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* State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, People’s Republic of China;
Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI 53226;
Department of Medicine, Case Western Reserve University, Cleveland, OH 44106;
Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; and
¶ Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Several important transcription factors, including PU.1, E2A, EBF, and Pax5, are essential for B cell lymphopoiesis that initiates following the commitment of the common lymphoid progenitors into earliest B cell progenitors (4, 5, 6, 7). PU.1 plays a critical role in the development of the common lymphoid progenitors (5, 6, 7) whereas E2A, EBF, and Pax5 are required for the development of the early B progenitors (4, 5). E2A functions upstream of EBF and both transcription factors coordinate to activate the expression of Pax5 (5, 6, 8, 9). Moreover, cytokines, including IL-7, play a critical role in B cell development (10, 11). IL-7 can promote proliferation, survival, and differentiation of B cell progenitors and regulate IgH gene rearrangement (12, 13, 14, 15). IL-7R deficiency impairs the expression of EBF and its target genes and results in arrest of B cell development at early pro-B and even the common lymphoid progenitor stage (13, 14, 15, 16). Although it is clear that IL-7 plays a crucial role in early B cell development, the molecular mechanism of its function remains unclear.
By transmitting signals from cytokine receptors to the nucleus and regulating cytokine-inducible gene expression, the Stat family of molecules is important for a variety of cytokine-mediated cellular responses, including cell growth, survival, differentiation, and function (17, 18, 19). There are seven mammalian Stat family members, including Stat1, 2, 3, 4, 5A, 5B, and 6, and each Stat member has unique functions in cytokine signaling (19). The two closely related Stat5 proteins (Stat5A and Stat5B) have been of particular interest because of the broad spectrum of hemopoietic cytokines and growth factors that induce their activation (19). Gene disruptions in mice have highlighted important functions for Stat5 proteins.
Disruption of Stat5A impairs prolactin-mediated functions in the lactating mammary gland (20, 21) whereas disruption of Stat5B blocks growth hormone-regulated functions in the liver (21, 22). Mice with both Stat5A and Stat5B alleles targeted at the N terminus of the proteins retain N-terminally truncated Stat5 proteins and are hence termed as Stat5A/5B
N. Studies of Stat5A/5BN mice have illustrated key roles for Stat5A and Stat5B in prolactin-regulated ovarian function (21), the repopulating potential of hemopoietic stem cells (23, 24, 25), IL-2-induced T cell proliferation (26), IL-3-mediated mast cell development and survival (27), and IL-5-driven differentiation of Th2-type eosinophils (28). IL-7 predominantly activates Stat5 (29). IL-7R consists of a ligand-specific subunit (IL-7R
) and a shared receptor subunit (common 
c) (30, 31, 32). Upon IL-7 binding, IL-7Rs aggregate and activate Jak1 and Jak3 (33). Activated Jaks phosphorylate Stat5 proteins, which in turn dimerize, translocate to the nucleus, and activate transcription of a variety of genes (17, 18, 19). Disruption of IL-7R, c, Jak1, or Jak3 severely impairs early lymphoid, including B cell, development (14, 34, 35, 36, 37, 38). Surprisingly, previous studies of Stat5A- or Stat5B-deficient or Stat5A/5BN mice do not detect marked defect in early development of lymphocytes (20, 21, 22, 26, 39, 40). The Stat5A/5BN mice only have slightly reduced BM B cell progenitors and peripheral B cells, suggesting a role of Stat5 in B cell development (21, 41, 42). The Stat5A/5BN mice have a normal number of thymocytes, although a marked reduction in thymocytes is observed during fetal development (24, 26, 43). It is puzzling that Stat5A/5BN mice have no severe impairment of IL-7-mediated early lymphocyte development.
Recently, new Stat5A/5B double-deficient mice with deletion of the entire Stat5A/5B locus have been developed and are called Stat5A/5B–/– (44). These new Stat5A/5B–/– mice are perinatal lethal, a more severe phenotype than the original Stat5A/5BN mice (44). Interestingly, most recent studies showed that these new mutant mice had severely impaired development of T and B cells (45, 46). Nonetheless, these studies did not exclude the possibility that the impaired lymphoid development was simply due to defective stem cells in the new mutant mice, as Stat5A/5B play a critical role in stem cell functions (23, 24, 25). In addition, the fact that Stat5A/5B–/– fetal liver cells failed to reconstitute T and B cells in partially irradiated Rag2–/– mice could be explained by the failure of Stat5A/5B–/– stem cells to compete with the remaining Rag2–/– stem cells to repopulate the recipients (45). In the current report, we demonstrated that Stat5A/5B directly plays a central role in IL-7-mediated early B cell development. Moreover, we discovered that Stat5A/5B are not required for late B cell maturation and functions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Stat5A/5B–/– mice and mice with the entire germline Stat5a/5b locus gene flanked with loxP sites (designated fl, flox allele) were as previously described (44). The Stat5A/5Bfl/+ mice were bred with Stat5A/5B+/– mice to generate Stat5A/5Bfl/– mice. Stat5A/5Bfl/– mice were bred with CD19Cre mice that express Cre under the control of the CD19 promoter (47) to generate Stat5A/5Bfl/–CD19Cre mice.
Retroviral transduction and fetal liver transplantation
The mouse Stat5A gene was cloned into a bicistronic retrovirus MSCV-IRES-GFP vector, which contains an internal ribosome entry site (IRES) downstream of the cloned gene and upstream of GFP (48). The expression of the cloned Stat5A and GFP is under control of the murine stem cell promoter (MSCV). GFP serves as a marker for the identification of retrovirally transduced cells. Conditioned medium containing high-titer, amphotropic retrovirus particles was derived by cotransfection of 293T cells with the MSCV-Stat5A-IRES-GFP retrovirus vector and a helper plasmid, pEQPAM3, containing the required gag, pol, and env retroviral genes. These media were filtered and used to transduce ecotropic packaging cells GP plus E86 with 6 µg/ml polybrene (Sigma-Aldrich) a total of six times over 3 days. The highest expressing GFP cells were sorted under sterile conditions and subsequently expanded as virus-producing cells.
Mouse fetal liver cells from 13- to 14-day-old wild-type or Stat5A/5B–/– embryos were prestimulated with 20 ng/ml mouse IL-3, 50 ng/ml human IL-6, and 50 ng/ml rat stem cell factor (SCF) for 2 days. Cells were then cocultured on irradiated ecotropic producer cells (GP plus E86) in the presence of IL-3, IL-6, SCF, and polybrene (6 µg/ml). After 2 days, 2–5 x 106 nucleated BM cells were injected via retro-orbital injection into lethally irradiated (1100 rad) recipient Jak3-deficient mice. Eight to 12 wk later, the recipients were analyzed.
For direct fetal liver transplantation, fetal liver cells were isolated from 13- to 14-day-old wild-type or Stat5A/5B–/– embryos and subsequently transplanted into lethally irradiated (1100 rad) Jak3–/– or CD45.1 wild-type recipient mice via retro-orbital injection (2 x 106 nucleated fetal liver cells/recipient). Eight to 12 wk following transplantation, the development of B cells in the BM and spleen of the recipients was examined.
Flow cytometry
Single-cell suspensions of spleen and BM cells were treated with Gey’s solution to remove RBC and resuspended in PBS supplemented with 2% BSA. The cells were then stained with a combination of fluorescence-conjugated Abs. PE-conjugated anti-Mac-1 (12-0112-82), PE-conjugated anti-Gr-1 (12-5931-82), PE-conjugated anti-Ter-119 (12-5921-83), PE-conjugated c-Kit (12-1171-83), allophycocyanin-conjugated IgM (17-5790-82), CyChrome-conjugated (15-0452-82) and PE-Cy7-conjugated (20-0452-82) anti-B220 were purchased from eBioscience. FITC-conjugated anti-CD45.2 (553772), PE-conjugated anti-NK1.1 (0129513), PE-conjugated anti-CD43 (553271), PE-conjugated anti-Thy1.2 (01005B), and FITC-conjugated anti-IgD (553439) were purchased from BD Biosciences Pharmingen. PE-conjugated anti-IgM (1140-09) was purchased from Southern Biotechnology Associates. All Abs were monoclonal. Samples were applied to a flow cytometer (LSRII; BD Biosciences) and data were collected and analyzed using CellQuest software (BD Biosciences).
Semiquantitative RT-PCR analysis
Total RNA was isolated from fetal liver cells derived from 12- to 14-day-old wild-type or Stat5A/5B–/– embryos or from BM cells derived from Jak3–/– recipients of wild-type or Stat5A/5B–/– fetal liver cells by RNAzol (Tel-Test). Synthesis of cDNA from the total RNA was conducted with the Omniscript RT kit (Qiagen). For semiquantitative PCR, serial dilutions of the cDNA templates were subjected to PCR amplification of indicated genes. Amplification of the 
-actin gene served as an input control of cDNA templates. The primers and the conditions for the PCR were described in Table I.
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Freshly isolated BM cells from Stat5A/5B+/– or Stat5A/5Bfl/– mice (8–10 wk old) were cultured in RPMI 1640 medium with 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 5 x 10–5 M 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine (all obtained from Invitrogen Life Technologies), 10% heat-inactivated FBS (HyClone) and IL-7 (2 ng/ml; R&D Systems) for 5 days. Subsequently, these cell progenitors were cocultured with the irradiated (1500 rad) bicistronic retrovirus MSCV-Cre-IRES-GFP-producing cells (GP plus E86), in which the expression of Cre recombinase and GFP is under control of the MSCV promoter, in the presence of IL-7 and polybrene (6 µg/ml) for 48 h. Subsequently, the cells were continuously cultured in IL-7-containing medium and at the indicated time points, the percentages of GFP+ cells were determined by FACS analysis.
Western blot analysis
BM B cells at different developmental stages were purified by FACS sorting. Splenic B and T cells were purified by using anti-B220 Ab-coated magnetic beads through procedure of positive and negative selections, respectively, according to the manufacturer’s instructions (Miltenyi Biotec). Cells were lysed in lysis buffer and centrifuged to remove debris as previously described (49). Cell lysates (20 µg) were subjected to SDS-PAGE and Western blot analysis with anti-Stat5A (50) or anti-actin (MAB1501; Chemicon International).
TUNEL assay
Splenocytes (1 x 106 cells/ml) from Stat5A/5B+/–CD19Cre or Stat5A/5Bfl/–CD19Cre mice (8–10 wk old) were stained with CyChrome-conjugated anti-B220 and PE-conjugated anti-Thy1.2 Abs. Then, cells were fixed, permeabilized, and labeled with FITC-conjugated TUNEL enzyme according to the manufacturer’s instructions (In Situ Cell Death Detection kit; Roche) and analyzed by FACS.
Cell cycle distribution
Splenic B cells were purified from Stat5A/5B+/–CD19Cre or Stat5A/5Bfl/–CD19Cre mice (8–10 wk old) by anti-B220-coated magnetic beads and then stimulated with anti-IgM (10 µg/ml) plus IL-4 (10 ng/ml). At the indicated time points, the cells were collected and resuspended at 1 x 106 cells/ml in propidium iodide solution (50 µg/ml propidium iodide, 0.1% sodium citrate, 0.1% Triton X-100, 15 mM NaCl, 10 mM Tris-HCl (pH 7.5), 200 µg/ml RNase) at room temperature for 30 min. DNA fluorescence of the cells was measured by FACS. The percentages of cells within the G1, S, and G2/M phases of the cell cycle were calculated by use of CellQuest software.
Measurement of IgG1 production in vitro and in vivo
IgG1 production in vitro: splenic B cells were purified from Stat5A/5B+/–CD19Cre or Stat5A/5Bfl/–CD19Cre mice (8–10 wk old) by anti-B220-coated magnetic beads. The purified cells were cultured at a density of 1 x 106 cells/well in 1 ml of RPMI 1640 with 10% FBS in the presence or absence of IL-4 (10 ng/ml) plus LPS (10 µg/ml). At the indicated time points, the percentages of IgG1-producing B cells were determined by FACS analysis.
IgG1 production in vivo: Stat5A/5B+/–CD19Cre or Stat5A/5Bfl/–CD19Cre mice (8–10 wk old) were immunized i.p. with 100 µg of NP16-CG (Biosearch Technologies) conjugates precipitated in alum. Sera were collected 10 days after immunization. Levels of Ag-specific IgG1 in the sera were measured by ELISA using either NP3-BSA or NP30-BSA as the plate-bound capture Ags as previously described (51).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Stat5A/5B play an important role in stem cell function. BM and fetal liver stem cells from the original Stat5A/5BN mice have severely impaired ability to repopulate irradiated recipients when compete with wild-type stem cells (23, 24, 25). Thus, the severe reduction of T and B cells observed in the new Stat5A/5B–/– fetuses and few surviving mice could be due to reduced numbers or function of stem cells (23, 24, 25, 45, 46). To address whether complete Stat5A/5B deficiency affects early lymphoid development, fetal liver cells from the new Stat5A/5B–/– fetuses were examined for the expression of a variety of genes associated with erythroid, myeloid and lymphoid progenitors. The expression of genes linked to hemopoietic stem cells and early progenitors of all hemopoietic lineages, including c-Kit (52), c-myb (53), PU.1 (54, 55), AML-1 (56), rhombotin-2 (RBTN) (57), and GATA-2 (58), was comparable between wild-type and Stat5A/5B–/– fetal liver cells (Fig. 1A). The expression of a number of genes characteristic of early erythroid progenitors, including the erythropoietin receptor (EpoR) (59), EKLF (60, 61), and GATA-1 (62), was also normal in Stat5A/5B–/– relative to wild-type fetal liver cells (Fig. 1A). In addition, expression of all the globin genes, including the predominant embryonic 
, 
, and h1 genes, the gene expressed in embryonic and definitive erythropoiesis, and the maj gene expressed in fetal liver-definitive erythropoiesis (63), was comparable between wild-type and Stat5A/5B–/– fetal liver cells (Fig. 1A). Of note, the expression of genes in myeloid cells, including c-Fes and G-CSFR, was reduced but detectable in Stat5A/5B–/– relative to wild-type fetal liver cells (Fig. 1A). By contrast, the expression of genes of very early T and B cell progenitors, including GATA-3 (64) and E2A (4, 5), was markedly decreased in Stat5A/5B–/– relative to wild-type fetal liver cells (Fig. 1A). Importantly, the expression of genes characteristic of early B cell progenitors, including EBF, Pax5, and Mb-1 (4, 5), was barely detectable in Stat5A/5B–/– fetal liver cells (Fig. 1A). Taken together, these data indicate that complete Stat5 deficiency specifically results in disappearance of lymphoid progenitors.
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Stat5A/5B–/– fetal liver cells could give rise to myeloid cells but not T and B cells in vivo
The original Stat5A/5BN stem cells have markedly decreased ability to compete with wild-type stem cells to repopulate irradiated recipients (23, 24, 25). Thus, the Stat5A/5B–/– stem cells might not be able to compete with the remaining Rag2-deficient stem cells, resulting in failure to reconstitute T and B cells in partially irradiated Rag2-deficient mice (45). To determine whether Stat5A/5B specifically play an essential role in lymphoid development in vivo, we examined the ability of the new Stat5A/5B–/– fetal liver cells to repopulate lethally, instead of partially, irradiated recipients. Fetal liver cells from 13- to 14-day old Stat5A/5B–/– or wild-type embryos of C57BL/6 genetic background (CD45.2) were transplanted into lethally irradiated CD45.1 congenic wild-type recipients. Eight to 12 wk after transplantation, the development of myeloid and lymphoid cells in the recipients was examined. BM cells from the recipients of Stat5A/5B–/– or wild-type fetal liver cells were mainly donor origin as they were predominantly CD45.2+ (Fig. 2A). Within the CD45.2+ cells, the recipients of Stat5A/5B–/– fetal liver cells had a marginal fraction of B220+ B cells (Fig. 2B), which were largely B220+CD43+IgM– B cell progenitors (Fig. 2C). In contrast, the recipients of wild-type fetal liver cells had marked portion of B220+ B cells (Fig. 2B), which contained early B220+CD43+IgM– and as well as late B220+CD43–IgM+ B cells (Fig. 2C). Of note, BM cells from the recipients of Stat5A/5B–/– or wild-type fetal liver cells had comparable populations of c-Kit+ hemopoietic progenitors, Mac-1+ or Gr-1+ myeloid cells, and Ter-119+ erythroid cells (Fig. 2, D–G).
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Stat5A/5B deficiency impairs IL-7 signaling
IL-7 is essential for early development of lymphocytes and predominantly activates Stat5 (10, 11, 29). However, complete Stat5A/5B deficiency leads to disappearance of early B cell progenitors (Fig. 1A and 4C), making it difficult to directly examine the role of Stat5A/5B in IL-7-mediated early B cell development. To facilitate direct analysis of the effect of Stat5A/5B deficiency on IL-7-mediated B cell progenitor expansion, we used mice with the entire germline Stat5a/5b locus gene flanked with loxp sites (designated fl, flox allele). The Stat5A/5Bfl/+ mice were bred with Stat5A/5B+/– mice to generate Stat5A/5Bfl/– mice that were normal, indicating normal expression and function of the floxed Stat5a/5b allele throughout development (data not shown). BM from Stat5A/5B+/– or Stat5A/5Bfl/– mice was cultured with IL-7 to generate B cell progenitors, and subsequently transfected with a bicistronic retrovirus MSCV-Cre-IRES-GFP, in which the expression of Cre recombinase and GFP is under control of the MSCV promoter. GFP serves as a marker for the identification of retrovirally transduced Cre-expressing cells. Two days after infection, the retrovirus was removed and the IL-7-responsive B cell progenitors were continuously cultured in IL-7. At the indicated time points, the cells were collected and the percentages of GFP+ cells were determined by FACS. Initially, the percentages of GFP+ IL-7-responsive Stat5A/5B+/– and Stat5A/5Bfl/– B cell progenitors were comparable, indicating that the retrovirus transfected both cells with equal efficiency (Fig. 4D, upper). Over time, the levels of GFP+ IL-7-responsive Stat5A/5B+/– cells remained relatively consistent whereas the levels of GFP+ IL-7-responsive Stat5A/5Bfl/– B cell progenitors gradually declined (Fig. 4D, upper). By 10 days after the retrovirus transfection, the level of GFP+ IL-7-responsive Stat5A/5Bfl/– cells was close to background levels (Fig. 4D, upper). In contrast, following transfection of control GFP alone retrovirus (MSCV-IRES-GFP), the levels of both GFP+ IL-7-responsive Stat5A/5B+/– and GFP+ IL-7-responsive Stat5A/5Bfl/– B cell progenitors steadily increased and remained equally high (Fig. 4D, lower). Thus, following transfection of the MSCV-Cre-IRES-GFP retrovirus, the nearly complete outgrowth of GFP– Stat5A/5Bfl/– B cells with nondeleted floxed Stat5A/5B indicates that deletion of Stat5A/5B by Cre rendered the B cell progenitors at a disadvantage for IL-7-mediated expansion. These data demonstrate that Stat5A/5B deficiency directly impairs IL-7-mediated B cell expansion.
Reconstitution of Stat5A in Stat5A/5B–/– fetal liver cells rescues B cell development in vivo
Importantly, the impaired early B cell development by complete deficiency of Stat5A/5B could potentially be due to that other genes responsible for the defects were affected during the targeted disruption of the entire Stat5A/5B locus. To exclude this possibility that previous studies did not rule out (45, 46), we assessed the ability of Stat5A to restore development of Stat5A/5B–/– B cells in vivo. We used a retrovirus-mediated gene transfer with fetal liver reconstitution strategy (48, 66). Stat5A/5B–/– fetal liver cells were infected in vitro with a retrovirus, MSCV-Stat5A-IRES-GFP, encoding Stat5A. As controls, Stat5A/5B–/– fetal liver cells transduced with a retrovirus, MSCV-IRES-GFP, encoding GFP alone served as a negative control, whereas wild-type fetal liver cells infected with MSCV-IRES-GFP served as a positive control. Subsequently, the retrovirally transduced fetal liver cells were transplanted into lethally irradiated Jak3-deficient mice, which have virtually no B cells and dramatically decreased T cells (35, 65). Virus-transduced cells were identified by expression of the GFP gene. GFP+ large myeloid cells were FACS sorted out from BM cells of the recipients and direct Western blot analysis of the cell lysates demonstrated that expression of Stat5A was reintroduced into Stat5A/5B–/– cells by the MSCV-Stat5A-IRES-GFP but not MSCV-IRES-GFP retrovirus in vivo (data not shown). As expected, BM derived from Jak3-deficient recipients that were transplanted with GFP-transduced Stat5A/5B–/– fetal liver cells had few detectable total lymphoid cells (data not shown) or GFP+ cells among lymphoid cells (Fig. 5A). In addition, BM from these recipients had barely detectable GFP+ B cells (Fig. 5B) but marked population of GFP+ nonlymphoid cells (Fig. 5C). In contrast, BM derived from Jak3-deficient recipients that were transplanted with Stat5A-transduced Stat5A/5B–/– fetal liver cells had obvious population of lymphoid cells (data not shown) and GFP+ lymphoid cells (Fig. 5A). BM from these recipients also displayed a marked portion of B cells among the GFP+ lymphoid cells (Fig. 5B) and an apparent portion of GFP+ nonlymphoid cells (Fig. 5C). Similarly, BM from control Jak3-deficient recipients that were transplanted with GFP-transduced wild-type fetal liver cells had abundant lymphoid cells (data not shown) and GFP+ lymphoid cells (Fig. 5A). As well, BM from these recipients had a great portion of B cells among the GFP+ lymphoid cells (Fig. 5B) and a large portion of GFP+ cells among nonlymphoid cells (Fig. 5C).
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B cell-specific deletion of Stat5A/5B impairs early B cell development but not B cell maturation and function
To further study the role of Stat5A/5B in B cell development and function and to determine effect of complete Stat5A/B deficiency on B cell development is B cell autonomous, we used Stat5A/5Bfl/–CD19Cre mice. These mice express the Cre recombinase under the control of the promoter of CD19, which begins to be active at the early pro-B stage. Deletion of Stat5A/B was examined by Western blot analysis of FACS-sorted B cells at different developmental stages. CD19Cre-mediated deletion of the floxed Stat5A/5B initiated at the pro-B cell stage and continued along with the B cell maturation (Fig. 6A). At the immature and mature B cell stages, the deletion of StatA/5B was almost complete (Fig. 6A). Moreover, CD19Cre-mediated deletion of the floxed Stat5A/5B maintained in the splenic B cells from Stat5A/5Bfl/–CD19Cre, relative to Stat5A/5B+/–CD19Cre, mice (Fig. 6A). In contrast, the splenic T cells from both Stat5A/5Bfl/–CD19Cre and Stat5A/5B+/–CD19Cre mice had comparable expression levels of Stat5 (Fig. 6A). Therefore, CD19Cre mediates deletion of the floxed Stat5A/5B in B cells but not T cells.
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Furthermore, we examined the effects of complete Stat5A/B deficiency on survival of splenic B cells during maturation. As Stat5A/5Bfl/–CD19Cre and Stat5A/5B+/–CD19Cre mice had comparable proportions of T1, T2, and FO B cells in the spleens (Fig. 6E), the degree of apoptotic cells in total splenic B220+ B cells from these mice was examined by the TUNEL assay. As shown in Fig. 7A, the splenic B220+ B cells from both Stat5A/5B+/–CD19Cre and Stat5A/5Bfl/–CD19Cre mice had comparable low levels of TUNEL-positive apoptotic cells. As a control, the degree of TUNEL-positive apoptotic Thy1.2+ splenic T cells was also comparable between Stat5A/5B+/–CD19Cre and Stat5A/5Bfl/–CD19Cre mice (Fig. 7A). Therefore, specific deletion of Stat5A/5B in B cell compartment has no effect on B cell survival in vivo.
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Lastly, Stat5A/5B have been implicated in Ig class switching and Ig production (67, 68, 69). Thus, we examined the role of Stat5A/5B in B cell function in terms of Ig production. Nonmanipulated Stat5A/5Bfl/–CD19Cre mice had normal levels of IgM, IgA, IgG1, IgG2b, and IgG3 and a slightly increased level of IgG2a in their sera compared with Stat5A/5B+/–CD19Cre controls (Fig. 7C). Moreover, splenic B cells from Stat5A/5Bfl/–CD19Cre mice switched normally to IgG1-positive cells, in terms of percentage of cells switching and kinetics of switching, upon stimulation with LPS plus IL-4 in vitro compared with the cells from Stat5A/5B+/–CD19Cre mice (Fig. 7D). More importantly, following immunization with Ag NP-CG, both Stat5A/5Bfl/–CD19Cre and Stat5A/5B+/–CD19Cre mice produced comparable levels of low-affinity and high-affinity NP-specific IgG1, detected by NP30-BSA and NP3-BSA, respectively (Fig. 7E). Therefore, these data demonstrate that lack of Stat5A/5B has little effect on B cell Ig class switching and Ig production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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N mice, which retain N-terminally truncated Stat5 proteins, have revealed the critical roles of Stat5A and Sta5B in prolactin-regulated ovarian function (21), hemopoietic stem cell repopulating potential (23, 24, 25), IL-2-induced T cell proliferation (26), IL-3-mediated mast cell development and survival (27), and IL-5-driven differentiation of Th2-type eosinophils (28). However, it is surprising that Stat5A/5BN mice only have minor disturbance of early B cell progenitors (21, 41, 42) and a normal number of thymocytes (24, 26, 43). These mice also have normal B cell proliferation, Ig production, and class switching (26, 41). It is well-known that IL-7 predominantly activates Stat5 (29) and is essential for early development of lymphocytes (11). Disruption of one of IL-7-signaling components, e.g., IL-7R, c, Jak1, or Jak3, severely impairs early lymphoid development (14, 34, 35, 36, 37, 38). The limited disturbance of lymphoid development in Stat5A/5BN mice has been attributed to substantial roles of Stat5-independent pathways in early lymphoid cell development. However, the other possible explanation for the lack of severe early lymphoid defects in Stat5A/5BN mice is that these mice retain N-terminally truncated Stat5 proteins, which might be partially functional. Stat5A/5BN mice were created by targeting the exon containing the initiation methionine, leaving a possibility of generating N-terminally truncated Stat5 proteins from downstream in-frame methionines (21). In fact, N-terminally truncated Stat5 proteins have been detected in the cells derived from Stat5A/5BN mice (45, 70). Consistent with the notion that the truncated Stat5 proteins might be partially functional, the new Stat5A/5B–/– mice have more severe phenotypes than those of the Stat5A/5BN mice (44, 45). Thus, it is necessary to re-examine lymphoid development and function in the new Stat5A/5B–/– mice. A recent study has reported that the new Stat5A/5B–/– mice have severely impaired development of T and B cells (45). However, this study does not exclude the possibility that the observed impairment of early lymphoid development is simply due to defective functions of stem cells in the Stat5A/5B–/– mice, as Stat5A/5B play a critical role in stem cell functions (23, 24, 25). The observed failure of Stat5A/5B–/– fetal liver cells to repopulate the lymphoid compartment in partially irradiated Rag2–/– recipients could be explained by the poor repopulating potential of Stat5A/5B–/– relative to Rag2–/– stem cells. Our current studies directly focus on the role of Stat5A/5B in B cell development and reveal that the severe diminution of B cell progenitors in Stat5A/5B–/– fetal liver cells is not simply due to impaired stem cell functions, as the mutant fetal liver cells clearly have myeloid progenitors. Stat5A/5B–/– fetal liver cells could give rise to hemopoietic progenitors and myeloid cells but not B cells beyond pro-B cell progenitors in lethally irradiated wild-type or Jak3–/– mice, which exclude the complication of the role of Stat5A/5B in stem cell repopulating potential. Moreover, deletion of Stat5A/5B in vitro directly impairs IL-7-mediated B cell expansion and CD19-Cre-mediated deletion of Stat5A/5B in B cell compartment specifically impairs early B cell development. Importantly, reintroduction of Stat5A back into Stat5A/5B–/– fetal liver cells restored their abilities to develop B cells, excluding the possibility that other genes responsible for the defects are affected during the targeted disruption of the Stat5A/5B genes. Taken together, our present studies clearly demonstrate that complete Stat5 deficiency directly blocks early B development at the pro-B cell stage, matching the phenotype of Il7r–/– mice (16). Therefore, Stat5 is a major component of IL-7 signals in terms of early B cell development.
The hemopoietic stem cells commit into the common lymphoid progenitors and transcription factor PU.1 is essential for the development of these progenitors (5, 6, 7). Subsequent differentiation of the common lymphoid progenitors into the early B progenitors requires the transcription factors E2A, EBF, and Pax5 (4, 5). E2A functions upstream of EBF, which coordinates with E2A to further activate the expression of Pax5 and other B cell-specific genes, including Ig, Ig, 
5, Vpre-B (5, 6, 8, 9). Importantly, EBF expression is up-regulated by IL-7 stimulation and deficiency of IL-7R ablates the expression of EBF and Pax5 but not PU.1 or E2A in pre-pro-B cells (16). Our present studies demonstrate that complete Stat5 deficiency slightly affects expression levels of PU.1 and E2A but abolishes expression of EBF and Pax5. Consistently, constitutively active Stat5 is able to restore expression of EBF as well as its downstream target genes, such as Pax5, Ig, and 5, in IL-7R-deficient pre-pro-B cells (16). In addition, Stat5 can synergize with EBF to directly up-regulate Pax5 transcription in early B cells via IL-7 (71, 72). Thus, Stat5 plays a central role in IL-7 signaling to activate EBF expression and its downstream target genes, including Pax5. Both EBF and Pax5 are essential for early B cell development and deficiency of EBF or Pax5 blocks B cell development at the pre-pro-B to pro-B transition stage (73, 74). Notably, Stat5A/5B–/– fetal liver cells express normal or detectable levels of the genes linked to hemopoietic stem cells, associated with early erythroid progenitors, or related to myeloid cells. Therefore, the complete dependence of Stat5A/5B for the expression of EBF and Pax5 might account for the absolute requirement of Stat5A/5B for early B cell development. Moreover, Stat5 controls early B cell proliferation by induction of cyclin D2 and early B cell survival by up-regulation of pim-1 and bcl-xL (75).
Following progression from pro-B cell stage, late B cells maintain abundant expression of Stat5. During late B cell development, cytokines, including IL-2 and IL-4, play importance roles in mature B cell proliferation and Ig production (67, 68, 75, 76). IL-2 predominantly activates Stat5 (19) and IL-2-induced T cell proliferation completely depends on Stat5 (26). IL-4 is able to activate Stat5, although it mainly activates Stat6 (19, 77). In addition, previous studies have reported that engagement of BCR activates Stat5 via Bruton’s tyrosine kinase (78). Thus, deletion of Stat5A/5B in B cells at late developmental stages should provide important insights into their roles in B cell maturation and function. CD19Cre-mediated deletion of the floxed Stat5A/5B initiated at pro-B cell stage and continued along the B cell maturation, resulting in nearly complete deletion of Stat5A/5B in the immature and mature B cells. The substantial presence of T1, T2, and mature B cells as well as their normal ratio in Stat5A/5Bfl/– CD19Cre mice demonstrate that Stat5A/5B are not required for B cell maturation and homeostasis of B cells at late developmental stages. In addition, Stat5A/5B are not required for the proliferation of mature B cells, as Stat5A/5Bfl/–CD19Cre mature B cells proliferate normally in response to IL-4 plus anti-IgM stimulation. Lastly, although Stat5A/5B are implicated in Ig class switching and Ig production (67, 68, 69), studies of Stat5A/5BN mice discover that Ig class switching toward IgG1 is normal (41). Our current findings clearly demonstrate that deficiency of Stat5A/5B has little effect on basal levels of Ig production or IgG1 production in vitro and in vivo by B cells. In conclusion, our present studies demonstrate Sta5A/5B are major components of IL-7 signaling in regulating early B cell development but are not required for B cell maturation and Ig production.
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1 This work was supported in part by National Institutes of Health Grants R01 DK059380 (to K.D.B.), R01 AI52327 (to R.W.), R01 HL073284 (to D.W.), and by American Cancer Society Grant RSG CCG-106204 (to D.W.). 
2 Address correspondence and reprint requests to Dr. Demin Wang, Blood Research Institute, 8727 Watertown Plank Road, Milwaukee, WI 53226. E-mail address: demin.wang{at}bcw.edu
3 Abbreviations used in this paper: BM, bone marrow; T1, transitional B cells of type 1; T2, transitional B cells of type 2; FO, follicular; IRES, internal ribosome entry site; MSCV, murine stem cell promoter; SCF, stem cell factor.
Received for publication February 16, 2007. Accepted for publication April 30, 2007.
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