Peripheral blood mononuclear cells (PBM) from normal donors and patients with recurrent glioma were activated initially for 48-72 h with phytohemagglutinin-P (PHA) and recombinant human interleukin-2 (IL-2), and then proliferated in vitro for up to 5 months with IL-2. These cells are termed mitogen-stimulated lymphokine-activated T killer (T-LAK) cells. We measured patterns of T-LAK cell growth, in vitro cytolytic activity on a panel of continuous and primary tumor cells, and the phenotypes of the cells in these cultures. Lymphocyte viability declined dramatically over the first 3-5 days; and then the remaining cells in these cultures began to divide and maintained a constant 30-36 h doubling time for long periods in vitro. Phenotyping revealed that cells in the initial few days of culture were heterogeneous, but became almost totally CD3 T cells after 7-10 days in culture. The T-LAK cells from individual normal donors and cancer patients demonstrated a non-genetically restricted cytolytic ability against a panel of both continuous cell lines and primary autologous and allogeneic glioblastoma cells in vitro. This technique provides a method of generating large numbers of autologous cytolytic T cells with non-restricted anti-tumor activity that can be derived from peripheral blood mononuclear cells.

Nineteen patients with recurrent high grade gliomas were treated in a phase I/II trial with aggressive debulking of the tumor, mitogen activated IL-2 stimulated peripheral blood lymphocytes (MAK cells), and rIL-2. Phytohemagglutin (PHA) was introduced into the tumor site in 16 patients prior to implanting MAK cells and IL-2 in an attempt to trigger more effective lysis of the tumor in vivo. In vitro both TNF bioactivity and cytolytic activity of long term cultured MAK (LMAK) cells were dramatically enhanced by adding PHA to the cultures of these activated PBL. Three of eleven patients (27%) had a decrease in size of the enhancing lesion on CT and/or MRI. Seven (37%) patients clinically improved. Median survival after therapy was 30 weeks. PHA was shown to be safe in vivo and more effective than IL-2 triggering enhanced effector function in vitro.