Inhibition of pre-BCR relocalization impairs pre-BCR activation and pre-BII-cell differentiation. (A) Pro-B/pre-BI cells were allowed to differentiate as described in Figure 4. Differentiation was evaluated by following CD2 and IgL (Igκ + Igλ) expression, by flow cytometry (top). The histogram shows the percentage of inhibition (± SD) of total CD2⁺ cells compared with the mSLCΔΔ control (bottom). For each inhibitory condition, the percentage of CD2⁺ and CD2⁺IgL⁺ subpopulations was compared with the control situation (OP9-pSR+mSLCΔΔ) and P values were determined using the Mann-Whitney unpaired test with a risk of 5% (P ≤ .032). (B) Cellular apoptosis was evaluated by annexin V/7-AAD staining after 1 day of culture. The percentage of apoptotic cells (annexin V⁺ 7-AAD⁻) and dead cells (annexin V⁺ 7-AAD⁺) is shown. For panels A and B, data are representative of at least 4 independent experiments. (C) Sorted CD2⁻ large pre-BII cells were incubated for 1 hour with OP9-pSR cells in the presence of mSLCΔΔ, with OP9-shG1 cells in the presence of mSLC, or without stromal cells. The percentage of phospho-BLNK⁺ pre-BII cells plus or minus SD was determined by flow cytometry (3 independent experiments).