Ex Vivo-Expanded Vγ9Vδ2 T Cells Can Efficiently Kill Human Acute Myeloid Leukemia Cells Via Trogocytosis
Authors: Wu Kangni , Hu Yongxian , Fu Huarui , Sheng Lixia , Huang He 
Published: October 2019

Abstract
Abstract 580
Cellular immunotherapy of hematopoietic malignancies is regarded as one of the most promising approaches to deal with the common relapse or resistance to conventional treatments. γδT cells are innate-like lymphocytes capable of potent antitumor activity toward a variety of malignant cell types and have been applied to clinical trials with positive effects. But it still lacks basis whether these cells coule be used in the treatment of acute myeloid leukemia(AML).Here we set out to determine the potent cytotoxicity of ex vivo-expanded Vγ9Vδ2 T cells against AML cells and the underlying mechanisms. To expand human Vγ9Vδ2 T cells, peripheral blood mononuclear cells(PBMCs)from healthy donors(HD)were cultured in the presence of 2uM zoledronate together with recombinant human IL-2 ( 250 IU/ml). After culture in vitro for 12 days, 90–95% Vγ9Vδ2 T cells were detected by flow cytometry(FACS). Cell viability was determined using trypan blue exclusion and >99% cells were viable. We selected 6 AML cell lines as target cells including Kasumi-1 and HL-60 cell lines as the AML M2 type, NB4 as the AML M3 type, K562 as the AML M6 type, HL60/ADR and K562/AO2 cell lines as the chemotherapeutics-resistant AML. First, we evaluated the cytotoxic activity of Vγ9Vδ2 T cells against different AML cell lines. Cytotoxicity was measured by FACS analysis using CFSE and 7-AAD. AML cell lines above were labeled with 1uM CFSE and then incubated with Vγ9Vδ2 T cells for 4h or 8h at the effect: target (E:T) ratios of 5:1, 10:1, and 20:1. 7-AAD was added before acquisition on the FACSCalibur cytometer. The calculation of cytolytic activity was based on the degree of reduction of viable target cells (VTC) with the ability to retain CFSE and exclude 7-AAD (CFSEhigh 7-AAD−). We found Vγ9Vδ2 T cells exerted cytotoxicity on different AML cell lines in varying degrees. At an E:T ratio of 20:1, the cytotoxicity of Vγ9Vδ2 T cells on Kasumi-1, HL-60, HL-60R, NB4, K562, K562-AO2 cell lines for 4h were: 53.27%±18.43% A12.03%±18.16 A13.47%±10.36% A17%±11.01% A29.53%±12.22% A26.4%±0.71%, and for 8h could increase to 56.86%±16.92% A32.25%±6.71% A25.9%±0.99% A41.2%±8.22% A24.27%±8.51% A24.35%±13.51% respectively(P<0.05). Interestingly, Vγ9Vδ2 T cells did not kill normal allogeneic bone marrow mononuclear cells. Then we chose Kasumi-1 cell line, which was the most sensitive to Vγ9Vδ2 T cells' cytotoxicity, as target cells for the study of mechanism below. Kasumi-1 cells were cocultured with 10 folds of Vγ9Vδ2 T cells for 24h and cell morphology was observed by phase contrast microscope. We noted the refractive index of Kasumi-1 cells was significantly decreased, some cells became smaller, shrunken and disintegrated. Also some irregular cells could be seen. It has been proposed that human Vγ9Vδ2 T cells trigger several distinct pathways for killing tumor cells, which include secretion of proinflammatory cytokines and proapoptoticmolecules or cell contact-dependent lysis. Trogocytosis has been reported to play vital roles in killing chronic myeloid leukemia by Vγ9Vδ2 T cells. To know whether Vγ9Vδ2 T cells kill the AML cells in the same way, Vγ9Vδ2 T lymphocytes were further loaded with green-CFSE and coincubated for 4h with Kasumi-1 cells, which were previously labeled with orange-DiI, to measure Vγ9Vδ2 T lymphocytes' ability to conjugate with target cells into lytic synapses by fluorescence microscope. The result revealed that Vγ9Vδ2 T cells were significantly activated after 1h coincubation, and the target cells staying in the middle were surrounded by metatypical Vγ9Vδ2 T cell populations. When the two groups of cells were incubated for 4h, we observed Vγ9Vδ2 T lymphocytes captured the Kasumi-1 cells' membrane fragments by direct contact. In summary, our findings indicate that ex vivo-expanded Vγ9Vδ2 T cells possess a promising cytotoxic activity against not only AML cells, but also chemotherapeutics resistant cells, especially AML M2-type cell line-Kasumi-1, with t (8,21) characteristics. We also discover that the death of target cells relies much upon cell contact with Vγ9Vδ2 T lymphocytes. The further mechanisms involved in Vγ9Vδ2 T cell-mediated cytotoxicity following direct contact and the reason of varying sensitivity still remain unclear. We will clarify all these in the future.

Disclosures:
No relevant conflicts of interest to declare.