Expression of a CD3ε FRET probe in a human T cell clone. HA-tagged CD3ε chain was expressed through lentiviral infection in human T cell clone B-A8 specific for the influenza HA306-318 peptide. The two ITAM tyrosines were mutated to phenylalanine (YF). TFP was added to the C terminus for FRET measurements (CD3ε-YF-TFP). Data are representative of n > 3 experiments. (A) T cells expressing CD3ε-YF-TFP were applied on lipid bilayers displaying HA-DR4 and ICAM-1. Synapse formation was imaged by TRIFM at 30-s intervals; shown are representative examples of synapses at early (60 s) and later (300 s) time points. Bars, 10 µm. (B) FACS analysis of CD3ε and TFP expression, representative of three separate experiments. (C) B-A8 T cells expressing CD3ε WT or YF mutant were stained with HA epitope tag or isotype control antibody (Dylight649 labeled) to measure surface expression of CD3ε constructs. (D) Localization of CD3ε WT or YF mutant protein was determined by confocal microscopy; pseudo-color gradient indicates signal intensity. Bars, 10 µm.
