Abstract
Background/Aim: Malignant ascites contain many tumour-infiltrating lymphocytes. γδ T cells with antitumour activity have attracted attention as effector cells in cancer immunotherapy. Vδ2+ T cells were cultured from peripheral blood mononuclear cells (PBMCs) and ascites-infiltrating lymphocytes (AILs) to compare the differences in response to 2-methyl-3-butenyl-1-pyrophosphate (2M3B1-PP) and zoledronate (Zol) as antigens in vitro. Materials and Methods: To expand Vδ2+ T cells from PBMCs and AILs from 29 patients with cancer, these cells were cultured and subjected to analysis. Results: The proliferation rate of Vδ2+ T cells was higher in both PBMCs and AILs when cultured with Zol than with 2M3B1-PP. Although Vδ2+ T cells show a higher rate of expansion in AILs compared to PBMCs, the number of mixed tumour cells in ascites was decreased when cultured with Zol. Conclusion: Vδ2+ T cells in AILs are cytotoxic to tumour cells in ascites and may be considered in adoptive immunotherapy.
- γδ T cells
- ovarian cancer
- ascites
- tumour-infiltrating lymphocytes
- ascites-infiltrating lymphocytes
The usefulness of immunotherapy using tumour-specific tumour-infiltrating lymphocytes (TIL) has been reported (1), but it is difficult to obtain sufficient TIL for therapeutic purposes in solid cancers. Therefore, their use under these circumstances is not yet widespread. However, ascites contains abundant TIL, called ascites-infiltrating lymphocytes (AILs), that are easier to obtain than TILs. A previous report on patients with ovarian cancer and malignant ascites who received treatment with AILs and/or chemotherapy showed that the combination therapy extended time to progression compared to chemotherapy alone (2).
Ovarian cancer is often diagnosed at an advanced stage, and cases of decreased quality of life due to malignant ascites accumulation are common; therefore, ascites control is important. Although puncture drainage of ascites is effective in alleviating symptoms, the nutritional status declines due to a loss of protein. The ascites get accumulated within a few days, and frequent puncturing is necessary (3).
In addition, human γδ T cells expressing Vγ9Vδ2 (also known as Vγ2Vδ2) recognize nonpeptide antigens derived from pyrophosphomonoester compounds (4, 5). Vδ2+ T cells, which possess antitumour activity, are attracting attention as effector cells in cancer immunotherapy; various clinical examinations have been performed, and their usefulness has been reported (6). Vδ2+ T cells respond to the presence of small isoprenoid metabolites, such as self-isopentenyl pyrophosphonate (IPP) with butyrophilin 3A1(BFN3A1) conformation on normal and tumor cells (7). Nitrogen-containing bisphosphonates (N-BPs) inhibit farnesyl pyrophosphate synthetase in normal and tumor cells, thereby resulting in intracellular accumulation of IPP, and subsequently, Vδ2+ T cells respond.
The present study focuses on Vδ2+ T cells in (AILs) for possible clinical application.
Materials and Methods
Reagents. 2-Methyl-3-butenyl-1-pyrophosphate (2M3B1-PP), a pyrophosphomonoester that acts on Vδ2+ T cells with an effect 100 times stronger than IPP, was prepared at our institution as described previously (5, 8). Recombinant human interleukin-2 (Proleukin) and Zoledronate (Zol) were obtained from Novartis (Basel, Switzerland). ALyS505 NO medium (Iscove's MEM-based serum-free medium) was obtained from the Cell Science & Technology Institute (Sendai, Miyagi, Japan).
Antibodies. Anti-Vδ2 (FITC), anti-CD56 (PE), and anti-CD3 (Phycoerythrin-Cy5) were obtained from Beckman Coulter (Marseille, France). PE mouse IgG1κ (isotype control) was obtained from Becton Dickinson Biosciences (Franklin Lakes, NJ, USA).
Cell culture, stimulation, and Vδ2+ T cell expansion. All donors gave informed consent in accordance with the Declaration of Helsinki for the use of their blood and ascites for research purposes. Approval was obtained from the Review Board of the Tokyo Women's Medical University of Innsbruck (Approval number: 3126). Peripheral blood was obtained from heparinized blood samples, and patient ascites were obtained by peritoneal puncture or from intraoperative ascites. Peripheral blood was diluted with PBS and layered on Lymphoprep. PBMCs were isolated by centrifugation (1,500 rpm, 35 min) and washed three times.
The cellular component of ascites was isolated by centrifugation (2,000 rpm, 10 min). Cellular component was diluted with PBS and layered on Lymphoprep. AILs/cancer cell mixtures (CCs) were purified from ascites by centrifugation (1,500 rpm, 35 min).
In total, 23 patients with ovarian cancer; one patient with fallopian tube cancer; and six patients with primary peritoneal cancer who were at stages 1 c, 2 c, 3 c, and 4 b were included in the present study (Table I).
PBMCs and AILs/CCs were cultured in Alys505 medium and incubated at 37°C/5% CO2 atmosphere.
To expand Vδ2+ T cells, total PBMCs or AILs (1-1.3×106/ml) were stimulated with 2M3B1-PP (100 μM) or Zol (5 μM). The next day, 10% human-pooled type AB serum was added along with 200 U/ml of rhIL-2. rhIL-2 was added every 2-3 days, and the cells were cultured for a total of 14 days. These cultured cells were stained with anti-Vδ2 (FITC), anti-CD56 (PE), and anti-CD3 (Phycoerythrin-Cy5) for 20 min on ice, as described previously (9). These cells were subjected to flow cytometry (FACS Calibur, Becton Dickinson Biosciences).
Mouse IgGs were used as isotype controls.
Statistical analysis. Data were analysed using a Wilcoxon signed-rank test. A p-value <0.05 was considered significant. JMP version 13.0 software was used for statistical analysis.
Results
Twenty-two patients with ovarian cancer, one patient with fallopian tube cancer, and six patients with primary peritoneal cancer were examined in the present study (Table I). The median patient age was 65.0 years, and stage 3c cancers were the most common (17 cases).
First of all, PBMCs and AILs were cultured with either 2M3B1-PP or Zol to confirm whether Vδ2+ T cells in AILs were able to respond to these reagents and expand.
After the culture, the proportion of Vδ2+ T cells in CD3+ cells was calculated. No significant difference was observed, with the median for blood being 1.42% and that for ascites being 1.35% prior to culturing (Figure 1A). The proportion of Vδ2+ T cells after the culture with Zol increased significantly in peripheral blood (92.7%; Figure 1B), and there was an accompanying significant increase in the proliferation rate (204.9-fold; Figure 1C) compared with the culture with 2M3B1-PP (39.1% and 58.8-fold).
The proportions of Vδ2+ T cells in PBMCs and AILs were significantly higher in the presence of either 2M3B1-PP or Zol, whereas in AILs, the ratio tended to be higher with Zol than that with 2M3B1-PP, although this difference was not significant (Wilcoxon signed-rank test, p=0.0645; Figure 1B).
In addition, the growth rate of Vδ2+ T cells cultured with 2M3B1-PP was significantly greater in PBMCs (58.8 times) than in AILs (1.9 times).
In the ascites, the median growth rate of Vδ2+ T cells cultured in Zol was 3.8 times and that of those cultured in 2M3B1-PP was 1.9 times. Zol seemed to be a more potent stimulator for Vδ2+ T cells in AILs than 2M3B1-PP, but no significant difference was observed between these reagents. During review of individual cases, 4 of 16 cases (25%) in which AILs were cultured with 2M3B1-PP and 6 out of 14 cases (43%) in which AILs were cultured with Zol showed more than 50 times increased numbers of Vδ2+ T cells.
Next, the impact of the two reagents to CCs mixed in ascites during the culture was observed. Representative data are shown in Figure 2. The proportion of Vδ2+ T cells in freshly isolated AILs was 0.9% and following culture increased up to 10.8% when stimulated with 2M3B1-PP and up to 61.0% when stimulated with Zol (Figure 2A). The number of Vδ2+ T cells increased 52-fold when stimulated with 2M3B1-PP and 426- fold when stimulated with Zol. (Figure 2B).
Surprisingly, because Vδ2+ T cells in AILs were growing in culture (Figure 2B), the number of mixed CCs decreased and eventually disappeared (Figure 2C). This phenomenon was confirmed since the results showed that mixed CCs disappeared in two of 16 cases (1.5%) whose AILs were stimulated with 2M3B1-PP and in three of 14 cases (21.4%) whose AILs were stimulated with Zol.
Discussion
It has been reported that the ratio of Vδ2+ T cells in peripheral blood is 1%-10%. No clear correlation was found between the Vδ2+ T cells ratio in peripheral blood and those in ascites in the present study.
Some studies have investigated the relationship between the number and type of TILs in ovarian cancer (10); however, the proportion of Vδ2+ T cells in TILs has not been studied. Here it is shown that the proportion of Vδ2+ T cells in AILs is 1.35%. This ratio increased significantly in peripheral blood after culture in Zol compared with culture in 2M3BI1-PP, and the proliferation rate also increased. The same tendency was observed in ascites. The mechanism of activation of Vδ2+ T cells by Zol was slightly more complex; Zol inhibits farnesyl pyrophopate synthase in mevalonic acid metabolism in normal monocyte lineage cells and tumor cells, which results in intracellular accumulation of IPP. IPP binds to the B30.2 intracellular domains of BTN3A and activates Vδ2+ T cells.
Our findings and previous studies suggest that Zol acts on co-cultured tumour cells and antigen presenting cells Vδ2+ T cells in ascites, and it is thought to further increase γδ T cell proliferative capacity (11, 12). In turn, activated Vδ2+ T cells showed potent cytotoxic activity to tumour cells. In ascites, no significant increase in the median proportion or proliferation rate was observed when Zol was used compared with 2M3B1-PP. It may depend on the patients; a review of individual cases demonstrated that 43% (six of 14 cases) of patients showed increased number of Vδ2+ T cells, which was greater than 50 times the number when Zol was used. Recent studies report that Zol and IL-18 promote vigorous proliferation of Vδ2+ T cells (13, 14), and IL-18 will be useful for the expansion of Vδ2+ T cells in AILs. However, it is unclear which patients showed vigorous increase in Vδ2+ T cells in AILs and, therefore, further examination is needed. Regarding clinical application, peripheral blood Vδ2+ T cells have a higher median proliferation, but the amount of peripheral blood Vδ2+ T cells that can be taken from one apheresis is less and invasive; however, there is a greater amount of Vδ2+ T cells in ascites that can be recovered by a single ascitic puncture. There is a possibility that more Vδ2+ T cells may be obtained by preselecting and culturing cases that grow well in Vδ2+ T cells in ascites than in peripheral blood.
In addition, when using AILs for immunotherapy, it is difficult to remove all CCs from ascites before the culture. In the present study, even if the CCs were mixed before the culture, during the culture of Vδ2+ T cells stimulated by Zol, the CCs acted as antigen presenting cells to Vδ2+ T cells and were in turn injured as target cells by Vδ2+ T cells, and finally, they disappeared from the culture. In bacterial peritonitis, there is a possibility of bacterial contamination of ascites. When using AIL for the treatment of cancer, the prevention of infection of recipients can be achieved by sterility and endotoxin testing of the material at the time of shipment. Such treatments hold promises for treating other cancer types such as ovarian cancer, gastric cancer, and colon cancer, that are likely to accumulate malignant ascites (2, 15).
In conclusion, Vδ2+ T cells in ascites can be sufficiently expanded, depending on the patient, opening up the possibility of culturing and usage as a source of cells for adoptive immunotherapy.
Acknowledgements
The Authors thank Ms. Shoko Inada for her expert technical assistance.
The Authors would like to thank Enago (www.enago.jp) for the English language review.
Footnotes
Conflicts of Interest
The Authors have no financial conflicts of interest regarding this study.
- Received April 11, 2018.
- Revision received May 21, 2018.
- Accepted May 23, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved