Effects Of Nicotine On Anti-tumor Activity Of Human Vγ9Vδ2-T Cells

Objective: To study whether the nicotine could regulate anti-tumor activity of human Vγ9Vδ2-T cells, and to investigate the underlying mechanisms could be involved in these effects. Methods:The peripheral blood mononuclear cells(PBMC) were isolated from healthy donors by density gradient centrifugation. Pamidronate in presence of IL-2 selectively expanded Vγ9Vδ2-T cells in vitro. The immunological phenotypes of resting and in vitro expanded-Vγ9Vδ2-T cells were determined by using flow cytometry. Subsequently, expanded-Vγ9Vδ2-T cells were purified by negative selection using TCRγ/δ+ T cell isolation kit. The expression level of α7-nAchR in Vγ9Vδ2-T cells was measured by western blot and immunofluorescence assay. Then, the purified-Vγ9Vδ2-T cells were treated by gradient concentrations of nicotine, and using AnnexinV-PI assay to evaluate the percentage of apoptotic cells after nicotine treatment. PBMCs of healthy-non-smoking donors were selected for expansion of Vγ9Vδ2-T cells in the following experiments. The percentage, absolute number and immunological phenotypes of Vγ9Vδ2-T cells were determined after expansion with or without nicotine treatment(100μM). After 14 days expansion, Vγ9Vδ2-T cells were purified by negative selection then co-cultured with K562 cells at different E:T ratios for 4 h. The percentages of dead K562 cells among the whole target cells were identified as DIOC+PI+. After co-culture with K562 at a ratio of 1:1, the cytotoxic and cytolytic activity of Vγ9Vδ2-T cells were further investigated by using flow cytometry.Results: Pamidronate in presence of IL-2 could selectively expand and activate Vγ9Vδ2-T cells in vitro. After negative selection, the purity of Vγ9Vδ2-T cells was consistently >97%. Using western blot and immunofluoroscence, we found that Vγ9Vδ2-T cells could express α7-nAchR. High concentration of nicotine(1mM) could affect the viability of Vγ9Vδ2-T cells. However, we could not observe this phenomenon in low concentration of nicotine(100μM) treatment. Therefore, low concentration of nicotine(100μM) was selected in the following functional experiments. Accordingly, 100μM nicotine treatment could significantly inhibit the proliferative ability of Vγ9Vδ2-T cells, and also reduced the percentage and absolute number of Vγ9Vδ2-T cells(p <0.05) in the cell culture. Moreover, nicotine could significantly reduce killing ability of Vγ9Vδ2-T cells against K562 cells at E:T ratios of 10:1 and 20:1. Finally, after co-culture with K562 at 1:1 ratio, the expression level of FasL, CD107 a, TRAIL and Perforin were obviously down-regulated.. Conclusion: Here we demonstrated that Vγ9Vδ2-T cells can express α7-n AchR. Low concentrations of nicotine(100μM) couldn’t induced apoptosis of Vγ9Vδ2-T cells. According to in vitro results, we found that nicotine treatment could impair the proliferative ability of Vγ9Vδ2-T cells. Most importantly, the cytotoxic activity of Vγ9Vδ2-T cells was also inhibited, because we found that down-regulation of FasL, TRAIL, CD107 a and Perforin expression levels in Vγ9Vδ2-T cells after by treatment of nicotine. These results suggested that nicotine could inhibit the cytotoxic activity of Vγ9Vδ2-T cells. And our investigation also indicated that the tumor invasion is caused by the immune surveillance of Vγ9Vδ2-T cells was hampered in the smokers. This was the leading cause for high incidence of cancer were found in the population of smokers.