The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 1068 -1079
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dai, X.
Right arrow Articles by Wang, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dai, X.
Right arrow Articles by Wang, D.

Stat5 Is Essential for Early B Cell Development but Not for B Cell Maturation and Function1

Xuezhi Dai*,{dagger}, Yuhong Chen{dagger}, Lie Di{dagger}, Andrew Podd{dagger},¶, Geqiang Li{ddagger}, Kevin D. Bunting{ddagger}, Lothar Hennighausen§, Renren Wen{dagger} and Demin Wang2,*,{dagger},¶

* State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, People’s Republic of China; {dagger} Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI 53226; {ddagger} 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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The two closely related Stat5 (Stat5A and Stat5B) proteins are activated by a broad spectrum of cytokines. However, with the complication of the involvement of Stat5A/5B in stem cell function, the role of Stat5A/5B in the development and function of lymphocytes, especially B cells, is not fully understood. In this study, we demonstrated that Stat5A/5B–/– fetal liver cells had severe diminution of B cell progenitors but clearly had myeloid progenitors. Consistently, the mutant 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. Deletion of Stat5A/5B in vitro directly impaired IL-7-mediated B cell expansion. Of note, reintroduction of Stat5A back into Stat5A/5B–/– fetal liver cells restored their abilities to develop B cells. Importantly, CD19-Cre-mediated deletion of Stat5A/5B in the B cell compartment specifically impaired early B cell development but not late B cell maturation. Moreover, the B cell-specific deletion of Stat5A/5B did not impair splenic B cell survival, proliferation, and Ig production. Taken together, these data demonstrate that Stat5A/5B directly control IL-7-mediated early B cell development but are not required for B cell maturation and Ig production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
One central event for B cell development is rearrangement of the Ig H and L chain genes. Based on the sequential and proper rearrangements of the Ig H and L chain genes and the controlled expression of transcription factors and cell surface markers, B cell development can be defined into multiple stages (1, 2). The earliest B cell progenitors, pre-pro-B cells, maintain germline IgH and IgL loci whereas pro-B cells initiate IgH gene chain gene rearrangement. Successful rearrangement of IgH leads to the formation of the pre-BCR, which directs the expansion of pre-B cells (1, 2). Subsequently, the successfully rearranged L chain complexes with the H chain to generate the BCR, a hallmark of immature B cells (1, 2). Newly formed immature B cells from the bone marrow (BM)3 emerge into the spleen as transitional B cells of type 1 (T1), which develop into transitional B cells of type 2 (T2). Ultimately, T2 B cells give rise to long-lived mature follicular (FO) B cells (3).

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{Delta}N. Studies of Stat5A/5B{Delta}N 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{alpha}) and a shared receptor subunit (common {gamma}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{alpha}, {gamma}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/5B{Delta}N mice do not detect marked defect in early development of lymphocytes (20, 21, 22, 26, 39, 40). The Stat5A/5B{Delta}N 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/5B{Delta}N 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/5B{Delta}N 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/5B{Delta}N 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

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 beta-actin gene served as an input control of cDNA templates. The primers and the conditions for the PCR were described in Table I.


View this table:
[in this window]
[in a new window]

  		Table I. The primers and the conditions for the semiquantitative RT-PCRa

 
Cre-mediated deletion of Stat5A/5B in vitro

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).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Stat5A/5B–/– fetal liver cells lack lymphoid progenitors and fail to develop into B cells in vitro

Stat5A/5B play an important role in stem cell function. BM and fetal liver stem cells from the original Stat5A/5B{Delta}N 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 {epsilon}, {zeta}, and betah1 genes, the {alpha} gene expressed in embryonic and definitive erythropoiesis, and the betamaj 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.


Figure 1
View larger version (49K):
[in this window]
[in a new window]

  		FIGURE 1. Impairment of lymphoid progenitors in Stat5A/5B–/– fetal liver cells results in their inability to develop into B cells in vitro. A, Stat5A/5B–/– fetal liver cells have impaired expression of lymphoid, but not myeloid, lineage marker genes. Total RNA was isolated from fetal liver cells of 12- to 14-day-old Stat5A/5B+/+ or Stat5A/5B–/– embryos. Subsequently, the RNA was subjected to semiquantitative RT-PCR to detect the presence of a variety of the indicated genes that are associated with lymphoid or myeloid differentiation. B, Stat5A/5B–/– fetal liver cells fail to expand in response to IL-7. Fetal liver cells from 12- to 14-day-old Stat5A/5B+/+ or Stat5A/5B–/– embryos were cultured in medium supplemented with IL-7 or a cytokine mixture containing SCF, IL-3, and IL-6 for 5 days. Subsequently, the numbers of live cells were counted. C, Stat5A/5B–/– fetal liver cells fail to develop into B cells in response to IL-7. Fetal liver cells from 12- to 14-day-old Stat5A/5B+/+ or Stat5A/5B–/– embryos were cultured in medium supplemented with IL-7 for 5 days and subsequently subjected to FACS analyses with B220 staining. D, Stat5A/5B–/– fetal liver cells develop into myeloid lineage cells in response to a cytokine mixture containing SCF, IL-3, and IL-6. Fetal liver cells from 12- to 14-day-old Stat5A/5B+/+ or Stat5A/5B–/– embryos were cultured in medium supplemented with a cytokine mixture containing SCF, IL-3, and IL-6 for 5 days and subsequently subjected to FACS analyses with Gr-1 or Mac-1 staining. The figure shown is the representative of two (A) or three (B–D) independent experiments.

 
To confirm that Stat5A/5B–/– fetal liver cells lack lymphoid progenitors, we conducted the IL-7-induced expansion of B cell progenitors in the in vitro culture. After 5 days culture in IL-7, wild-type fetal liver cells expanded to a large number of cells that were predominantly B220+ B cell progenitors whereas Stat5A/5B–/– fetal liver cells yielded a few cells that were B220+ cells (Fig. 1, B and C). In contrast, both wild-type and Stat5A/5B–/– fetal liver cells gave rise to large number of cells that were either Gr-1+ or Mac+ myeloid cells after 5 days culture in IL-3, IL-6, and SCF (Fig. 1, B and D). Therefore, Stat5A/5B–/– fetal liver cells had myeloid progenitors but reduced or impaired IL-7-responsive B cell progenitors.

Stat5A/5B–/– fetal liver cells could give rise to myeloid cells but not T and B cells in vivo

The original Stat5A/5B{Delta}N 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).


Figure 2
View larger version (45K):
[in this window]
[in a new window]

  		FIGURE 2. BM derived from lethally irradiated wild-type recipient mice of Stat5A/5B–/– fetal liver cells have normal myelopoiesis but impaired B cell lymphopoiesis. 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 10 wk after transplantation, BM cells from the recipients were stained with a combination of Abs to CD45.2, B220, CD43, and IgM, to CD45.2 and c-Kit, to CD45.2 and Mac-1, CD45.2 and Gr-1, or to CD45.2 and Ter-119. A, FACS analysis with CD45.2 staining. Histograms show the percentages of CD45.2+ cells within the total live BM cells. B, FACS analysis with B220 staining of CD45.2+ gated cells. Histograms show the percentages of B220+ cells within the CD45.2+ gated cells. C, FACS analysis with CD43 and IgM staining of CD45.2+B220+ gated cells. The percentages of cells in the CD45.2+B220+ gated cells are indicated. D, FACS analysis with c-Kit staining of CD45.2+ gated cells. Histograms show the percentages of c-Kit+ cells within the CD45.2+ gated cells. E, FACS analysis with Mac-1 staining of CD45.2+ gated cells. Histograms show the percentages of Mac-1+ cells within the CD45.2+ gated cells. F, FACS analysis with Gr-1 staining of CD45.2+ gated cells. Histograms show the percentages of Gr-1+ cells within the CD45.2+ gated cells. G, FACS analysis with Ter-119 staining of CD45.2+ gated cells. Histograms show the percentages of Ter-119+ cells within the CD45.2+ gated cells. The figure shown is representative of three independent analyses.

 
Moreover, the proportion of CD45.2+ donor origin cells in the spleens from the recipients of Stat5A/5B–/–, relative to wild-type fetal liver cells, was markedly reduced (Fig. 3A). Within the CD45.2+ splenocytes, the recipients of Stat5A/5B–/– relative to wild-type fetal liver cells had even fewer fractions of B220+ B cells, Thy1.2+ T cells, and NK1.1+ NK cells (Fig. 3, B–D). In contrast, proportions of c-Kit+ hemopoietic progenitors and Mac-1+ or Gr-1+ myeloid cells were increased whereas proportions of Ter-119+ erythroid cells were decreased in the CD45.2+ splenocytes from the recipients of Stat5A/5B–/– relative to wild-type fetal liver cells (Fig. 3, E–H). Importantly, the total numbers of CD45.2+ cells were dramatically decreased and the absolute numbers of CD45.2+ B, T, and NK cells were barely detectable in the spleens from the recipients of Stat5A/5B–/– relative to wild-type fetal liver cells (Fig. 3I). In contrast, the absolute numbers of the donor-derived (CD45.2+) c-Kit+ hemopoietic progenitors, Mac-1+ or Gr-1+ myeloid cells, and Ter-119+ erythroid cells were decreased but sizeable in the spleens from the recipients of Stat5A/5B–/– relative to wild-type fetal liver cells (Fig. 3J). Taken together, these data demonstrate that complete Stat5 deficiency affects myeloid development but completely blocks early B and possible T cell development in vivo.


Figure 3
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 3. The spleens derived from lethally irradiated wild-type recipient mice of Stat5A/5B–/– fetal liver cells have impaired myelopoiesis and barely detectable lymphopoiesis. 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 10 wk after transplantation, splenic cells from the recipients were stained with a combination of Ab to CD45.2 and Ab to B220, Thy1.2, NK1.1, c-Kit, Mac-1, Gr-1, or Ter-119. A, FACS analysis with CD45.2 staining. Histograms show the percentages of CD45.2+ cells within the total live splenic cells. B, FACS analysis with B220 staining of CD45.2+ gated lymphoid cells. Histograms show the percentages of B220+ cells within the CD45.2+ gated lymphoid cells. C, FACS analysis with Thy1.2 staining of CD45.2+ gated lymphoid cells. The percentages of cells in the CD45.2+ gated lymphoid cells are indicated. D, FACS analysis with NK1.1 staining of CD45.2+ gated lymphoid cells. Histograms show the percentages of NK1.1+ cells within the CD45.2+ gated lymphoid cells. E, FACS analysis with c-Kit staining of CD45.2+ gated cells. Histograms show the percentages of c-Kit+ cells within the CD45.2+ gated cells. F, FACS analysis with Mac-1 staining of CD45.2+ gated cells. Histograms show the percentages of Mac-1+ cells within the CD45.2+ gated cells. G, FACS analysis with Gr-1 staining of CD45.2+ gated cells. Histograms show the percentages of Gr-1+ cells within the CD45.2+ gated cells. H, FACS analysis with Ter-119 staining of CD45.2+ gated cells. Histograms show the percentages of Ter-119+ cells within the CD45.2+ gated cells. I, The total numbers of CD45.2+ cells as well as the absolute numbers of CD45.2+B220+, CD45.2+Thy1.2+, and CD45.2+NK1.1+ cells in the spleens from the recipients of wild-type or Stat5A/5B–/– fetal liver cells. J, The absolute numbers of CD45.2+Mac-1+, CD45.2+Gr-1+, CD45.2+Ter-119+, and CD45.2+c-Kit+ cells in the spleens from the recipients of wild-type or Stat5A/5B–/– fetal liver cells. The figure shown is representative of three independent analyses.

 
To further study role of Stat5 in lymphoid development, we also transplanted fetal liver cells from 13- to 14-day old Stat5A/5B-deficient or wild-type embryos into lethally irradiated B cell-null and T cell-few Jak3-deficient mice (35, 65). The lymphocytes, especially B cells, which developed in the recipients were derived from donor fetal liver cells and were analyzed 8–10 wk after transplantation. In BM, the recipients of wild-type fetal liver cells had marked B220+ B cells whereas the recipients of Stat5A/5B–/– fetal liver cells or the Jak3-deficient mice without transplantation contained barely detectable B cells (Fig. 4A, upper). The marginal BM B cells in the recipient of Stat5A/5B–/– fetal liver cells were mainly B220+CD43+ B cell progenitors, similar to those of the Jak3-deficient mice without transplantation (Fig. 4A, lower). In addition, wild-type fetal liver cells reconstituted B cells in the spleens of the recipients (Fig. 4B). In contrast, the recipients of Stat5A/5B–/– fetal liver cells had few B cells in the spleens as the Jak3-deficient mice without transplantation (Fig. 4B).


Figure 4
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 4. The lethally irradiated Jak3–/– recipient mice of Stat5A/5B–/– fetal liver cells have severely impaired B cell lymphopoiesis. Lethally irradiated Jak3–/– mice were transplanted with fetal liver cells from 13- to 14-day-old wild-type (Jak3–/– + Stat5A/5B+/+) or Stat5A/5B–/– (Jak3–/– + Stat5A/5B–/–) embryos. Eight to 10 wk after transplantation, B cell development was analyzed in the recipients. Jak3–/– mice without transplantation served as a negative control. A, FACS analysis with B220 and CD43 staining of BM cells of the recipients. Upper panels, Histograms that show the percentages of B220+ cells within the total live BM cells. Lower panels, The percentages of CD43+ cells within the B220+ gated cells. The figure shown is the representative of four independent analyses. B, FACS analysis with B220 and Thy1.2 staining of splenocytes of the recipients. The percentages of cells in the live cell population are indicated. The figure shown is the representative of four independent analyses. C, BM cells from Jak3–/– recipients of Stat5A/5B–/– fetal liver cells have impaired expression of B cell, but not hemopoietic, lineage marker genes. Total RNA was isolated from BM of Jak3–/– recipients of 13- to 14-day-old Stat5A/5B+/+ or Stat5A/5B–/– embryos and subjected to semiquantitative RT-PCR to detect the presence of the indicated genes. D, Cre-mediated deletion of Stat5A/5B directly impairs IL-7-driven B cell expansion in vitro. BM cells from Stat5A/5B+/– or Stat5A/5Bfl/– mice were cultured in IL-7-containing medium for 5 days and then transfected with a bicistronic retrovirus MSCV-Cre-IRES-GFP encoding Cre (Cre) (upper) or a control retrovirus MSCV-IRES-GFP encoding GFP alone (GFP) (lower). Two days after infection, the retrovirus was removed and the cells were continuously cultured in IL-7. At indicated time points, the cells were collected and the percentages of GFP+ live cells were determined by FACS. Presented are the combined results of three independent analyses.

 
To further confirm that complete Stat5 deficiency blocks B cell development in the BM environment, we examined the expression of several genes associated with hemopoietic and B cell progenitors in the BM of the recipients. The expression of genes linked to early progenitors of all hemopoietic lineages, including PU.1 (54, 55) and AML-1 (56), and the expression of genes associated with very early B cell progenitors, like E2A (4, 5), were comparable between the BM from the recipients of wild-type and Stat5A/5B-deficient fetal liver cells (Fig. 4C). In contrast, the expression of genes characteristic of early B cell progenitors, including EBF and Pax5 (4, 5), was barely detectable in the BM from the recipients of Stat5A/5B-deficient relative to wild-type fetal liver cells (Fig. 4C). Again, these data demonstrate that complete Stat5 deficiency blocks early B development, resulting in disappearance of early B cell progenitors in vivo.

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).


Figure 5
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 5. Reconstitution of Stat5A restores development of Stat5A/5B–/– B cells in vivo. Lethally irradiated JAK3–/– recipients were transplanted with Stat5A/5B–/– fetal liver cells transfected with a retrovirus MSCV-Stat5A-IRES-GFP encoding Stat5A (Stat5A/5B–/– + Stat5A) or a retrovirus MSCV-IRES-GFP encoding GFP alone (Stat5A/5B–/– + GFP) or wild-type fetal liver cells transfected with a retrovirus MSCV-IRES-GFP (Stat5A/5B+/+ + GFP). Eight to 10 wk after the reconstitution, BM cells and splenocytes derived from the recipients were analyzed. A, FACS analysis of GFP+ BM lymphoid cells. Histograms show the percentage of GFP+ cells within the gated lymphoid population. B, FACS analysis with B220 staining of GFP+ gated BM lymphoid cells. Histograms show the percentage of B220+ cells within the GFP+ gated BM lymphoid cells. C, FACS analysis of GFP+ BM nonlymphoid cells. Histograms show the percentage of GFP+ cells within the gated large nonlymphoid population. D, FACS analysis of GFP+ splenic lymphoid cells. Histograms show the percentage of GFP+ cells within the gated lymphoid population. E, FACS analysis with B220 and Thy1.2 staining of GFP+ splenic lymphoid cells. The percentages of cells in the gated GFP+ lymphoid population are indicated. The figure shown is representative of two independent analyses.

 
Moreover, the spleen derived from Jak3-deficient recipients that were transplanted with GFP-transduced Stat5A/5B–/– fetal liver cells exhibited hardly detectable lymphoid cells, GFP+ lymphoid cells, or T or B cells among the GFP+ lymphoid cells (data not shown and Fig. 5, D and E). In contrast, the spleen derived from Jak3-deficient recipients that were transplanted with Stat5A-transduced Stat5A/5B–/– fetal liver cells had easily detectable lymphoid cells (data not shown) and GFP+ lymphoid cells, which consisted of B and T cells (Fig. 5, D and E). As positive control, the spleen from Jak3-deficient recipients that were transplanted with GFP-transduced wild-type fetal liver cells had plentiful lymphoid cells and GFP+ lymphoid cells, which consisted of B and T cells (data not shown and Fig. 5, D and E). Taken together, these results demonstrate that reconstitution of Stat5A rescues development of Stat5A/5B-deficient B cells. Thus, Stat5A/5B deficiency directly disrupts B cell development.

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.


Figure 6
View larger version (47K):
[in this window]
[in a new window]

  		FIGURE 6. Deficiency of Stat5A/5B impairs early B cell development but not B cell maturation. A, Deletion of Stat5A/B during B cell development in Stat5A/5Bfl/–CD19Cre mice. Pro-B (B220+CD43+IgM–), pre-B (B220+CD43–IgM–), immature (B220+IgM+) and mature (B220highIgM+) B cells were FACS sorted from BM of Stat5A/5B+/–CD19Cre (+/–) or Stat5A/5Bfl/–CD19Cre (fl/–) mice whereas splenic T and B cells were purified by anti-B220-coated magnetic beads through positive and negative selections from the splenocytes of these mice. Total cell lysates were subjected to direct Western blot analysis with anti-Stat5A ({alpha}-Stat5a) or anti-actin ({alpha}-Actin) Abs. B, Reduction of B220+ B cells in BM of Stat5A/5Bfl/–CD19Cre mice. FACS analysis with B220 staining of BM cells from Stat5A/5B+/–CD19Cre or Stat5A/5Bfl/–CD19Cre mice. Histograms show the percentage of B220+ cells within the gated lymphoid cells. C, Reduction of mature B cells and increase of pro-B cells in BM of Stat5A/5Bfl/–CD19Cre mice. BM cells from Stat5A/5B+/–CD19Cre or Stat5A/5Bfl/–CD19Cre mice were FACS analyzed with B220, CD43, and IgM staining. The percentages of cells in the gated B220+ lymphoid population are indicated. D, Reduction of B cells in the spleen of Stat5A/5Bfl/–CD19Cre mice. Splenocytes from Stat5A/5B+/–CD19Cre or Stat5A/5Bfl/–CD19Cre mice were FACS analyzed with B220 and Thy1.2. The percentages of cells in the gated lymphoid population are indicated. E, Normal maturation of B cells in the spleen of Stat5A/5Bfl/–CD19Cre mice. Splenocytes from Stat5A/5B+/–CD19Cre or Stat5A/5Bfl/–CD19Cre mice were FACS analyzed with B220, IgD, and IgM staining. The percentages of cells in the gated B220+ lymphoid population are indicated. The figure shown is the representative of three (A) to five (B–E) independent analyses.

 
BM had a decrease of total B220+ B cells in Stat5A/5Bfl/–CD19Cre relative to Stat5A/5B+/–CD19Cre mice (Fig. 6B). Detailed FACS analysis revealed that, compared with Stat5A/5B+/–CD19Cre mice, Stat5A/5Bfl/–CD19Cre mouse BM had a reduction in B220highIgM+ mature B cells and an increase in B220+IgM– B cell progenitors (Fig. 6C, upper), which was mainly CD43+IgM– pro-B cells (Fig. 6C, lower). Moreover, the spleen of Stat5A/5Bfl/–CD19Cre mice also had an evident reduction in the population of B220+ B cells, compared with that of Stat5A/5B+/–CD19Cre mice (Fig. 6D). This observation is consistent with the finding of reduced mature B cells in BM. However, the proportions of IgM+IgD– T1, IgM+IgD+ T2, and IgMlowIgD+ FO B cells were normal in Stat5A/5Bfl/–CD19Cre relative to Stat5A/5B+/–CD19Cre mice (Fig. 6E). Taken together, these data demonstrate that specific deletion of Stat5A/5B in the B cell compartment impairs early B cell development but not B cell maturation.

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.


Figure 7
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 7. Deficiency of Stat5A/5B does not affect B cell apoptosis, cell cycle entry or Ig production. A, Normal cell apoptosis in Stat5A/5Bfl/–CD19Cre splenic B cells. Freshly isolated splenocytes from Stat5A/5Bfl/–CD19Cre and Stat5A/5B+/–CD19Cre mice were stained with fluorescence-conjugated anti-B220 and anti-Thy1.2 Abs and subjected to the TUNEL assays. Subsequently, the degrees of TUNEL positive in B220+ B cells (upper) and Thy1.2+ T cells (lower) were determined by FACS analysis. The figure shown is representative of three independent analyses. B, Normal B cell cycle entry of Stat5A/5Bfl/–CD19Cre splenic B cells upon anti-IgM plus IL-4 stimulation. Splenic B cells were isolated from Stat5A/5Bfl/–CD19Cre and Stat5A/5B+/–CD19Cre mice and were stimulated with anti-IgM plus IL-4. Subsequently, cells were collected, stained with propidium iodide, and analyzed for their position in cell cycle by FACS. The percentage of cells in G0/G1, S, and G2/M were indicated in each panel. The figure shown is representative of three independent analyses. C, Largely normal serum Ig levels in Stat5A/5Bfl/–CD19Cre mice. Sera were collected from naive Stat5A/5Bfl/–CD19Cre and Stat5A/5B+/–CD19Cre mice. Then, serum Ig isotype level in each mouse was determined by ELISA (upper). The mean value and SD of the serum Ig levels in Stat5A/5Bfl/–CD19Cre and Stat5A/5B+/–CD19Cre mice were calculated and statistical analysis was performed (lower); (n = 8 for Stat5A/5Bfl/–CD19Cre mice and n = 6 for Stat5A/5B+/–CD19Cre mice; *, p < 0.05). D, Stat5A/5Bfl/–CD19Cre splenic B cells have normal IgG1 class switch in vitro. Splenic B cells were isolated from Stat5A/5Bfl/–CD19Cre and Stat5A/5B+/–CD19Cre mice and stimulated with LPS plus IL-4 for the indicated times. Then, the cells were collected and IgG1-positive cells were determined by FACS analysis. The figure shown is representative of three independent analyses. E, Stat5A/5Bfl/–CD19Cre mice produce normal levels of Ag-specific high- and low-affinity IgG1 following immunization. Stat5A/5Bfl/–CD19Cre and Stat5A/5B+/–CD19Cre mice were immunized with soluble Ag NP-CG. Ten days after immunization, the titer of high- and low-affinity anti-NP IgG1 in sera were measured by ELISA using the capture Ag NP3-BSA (left) and NP30-BSA (right), respectively.

 
In addition, we examined the effect of complete Stat5A/B deficiency on B cell proliferation. Splenic B cells from Stat5A/5Bfl/–CD19Cre and Stat5A/5B+/–CD19Cre mice were stimulated with anti-IgM plus IL-4 and subsequently, the cell cycle distribution of these B cells was examined. As shown in Fig. 7B, splenic B cells from Stat5A/5Bfl/–CD19Cre mice entered the cell cycle the same as cells from normal Stat5A/5B+/–CD19Cre mice upon stimulation. Therefore, specific deletion of Stat5A/5B in the B cell compartment has no effect on B cell proliferation following anti-IgM plus IL-4 stimulation.

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.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Stat5A and Stat5B are activated by a broad spectrum of hemopoietic cytokines and growth factors, including prolactin, erythropoietin, IL-2, IL-3, IL-5, and IL-7 (19). Gene disruptions of Stat5A and Stat5B in mice have highlighted the important functions of Stat5A and Stat5B in cytokine signaling (19). Studies of the Stat5A/5B{Delta}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/5B{Delta}N 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{alpha}, {gamma}c, Jak1, or Jak3, severely impairs early lymphoid development (14, 34, 35, 36, 37, 38). The limited disturbance of lymphoid development in Stat5A/5B{Delta}N 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/5B{Delta}N mice is that these mice retain N-terminally truncated Stat5 proteins, which might be partially functional. Stat5A/5B{Delta}N 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/5B{Delta}N 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/5B{Delta}N 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{alpha}, Igbeta, {lambda}5, Vpre-B (5, 6, 8, 9). Importantly, EBF expression is up-regulated by IL-7 stimulation and deficiency of IL-7R{alpha} 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{alpha}, and {lambda}5, in IL-7R{alpha}-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/5B{Delta}N 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.


   Acknowledgments
 
We gratefully acknowledge the technical support of Shoua Yang and Guoping Fu.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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.). Back

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 Back

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. Back

Received for publication February 16, 2007. Accepted for publication April 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Healy, J. I., C. C. Goodnow. 1998. Positive versus negative signaling by lymphocyte antigen receptors. Annu. Rev. Immunol. 16: 645-670. [Medline]
  2. Hardy, R. R., K. Hayakawa. 2001. B cell development pathways. Annu. Rev. Immunol. 19: 595-621. [Medline]
  3. Loder, F., B. Mutschler, R. J. Ray, C. J. Paige, P. Sideras, R. Torres, M. C. Lamers, R. Carsetti. 1999. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J. Exp. Med. 190: 75-89. [Abstract/Free Full Text]
  4. Busslinger, M., S. L. Nutt, A. G. Rolink. 2000. Lineage commitment in lymphopoiesis. Curr. Opin. Immunol. 12: 151-158. [Medline]
  5. Busslinger, M.. 2004. Transcriptional control of early B cell development. Annu. Rev. Immunol. 22: 55-79. [Medline]
  6. Quong, M. W., W. J. Romanow, C. Murre. 2002. E protein function in lymphocyte development. Annu. Rev. Immunol. 20: 301-322. [Medline]
  7. Kondo, M., I. L. Weissman, K. Akashi. 1997. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91: 661-672. [Medline]
  8. Sigvardsson, M., M. O’Riordan, R. Grosschedl. 1997. EBF and E47 collaborate to induce expression of the endogenous immunoglobulin surrogate light chain genes. Immunity 7: 25-36. [Medline]
  9. O’Riordan, M., R. Grosschedl. 1999. Coordinate regulation of B cell differentiation by the transcription factors EBF and E2A. Immunity 11: 21-31. [Medline]
  10. von Freeden-Jeffry, U., P. Vieira, L. A. Lucian, T. McNeil, S. E. Burdach, R. Murray. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181: 1519-1526. [Abstract/Free Full Text]
  11. Khaled, A. R., S. K. Durum. 2002. Lymphocide: cytokines and the control of lymphoid homeostasis. Nat. Rev. Immunol. 2: 817-830. [Medline]
  12. Hardy, R. R.. 2003. B-cell commitment: deciding on the players. Curr. Opin. Immunol. 15: 158-165. [Medline]
  13. Corcoran, A. E., A. Riddell, D. Krooshoop, A. R. Venkitaraman. 1998. Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor. Nature 391: 904-907. [Medline]
  14. Peschon, J. J., P. J. Morrissey, K. H. Grabstein, F. J. Ramsdell, E. Maraskovsky, B. C. Gliniak, L. S. Park, S. F. Ziegler, D. E. Williams, C. B. Ware, et al 1994. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180: 1955-1960. [Abstract/Free Full Text]
  15. Miller, J. P., D. Izon, W. DeMuth, R. Gerstein, A. Bhandoola, D. Allman. 2002. The earliest step in B lineage differentiation from common lymphoid progenitors is critically dependent upon interleukin 7. J. Exp. Med. 196: 705-711. [Abstract/Free Full Text]
  16. Kikuchi, K., A. Y. Lai, C.-L. Hsu, M. Kondo. 2005. IL-7 receptor signaling is necessary for stage transition in adult B cell development through up-regulation of EBF. J. Exp. Med. 201: 1197-1203. [Abstract/Free Full Text]
  17. Darnell, J. E., Jr. 1997. STATs and gene regulation. Science 277: 1630-1635. [Abstract/Free Full Text]
  18. Ihle, J. N.. 1996. STATs: signal transducers and activators of transcription. Cell 84: 331-334. [Medline]
  19. Ihle, J. N.. 2001. The Stat family in cytokine signaling. Curr. Opin. Cell. Biol. 13: 211-217. [Medline]
  20. Liu, X., G. W. Robinson, K. U. Wagner, L. Garrett, A. Wynshaw-Boris, L. Hennighausen. 1997. Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev. 11: 179-186. [Abstract/Free Full Text]
  21. Teglund, S., C. McKay, E. Schuetz, J. M. van Deursen, D. Stravopodis, D. Wang, M. Brown, S. Bodner, G. Grosveld, J. N. Ihle. 1998. Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell 93: 841-850. [Medline]
  22. Udy, G. B., R. P. Towers, R. G. Snell, R. J. Wilkins, S. H. Park, P. A. Ram, D. J. Waxman, H. W. Davey. 1997. Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc. Natl. Acad. Sci. USA 94: 7239-7244. [Abstract/Free Full Text]
  23. Bradley, H. L., T. S. Hawley, K. D. Bunting. 2002. Cell intrinsic defects in cytokine responsiveness of STAT5-deficient hematopoietic stem cells. Blood 100: 3983-3989. [Abstract/Free Full Text]
  24. Bunting, K. D., H. L. Bradley, T. S. Hawley, R. Moriggl, B. P. Sorrentino, J. N. Ihle. 2002. Reduced lymphomyeloid repopulating activity from adult bone marrow and fetal liver of mice lacking expression of STAT5. Blood 99: 479-487. [Abstract/Free Full Text]
  25. Snow, J. W., N. Abraham, M. C. Ma, N. W. Abbey, B. Herndier, M. A. Goldsmith. 2002. STAT5 promotes multilineage hematolymphoid development in vivo through effects on early hematopoietic progenitor cells. Blood 99: 95-101. [Abstract/Free Full Text]
  26. Moriggl, R., D. J. Topham, S. Teglund, V. Sexl, C. McKay, D. Wang, A. Hoffmeyer, J. van Deursen, M. Y. Sangster, K. D. Bunting, et al 1999. Stat5 is required for IL-2-induced cell cycle progression of peripheral T cells. Immunity 10: 249-259. [Medline]
  27. Shelburne, C. P., M. E. McCoy, R. Piekorz, V. Sexl, K. H. Roh, S. M. Jacobs-Helber, S. R. Gillespie, D. P. Bailey, P. Mirmonsef, M. N. Mann, et al 2003. Stat5 expression is critical for mast cell development and survival. Blood 102: 1290-1297. [Abstract/Free Full Text]
  28. Zhu, Y., L. Chen, Z. Huang, S. Alkan, K. D. Bunting, R. Wen, D. Wang, H. Huang. 2004. Cutting edge: IL-5 primes Th2 cytokine-producing capacity in eosinophils through a STAT5-dependent mechanism. J. Immunol. 173: 2918-2922. [Abstract/Free Full Text]
  29. Lin, J. X., T. S. Migone, M. Tsang, M. Friedmann, J. A. Weatherbee, L. Zhou, A. Yamauchi, E. T. Bloom, J. Mietz, S. John. 1995. The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. Immunity 2: 331-339. [Medline]
  30. Park, L. S., D. J. Friend, A. E. Schmierer, S. K. Dower, A. E. Namen. 1990. Murine interleukin 7 (IL-7) receptor: characterization on an IL-7-dependent cell line. J. Exp. Med. 171: 1073-1089. [Abstract/Free Full Text]
  31. Kondo, M., T. Takeshita, N. Ishii, M. Nakamura, S. Watanabe, K. Arai, K. Sugamura. 1993. Sharing of the interleukin-2 (IL-2) receptor {gamma} chain between receptors for IL-2 and IL-4. Science 262: 1874-1877. [Abstract/Free Full Text]
  32. Noguchi, M., Y. Nakamura, S. M. Russell, S. F. Ziegler, M. Tsang, X. Cao, W. J. Leonard. 1993. Interleukin-2 receptor {gamma} chain: a functional component of the interleukin-7 receptor. Science 262: 1877-1880. [Abstract/Free Full Text]
  33. Russell, S. M., J. A. Johnston, M. Noguchi, M. Kawamura, C. M. Bacon, M. Friedmann, M. Berg, D. W. McVicar, B. A. Witthuhn, O. Silvennoinen, et al 1994. Interaction of IL-2R beta and {gamma}c chains with Jak1 and Jak3: implications for XSCID and XCID. Science 266: 1042-1045. [Abstract/Free Full Text]
  34. Noguchi, M., H. Yi, H. M. Rosenblatt, A. H. Filipovich, S. Adelstein, W. S. Modi, O. W. McBride, W. J. Leonard. 1993. Interleukin-2 receptor {gamma} chain mutation results in X-linked severe combined immunodeficiency in humans. Cell 73: 147-157. [Medline]
  35. Nosaka, T., J. M. van Deursen, R. A. Tripp, W. E. Thierfelder, B. A. Witthuhn, A. P. McMickle, P. C. Doherty, G. C. Grosveld, J. N. Ihle. 1995. Defective lymphoid development in mice lacking Jak3. Science 270: 800-802. [Abstract/Free Full Text]
  36. Puel, A., S. F. Ziegler, R. H. Buckley, W. J. Leonard. 1998. Defective IL7R expression in T–B+NK+ severe combined immunodeficiency. Nat. Genet. 20: 394-397. [Medline]
  37. Rodig, S. J., M. A. Meraz, J. M. White, P. A. Lampe, J. K. Riley, C. D. Arthur, K. L. King, K. C. Sheehan, L. Yin, D. Pennica, et al 1998. Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic responses. Cell 93: 373-383. [Medline]
  38. Thomis, D. C., C. B. Gurniak, E. Tivol, A. H. Sharpe, L. J. Berg. 1995. Defects in B lymphocyte maturation and T lymphocyte activation in mice lacking Jak3. Science 270: 794-797. [Abstract/Free Full Text]
  39. Nakajima, H., X. W. Liu, A. Wynshaw-Boris, L. A. Rosenthal, K. Imada, D. S. Finbloom, L. Hennighausen, W. J. Leonard. 1997. An indirect effect of Stat5a in IL-2-induced proliferation: a critical role for Stat5a in IL-2-mediated IL-2 receptor {alpha} chain induction. Immunity 7: 691-701. [Medline]
  40. Imada, K., E. T. Bloom, H. Nakajima, J. A. Horvath-Arcidiacono, G. B. Udy, H. W. Davey, W. J. Leonard. 1998. Stat5b is essential for natural killer cell-mediated proliferation and cytolytic activity. J. Exp. Med. 188: 2067-2074. [Abstract/Free Full Text]
  41. Sexl, V., R. Piekorz, R. Moriggl, J. Rohrer, M. P. Brown, K. D. Bunting, K. Rothammer, M. F. Roussel, J. N. Ihle. 2000. Stat5a/b contribute to interleukin 7-induced B-cell precursor expansion, but abl- and bcr/abl-induced transformation are independent of stat5. Blood 96: 2277-2283. [Abstract/Free Full Text]
  42. Bertolino, E., K. Reddy, K. L. Medina, E. Parganas, J. Ihle, H. Singh. 2005. Regulation of interleukin 7-dependent immunoglobulin heavy-chain variable gene rearrangements by transcription factor STAT5. Nat. Immunol. 6: 836-843. [Medline]
  43. Kang, J., B. DiBenedetto, K. Narayan, H. Zhao, S. D. Der, C. A. Chambers. 2004. STAT5 is required for thymopoiesis in a development stage-specific manner. J. Immunol. 173: 2307-2314. [Abstract/Free Full Text]
  44. Cui, Y., G. Riedlinger, K. Miyoshi, W. Tang, C. Li, C.-X. Deng, G. W. Robinson, L. Hennighausen. 2004. Inactivation of Stat5 in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation. Mol. Cell. Biol. 24: 8037-8047. [Abstract/Free Full Text]
  45. Yao, Z., Y. Cui, W. T. Watford, J. H. Bream, K. Yamaoka, B. D. Hissong, D. Li, S. K. Durum, Q. Jiang, A. Bhandoola, et al 2006. Stat5a/b are essential for normal lymphoid development and differentiation. Proc. Natl. Acad. Sci. USA 103: 1000-1005. [Abstract/Free Full Text]
  46. Hoelbl, A., B. Kovacic, M. A. Kerenyi, O. Simma, W. Warsch, Y. Cui, H. Beug, L. Hennighausen, R. Moriggl, V. Sexl. 2006. Clarifying the role of Stat5 in lymphoid development and Abelson-induced transformation. Blood 107: 4898-4906. [Abstract/Free Full Text]
  47. Rickert, R. C., J. Roes, K. Rajewsky. 1997. B lymphocyte-specific, Cre-mediated mutagenesis in mice. Nucleic Acids Res. 25: 1317-1318. [Abstract/Free Full Text]
  48. Bunting, K. D., M. Y. Sangster, J. N. Ihle, B. P. Sorrentino. 1998. Restoration of lymphocyte function in Janus kinase 3-deficient mice by retroviral-mediated gene transfer. Nat. Med. 4: 58-64. [Medline]
  49. Chen, Y., R. Wen, S. Yang, J. Schuman, E. E. Zhang, T. Yi, G. S. Feng, D. Wang. 2003. Identification of Shp-2 as a Stat5A phosphatase. J. Biol. Chem. 278: 16520-16527. [Abstract/Free Full Text]
  50. Wang, D., R. Moriggl, D. Stravopodis, N. Carpino, J. C. Marine, S. Teglund, J. Feng, J. N. Ihle. 2000. A small amphipathic {alpha}-helical region is required for transcriptional activities and proteasome-dependent turnover of the tyrosine-phosphorylated Stat5. EMBO J. 19: 392-399. [Medline]
  51. Takahashi, Y., P. R. Dutta, D. M. Cerasoli, G. Kelsoe. 1998. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. V. Affinity maturation develops in two stages of clonal selection. J. Exp. Med. 187: 885-895. [Abstract/Free Full Text]
  52. Geissler, E. N., M. A. Ryan, D. E. Housman. 1988. The dominant-white spotting (W) locus of the mouse encodes the c-kit proto-oncogene. Cell 55: 185-192. [Medline]
  53. Mucenski, M. L., K. McLain, A. B. Kier, S. H. Swerdlow, C. M. Schreiner, T. A. Miller, D. W. Pietryga, W. J. Scott, Jr, S. S. Potter. 1991. A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell 65: 677-689. [Medline]
  54. Klemsz, M. J., S. R. McKercher, A. Celada, C. Van Beveren, R. A. Maki. 1990. The macrophage and B cell-specific transcription factor PU. 1 is related to the ets oncogene. Cell 61: 113-124. [Medline]
  55. Scott, E. W., M. C. Simon, J. Anastasi, H. Singh. 1994. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 265: 1573-1577. [Abstract/Free Full Text]
  56. Okuda, T., J. van Deursen, S. W. Hiebert, G. Grosveld, J. R. Downing. 1996. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell 84: 321-330. [Medline]
  57. Warren, A. J., W. H. Colledge, M. B. Carlton, M. J. Evans, A. J. Smith, T. H. Rabbitts. 1994. The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell 78: 45-57. [Medline]
  58. Tsai, F. Y., G. Keller, F. C. Kuo, M. Weiss, J. Chen, M. Rosenblatt, F. W. Alt, S. H. Orkin. 1994. An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 371: 221-226. [Medline]
  59. Wu, H., X. Liu, R. Jaenisch, H. F. Lodish. 1995. Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell 83: 59-67. [Medline]
  60. Nuez, B., D. Michalovich, A. Bygrave, R. Ploemacher, F. Grosveld. 1995. Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene. Nature 375: 316-318. [Medline]
  61. Perkins, A. C., A. H. Sharpe, S. H. Orkin. 1995. Lethal beta-thalassaemia in mice lacking the erythroid CACCC-transcription factor EKLF. Nature 375: 318-322. [Medline]
  62. Pevny, L., M. C. Simon, E. Robertson, W. H. Klein, S. F. Tsai, V. D’Agati, S. H. Orkin, F. Costantini. 1991. Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature 349: 257-260. [Medline]
  63. Whitelaw, E., S. F. Tsai, P. Hogben, S. H. Orkin. 1990. Regulated expression of globin chains and the erythroid transcription factor GATA-1 during erythropoiesis in the developing mouse. Mol. Cell Biol. 10: 6596-6606. [Abstract/Free Full Text]
  64. Ting, C. N., M. C. Olson, K. P. Barton, J. M. Leiden. 1996. Transcription factor GATA-3 is required for development of the T-cell lineage. Nature 384: 474-478. [Medline]
  65. Park, S. Y., K. Saijo, T. Takahashi, M. Osawa, H. Arase, N. Hirayama, K. Miyake, H. Nakauchi, T. Shirasawa, T. Saito. 1995. Developmental defects of lymphoid cells in Jak3 kinase-deficient mice. Immunity 3: 771-782. [Medline]
  66. Wen, R., D. Wang, C. McKay, K. D. Bunting, J. C. Marine, E. F. Vanin, G. P. Zambetti, S. J. Korsmeyer, J. N. Ihle, J. L. Cleveland. 2001. Jak3 selectively regulates Bax and Bcl-2 expression to promote T-cell development. Mol. Cell. Biol. 21: 678-689. [Abstract/Free Full Text]
  67. Rinkenberger, J. L., J. J. Wallin, K. W. Johnson, M. E. Koshland. 1996. An interleukin-2 signal relieves BSAP (Pax5)-mediated repression of the immunoglobulin J chain gene. Immunity 5: 377-386. [Medline]
  68. Kang, C. J., C. Sheridan, M. E. Koshland. 1998. A stage-specific enhancer of immunoglobulin J chain gene is induced by interleukin-2 in a presecretor B cell stage. Immunity 8: 285-295. [Medline]
  69. Snapper, C. M., K. B. Marcu, P. Zelazowski. 1997. The immunoglobulin class switch: beyond "accessibility". Immunity 6: 217-223. [Medline]
  70. Moriggl, R., V. Sexl, L. Kenner, C. Duntsch, K. Stangl, S. Gingras, A. Hoffmeyer, A. Bauer, R. Piekorz, D. Wang, et al 2005. Stat5 tetramer formation is associated with leukemogenesis. Cancer Cell 7: 87-99. [Medline]
  71. Goetz, C. A., I. R. Harmon, J. J. O’Neil, M. A. Burchill, T. M. Johanns, M. A. Farrar. 2005. Restricted STAT5 activation dictates appropriate thymic B versus T cell lineage commitment. J. Immunol. 174: 7753-7763. [Abstract/Free Full Text]
  72. Hirokawa, S., H. Sato, I. Kato, A. Kudo. 2003. EBF-regulating Pax5 transcription is enhanced by STAT5 in the early stage of B cells. Eur. J. Immunol. 33: 1824-1829. [Medline]
  73. Lin, H., R. Grosschedl. 1995. Failure of B-cell differentiation in mice lacking the transcription factor EBF. Nature 376: 263-267. [Medline]
  74. Urbanek, P., Z. Q. Wang, I. Fetka, E. F. Wagner, M. Busslinger. 1994. Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP. Cell 79: 901-912. [Medline]
  75. Goetz, C. A., I. R. Harmon, J. J. O’Neil, M. A. Burchill, M. A. Farrar. 2004. STAT5 activation underlies IL7 receptor-dependent B cell development. J. Immunol. 172: 4770-4778. [Abstract/Free Full Text]
  76. Kopf, M., G. Le Gros, A. J. Coyle, M. Kosco-Vilbois, F. Brombacher. 1995. Immune responses of IL-4, IL-5, IL-6 deficient mice. Immunol. Rev. 148: 45-69. [Medline]
  77. Lischke, A., R. Moriggl, S. Brandlein, S. Berchtold, W. Kammer, W. Sebald, B. Groner, X. Liu, L. Hennighausen, K. Friedrich. 1998. The interleukin-4 receptor activates STAT5 by a mechanism that relies upon common {gamma}-chain. J. Biol. Chem. 273: 31222-31229. [Abstract/Free Full Text]
  78. Mahajan, S., A. Vassilev, N. Sun, Z. Ozer, C. Mao, F. M. Uckun. 2001. Transcription factor STAT5A is a substrate of Bruton’s tyrosine kinase in B cells. J. Biol. Chem. 276: 31216-31228. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
Y. K. Parrish, I. Baez, T.-A. Milford, A. Benitez, N. Galloway, J. W. Rogerio, E. Sahakian, M. Kagoda, G. Huang, Q.-L. Hao, et al.
IL-7 Dependence in Human B Lymphopoiesis Increases during Progression of Ontogeny from Cord Blood to Bone Marrow
J. Immunol., April 1, 2009; 182(7): 4255 - 4266.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
L. Kearney, D. Gonzalez De Castro, J. Yeung, J. Procter, S. W. Horsley, M. Eguchi-Ishimae, C. M. Bateman, K. Anderson, T. Chaplin, B. D. Young, et al.
Specific JAK2 mutation (JAK2R683) and multiple gene deletions in Down syndrome acute lymphoblastic leukemia
Blood, January 15, 2009; 113(3): 646 - 648.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. A. Kerenyi, F. Grebien, H. Gehart, M. Schifrer, M. Artaker, B. Kovacic, H. Beug, R. Moriggl, and E. W. Mullner
Stat5 regulates cellular iron uptake of erythroid cells via IRP-2 and TfR-1
Blood, November 1, 2008; 112(9): 3878 - 3888.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
A. Hosui and L. Hennighausen
Genomic dissection of the cytokine-controlled STAT5 signaling network in liver
Physiol Genomics, July 1, 2008; 34(2): 135 - 143.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
Y. Chen, M. Yu, A. Podd, R. Wen, M. Chrzanowska-Wodnicka, G. C. White, and D. Wang
A critical role of Rap1b in B-cell trafficking and marginal zone B-cell development
Blood, May 1, 2008; 111(9): 4627 - 4636.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dai, X.
Right arrow Articles by Wang, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dai, X.
Right arrow Articles by Wang, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS