OT-I–GFP TCR is expressed on the cell surface and is functional. (a) Flow cytometric analysis of GFP fluorescence in OT-I–GFP LN cell subsets expressing the indicated surface markers. (b) Flow cytometric analysis of GFP fluorescence and TCR surface expression in OT-I–GFP Rag2^−/− LN cells. Anti-Vα2 and anti-Vβ5 stain the OT-I–GFP α and β chains, respectively. Percentages are shown in each quadrant. (c and d) Naive WT OT-I or OT-I–GFP T cells were surface labeled with anti–TCR-β antibody, placed on a lipid bilayer presenting pMHC and ICAM-1, and imaged by TIRF microscopy. (c) Dynamics of microcluster and cSMAC formation. See Video 1 for full time lapse. Data are representative of two experiments. Bar, 10 µm. (d) Quantification of fluorescent intensity of surface labeling of TCR-β (H57) and GFP of OT-I–GFP TCR microclusters. (e) Proliferation of WT OT-I CD45.1 T cells versus OT-I–GFP Rag2^−/− T cells. CFSE dilution of cotransferred T cells 66 h after SL8 peptide + CFA immunization. (a, b, and e) Data are representative of at least three experiments. (f) TCR down-regulation in naive T cells after stimulation with antigen-pulsed DCs. Error bars represent SEM. (d and f) Data were pooled from two experiments.