Figure 4.
Figure 4. Blocked T cell development in DuxblindxpTαCre mice. (A) Total numbers of live, nucleated cells in the thymus, spleen, and bone marrow of WT (n = 6), Duxblind (n = 5), and DuxblindxpTαCre (n = 6) mice. Data were collected from three independent experiments. (B) Representative FACS plots of CD4 and CD8 expression in total live cells (top) and CD44 and CD25 expression in DN cells (bottom) of WT, Duxblind, and DuxblindxpTαCre mice. DN cells were gated as CD4, CD8, CD3, and B220 negative. Three independent experiments were performed, with representative data from one experiment shown. (C) Numbers of DN, DP, CD4 T cells, CD8 T cells, and γδ-T cells in WT (n = 6), Duxblind (n = 5), and DuxblindxpTαCre (n = 6) mice. (D) Frequencies of DN1, DN2, DN3, DN3-4, and DN4 cells as percentage of DN cells in WT (n = 6), Duxblind (n = 5), and DuxblindxpTαCre (n = 6) mice. (C and D) Data were collected from three independent experiments. (E) Representative histogram showing the size (FSC-A) of DN3-4 cells in WT, Duxblind, and DuxblindxpTαCre mice. Three independent experiments were performed, with representative data from one experiment shown. (F and G) Histograms showing the DNA content (F) and intracellular β-chain expression (G) of DN3, DN3-4, and DN4 cells in WT, Duxblind, and DuxblindxpTαCre mice. Four independent experiments were performed, with representative data from one experiment shown. (H) Numbers of sorted DN3 cells from WT and DuxblindxpTαCre mice as percentage of input after 3 and 5 d of culture in wells coated with Dll 4 and supplemented with Cxcl12. (I) Representative FACS plots of sorted DN3 cells from WT and DuxblindxpTαCre mice showing CD4 and CD8 expression after 5 d of culture. (J) Frequencies of living, early apoptotic, and late apoptotic cells after 3 d of culture of sorted DN3 cells from WT and DuxblindxpTαCre mice. Living cells were defined as annexinV− 7AAD−, early apoptotic cells as annexinV+ 7AAD−, and late apoptotic cells as annexinV+ 7AAD+. (H–J) Three independent experiments were performed, with representative data from one experiment shown. Gate numbers in FACS plots and histograms indicate frequencies of parent gate. DN stages were identified by FACS as shown in Fig. S1. Statistical analysis was done with two-tailed unpaired Student’s t test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. Error bars indicate standard deviation.

Blocked T cell development in DuxblindxpTαCre mice. (A) Total numbers of live, nucleated cells in the thymus, spleen, and bone marrow of WT (n = 6), Duxblind (n = 5), and DuxblindxpTαCre (n = 6) mice. Data were collected from three independent experiments. (B) Representative FACS plots of CD4 and CD8 expression in total live cells (top) and CD44 and CD25 expression in DN cells (bottom) of WT, Duxblind, and DuxblindxpTαCre mice. DN cells were gated as CD4, CD8, CD3, and B220 negative. Three independent experiments were performed, with representative data from one experiment shown. (C) Numbers of DN, DP, CD4 T cells, CD8 T cells, and γδ-T cells in WT (n = 6), Duxblind (n = 5), and DuxblindxpTαCre (n = 6) mice. (D) Frequencies of DN1, DN2, DN3, DN3-4, and DN4 cells as percentage of DN cells in WT (n = 6), Duxblind (n = 5), and DuxblindxpTαCre (n = 6) mice. (C and D) Data were collected from three independent experiments. (E) Representative histogram showing the size (FSC-A) of DN3-4 cells in WT, Duxblind, and DuxblindxpTαCre mice. Three independent experiments were performed, with representative data from one experiment shown. (F and G) Histograms showing the DNA content (F) and intracellular β-chain expression (G) of DN3, DN3-4, and DN4 cells in WT, Duxblind, and DuxblindxpTαCre mice. Four independent experiments were performed, with representative data from one experiment shown. (H) Numbers of sorted DN3 cells from WT and DuxblindxpTαCre mice as percentage of input after 3 and 5 d of culture in wells coated with Dll 4 and supplemented with Cxcl12. (I) Representative FACS plots of sorted DN3 cells from WT and DuxblindxpTαCre mice showing CD4 and CD8 expression after 5 d of culture. (J) Frequencies of living, early apoptotic, and late apoptotic cells after 3 d of culture of sorted DN3 cells from WT and DuxblindxpTαCre mice. Living cells were defined as annexinV− 7AAD−, early apoptotic cells as annexinV+ 7AAD−, and late apoptotic cells as annexinV+ 7AAD+. (H–J) Three independent experiments were performed, with representative data from one experiment shown. Gate numbers in FACS plots and histograms indicate frequencies of parent gate. DN stages were identified by FACS as shown in Fig. S1. Statistical analysis was done with two-tailed unpaired Student’s t test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. Error bars indicate standard deviation.

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