A D^b-PE tetramer binds and is internalized by cognate T cells. (A) Bound tetramer is visible on adoptively transferred P14.GFP T cells in the lymph node after injection of gpC9M-PE, causing activation. Histograms are gated on GFP⁺ CD8⁺ T cells; there was no increase in CD25 expression on host GFP⁻ CD8⁺ T cells. (B) Internalized tetramer colocalizes with LAMP-1 (CD107a), as visualized by confocal microscopy of acid-stripped P14 T cells. Both surface and internal gpC9M-PE tetramers were observed with nonstripped cells (not shown). In all panels, original magnification was × 630. (C) Bound gpC9M-PE tetramer and the TCR are internalized rapidly at 37°C, with identical kinetics. For clarity, only data from acid-stripped cells (ie, internal fluorescence) is shown. (D) All metabolically active T cells have internalized tetramer by 30 minutes. P14 T cells incubated with gpC9M-PE show a progressive increase in acid-resistant fluorescence over time (time points > 60 minutes not shown). Results represent 2 independent experiments. In all experiments, control tetramers flu-PE (A) and hyC2A-PE (B-D) and the control FITC-labeled hamster anti-TNP mAb (C-D), did not bind, activate, or enter P14.GFP or P14 T cells (not shown).