Chimeric receptor response to low-affinity ligand.
(A) Transduced 4G4 cells were incubated with 50 μg/mL ovalbumin p257-264 and OT-1 TCR transgenic lymph node T cells in the presence of brefeldin A. Alternatively, 4G4-transduced cells were added to 96-well plates coated with H-2Kb–specific antibody, also with brefeldin A. After 6 hours incubation, cells were fixed, permeabilized, and stained for intracytoplasmic interleukin-2. The 4G4 cells could be readily distinguished from the transgenic lymph node cells during flow cytometric analysis based on cell size and due to their expression of GFP. Comparable results were obtained by preincubating 4G4 T cells with ovalbumin peptide, and washing unbound peptide prior to coincubation with OT-1 cells (data not shown). (B) Transduced 4G4 T cells were preincubated with p257-264, washed, and coincubated for 20 hours with OT-1 T cells. Surface expression of the early activation marker CD69 was determined by staining with CD69 and CD8 followed by flow cytometric analysis with gating for CD8+ (OT-1) cells. * indicates not tested.
