Fig 2: The transcriptome of the pre-BCR-dependent expansion stage is governed by MYC-associated gene expression networks and requires repression of Ebf1 expression.a Highlighted population of the pre-BCR-dependent cluster (left) and feature plots for the pre-BCR-dependent markers highly expressed during this stage (right). b Violin plot of B-lymphoid transcription-factor expression, including Ebf1, Pax5, and Ikzf1 across B-cell development. c Violin plot of additional genes that are downregulated during pre-BCR-dependent proliferation, which includes Il7r, and EBF1-target genes such as Cd79a and Cd79b. d Landscape In silico Deletion Analysis (LISA) to predict the transcriptional regulator of the top 100 differentially upregulated genes during pre-BCR-dependent proliferation (left table). Gene-set enrichment analysis was performed to identify gene sets from the Molecular Signature Database that were enriched in the pre-BCR-dependent cells. Comparison between the pre-BCR-independent cells (left side) and the pre-BCR-dependent cells was performed (right side). Statistical significance was calculated using one-sided Wilcoxon rank-sum test. e EBF1-binding sites at the promoters of Nrgn, Slc7a5 and Slc3a2 with indicated MACS peak calls20. Data were obtained from GSM2863146. f Log2-transformed FPKM expression values obtained from RNA-seq of wild-type and Pax5+/− × Ebf1+/− (PE) leukemic progenitor B cells. Progenitor B cells were obtained via negative selection of CD11c, TER119, Ly6G, Ig Kappa, and Ig Lambda and positive selection of CD19. Statistical significance was determined using a two-tailed unpaired student t-test for Ybx3 (P = 0.0024) and Slc7a5 (P = 0.0001). RNA-seq data were obtained from GSE148680. A two-tailed Mann–Whitney test was performed for Slc3a2 (P = 0.0083) due to non-normal distribution. n = 7 biologically independent samples over one independent experiment. Color scales in a, b, and c represent natural log-transformed SCTransform-corrected counts.

Fig 3: Schematic diagram of generating functional T lymphocytes by reprogramming in vivo. The recombinant Hoxb5 vector or GFP empty vector control was transduced into Plat-E cells to produce viruses. Pro/pre-B cells (Ter119–Mac1–CD3–CD4–CD8–B220+CD19+CD93+IgM–) were sorted from the bone marrow and then cultured in medium (15%FBS, 100 mM GlutaMAX, 10−4 M β-ME, 10 ng/ml mSCF, 10 ng/ml Flt3L and 10 ng/ml IL7) for 12–16 h before virus transduction. Pro/pre-B cells (1 × 106 per ml) were transduced with the mixed viruses with equal pre-adjusted titers. The pro/pre-B cells transduced with retro-Hoxb5 virus were collected for transfection efficiency analysis and then were transplanted into sublethally irradiated syngeneic recipient mice via orbital veins. Four weeks after transplantation, cells harvested from PB, spleen, lymph node and thymus were analyzed by flow cytometry.

Fig 4: Flow cytometry analysis of induced T lymphocytes in irradiated recipients injected with Hoxb5-retrovirus transduced pro/pre-B cells. GFP+Ter119–Mac1– populations obtained from the PB, spleen, and LNs (a) or GFP+Ter119–Mac1–CD19– iDN populations from the thymus (b) of recipient mice were analyzed. Three million pro/pre-B cells transduced with the retro-Hoxb5 virus were transplanted into sublethally irradiated syngeneic mice. Four weeks after transplantation, the single nucleated blood cells harvested from PB, spleen, lymph node and thymus were analyzed by flow cytometry. Representative plots were shown.