IL-22/2 mice exhibit essentially normal T and B cell re- sponses but compromised NK cell responses (32). Indeed, flow cytometric analysis revealed that the percentage of NK (CD32DX51) cells was uniformly diminished in Stat5b2/2 mice (z55% of the percentage seen in wild-type mice; Fig. 3 B). Interestingly, the percentage of NK cells in Stat5a2/2 mice was also diminished, although to a lesser ex- tent (z70% of the percentage seen in wild-type mice). However, the much greater defect in proliferation in Stat5b2/2 compared with Stat5a2/2 mice indicated that the defect was not proportional to the number of NK cells but rather reflected a greater functional defect in Stat5b2/2 mice. Corresponding to the greater defect in proliferation in freshly isolated splenocytes from Stat5b2/2 versus Stat5a2/2 mice (Fig. 3 A), NK cytolytic activity was much more im- paired in Stat5b2/2 than in Stat5a2/2 splenocytes (Fig. 3 C). The defect in Stat5b2/2 mice was even more evident when either IL-2 (Fig. 3 D) or IL-15 (Fig. 3 E) was added during the NK assay (note the difference in the scale on the ordi- nate in Fig. 3, C–E). Thus, Stat5b2/2 mice exhibited sub- stantial defects in both proliferative and cytolytic activities. Because of these IL-2 and IL-15 signaling defects, and because the IL-2 and IL-15 receptors both contain IL-2Rb and gc as critical receptor components (33, 34), we evalu- ated expression of these proteins in NK cells. No defect in gc expression was observed in either Stat5a2/2 or Stat5b2/2 mice (data not shown). Interestingly, however, the number of NK cells expressing IL-2Rb expression was uniformly decreased in all Stat5b2/2 mice examined (n 5 17; Fig. 3 F); in contrast, in Stat5a2/2 NK cells, IL-2Rb expression was typically normal, although 2 out of 14 mice examined had a significantly diminished percentage of IL-2Rb–express- ing NK cells. Overall, the decreased number of IL-2Rb1 NK cells in Stat5b2/2 mice generally correlated with the decreased proliferative response to IL-2 and IL-15 (Fig. 3 A) as well as the marked decrease in IL-2– and IL-15–medi- ated boosting of NK cytolytic activity (Fig. 3, D and E). Again, however, the defects in IL-2– and IL-15–induced proliferation and cytolytic activity were more severe than would have been predicted based solely on the decreased number of CD32DX51IL-2Rb1 cells, suggesting that Stat5b plays an additional important and specific role(s) in IL-2– and IL-15–induced signaling in NK cells. To further investigate the basis for defective NK func- tion, we examined b2-integrin (CD18) and perforin ex- pression. CD18 is known to be important for recognition of target cells by NK cells (35), and perforin is known to be one of the key molecules for NK cell–mediated cytolysis (36). Interestingly, although cell surface CD18 expression was not diminished (data not shown), induction of perforin mRNA by IL-2 and IL-15 was markedly decreased in Stat5b2/2 splenocytes (Fig. 4). The mechanism of NK cell development has been an area of active investigation. NK cells do not develop in sev- eral different knockout mice, including those lacking IL- Figure 3. (A) Diminished proliferation of freshly isolated splenocytes from Stat5b2/2 mice to 2 nM IL-2 or 7.75 nM IL-15. Shown are the mean 6 SD from a compilation of two independent experiments. (B) Flow cytometric analysis of CD32DX51 NK cells in splenocytes from wild type, Stat5a2/2, and Stat5b2/2 mice. (C–E) Decreased NK cytolytic activity in Stat5b2/2 mice in the presence of no cytokine (C), IL-2 (D), or IL-15 (E). Shown are the mean 6 SEM from combining two or three in- dependent experiments, each of which had three mice of each genotype. (F) Defective IL-2Rb expression in NK cells in Stat5b2/2 mice, as com- pared with levels in wild type or Stat5a2/2 mice. A total of 14 Stat5a2/2 and 17 Stat5b2/2 mice were examined. 2071 Imada et al. Figure 4. (A) Northern blotting for perforin or control pHe7 using RNA from fresh splenocytes or from splenocytes treated with IL-2 or IL- 15 for 90 h. (B) For each condition, the relative level of perforin mRNA expression in A was normalized for pHe7 expression.