Figure 1. (A) Extracts from thymuses (lanes 1–3) or spleens (lanes 4–6) were isolated from wild-type mice (lanes 1, 4), Stat5a2/2 mice (lanes 2, 5), or Stat5b2/2 mice (lanes 3, 6), and proteins were then separated by SDS-PAGE and Western blotted with antisera specific for Stat5a (top) or Stat5b (bottom). (B) Diminished thymocyte numbers in Stat5b2/2mice (7–10-wk-old mice were analyzed). (C and D) Flow cytometric analysis of thymocytes from 8-wk- old mice. (C) CD4 versus CD8 flow cytometric profiles. (D) CD25 versus CD44 flow cytometric profiles of double negative thymocytes. (E) Diminished numbers of splenocytes in Stat5b2/2 mice (7–10-wk-old mice were analyzed). (F–H) Flow cytometric profiles (8-wk-old mice) show a decrease in the ra- tio of CD31 to B2201 cells (F) and a decrease in the percentage of CD81 single positive T cells (G) in Stat5b2/2 mice. The percentage of TCR-a/b and TCR-g/d cells was normal in Stat5b2/2 mice (H). tive cells there was a moderate decrease in the percentage of CD441CD252 cells and increase in the percentage of CD442CD251 cells (Fig. 1 D). In the periphery, Stat5b2/2 mice tended to have de- creased numbers of splenocytes (P , 0.001; Fig. 1 E), as is observed in Stat5a2/2 mice (24). The T/B cell ratio was slightly decreased (Fig. 1 F). Flow cytometric analysis re- vealed diminished numbers of single positive splenocytes; the number of CD42CD81 splenocytes was substantially reduced, whereas the number of CD41CD82 cells was at most only slightly affected (Fig. 1 G). Interestingly, whereas Stat5a2/2 mice have decreased numbers of TCR-g/d cells in the spleen (24), this was not the case for Stat5b2/2 mice (Fig. 1 H). We next evaluated immune function in the periphery in Stat5b2/2 mice and found a defect in the responsiveness of 2069 Imada et al. splenocytes to mitogenic stimulation. Previously, we had found that Stat5a2/2 splenocytes exhibited a defect in IL-2– induced proliferation of splenocytes that had been preacti- vated for 48 h with anti-CD3. However, the diminished proliferation in Stat5a2/2 splenocytes could be overcome with high concentrations of IL-2 (24), suggesting that this defect primarily related to impaired IL-2–mediated in- duction of IL-2Ra (24), a protein that is required for for- mation of high-affinity IL-2 receptors (28). In Stat5b2/2 splenocytes, we observed a similar defect in IL-2–induced IL-2Ra expression, both at the protein (Fig. 2 A) and mRNA (Fig. 2, B and C) levels. This defect in IL-2– induced IL-2Ra expression seen in both Stat5a2/2 and Stat5b2/2 mice can be explained by the presence of an IL-2 response element in the IL-2Ra gene that is dependent on Stat5 proteins (29–31). However, in contrast to the ability