Figure 4

Retroviral transduction of PBMC with a cloned NY-ESO-1 specific T cell receptor allows for T cell recognition of decitabine-treated human T98 glioma cells. Normal donor peripheral blood mononuclear cells (PBMCs) were activated with OKT-3 at a concentration of 50 ng/mL for two days and then harvested for transduction. A) PBMCs were transduced with retroviral constructs encoding a HLA-A*0201-restricted NY-ESO-1 T cell receptor, and stained with fluorescent mAbs and tetramer NY-ESO-1 percentage. B) Representative flow cytometric analysis of CD107A of decitabine-treated and untreated T98G glioma cells (solid filled, NY-ESO-1 specific T cells alone; dashed line, NY-ESO-1 specific T cells co-cultured with untreated T98G glioma cells; solid line, NY-ESO-1 specific T cells co-cultured with decitabine-treated T98G glioma cells). Golgi-plug was added to co-cultures and stained with CD107A before incubation. The lymphocytes were stained with antibodies to CD3, CD4, and CD8 and subsequently fixed. The experiment was repeated three times with similar findings.