A simple method for the propagation and purification of γδT cells from the peripheral blood of glioblastoma patients using solid-phase anti-CD3 antibody and soluble IL-2

Abstract

Although γδT cells make up no more than 10% of the peripheral blood mononuclear (PBM) cells, they appear to play an important role in host defense against tumor growth. In order to evaluate their functional activity against tumors and their response to various cytokines, large numbers of cells are required. Here, we describe a newly-devised method for the isolation and expansion of γδT cells from the peripheral blood of cancer patients, in particular those with glioblastoma. Using this approach, a 1000–1500-fold increase in total cell numbers was achieved in two weeks, the proportion of γδT cells in the expanded population being, on average, approximately 30% after 14 days of culture. The method therefore gives a yield of approximately 10–15×10 8 γδT cells from only 5 ml of peripheral blood from glioblastoma patients and normal controls. The highly purified γδT cells of glioblastoma patients were shown to bear both a high-affinity interleukin-2 receptor (IL-2R) and a low-affinity IL-12 receptor (IL-12R). They also displayed significant cytotoxicity against autologous tumor cells, but not against autologous fresh or IL-2-treated lymphocytes, and proliferated in response to IL-2, both effects being dependent on the dose of IL-2 used for activation. In addition, overnight incubation with 700 U/ml of IL-2 or 50 ng/ml of IL-12 resulted in significant cytotoxic activity of patients' γδT cells against K562 target cells, the level of activity being almost the same as with similarly-treated γδT cells from normal controls ( P>0.05). These results demonstrate that the patients' γδT cells obtained using this method are intact in terms of cytotoxic function. Thus, this method not only makes it possible to produce large numbers of purified γδT cells but also to produce populations containing both γδT cells and NK cells, both active against tumor targets which might be suitable for clinical trials of adoptive immunotherapy, especially in cancer patients for whom no effective therapy is available.