T-cell specification fails to occur in Cbfbrss/− cells. (A) Lin− FL cells cocultured on OP9-DL1 in the presence of Flt3L and IL-7 for 7 days. CD8−Gr-1−GFP− cells (GFP gating was used to eliminate OP9-DL1) were analyzed for CD44 and CD25 expression. (B) Percentages of DN1, DN2, and DN3 cells (from panel A) averaged from 12 independent experiments. Error bars represent SEM. *indicates significant difference (P ≤ .01) between the percentages of Cbfb+/+ and Cbfbrss/− cells. (C) CD45+ Lin− (Lin = CD8, CD3, TCRβ, TCRγ, CD11c, B220, Mac1, NK1.1, Ter119) 17.5 dpc fetal thymocytes analyzed for CD44 and CD25 expression. (D) Data from 6 animals of each genotype (from panel C). *indicates significant difference (P ≤ .03) between the percentages of Cbfb+/+ and Cbfbrss/− cells. (E) Expression of cell surface Thy1.2 on 17.5 dpc DN1 (Lin−CD45+CD44+CD25−) thymocytes, and on DN1 cells (CD8−Gr-1−CD45+CD44+CD25−) from day 6 OP9-DL1 cultures. The thymocyte data were averaged from 4 Cbfb+/+ and 4 Cbfbrss/− fetuses, and the culture data were averaged from 4 Cbfb+/+ and 3 Cbfbrss/− fetuses. *indicates significant difference (P = .01) between Cbfb+/+ and Cbfbrss/− samples. (F) Percentage of 17.5 dpc DN thymocytes (CD45+ Lin−, Lin = CD8, CD3, CD11c, B220, Mac-1, Ter119) expressing intracellular TCRβ (iTCRβ) and iTCRγ. Data are averaged from 3 Cbfb+/+ and 3 Cbfbrss/− fetuses. (G) Percentage of Thy1.2+ DN2 cells after 7 days of OP9-DL1 culture. (H) Cd3e and Cd3g expression by qRT-PCR in DN2 cells sorted from day 7 OP9-DL1 cultures (culture conditions and staining as described in panel A). The purity of postsort populations was more than 95%. Expression of individual genes was normalized to Hprt. Data were derived from triplicate amplifications from 3 independent samples. *indicates significant difference (P≤ .01) between Cbfb+/+ versus Cbfbrss/− values. (I) Lin− FL cells cultured on OP9-DL1 cells, harvested at indicated time points. DN1 cells (Lin−CD45+CD44+CD25−) were isolated by cell sorting (purity > 95%). The expression of individual genes was normalized to Hprt (note log scale for Tcf7) and displayed as relative to day 0 values of Cbfb+/+ DN1 cells. Values are averaged from triplicate samples isolated from 3 independent experiments. *indicates significant difference (P ≤ .05) between Cbfb+/+ versus Cbfbrss/− values. (J) Rescue of T-cell development with CBFβ in 17.5 dpc Cbfbrss/− FL cells. Lin− FL cells (E17.5) were transduced with the indicated retroviruses and cocultured on OP9-DL1. Cells were harvested after 2 weeks and GFP+CD45+ cells analyzed for CD4 and CD8 expression (top panels) and TCRγ expression (middle panels). The bottom 2 plots are DN cells analyzed after 1 week of coculture. Gated GFP+Lin−CD45+ cells were analyzed for expression of CD44 and CD25 (nexperiments = 11). (K) Western blot showing CBFβ (p22) protein levels resulting from retroviral expression relative to endogenous protein levels, in whole cell extracts prepared from GFP+CD45+ cells purified from the OP9-DL1 cultures of Lin− FL cells (CD45+ cell purity ≥ 99.9%). The blot was probed with a monoclonal antibody to CBFβ. The 2 endogenous CBFβ isoforms generated as a result of alternative splicing (p21.5 and p22) are both visible on this gel. Samples were normalized for actin expression, and the relative amounts of CBFβ determined from a dilution series (not shown). CBFβ levels in retrovirally transduced cells were 5-fold higher than endogenous levels. Lane 1, Cbfbrss/− + MigR1; lane 2, Cbfbrss/− + CBFβ; lane 3, Cbfb+/+ + MigR1; lane 4, Cbfb+/+ + CBFβ. (L) Inefficient rescue of T-cell development on restoring CBFβ expression in 17.5 dpc Cbfbrss/− thymocytes. Thymocytes were transduced with the indicated retroviruses and cocultured for 7 days on OP9-DL1. Gated GFP+CD45+ cells were analyzed for CD4 and CD8 expression in the plots below. GFP+CD45− cells are predominantly OP9-DL1 cells.16 (M) Cell-cycle status of CD45+ Lin− (Lin = CD8, TCRβ, TCRγ, CD11b, Ter119, B220) 17.5 dpc fetal thymocytes. (N) Summary of cell-cycle data from 5 individual samples per genotype. The differences between Cbfb+/+ and Cbfbrss/− cells in G0/G1 and G2/M were significant at P≤ .01.
