ally showed a greater defect in growth in mice lacking both genes (22). Thus, Stat5a and Stat5b have distinctive roles but appear also to have partially redundant roles (22). Interest- ingly, the double knockout mice also exhibited a significant decrease in expression of Stat3 (which results in fetal lethality when expression is totally eliminated; reference 11), raising the possibility that diminished Stat3 levels contributed to some of the abnormalities in these mice, such as the dimin- ished response of bone marrow cells to G-CSF (22). In Stat5a2/2 mice, bone marrow–derived macrophages exhibit defective GM-CSF–induced proliferation and gene expression (23); moreover, these mice also exhibit several immunological defects, including reduced expansion of T cells in vivo (24). This latter defect is associated with di- minished IL-2–mediated induction of the IL-2Ra chain, although TCR-mediated IL-2Ra induction appears to be relatively normal; thus, impaired IL-2 signaling is believed to at least partially explain the defects in these mice. We have now analyzed the immunological properties in Stat5b2/2 mice and shown that they have a modest de- crease in both thymic and splenic cellularity. Moreover, Stat5b2/2 splenocytes exhibit a marked decrease in IL-2– mediated proliferation and a more moderate decrease in the number of NK cells. Although Stat5a2/2 mice also exhibit a partial decrease in the number of NK cells, only the Stat5b2/2 mice have a dramatic defect in NK cell function. The implications of these findings are discussed below. Materials and Methods Mice. The Stat5a2/2 (20) and Stat5b2/2 mice (21) have been described. These mice were backcrossed with C57BL/6 once, and then littermates were intercrossed for this study. All experi- ments were performed under protocols approved by National In- stitutes of Health Animal Use and Care Committee and followed the National Institutes of Health guidelines “Using Animals in Intramural Research.” Flow Cytometric Analysis. Single-cell suspensions from the thymus and spleen were stained and analyzed using a FACSort (Becton Dickinson, San Jose, CA) with CELLQuest software (Bec- ton Dickinson). The following antibodies were purchased from PharMingen (San Diego, CA): PE-conjugated anti–TCR-a/b (anti-TCR-a/b–PE; H57-597 to TCR-b); anti-TCR-g/d–FITC (GL3); anti-CD3e–allophycocyanin (145-2C11); anti-CD4–FITC, –PE, and –Cy-Chrome (RM4-5); anti-CD8–PE, –Cy-Chrome, and –allophycocyanin (53-6.7); anti-IL-2Ra (CD25)-FITC (7D4); anti-CD44–Cy-Chrome (IM7); anti-CD45R (B220)–PE (RA3- 6B2); anti-IL-2Rb–FITC and –PE (TM-b1); anti-Pan-NK cells– FITC (DX5); and anti-gc–PE (4G3). In some experiments, dead cells were excluded by staining with propidium iodide. RNA Analysis. To evaluate IL-2–induced IL-2Ra and IL-2Rb mRNA expression in activated lymphocytes, fresh splenocytes were stimulated in Falcon 3003 plates (Becton Dickinson) coated with 10 mg/ml anti-CD3e mAb (145-2C11) for 48 h, washed twice with PBS, and then cultured with or without 2 nM recombinant hu- man IL-2 (Hoffmann-La Roche, Nutley, NJ) for 10 h. RNA was extracted using Trizol (GIBCO BRL, Gaithersburg, MD), fractionated on a 0.8% formaldehyde/agarose gel, and transferred to nylon membranes. Blots were hybridized with 1.2 kb IL-2Ra, 1.7 kb IL-2Rb (24), or control probe (0.3 kb pHe7 cDNA; refer- ence 25) labeled with 32P using the Prime-It Rmt Kit (Stratagene, La Jolla, CA). To evaluate perforin mRNA expression, RNA was extracted from fresh splenocytes or splenocytes cultured for 90 h at 2 3 106/ml in RPMI 1640 medium containing 10% fetal bo- vine serum, 50 mM 2-ME, and either IL-2 (10 nM) or IL-15 (15.5 nM). The blot was sequentially hybridized with murine perforin (26) and pHe7 probes. Western Blotting. Whole cell extracts (15 mg/sample) were fractionated on 8% SDS polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Blots were processed as previously described (24). In brief, after blocking, blots were incubated with antisera specific for Stat5a or Stat5b (17), followed by the incubation with anti–rabbit IgG conjugated with horseradish peroxidase (Nycomed Amersham, Little Chal- font, Buckinghamshire, UK). Blots were washed and developed with an enhanced chemiluminescent substrate (Pierce Chemical Co., Rockford, IL). To examine Stat6 expression, anti-Stat6 anti- body (Transduction Laboratories, Lexington, KY) was used. Proliferation Assay. Fresh splenocytes (2 3 105 cells/well) were cultured in RPMI 1640 medium containing 10% fetal bo- vine serum, 2 mM l-glutamine, and antibiotics with or without 2 nM IL-2 or 100 ng/ml (7.75 nM) IL-15 (Peprotech, Rocky Hill, NJ) for 48 h and pulsed with 1 mCi of [3H]thymidine (6.7 Ci/mmol, NEN, Boston, MA) for the last 12 h of culture. For proliferation of anti-CD3-stimulated splenocytes, cells (105/well) were cultured for 24 h in the presence or absence of 1 nM IL-2 with a 1 mCi [3H]thymidine pulse for the last 10 h. Because IL-4 alone cannot potently stimulate splenocytes, we stimulated sple- nocytes with a combination of PMA (1 ng/ml) and murine IL-4 (1,000 U/ml) for 48 h and pulsed them with 1 mCi of [3H]thymi- dine for the last 12 h of culture in order to evaluate the ability of IL-4 to induce proliferation in Stat5-deficient mice. Cytotoxicity Assay. 51Cr-release assays were performed by standard methods in final volumes of 200 ml. YAC-1 (H-2A), an NK-sensitive cell, was used to assess NK activity with effector/ target ratios of 100, 50, 25, and 12.5:1 and 40, 10, 2.5, and 0.6:1, respectively. 10 nM IL-2 or 15.5 nM IL-15 were added to wells. Data are expressed as lytic units (LU) in 107 cells (1 LU corre- sponds to the number of effector cells lysing 20% of the targets) and obtained by fitting the titration curves to scale families of curves, as previously described (27). Results and Discussion The majority of mice lacking Stat5a have normal levels of Stat5b in thymus and spleen (24). Correspondingly, Stat5b2/2 mice express relatively normal levels of Stat5a. Thus, in both Stat5a2/2 and Stat5b2/2 mice, the loss of ex- pression of one Stat5 gene did not typically affect the ex- pression of the other Stat5 gene in either thymus or spleen, although occasional variations were seen (Fig. 1 A, refer- ence 24, and data not shown). Furthermore, both Stat5a2/2 (24) and Stat5b2/2 mice had normal levels of Stat3 (data not shown). We first analyzed lymphoid development in Stat5b2/2 mice. In these mice, thymocyte numbers were modestly diminished (P , 0.01; Fig. 1 B). Flow cytometric analysis of thymocytes from these mice revealed that all populations were represented, with almost normal numbers of CD4 and CD8 single positive and CD41CD81 double positive thymocytes (Fig. 1 C). However, among the double nega- 2068 Defective NK Function in Stat5b-deficient Mice