such as CD226, TRAIL, and FAS-L. To be presented to Vδ2 γδ T cells, ZOL is metabolized to isopentenyl pyrophosphate (IPP) in antigen-presenting cells and tumor cells by the mevalonate pathway, which is an essential metabolic pathway in all eukaryotes (5). Although Vδ1 γδ T cells do not recognize IPP as an antigen, Vδ1 γδ T cells might be affected by the mevalonate pathway in the presence of ZOL. There is a report showing that IPP-analogues can upregulate Ras-related protein expression in a human leukemia cell line (22). Nonetheless, it is yet to be elucidated how phospho-antigens affect Vδ1 γδ T cells. Among the receptors assessed, only FAS-L+ Vδ2 γδ T cells were increased by ZOL. Importantly, none of the activating receptors and death ligands was downregulated in culture, implying the cytotoxicity of expanded γδ T cells would not be hampered. It should also be noted that both PD-1 and BTLA were not upregulated in culture, implying that the potency of expanded γδ T cells could be maintained without increased exhaustion.

Expansion rates of γδ T cells in response to phospho-antigen does not correlate with their phenotypes, such as naïve, central memory, effector memory, terminally differentiated T cells (23). The use of HVEM/BTLA blockade during expansion could reduce terminally differentiated T cell population (7), potentially improving the efficacy of immune cell therapy.

CAR-γδ T cells have only been recently suggested as a novel cancer therapeutic (24, 25). Capsomidis et al. evaluated GD2-targeting CAR-γδ T cells against neuroblastoma cells in vitro, and Rozenbaum, et al. produced and tested CD19 CAR-γδ T cells in vitro and in vivo. The latter group transduced CD19 CAR retroviral particles to total T cells on day 5 of culture and found that up to 60% of cells were CAR+ on day 14. Depletion of αβ T cells was performed on day 9 (24). In this study, depletion of αβ T cells was performed on day 0, and CD19 lentiviral particles were transduced on day 10 and 11. In our experience, γδ T cells were more 80% in the presence of ZOL on day 14 even without depletion (Data not shown). The proportion of CAR+ γδ T cells was observed to be very low. It remains uncertain whether lentiviral or retroviral vectors would be more suitable to produce CAR-γδ T cells. Beginning transduction earlier might improve the production of CAR-γδ T cells by letting them expand multiple times. Nevertheless, the elimination of CD19+ B cell lymphoma cells by CD19 CAR-γδ T cells was rather clear in the peritoneal lavage in vivo. With improved protocols to produce CAR-γδ T cells, they could be more effective against B cell lymphoma without the potential risk of GVHD. In conclusion, ex vivo expanded γδ T cells could be a promising candidate for cancer immunotherapy.

Acknowledgements

The Authors thank Dr. Byoung Y. Ryu (St. Jude Hospital, Memphis, Tennessee, USA) for kindly sharing materials. This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2018R1C1B6008852).

Footnotes

  • Authors’ Contributions

    N. Kim and K.-N. Koh designed and supervised the research. M. Kim, M.Han, and H. J. Hwang conducted the research. Hyori Kim, Hyeri Kim, H. J. Im provided research materials and clinical samples. M. Kim, N. Kim, and K.-N. Koh wrote the manuscript. K.-N. Koh obtained funding.

  • Conflicts of Interest

    The Authors report no conflicts of interest in relation to this study.

  • Received September 8, 2021.
  • Revision received September 22, 2021.
  • Accepted October 23, 2021.

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Characteristics of Human Peripheral Blood γδ T Cells Expanded With Zoledronate
MINSONG KIM, HYORI KIM, MINA HAN, HYUN JU HWANG, HYERY KIM, HO JOON IM, NAYOUNG KIM, KYUNG-NAM KOH
Anticancer Research Dec 2021, 41 (12) 6031-6038; DOI: 10.21873/anticanres.15422
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