Evidence for a critical role for the cytoplasmic region of the interleukin 2 (IL-2) receptor gamma chain in IL-2, IL-4, and IL-7 signalling.

The high-affinity interleukin 2 receptor (IL-2R) consists of at least three distinct subunits: the IL-2R alpha chain (IL-2R alpha), beta chain (IL-2R beta), and gamma chain (IL-2R gamma). It has been shown that the cytoplasmic region of IL-2R beta, but not of IL-2R alpha, is essential for IL-2 signalling to the cell interior. In the present study, we examined the functional role of the IL-2R gamma cytoplasmic region in the IL-3-dependent mouse hematopoietic cell line BAF-B03, which expresses the endogenous IL-2R alpha and IL-2R gamma, or its subline F7, which additionally expresses human IL-2R beta cDNA. We show that overexpression of a mutant IL-2R gamma, lacking all but 7 amino acids of its cytoplasmic region, results in the selective inhibition of IL-2-induced c-fos gene activation and cellular proliferation in F7 cells. When two chimeric receptor molecules in which the cytoplasmic regions of IL-2R beta and IL-2R gamma had been swapped with each other (IL-2R beta/gamma and IL-2R gamma/beta) were coexpressed in BAF-B03, the cells responded to IL-2. These results indicate the critical importance of the IL-2-induced functional cooperation of the two cytoplasmic regions. Finally, we provide evidence that the IL-2R gamma cytoplasmic region is also critical for the IL-4 and IL-7-induced growth signal transduction in BAF-B03.     

Control of T cell development in vivo by subdomains within the IL-7 receptor alpha-chain cytoplasmic tail

IL-7/IL-7R signaling functions in both growth and differentiation during T cell development. In this study, we examined the extent these activities were controlled by signaling associated with distinct IL-7R alpha cytoplasmic domains by transgenic expression of wild-type or cytoplasmic deletion mutants of IL-7R alpha in the thymi of IL-7R alpha(-/-) mice. We show an essential requirement for the tyrosine-containing carboxyl-terminal T domain in restoring thymic cellularity, pro-/pre-T cell progression, and survival. In contrast, the functional differentiation of TCR alpha beta cells and the development of TCR gamma delta cells are partially independent of the T domain. Thus, separate cytoplasmic domains of the IL-7R alpha chain differentially control distinct functions during T cell development, whereas normal IL-7R-dependent thymic development requires the integrated activity of all these domains.     

Stat-1 is not essential for inhibition of B lymphopoiesis by type I IFNs

Type I IFNs, IFN-alpha, -beta, and -omega, are cytokine family members with multiple immune response roles, including the promotion of cell growth and differentiation. Conversely, the type I IFNs are potent inhibitors of IL-7-dependent growth of early B lineage progenitors, effectively aborting further B lineage differentiation at the pro-B cell stage. Type I IFNs alpha and beta function via receptor-mediated activation of a Jak/Stat signaling pathway in which Stat-1 is functionally important, because many IFN-induced responses are abrogated in Stat-1-deficient mice. To the contrary, we show here that the inhibition of IL-7-dependent B lymphopoiesis by IFN-alphabeta is unaffected in Stat-1-deficient mice. The present data indicate that the type I IFNs can activate an alternative signaling pathway in which neither Stat-1 nor phosphatidylinositol 3'-kinase are essential components.     

STAT5 activation underlies IL7 receptor-dependent B cell development

Signals initiated by the IL7R are required for B cell development. However, the roles that distinct IL7R-induced signaling pathways play in this process remains unclear. To identify the function of the Raf and STAT5 pathways in IL7R-dependent B cell development, we used transgenic mice that express constitutively active forms of Raf (Raf-CAAX) or STAT5 (STAT5b-CA) throughout lymphocyte development. Both Raf-CAAX and STAT5b-CA mice exhibit large increases in pro-B cells. However, crossing the Raf-CAAX transgene onto the IL7R(-/-) background fails to rescue B cell development. In contrast, STAT5 activation selectively restores B cell expansion in IL7R(-/-) mice. Notably, the expansion of pro-B cells in STAT5b-CA mice correlated with an increase in cyclin D2, pim-1, and bcl-x(L) expression, suggesting that STAT5 directly affects pro-B cell proliferation and survival. In addition, STAT5 activation also restored B cell differentiation in IL7R(-/-) mice as determined by 1) the restoration of V(H) Ig gene rearrangement and 2) the appearance of immature and mature B cell subsets. These findings establish STAT5 as the key player entraining B cell development downstream of the IL7R.     

Murine pro-B cells require IL-7 and its receptor complex to up-regulate IL-7R alpha, terminal deoxynucleotidyltransferase, and c mu expression

Phenotypic analysis of bone marrow cells from IL-7 knockout (KO) mice revealed that B cell development is blocked precisely at the transition between pro-B cells and pre-B cells. In contrast, the generation of pre-pro-B cells and pro-B cells appeared to be normal, as judged by total cell numbers, proliferative indexes, D-JH and V-DJH gene rearrangements, and mRNA for recombinase-activating gene-1 (RAG-1), RAG-2, TdT, Ig mu, lambda 5, and VpreB. However, upon closer inspection, several abnormalities in pro-B cell development were identified that could be corrected by injection of rIL-7 in vivo. These included the absence of the subset of late pro-B cells that initiates cmu expression for pre-B cell Ag receptor (BCR) formation, and the failure of pro-B cells to up-regulate TdT and the IL-7R alpha (but not the common gamma-chain) chain. Similar defects were present in common gamma-chain and Jak3 KO mice, but not in lambda 5 or (excluding cytoplasmic Ig mu heavy chain (c mu)) RAG-1 KO mice, all of which also arrest at the late pro-B cell stage. Consequently, up-regulation of TdT and IL-7R alpha expression requires signaling through the high affinity IL-7R, but does not require cmu expression or a functional pre-BCR. Taken together, these results suggest that IL-7 and its receptor complex are essential for 1) up-regulating the expression of TdT and IL-7R alpha, 2) initiating the production of cmu and 3) promoting the formation of a functional pre-BCR in/on pro-B cells. These key events, in turn, appear to be prerequisite both for differentiation of pro-B cells to pre-B cells and for proliferation of these cell subsets upon continued stimulation with IL-7.     

Absence of an essential role for thymic stromal lymphopoietin receptor in murine B-cell development

The murine cytokine thymic stromal lymphopoietin (TSLP) supports the development of B220+ IgM+ immature B cells and induces thymocyte proliferation in vitro. Human TSLP, by contrast, activates CD11c+ dendritic cells, but not B or T cells. Recent studies have demonstrated that the receptor for TSLP consists of a heterodimer of the interleukin 7 (IL-7) alpha chain and a novel protein that resembles the hematopoietic cytokine receptor common gamma chain. We examined signal transduction by the gamma-like chains using chimeric receptor proteins. The cytoplasmic domain of the human, but not of the murine, gamma-like chain, activates Jak2 and Stat5 and supports the proliferation of hematopoietic cell lines. In order to assess the role of the murine gamma-like chain in vivo, we generated gamma-like chain-deficient mice. Receptor-deficient mice are unresponsive to TSLP but exhibit no obvious phenotypic defects. In particular, hematopoietic cell development appeared normal. B-cell development, including the IgM+ compartment, was unaffected by loss of the TSLP pathway, as were T lymphopoiesis and lymphocyte proliferation in vitro. Cytokine receptors that utilize the common gamma chain signal through the lymphocyte-specific kinase Jak3. Mice deficient in Jak3 exhibit a SCID phenotype but harbor a residual B220+ splenic lymphocyte population. We demonstrate here that this residual lymphocyte population is lost in mice lacking both the gamma-like chain and Jak3.     

A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction.

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.     

The estrogen receptor cooperates with the TGF alpha receptor (c-erbB) in regulation of chicken erythroid progenitor self-renewal.

A unique combination of growth promoting factors is described that allows growth of large amounts (10(10)-10(11)) of normal erythroid progenitors from chick bone marrow. These erythroid progenitors express the estrogen receptor (ER) as well as the receptor tyrosine kinase TGF alpha R/c-erbB. They require both TGF alpha and estradiol for sustained self-renewal in vitro, but terminally differentiate upon withdrawal of TGF alpha and inactivation of the ER by an antagonist (ICI 164.384). Overexpression of the human ER in erythroblasts devoid of endogenous ER revealed that the hormone-activated ER alone arrested erythroid differentiation and repressed a large group of erythrocyte genes. When similarly overexpressed, TGF alpha R/c-erbB inhibited the expression of a distinct, but overlapping, set of genes. The endogenous ER and TGF alpha R/c-erbB affect erythrocyte gene expression in a similar, but less pronounced fashion. Surprisingly, suppression of ER function by antagonist efficiently inhibited erythroblast transformation by tyrosine kinase oncogenes, suggesting a role of the endogenous ER in leukemogenesis. We speculate that the oncogenes v-erbB and v-erbA cooperate in erythroleukemia induction by a mechanism that is employed by TGF alpha R/c-erbB and ER to regulate normal progenitor self-renewal in response to external signals.     

A stromal cell-specific monoclonal antibody augments the stromal cell-dependent B lymphopoiesis.

We produced the mAb R25 against the stromal cell line ST2, which could support B lymphopoiesis in vitro. R25 enhanced the ability of ST2 to support B lymphopoiesis and precipitated molecules of 110 and 120 kDa (p110/120) from ST2 cell lysates. p110/120 were also expressed on other stromal and fibroblast cell lines but not on freshly isolated bone marrow hematopoietic cells, spleen cells, and lymphoid cell lines. However, R25 had no or weak effect on the stromal cell-dependent myelopoiesis. Even under conditions in which bone marrow cells were separated from stromal cells with a membrane filter, R25 could augment the stromal cell-dependent B lymphopoiesis. However, R25 did not induce the increase of IL-7 mRNA of ST2 cells. Taken together, these findings suggest that the stromal cell surface molecules p110/120 are involved in the stromal cell-dependent B lymphopoiesis and that certain soluble factors distinct from IL-7 may contribute to the p110/120-mediated B cell generation.     

Osteoclast activity modulates B-cell development in the bone marrow

B-cell development is dependent on the interactions between B-cell precursors and bone marrow stromal cells, but the role of osteoclasts (OCLs) in this process remains unknown. B lymphocytopenia is a characteristic of osteopetrosis, suggesting a modulation of B lymphopoiesis by OCL activity. To address this question, we first rescued OCL function in osteopetrotic oc/oc mice by dendritic cell transfer, leading to a restoration of both bone phenotype and B-cell development. To further explore the link between OCL activity and B lymphopoiesis, we induced osteopetrosis in normal mice by injections of zoledronic acid (ZA), an inhibitor of bone resorption. B-cell number decreased specifically in the bone marrow of ZA-treated mice. ZA did not directly affect B-cell differentiation, proliferation and apoptosis, but induced a decrease in the expression of CXCL12 and IL-7 by stromal cells, associated with reduced osteoblastic engagement. Equivalent low osteoblastic engagement in oc/oc mice confirmed that it resulted from the reduced OCL activity rather than from a direct effect of ZA on osteoblasts. These dramatic alterations of the bone microenvironment were disadvantageous for B lymphopoiesis, leading to retention of B-cell progenitors outside of their bone marrow niches in the ZA-induced osteopetrotic model. Altogether, our data revealed that OCLs modulate B-cell development in the bone marrow by controlling the bone microenvironment and the fate of osteoblasts. They provide novel basis for the regulation of the retention of B cells in their niche by OCL activity.     

Contrasting responses of lymphoid progenitors to canonical and noncanonical Wnt signals

The Wnt family of secreted glycoproteins has been implicated in many aspects of development, but its contribution to blood cell formation is controversial. We overexpressed Wnt3a, Wnt5a, and Dickkopf 1 in stromal cells from osteopetrotic mice and used them in coculture experiments with highly enriched stem and progenitor cells. The objective was to learn whether and how particular stages of B lymphopoiesis are responsive to these Wnt family ligands. We found that canonical Wnt signaling, through Wnt3a, inhibited B and plasmacytoid dendritic cell, but not conventional dendritic cell development. Wnt5a, which can oppose canonical signaling or act through a different pathway, increased B lymphopoiesis. Responsiveness to both Wnt ligands diminished with time in culture and stage of development. That is, only hematopoietic stem cells and very primitive progenitors were affected. Although Wnt3a promoted retention of hematopoietic stem cell markers, cell yields and dye dilution experiments indicated it was not a growth stimulus. Other results suggest that lineage instability results from canonical Wnt signaling. Lymphoid progenitors rapidly down-regulated RAG-1, and some acquired stem cell-staining characteristics as well as myeloid and erythroid potential when exposed to Wnt3a-producing stromal cells. We conclude that at least two Wnt ligands can differentially regulate early events in B lymphopoiesis, affecting entry and progression in distinct differentiation lineages.     

Restricted STAT5 activation dictates appropriate thymic B versus T cell lineage commitment

The molecular mechanisms regulating lymphocyte lineage commitment remain poorly characterized. To explore the role of the IL7R in this process, we generated transgenic mice that express a constitutively active form of STAT5 (STAT5b-CA), a key downstream IL7R effector, throughout lymphocyte development. STAT5b-CA mice exhibit a 40-fold increase in pro-B cells in the thymus. As documented by BrdU labeling studies, this increase is not due to enhanced B cell proliferation. Thymic pro-B cells in STAT5b-CA mice show a modest increase in cell survival ( approximately 4-fold), which correlates with bcl-x(L) expression. However, bcl-x(L) transgenic mice do not show increases in thymic B cell numbers. Thus, STAT5-dependent bcl-x(L) up-regulation and enhanced B cell survival are not sufficient to drive the thymic B cell development observed in STAT5b-CA mice. Importantly, thymic pro-B cells in STAT5b-CA mice are derived from early T cell progenitors (ETPs), suggesting that STAT5 acts by altering ETP lineage commitment. Supporting this hypothesis, STAT5 binds to the pax5 promoter in ETPs from STAT5b-CA mice and induces pax5, a master regulator of B cell development. Conversely, STAT5b-CA mice exhibit a decrease in the DN1b subset of ETPs, demonstrating that STAT5 activation inhibits early T cell differentiation or lineage commitment. On the basis of these findings, we propose that the observed expression of the IL-7R on common lymphoid progenitors, but not ETPs, results in differential STAT5 signaling within these distinct progenitor populations and thus helps ensure appropriate development of B cells and T cells in the bone marrow and thymic environments, respectively.     

Dynamics of the Splenic Innate-like CD19+CD45Rlo Cell Population from Adult Mice in Homeostatic and Activated Conditions

In the adult spleen, CD19(+)CD45R(-/lo) (19(+)45R(lo)) lymphocytes of embryonic origin exist as a distinct population to that of the conventional B cell lineage. These cells display a plasmablast phenotype, and they spontaneously secrete IgG1 and IgA, whereas the bone marrow population of 19(+)45R(lo) cells contains B1 progenitors. In this study, we show that 19(+)45R(lo) cells are also present in Peyer's patches and in the spleen throughout the life span of wild-type mice, beginning at postnatal day 7. Although this population is heterogeneous, the surface phenotype of most of these cells distinguishes them from follicular, transitional, marginal zone, and B1 cells. In CBA/CaHN mice, few 19(+)45R(lo) cells were detected at postnatal day 7, and none was observed in the adult spleen. Splenic 19(+)45R(lo) cells exhibited homeostatic BrdU uptake in vivo and actively transcribed cell cycle genes. When transferred to immunodeficient RAG2(-/-)γchain(-/-) recipient mice, 19(+)45R(lo) cells survived and differentiated into IgG1- and IgA-plasma cells. Moreover, in vitro stimulation of splenic 19(+)45R(lo) cells with LPS, CpG, BAFF/IL4, and CD40/IL4 induced cell proliferation, IgG1/IgA secretion and the release of IL-10, suggesting a potential immunoregulatory role for this subset of innate-like B cells.     

Proteomics analysis of interleukin (IL)-7-induced signaling effectors shows selective changes in IL-7Ralpha449F knock-in T cell progenitors

Interleukin (IL)-7 is a cytokine that plays a central role in the development, survival, and proliferation of T and B cell lymphocytes. Overexpression of IL-7 in mice (transgenic (Tg) IL-7) leads to both increased proliferation of early T and B cell progenitors and T and B cell lymphomas. Genetic evidence indicates that known IL-7 receptor (IL-7R)-dependent proteins, including prosurvival protein BCL-2, may not be solely responsible for the effects of IL-7. Other studies indicate that known IL-7-induced signaling proteins dock to a specific tyrosine (Tyr(449)) residue on the alpha-subunit of the IL-7R. We have previously shown in an IL-7Ralpha(449F) knock-in model that IL-7-induced lymphomas require Tyr(449) phosphorylation and that loss of this phosphorylation confers protection from disease. However, the mechanism by which this lymphoma protection occurs remains unclear. Using this genetic model, we aimed to identify novel prosurvival factors important for IL-7-mediated lymphocyte development and lymphomagenesis. An iTRAQ (isobaric tags for relative and absolute quantitation) proteomics analysis was performed comparing CD4(-)CD8(-) double negative T cell progenitors from mice overexpressing IL-7 (Tg IL-7) (lymphoma-prone) with Tg IL-7 mice with a mutated IL-7 receptor (Tg IL-7/IL-7Ralpha(449F)) (lymphoma-protected). Several proteins involved in survival, proliferation, and apoptosis were found to be differentially expressed between the two samples, and three proteins of particular interest, GIMAP4, BIT1, and FKBP51, were validated by immunoblot analysis.     

Apurinic/apyrimidinic endonuclease 2 is necessary for normal B cell development and recovery of lymphoid progenitors after chemotherapeutic challenge

B cell development involves rapid cellular proliferation, gene rearrangements, selection, and differentiation, and it provides a powerful model to study DNA repair processes in vivo. Analysis of the contribution of the base excision repair pathway in lymphocyte development has been lacking primarily owing to the essential nature of this repair pathway. However, mice deficient for the base excision repair enzyme, apurinic/apyrimidinic endonuclease 2 (APE2) protein develop relatively normally, but they display defects in lymphopoiesis. In this study, we present an extensive analysis of bone marrow hematopoiesis in mice nullizygous for APE2 and find an inhibition of the pro-B to pre-B cell transition. We find that APE2 is not required for V(D)J recombination and that the turnover rate of APE2-deficient progenitor B cells is nearly normal. However, the production rate of pro- and pre-B cells is reduced due to a p53-dependent DNA damage response. FACS-purified progenitors from APE2-deficient mice differentiate normally in response to IL-7 in in vitro stromal cell cocultures, but pro-B cells show defective expansion. Interestingly, APE2-deficient mice show a delay in recovery of B lymphocyte progenitors following bone marrow depletion by 5-fluorouracil, with the pro-B and pre-B cell pools still markedly decreased 2 wk after a single treatment. Our data demonstrate that APE2 has an important role in providing protection from DNA damage during lymphoid development, which is independent from its ubiquitous and essential homolog APE1.