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. 2015 Jun 29;212(7):1109–1123. doi: 10.1084/jem.20132100

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© 2015 Ungerbäck et al.

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Figure 4. — Pax5^+/−Ebf1^+/− pro–B cells undergo lineage conversion into T cells via a bivalent state. (A) Bars describe the expression change of surface markers CD19, Thy1.2, and CD3 d 4–18 after that freshly sorted Lin⁻B220⁺CD19⁺CD43^highIgM⁻ Wt of Pax5^+/−Ebf1^+/−were placed on OP9-DL1 (day 0) stroma cells under conditions permissive for T cell development. 1,000,000 cells were sorted from 2 Pax5^+/−Ebf1^+/− (TH) and 2 Wt animals, which were further divided into 2 replicates per mouse before plating onto OP9-DL1. (B) Representative FACS plots of CD19 and Thy1.2 surface expression at days 4, 12, and 18 d after seeding onto OP9-DL1 as explained in A. (C) Changes of relative gene expression of Lambda5, Tcf7, and Gata3 0–18 d after Lin⁻B220⁺CD19⁺CD43^highIgM⁻ Wt or Pax5^+/−Ebf1^+/− (TH) were placed on OP9-DL1. Data are shown as median expression levels relative to Hprt1 from four samples analyzed in triplicate Q-PCR reactions (two of each genotype in duplicate). Error bars describe interquartile range. (D) Diagrams describing the percentage CD19⁺Thy1.2⁻, CD19⁺Thy1.2⁺, or CD19⁻Thy1.2⁺ of total CD45⁺ cells in OP9 (dotted bars) or OP9-DL1 (gray bars) co-cultures from sorted CD19⁺Thy1.2⁺ or CD19⁻Thy1.2⁺ cells generated by preincubation of primary sorted Pax5^+/−Ebf1^+/− (TH) pro–B cells on OP9-DL1 for 10 d. The experiment is based on independently sorted cells from two mice and analysis of a total of six co-cultures for each condition. ***, P < 0.001.