PMC

Cytotoxicity assays done with HLA-A2–restricted CTL or TIL effectors and peptide-loaded T2 cells or glioma target cells. CTL (top and middle rows) were directed against Mart-1, GP100, EphA2, and Her/neu peptides and incubated with peptide-loaded T2 target cells (top row) or glioma target cells (middle row). TIL (bottom row) were incubated with either peptide-loaded T2 cells or the glioma target cells. The effector TIL 1374 cells are specific for tyrosinase (366-377; left) and TIL 771 cells are specific for GP100 (154-162; right) and incubated with the target glioma cells. The phenotypes of the glioma cells used are, for SNB19: HLA-A2+, Mart-1+, GP100+, EphA2+, Her2/neu+; U-251: HLA-A2+, Mart-1+, GP100+, EphA2+, Her2/neu+, tyro+; U-373: HLA-A2+, Mart-1+, GP100+, EphA2+, Her2/neu+, tyro−; A172:HLA-A2−, Mart-1+, GP100+, EphA2+;T98G: HLA-A2+, tyro+; LN229: HLA-A2−, tyro+.

TAPP associated with U-251cells by immunofluorescence microscopy and by intracellular flow cytometry. A, immunofluorescence of U-251cells shows that the TAPP are present within their predicted intracellular locations. U-251gliomas were allowed to attach to coverslips overnight. The cells were fixed, permeabilized, and then stained with the specific antibodies. Top, left to right, Her2/neu, GnT-V, and survivin. Top middle, tyrosinase Mage-1andTrp-1 (which is as bright as the negative control; not shown). Bottom middle, EphA2, GP100, and IL13Rα2. Bottom, B-cyclin, Mart-1, and hTert. B, intracellular flow cytometry of TAPP within U-251glioma cells. U-251glioma cells (10⁶) were fixed and permeabilized. The permeabilized cells were incubated with the primary antibodies for 1h, washed, and subsequently incubated with a FITC-labeled secondary antibody directed against the primary antibody. Flow cytometric analysis of 10⁴ glioma cells is shown. The isotypic control (dark line) and the anti-TAPP staining (either a fine line or dashed line) are shown. a, Her2/neu; b, GnT-V and survivin; c, tyrosinase and Mage-1; d, Trp-1 and EphA2; e, GP100 and IL13Rα2; f, Mart-1; g, B-cyclin and hTert.