β-Selection stage thymic defect in Hq mice. (A) Representative analysis of CD44 and CD25 expression on gated DN thymocytes (negative for lineage markers CD3/CD4/CD8/B220/CD11b/CD11c/Gr-1) of 8–12-wk-old WT and Hq mice, showing frequencies of cells in quadrants. (B) Numbers of various thymocyte subsets per thymus in WT or Hq mice, from analyses as in A, shown as mean ± SE (n = 3). *, P < 0.01; **, P < 0.05. (C) Representative analysis of gated DN3 thymocytes from WT (thin line) or Hq (thick line) stained for CD27. Gray curve indicates isotype control. (D) Frequencies of CD27^lo and CD27^hi DN3 thymocytes from WT and Hq mice as indicated, from analyses as in C, shown as mean ± SE (n = 3). *, P < 0.01. (E–G) Magnetically sorted >90% pure DN1 + DN2 thymocytes from WT or Hq thymus were put in culture with the OP9DL1 stromal cell line. Cells were harvested and analyzed flow cytometrically on day 3 of culture. Two-color plots (E) show the expression profile of Thy-1 and CD44 on cells from WT or Hq cultures as indicated. Flow cytometric histograms (F) show the CD25 expression profile of gated Thy-1⁺CD44⁻ cells from WT (thin line) or Hq (thick line) cultures. Bar diagrams (G) show frequencies of DN3 (CD44⁻CD25⁺) and DN4 (CD44⁻CD25⁻) cells in the Thy-1⁺CD44⁻ compartment of WT or Hq cultures as mean ± SE (n = 3). *, P < 0.05. (H) B220 staining on bone marrow cells from WT (thin line) or Hq (thick line) mice. Filled histogram represents isotype control. (I) Representative two-color analysis of gated B220⁺ bone marrow cells from WT or Hq mice, stained for CD43 and IgM, showing frequencies of pro–B (CD43⁺IgM⁻), pre–B (CD43⁻IgM⁻), and B (CD43⁻IgM⁺) cells. (J) Numbers of B cell lineage subsets in bone marrow of WT or Hq mice, from analyses in H and I, shown as mean ± SE (n = 3). Data are representative of at least two independent experiments.