Antibody-induced segregation of truncated CD148 rescues TCR signaling. (A) Schematic depiction of forced segregation experiment. B3Z T cells expressing FLAG-tagged CD48 are plated onto surfaces presenting immobilized anti-FLAG antibody before stimulation with anti-mouse CD3ε beads. By accumulating FLAG-tagged CD148 on the basal side of the cell, this forces segregation from apical areas where the beads engage TCR. (B) B3Z cells expressing the indicated form of CD148 and plated onto anti-FLAG or control surfaces were exposed to anti-mouse CD3ε (stimulation) or control beads IL-2 secretion determined after 16 hours after stimulation. Data are normalized with IL-2 secretion from unstimulated and stimulated vector transduced cells set to 0% and 100%, respectively. ***P < .01, using paired Student t test. (C) B3Z cells expressing the truncated form of CD148 were plated onto surfaces coated with the indicated concentration of immobilized anti-FLAG, stimulation with anti-mouse CD3ε beads, and IL-2 secretion determined after 16 hours. Data normalized to IL-2 secretion observed with highest levels of anti-FLAG (100%) and IL-2 secretion from unstimulated cells was set to 0%. (D) Schematic depiction of forced colocalization experiment. B3Z cells expressing FLAG-tagged truncated CD148 was plated onto surfaces containing both immobilized anti-FLAG and anti-mouse CD3ε. (E) Control B3Z cells or B3Z cells expressing the truncated form of CD148 were plated onto surfaces coated with the indicated combination of anti-FLAG and anti-mouse CD3ε antibodies and IL-2 secretion determined after 16 hours of stimulation. Data are normalized with IL-2 secretion from unstimulated and stimulated vector-transduced cells set to 0% and 100%, respectively. Error bars represent the SD of the mean from at least 3 replicates.