Figure 5.

Pre-TCR assembly at DN3a is critical for transition beyond the β-selection checkpoint. Purified TCRβ⁺ and TCRβ⁻ DN3a cells were cocultured with OP9-DL1 for 5 d. (A) Cellularity was assessed by measuring fold increase in both cocultures (as shown in the column-bar plot), and proliferation was assessed using the intensity of CFSE (as shown in histogram, bottom left). (B) Flow cytometry was used to assess the differences between TCRβ⁺ and TCRβ⁻ DN3a cocultures progression and differentiation to subsequent developing stages (as shown by flow cytometry plots). (C) The percentages of DN3a, DN3b, and DP cells from three independent biological replicates were expressed as a ratio (TCRβ⁺/TCRβ⁻) as shown by dot-plot (top right). (D) Purified TCRβ⁺ DN3a (indicates assembled pre-TCR) and TCRβ⁻ DN3a (lacks pre-TCR) cells were cocultured with OP9-DL1 for 5 d. Annexin V dye was used to assess apoptosis, and PI dye was used to assess cell death as shown by the histograms. The data are representative of three independent biological replicates with similar results. **, P = 0.004 (unpaired t test). Error bars (A and C) represent SEM.