Figure 4.

Approximately 55% of TCR-signaled thymocytes die by Bim-dependent negative selection at the CCR7⁻ stage. (A) Analysis of nascent BrdU⁺ CCR7⁻ CD24⁺ thymocytes from wild-type mice 3 d after BrdU injection. Quadrants in top left panel define three major CD69 versus TCRβ subsets that are each analyzed for CD4 and CD8 expression on the right. Bottom panels reverse the gating order to show percentage of CD4^SP in the nascent pool and their expression of CD69 and TCRβ. Plots were concatenated from four samples, representative of two experiments each analyzing at least four mice individually. (B and C) Analysis of nascent BrdU⁺ CCR7⁻ CD24⁺ thymocytes concatenated from four Bim^+/+ or four Bim^−/− mice on B10.BR background 24 h (B) or 3 d (C) after BrdU treatment. Two Zap70^mrt/mrt mice were also analyzed to test TCR signaling. Top row: percentage of CD69⁺ cells. Second row: Helios and PD-1 expression on CD69⁺ subset. Bottom rows: CD4 and CD8 expression on Helios⁺ and Helios⁻ subsets of CD69⁺ cells. Percentage of gated cells is shown in the plots. Graphs show mean and SD percentage of CD69⁺ cells among BrdU⁺ CD24⁺ CCR7⁻ thymocytes from at least five mice per group compiled from two experiments in B and at least four mice per group from two experiments in C. Unpaired Student’s t test p-value: ***, P < 0.001.