TCR expression and signaling in a DP thymocyte line expressing hCD3δ and lacking CD3γ. (A) The AND.DP.γ^–/– line was surface stained with antibodies to CD4, CD8, TCRβ, and CD3γϵ and analyzed by flow cytometry. (B) The histogram shows surface TCR (TCRβ) expression in the AND.DP.γ^–/– line (solid line) compared with DP thymocytes from AND TCR transgenic mice (dotted line). (C) CD69 and CD5 up-regulation in AND TCR transgenic DP thymocytes (left panels) or the AND.DP.γ^–/– line (right panels) after 48-hour stimulation with 1 μg plastic-bound anti-TCRβ antibody or with DCEK cells preloaded with 2 μM pigeon cytochrome C (PCC) peptide 88-100. Shaded curves represent marker expression in unstimulated cells. (D) CD69 and CD5 up-regulation (left panels) in WT and γδ^– × hδTg DP thymocytes stimulated for 48 hours with 0.5 μg (solid line) or 5 μg (dotted line) anti-TCRβ antibody, or in unstimulated cells (shaded curves), as determined by 3-color flow cytometry. Numbers within histograms represent the percentage of cells in the marked region for unstimulated (upper value) or anti-TCR–stimulated (0.5 μg, middle value; 5 μg, lower value) DP thymocytes. MFI values of CD69 and CD5 expression in WT (□) and γδ^– × hδTg (▪) DP thymocytes stimulated with the indicated amount of anti-TCRβ antibody are plotted at the right.