Figure 2. (A) Diminished IL-2–induced IL-2Ra expression in both CD41 and CD81 subpopulations of T cells after their preactivation with anti- CD3. Fresh splenocytes were stimulated in Falcon 3003 plates coated with 10 mg/ml anti-CD3e mAb for 48 h, washed twice with PBS, and then cul- tured with or without 2 nM IL-2 for 48 h. Cells were stained with anti-CD25–FITC, anti-CD4–PE, and anti-CD8–Cy-Chrome. Shown are IL-2Ra expression and mean fluorescent intensity on CD41 or CD81 T cells from one representative mouse out of eight mice analyzed. (B) Northern blotting for IL-2Ra, IL-2Rb, or control pHe7 using RNA from preactivated splenocytes not treated or treated with IL-2. Shown are representative data from three mice. (C) The relative levels of IL-2Ra and IL-2Rb mRNA expression in B were normalized for pHe7 expression. (D) High dose IL-2 (1 nM) could not restore proliferation of Stat5b2/2 splenocytes preactivated with anti-CD3. Shown are the mean 6 SEM from a compilation of two indepen- dent experiments. (E) Proliferation to PMA plus IL-4 was done in Stat5b2/2 splenocytes. Shown are the mean 6 SD from a compilation of two experi- ments. In one experiment, we confirmed that treatment of cells with PMA alone or IL-4 alone yielded minimal proliferation even in wild type mice. (F) Equivalent expression of Stat6 in splenocytes from wild-type, Stat5a2/2 and Stat5b2/2 mice. of high doses of IL-2 (sufficient to titrate intermediate af- finity receptors) to normalize proliferation in Stat5a2/2 mice (reference 24 and data not shown), this was not the case for Stat5b2/2 mice (Fig. 2 D). The diminished expres- sion of IL-2Rb (Fig. 2, B and C) presumably contributed to this effect. Thus, although diminished proliferation in Stat5a2/2 mice is primarily related to diminished IL-2Ra expression (24), the Stat5b2/2 mice appeared to have a more defective IL-2-signaling pathway involving but not necessarily limited to defective IL-2Ra and IL-2Rb ex- pression. Indeed, additional defects are indicated given that the Stat5b2/2 splenocytes also exhibited defective prolifera- tion to a combination of PMA and IL-4 (Fig. 2 E), even though IL-4 activates Stat6 rather than Stat5 proteins. Be- cause Stat62/2 mice exhibit diminished IL-4–dependent proliferation (8–10), we confirmed that Stat6 expression was normal in Stat5b2/2 splenocytes (Fig. 2 F). These data are consistent with a more general defect or perhaps multi- ple defects that together diminish but not abrogate T cell proliferation. Considering the above partial defect in proliferation of preactivated T cells, we next tested the ability of freshly iso- lated splenocytes to proliferate in response to IL-2 and IL- 15. Proliferation of Stat5a2/2 splenocytes was at most slightly diminished, whereas IL-2– and IL-15–induced pro- liferation of Stat5b2/2 splenocytes was greatly diminished (Fig. 3 A). We speculated that this defect in proliferation might result from a decrease in the number and/or func- tion of NK cells, given that NK cells represent a prominent IL-2–responsive cell population in murine spleens and that 2070 Defective NK Function in Stat5b-deficient Mice