| Object: To investigate γδT cells’ cytotoxicity against SW620 and HCT116 cells and explore the effect of combination of γδT cells with interleukin-2(IL-2) and 5-fluorouracil(5-FU) on SW620 and HCT116 cells apoptosis, using co-culture system of SW620 or HCT116 cells and γδT cells which were cultured and amplified from healthy volunteers’ peripheral blood.Methods:(1) Peripheral blood were collected from healthy volunteers. Peripheral blood mononuclear cell(PBMNCs) were separated with Ficoll density gradient centrifugation and cultured in RPMI-1640 medium containing 1umol zoledronate(Zol) and 100 IU / ml rh IL-2 to amplify γδT cells. Cells at day 7-10 were collected and γδT cells were sorted using immunomagnetic beads. γδTCR expression of sorted cells was detected with flow cytometry.(2) Sorted γδT cells were co-cultured with colon cancer cells SW620 or HCT116 with different effectors and target cells ratio. Inhibition rate of cancer cells proliferation was measured with LDH release assay and apoptosis rate with flow cytometry.(3) γδT cells were co-cultured with SW620 or HCT116 cells in the ratio of 10:1 in the medium containing different concentrations of rh IL-2 and the inhibition rate was measured with LDH release assay.(4) Half lethal concentration of 5-FU on SW620 or HCT116 cells was determined Half lethal concentration 5-FU combined with γδT cells and/or IL-2, was applied to SW620 or HCT116 cells culture. Inhibition rate was measured with LDH release assay and apoptosis rate with flow cytometry.Results:(1) Culturing PBMNCs in medium containing zoledronate and rh IL-2 produced a large and stable number of γδT cells at day 10. γδTCR+ cells accounted for 98.45% in magnetic beads sorted cells from the cultured cells.(2) γδT cells proved to be cytotoxic against SW620 and HCT116 cells which was related to γδT cells and cancer cells ratio(effectors: target ratio). After 6 hour culturing, inhibition rate reached the top when effectors: target ratio was 40:1. The inhibition rate was 12.55 ± 0.76% and 9.36 ± 0.61% for SW620 and HCT116 cells, respectively, while the apoptosis rate was 18.26 ± 0.63% and 26.46 ± 0.33%, respectively.(3) When γδT cells were combined with rh IL-2, the cytotoxicity increased and depended on IL-2 concentration and exposure time. After SW620 and HCT116 cells was treated with 400 IU/ml of IL-2 for 24 hours, the inhibition rate was 8.48 ± 0.31% and 18.53 ± 0.75%, respectively, which was increased to 14.17 ± 0.60% and 20.82 ± 0.90%, respectively,when IL-2 and γδ T cells were combined.(4) Half lethal concentration of 5-FU at 48 hour culture for SW620 and HCT116 cells were 400ug/ml and 4ug/ml, respectively. 5-FU showed much more potent inhibition on HCT116 cells’ proliferation than on SW620 cells. Both γδ T cell s(at low effectors: target ratio) and IL-2(at low concentration) intensified the cytotoxicity of 5-FU. When 5-FU treatment(at the half lethal concentration) was combined with both γδT cells and IL-2, the inhibition rates for SW620 and HCT116 were 60.62 ± 1.14% and 61.20 ± 1.36%, respectively, while apoptosis rates were 60.08 ± 0.98% and 67.31 ± 0.11%, respectively.Conclusions: Culturing healthy volunteers’ PBMNCs with zoledronate and rh IL-2 might produced large and stable number of γδT cells. γδT cells appeared to be cytotoxic against SW620 and HCT116 cells,inhibiting the proliferation and inducing cell apoptosis. Both γδT cells(at low effectors:target ratio) and IL-2(at low concentration) might intensify the cytotoxicity of 5-FU against colon cancer cells.Combination of 5-FU with both γδT cells and IL-2 might be of most potent cytotoxicity. |
