Changes in EL-4 T Cell Properties Due to Oxygen Plasma Irradiation

Recently, research and commercialization of immunotherapy have been promoted as one of cancer treatment techniques. There is one problem to solve that the time required to culture white blood cells that enough for cancer treatment is as long as 3 weeks. From previous research, short-time irradiation of atmospheric pressure plasma can activate cells. Typical feature of the T-cell is to finally differentiate to kinds of T-cells like helper T-cell, killer T-cell or regulated T-cell. Helper T-cells can release some cytokines which regulate immune cell activation like proliferation or differentiation. The purpose of this study is to investigate the activation effects and cytokine release characteristics of the EL4 T-cell by the oxygen plasma irradiation.<br/>A cylindrical ceramic tube covered with a rectangular grounded electrode which has length of 130 mm and inner diameter of 4 mm is used as a plasma torch. A metal rod as powered electrode is set in the ceramic tube and connected to high voltage (order of kV) and high frequency (9-11 kHz) power supply to obtain a dielectric barrier discharge (DBD). The atmospheric plasma was supplied at flow rate of 0.6 L/m with discharge voltage of 4.2 kV. The irradiation time varies from 0 to 40 s. The material gas for plasma production is pure oxygen gas. The initial cell population is kept at constant 5×10⁵ cells per ml throughout the experiment. A 96-well plate was used for cell culture and the amount of culture medium is 0.1 ml in one well. After plasma irradiation, T-cells were cultured with 5 % CO₂ for 24 h. The cell number was quantified using cell counting reagent WST-8 that counts the living cell number. Amount of two kinds of cytokine released by EL4 T-cells are measured by using ELISA-kit to investigate T-cell activation and differentiation. The T-cells are sensitized by the T-cell activator, which is anti CD3/CD28, after 24 h incubation from plasma irradiation.<br/>Dependence of the sensitized T-cell cell number and the cytokine amount released by EL4 T-cell on the plasma irradiation period is investigated. The cell number increased 1.2 times of the control with plasma irradiation for 10 s. On the other hand, cells are damaged with plasma irradiation for over 20 s. Since distance from the discharge area to the T-cell is approximately 30 mm, T-cell would be affected by long-lived active oxygen like O* and OH* from O₃ and H₂O₂.<br/>One kind of cytokine released by Th1 cells is IFN-γ. The amount of IFN-γ from EL4 T-cells didn’t change significantly after the plasma irradiation. The concentration of IFN-γ was low, below 5 pg/ml. The ability of releasing IFN-γ was not activated by plasma irradiation, and therefore the cells were differentiated to the other cells like Th2 cells. The amount of IL-4 released by EL4 T-cells was increased by 3.5 times of the control with plasma irradiation for 20 and 30 s. The ability of IL-4 production by EL4 T-cell was activated by the plasma irradiation. Therefore, the EL4 T-cells would be differentiated to Th2 cells. The increase of IL-4 production would be due to recover the damage of cell by too high oxygen stress on EL4 T-cells.<br/>Active oxygen species produced in the atmospheric oxygen plasma affect the T-cells and the T-cells change to be easy to activate. The ability of releasing IFN-γ was not changed but its IL-4 was activated by plasma irradiation. It is considered that EL4 T-cells were differentiated to Th2 cells by using oxygen plasma irradiation.