Figure 3.

LAMTOR2 is required for differentiation of B cell progenitors but not their expansion. (A–C) Pre-B1 cells (B220⁺CD19⁺CD117⁺) were sorted from bone marrow of LAMTOR2^fl/fl or LAMTOR2^Cd19/Cd19 mice and cultured on semi-liquid methylcelullose substrate supplemented with murine IL-7. Nine days later cells from individual colonies were collected and analyzed by flow cytometry. (A) Experimental scheme and diagram illustrating development of B cell progenitors. (B) Percentages of early (B220⁺CD117⁺IgM⁻) and late (B220⁺CD117⁻IgM⁺) B cell progenitors. Each dot represents data from a single colony. (C) Flow cytometric analysis of cell surface expression of IL-7Ra (CD127) on pre-B1 cells from bone marrow of LAMTOR2^fl/fl or LAMTOR2^mb1/mb1 mice (left panel) and analysis of proliferative expansion of B cell progenitors (LAMTOR2^fl/fl or LAMTOR2^Cd19/Cd19) cultured on methylcellulose in the presence of IL-7. Individual colonies were retrieved from methylcellulose and quantitated by FACS. The y-axis denotes number of cells per isolated colony. (D) PCR analysis of genomic DNA for V_HJ558-to-J_H3 rearrangements from sorted pre-B1 cells. pre-B2 (B220⁺CD19⁺CD25⁺) from LAMTOR2^fl/fl mice cells were used as recombination positive control and thymocytes as negative control. Expression of recombination-independent C serves as loading control. PCRs were carried out on the DNA content of 60,000, 20,000, and 6,700 cells. (E) Intracellular staining of surrogate light chain components (VpreB1 and λ5) expressed by pre-B1 cells. (B,C) Pooled data of two independent experiments are shown. Each dot represents data from a single colony. (D) Results of an individual experiment are shown; FACS plots on (C,E) are representative of two independent experiments. Statistical analysis was performed using unpaired t-test.