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. 2024 Jan 14;14(1):1291.
doi: 10.1038/s41598-024-51794-1.

Human Vγ9Vδ2 T cell expansion and their cytotoxic responses against cholangiocarcinoma

Piamsiri Sawaisorn  1 , Ahmed Gaballa  2 , Kween Saimuang  3 , Chaniya Leepiyasakulchai  4 , Sakaorat Lertjuthaporn  3 , Suradej Hongeng  5 , Michael Uhlin  6   7   8 , Kulachart Jangpatarapongsa  9
Affiliations

Human Vγ9Vδ2 T cell expansion and their cytotoxic responses against cholangiocarcinoma

Piamsiri Sawaisorn et al. Sci Rep. .

Abstract

Human Vγ9Vδ2 T lymphocytes are regarded as promising effector cells for cancer immunotherapy since they have the ability to eliminate several tumor cells through non-peptide antigen recognition. However, the cytotoxic function and the mechanism of Vγ9Vδ2 T cells leading to specific killing of cholangiocarcinoma cells are yet to be confirmed. In this study, we established a protocol for ex vivo expansion of Vγ9Vδ2 T cells from healthy donors' peripheral blood mononuclear cells by culture with zoledronate and addition of IL-2, and IL-15 or IL-18 or neither. Testing the cytotoxic capacity of cultured Vγ9Vδ2 T cells against cholangiocarcinoma cell lines showed higher reactivity than against control cells. Surface expression of CD107 was detected on the Vγ9Vδ2 T cells, suggesting that these cells limit in vitro growth of cholangiocarcinoma cells via degranulation of the perforin and granzyme pathway. Analysis of molecular signaling was used to demonstrate expression of pro- and anti-survival genes and a panel of cytokine genes in Vγ9Vδ2 T cells. We found that in the presence of either IL-15 or IL-18, levels of caspase 3 were significantly reduced. Also, IL-15 and IL-18 stimulated cells contained cytotoxicity against cholangiocarcinoma cells, suggesting that stimulated Vγ9Vδ2 T cells may provide a feasible therapy for cholangiocarcinoma.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Immunophenotyping and viability of ex-vivo expanded Vγ9Vδ2 T cells. PBMCs from 6 healthy donors were cultured for 10 days with zoledronate and IL-2, and IL-15 or IL-18 or neither. The cells after culture were stained and analyzed for T cell markers by flow cytometry. (A) Data shown are the percentage of cells expressing T cell receptor markers including γδ, Vδ2 and Vδ1, co-inhibitory markers CTLA-4 and CD158 with viability determined by 7AAD staining and cells number. The lines represent the mean values. (B) Representative flow cytometry panels with the percentage of the Vγ9Vδ2 T cells after cultivation with zoledronate, IL-2, and IL-15.
Figure 2
Figure 2
Real-time PCR analysis of cytokine gene expression in expanded Vγ9Vδ2 T cells. Vγ9Vδ2 T cells from 6 healthy donors were propagated as described and analyzed for mRNA expression levels coding for IL-1β, IL-2, IL-6, IL-7, IL-8, IL-12β, IL-15 and IL-17. Data displayed are the results for all tested cases. The lines represent the mean values.
Figure 3
Figure 3
Real-time PCR analysis of the pro- and anti-apoptotic gene expression in expanded Vγ9Vδ2 T cells. Vγ9Vδ2 T cells from 6 healthy donors were propagated as described and analyzed for mRNA expression levels coding for caspase 3, caspase 8, caspase 9 and bcl-2. Data displayed are the results for all tested cases. The lines represent the mean values.
Figure 4
Figure 4
Cytotoxicity of Vγ9Vδ2 T cells against cholangiocarcinoma cells. Cytotoxic properties of cultivated Vγ9Vδ2 T cells against HuCCT1 and TFK-1 cholangiocarcinoma cell lines are displayed. (A) Percentages of apoptotic cells at 10:1 E:T ratio are shown, comparing addition or non-addition of zoledronate for 24 h. Data shown are the mean values of cytotoxicity ± SD from the 3 independent experiments performed with different Vγ9Vδ2 T cell lines. (B) Representative flow cytometry panels with gating for Cell trace-labeled tumor cells and histogram plots of Annexin V+ gated cells from a control (cholangiocarcinoma cell lines without Vγ9Vδ2 T cells) and co-cultures of cholangiocarcinoma cell lines and Vγ9Vδ2 T cells.
Figure 5
Figure 5
Percentage of Vγ9Vδ2 T cells expressing surface CD107a. (A) Percentage of CD107+ Vγ9Vδ2+ T cells at a given E:T ratio are shown as compared with control. Data shown are the mean values the mean ± SD from 3 independent experiments performed with different Vγ9Vδ2 T cell lines. (B) Representative flow cytometry panels with gating for CD107+ Vγ9Vδ2+ T cells, between inclusion and non-inclusion of a cholangiocarcinoma cell line after 4 h.
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