Figure 4.

Hq thymic defect is independent of TCR-β recombination and associated with cell death during β-selection. (A) Representative histogram overlay of gated DN3 thymocytes from WT (thin line) or Hq (thick line) mice, stained for intracellular TCR-β. Filled histogram represents isotype control. (B) Frequencies of intracellular TCR-β containing DN3 thymocytes in WT and Hq mice as calculated from the analysis shown in A. Data are shown as mean ± SE (n = 3). (C) Total thymocyte numbers in 4–6-wk-old F2 progeny littermate mice from Hq and DO11.10-TCR tg parentage. F1 mice generated from Hq × DO11.10 breeding were intercrossed. The resultant progeny were typed for the Hq allele and the TCR transgene and separated into four groups on that basis as shown. Data are shown as mean ± SE (n = 3–4). *, P < 0.01. (D) Representative histogram overlays of gated DN3 and DN4 thymocytes from WT (thin line) or Hq (thick line) mice given EdU in vivo and stained for EdU incorporation. Both EdU staining and cell size (FSC) are shown. Filled histograms represent isotype controls. (E) Frequencies of DN3 and DN4 thymocytes incorporating EdU in vivo in WT and Hq mice as calculated from the analysis shown in D. Data are shown as mean ± SE (n = 3). *, P < 0.01. (F) Annexin V staining on gated DN thymocytes from WT (thin line) or Hq (thick line) mice. Filled histogram represents isotype control. (G) Frequencies of annexin V–binding DN3 and DN4 thymocytes in WT and Hq mice. Data are shown as mean ± SE (n = 4). *, P < 0.01. (H) Frequencies of annexin V–binding DP thymocytes in WT and Hq mice. Data are shown as mean ± SE (n = 3). (I) DN1, DN3, and DN4 thymocytes from WT or Hq mice as indicated were electronically sorted and put in culture in vitro. After varying periods of time as shown, the frequencies of dead cells were estimated by Trypan blue staining. Data are shown as mean ± SE (n = 3). *, P < 0.05. Data are representative of at least three independent experiments.