Puppet masters of B-cell progenitor acute lymphoblastic leukemia: The preB cell receptor and the interleukin 7 receptor α

B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) is enriched for a preB cell phenotype, hinting at a specific vulnerability of this cell stage. Two signaling pathways via the preB cell receptor (preBCR) and the interleukin 7 receptor α (IL-7Rα) chain govern the balance between differentiation and proliferation at this stage and both receptor pathways are routinely altered in human BCP-ALL. Here, we review the immunobiology of both the preBCR as well as the IL-7Rα and analyze the human BCP-ALL spectrum in the light of these signaling complexes. Finally, we present a terminology for preBCR signaling modules that distinguishes a pro-proliferative “phase-I” module from a pro-differentiative “phase-II” module. This terminology might serve as a framework to better address shared oncogenic mechanics of preB cell stage BCP-ALL.     

CD28 induces cell cycle progression by IL-2-independent down-regulation of p27kip1 expression in human peripheral T lymphocytes

CD28 is the primary T cell costimulatory receptor, and upon ligation with its ligands, it enhances T cell proliferation and IL-2 synthesis. In this study we examined the role of CD28 in the initial proliferative response and cell cycle entry of T lymphocytes. Stimulation through CD3 alone resulted in a poor proliferative response, while in the presence of CD28 costimulation a strong increase in the number of cells in S-phase could be detected after 48 h of stimulation. CD28 costimulation enhanced expression of cyclin D3 and induced down-regulation of p27kip1 expression. Cross-linking CD28 was much more effective in inducing cyclin D3 expression and in down-regulating p27kip1 expression than addition of IL-2. Blocking experiments, using antibodies that neutralize IL-2 or the IL-2 receptor, showed that the effects induced by CD28 are independent of endogenous IL-2. Moreover, using a variety of immunosuppressants that interfere with IL-2 signaling pathways, we were able to show that IL-2 is not required for cell cycle entry induced by CD28 costimulation. From these experiments it can be concluded that CD28 and IL-2 use different signaling pathways for down-regulation of p27kip1 expression. We hypothesize that costimulation through CD28 is responsible for initial cell cycle entry of T lymphocytes, while IL-2, which is produced after costimulation, might be involved in sustaining proliferation.     

IRAK and TAK1 are required for IL-18-mediated signaling.

Interleukin-18 (IL-18), a pleiotropic cytokine produced by activated macrophages, plays significant roles in the immune response, inducing the secretion of IFN-gamma, TNF-alpha and IL-2, enhancing NK cell activity and potentiating the differentiation of Th1 cells. The intercellular signal transduction pathways through which IL-18 functions have not been thoroughly defined. We have generated a mutant cell line, I1A, that lacks the IRAK protein. In this line which has low or no expression of the other known IRAK family members, we find that the IL-1 receptor-associated kinase (IRAK) is essential for the activation of NFkappaB and JNK in response to IL-18. Furthermore, the death domain, but not the kinase activity of IRAK, is necessary for NFkappaB activation in response to IL-18. Interestingly, the N-proximal undetermined region of IRAK is necessary for NFkappaB activation, but not for JNK activation in response to IL-18, indicating IRAK may be a branchpoint in IL-18 signaling. In addition to IRAK, we implicate two other components in IL-18 signaling, TAK1 (TGF-beta-activated kinase 1) and its activator and substrate TAB1. A dominant negative mutant of TAK1 inhibits the IL-18-mediated NFkappaB activation, while IL-18 stimulation leads to the phosphorylation of TAB1. Finally, analysis of IL-18 signaling in IL-1-unresponsive mutant cell lines suggests that the IL-1- and IL-18-mediated pathways are similar, but may not be identical.     

Generation and characterization of stromal cell independent IL-7 dependent B cell lines

In-vitro B cell cultures have played a significant role in the study of B cell development. Their utility in developmental and biochemical studies, however, has been limited by the challenges associated with obtaining and maintaining adequate cell numbers of pure and/or rare populations. Although B cell lines allow for circumvention of some of these issues, they have traditionally been generated via viral infection or genetic transformation and are thus less representative of in-vivo cells. In order to avoid such alterations in cell state, we have designed a procedure for the creation of B cell lines directly from murine bone marrow. In this study, we describe the generation and characterization of these IL-7 dependent cell lines. Our lines, established from both wild type and mutant mice, do not require stromal cell support for generation or maintenance. In addition, clones survive repeated freeze/thaw cycles and, in the presence of IL-7, can be kept in culture indefinitely. Phenotypically, our lines resemble pro/pre-B cells and exhibit IL-7 and preBCR signaling profiles that mimic ex-vivo B cells. These lines promise to be useful in the study of the signaling pathways that regulate B cell development.     

Monoclonal antibodies targeting ST2L Domain 1 or Domain 3 differentially modulate IL-33-induced cytokine release by human mast cell and basophilic cell lines

The cell-surface receptor ST2L triggers cytokine release by immune cells upon exposure to its ligand IL-33. To study the effect of ST2L-dependent signaling in different cell types, we generated antagonist antibodies that bind different receptor domains. We sought to characterize their activities in vitro using both transfected cells as well as basophil and mast cell lines that endogenously express the ST2L receptor. We found that antibodies binding Domain 1 versus Domain 3 of ST2L differentially impacted IL-33-induced cytokine release by mast cells but not the basophilic cell line. Analysis of gene expression in each cell type in the presence and absence of the Domain 1 and Domain 3 mAbs revealed distinct signaling pathways triggered in response to IL-33 as well as to each anti-ST2L antibody. We concluded that perturbing the ST2L/IL-33/IL-1RAcP complex using antibodies directed to different domains of ST2L have a cell-type-specific impact on cytokine release, and may indicate the association of additional receptors to the ST2L/IL-33/IL-1RAcP complex in mast cells.     

Regulation of in vitro human T cell development through interleukin-7 deprivation and anti-CD3 stimulation

ABSTRACT: BACKGROUND: The role of IL-7 and pre-TCR signaling during T cell development has been well characterized in murine but not in human system. We and others have reported that human BM hematopoietic progenitor cells (HPCs) display poor proliferation, inefficient double negative (DN) to double positive (DP) transition and no functional maturation in the in vitro OP9-Delta-like 1 (DL1) culture system. RESULTS: In this study, we investigated the importance of optimal IL-7 and pre-TCR signaling during adult human T cell development. Using a modified OP9-DL1 culture ectopically expressing IL-7 and Fms-like tyrosine kinase 3 ligand (Flt3L), we demonstrated enhanced T cell precursor expansion. IL-7 removal at various time points during T cell development promoted a slight increase of DP cells; however, these cells did not differentiate further and underwent cell death. As pre-TCR signaling rescues DN cells from programmed cell death, we treated the culture with anti-CD3 antibody. Upon pre-TCR stimulation, the IL-7 deprived T precursors differentiated into CD3+TCRalphabeta+DP cells and further matured into functional CD4 T cells, albeit displayed a skewed TCR Vbeta repertoire. CONCLUSIONS: Our study establishes for the first time a critical control for differentiation and maturation of adult human T cells from HPCs by concomitant regulation of IL-7 and pre-TCR signaling.     

Ikaros sets thresholds for T cell activation and regulates chromosome propagation

T cell activation involves the sustained accumulation of T cell receptor (TCR) and IL-2 receptor (IL-2R) mediated signaling events that promote cell cycle entry and progression. The Ikaros family of nuclear factors regulate this process by providing thresholds overcome by receptor signaling. T cells with reduced levels of Ikaros activity require fewer TCR engagement events for activation, exhibit a greater proliferative response to IL-2, and are less sensitive to inhibitors of TCR and IL-2R signaling. Upon T cell activation, Ikaros proteins localize in a higher-order chromatin structure where they colocalize with components of the DNA replication machinery. Proliferating T cells with reduced Ikaros activity display chromosome abnormalities. We propose that participation of Ikaros in higher-order chromatin structures controls cell cycle transitions and restricts DNA replication.     

Interleukin-7 activates p56lck and p59fyn,two tyrosine kinases associated with the p90 interleukin-7 receptor in primary human T cells

We have investigated signaling events associated with the cloned 90-kDa (p90) interleukin-7 receptor (IL-7R) to determine whether changes in the signaling pathways initiated by this molecule can explain the ability of T cells to proliferate to IL-7 following activation. Using in vitro kinase assays we find that the p90 IL-7R in both unstimulated and activated human T cells is physically associated with two molecules with intrinsic kinase activity. Western blotting analysis reveals these proteins to be the src kinase enzymes, p59^fyn and p56^lck. Binding of human recombinant IL-7 to the p90 IL-7R results in increased activity of both receptor-associated kinases in both resting and activated mature T cells. Thus, the signaling pathways initiated via the p90 IL-7R-associated src kinases are unlikely to be solely responsible for the proliferation of only activated T cells in response to IL-7. Additional signals, which may derive from other IL-7R-associated molecules such as the γc, are clearly required for IL-7-driven proliferation of activated primary T cells.     

CD40 signaling induces B cell responsiveness to multiple members of the gamma chain-common cytokine family.

CD40 signaling induces B cell proliferative and differentiation responses that can be modulated by many different cytokines. Cytokines in the IL-2 receptor gamma chain (gammac)-common family are known to play an integral role in B cell development. Therefore, we investigated the possibility that CD40 signaling induced B cell responsiveness to multiple gammac-common cytokines and that individual gammac-common cytokines induced distinct B cell responses. B cells were isolated from lymphoid follicles of sheep Peyer's patches (PP) and co-cultured with murine CD40 ligand (mCD40L). CD40 signaling induced PP B cell responsiveness to recombinant human IL-2, IL-4, IL-7 and IL-15. mCD40L-induced B cell growth was enhanced by combining IL-4 with a second gammac-common cytokine and sustained B cell growth required co-stimulation with IL-4 plus IL-2, IL-7 and IL-15. gammac-common cytokine responsiveness remained dependent upon CD40 signaling, and removal of mCD40L resulted in B cell differentiation and cell death. Similar proliferative responses to mCD40L and gammac-common cytokines were observed for both immature (ileal) and mature (jejunal) PP B cells. Finally, the capacity of CD40-activated B cells to respond to multiple gammac-common cytokines was analyzed with individual PP B cell clones. All B cell clones displayed similar proliferative responses to IL-2 but quantitatively different responses to IL-4, IL-7 and IL-15. The biological significance of B cell responsiveness to multiple gammac-common cytokines is discussed.     

Molecular mechanisms involved in interleukin-4-induced human neutrophils: expression and regulation of suppressor of cytokine signaling

Interleukin-4 (IL-4) is a CD132-dependent cytokine known to activate the Jak-STAT pathway in different cells and cell lines. Although IL-4 has been demonstrated previously to be an agonist in human neutrophils, its capacity to activate different cell signaling pathways in these cells has never been investigated. Two types of IL-4 receptor (IL-4R) exist: the Type I (CD132/IL-4Ralpha heterodimer) and the Type II (IL-4Ralpha/IL-13Ralpha1 heterodimer). In a previous study, we demonstrated that neutrophils express the Type I receptor. Herein, using flow cytometry, we demonstrated that neutrophils, unlike U-937 cells, do not express IL-13Ralpha1 and IL-13Ralpha2 and confirmed the expression of CD132 and IL-4Ralpha on their surface. We also demonstrated that IL-4 induced phosphorylation of Syk, p38, Erk-1/2, JNK, Jak-1, Jak-2, STAT6, and STAT1 and that treatment of cells with the inhibitors piceatannol, SB203580, PD98059, or AG490 reversed the ability of IL-4 to delay neutrophil apoptosis. Using RT-PCR, we demonstrated for the first time that neutrophils express mRNA for all suppressor of cytokine signaling (SOCS) members, namely SOCS1-7 and cytokine-inducible Src homology 2 protein. It is interesting that IL-4 increased expression of SOCS3 at the mRNA and protein levels. The effect of IL-4 on SOCS3 protein expression was increased markedly when the proteasome inhibitor MG132 was added to the cultures, but this was inhibited by cycloheximide, suggesting that SOCS3 is de novo-synthesized in response to IL-4. We conclude that neutrophils express only the Type I IL-4R on their surface and that IL-4 signals via different cell signaling pathways, including the Jak/STAT/SOCS pathway.     

Interleukin-7 Receptor Signaling Network： An Integrated Systems Perspective

Interleukin-7 （IL-7） is an essential cytokine for the development and homeostatic maintenance of T and B lymphocytes. Binding of IL-7 to its cognate receptor, the IL-7 receptor （IL-7R）, activates multiple pathways that regulate lymphocyte survival, glucose uptake, proliferation and differentiation. There has been much interest in understanding how IL-7 receptor signaling is modulated at multiple interconnected network levels. This review examines how the strength of the signal through the IL-7 receptor is modulated in T and B cells, including the use of shared receptor components, signaling crosstalk, shared interaction domains, feedback loops, integrated gene regulation, multimerization and ligand competition. We discuss how these network control mechanisms could integrate to govern the properties of IL-7R signaling in lymphocytes in health and disease. Analysis of IL-7 receptor signaling at a network level in a systematic manner will allow for a comprehensive approach to understanding the impact of multiple signaling pathways on lymphocyte biology. Cellular ＆ Molecular Immunology.     

Analysis of murine CD22 during B cell development: CD22 is expressed on B cell progenitors prior to IgM

CD22 is a B cell-restricted glycoprotein involved in cell adhesion and signaling. Since CD22 is likely to play an important role in interactions between B cells and other cells, and in regulating signaling thresholds, we characterized the expression of murine CD22 during different stages of B cell development. In contrast to previous reports, we show that CD22 is expressed on B cell progenitors prior to expression of IgM. IL-7-responsive B cell precursors from the fetal liver and early B lineage cells (B220+IgM-) from the bone marrow both express a low density of surface CD22. The majority of the earliest B cell progenitors (B220+IgM-CD43+) in the bone marrow, however, do not express CD22. As B cells mature, the density of CD22 molecules on the cell surface increases. B220brightIgM+ bone marrow cells express high levels of CD22, as do splenic B cells. The correlation of CD22 levels with B cell maturation is replicated in an in vitro culture system, which distinguishes stages of B cell development based on function. Following activation of mature resting splenic B cells with anti-mu mAb or lipopolysaccharide (LPS), levels of CD22 decrease. Finally, we show that the addition of anti-CD22 mAb augments the proliferative response of both anti-mu- and LPS-stimulated B cells, suggesting a role for CD22 in diverse signaling pathways.      

Interleukin-7 mediates the homeostasis of naïve and memory CD8 T cells in vivo

The na?ve and memory T lymphocyte pools are maintained through poorly understood homeostatic mechanisms that may include signaling via cytokine receptors. We show that interleukin-7 (IL-7) plays multiple roles in regulating homeostasis of CD8+ T cells. We found that IL-7 was required for homeostatic expansion of na?ve CD8+ and CD4+ T cells in lymphopenic hosts and for CD8+ T cell survival in normal hosts. In contrast, IL-7 was not necessary for growth of CD8+ T cells in response to a virus infection but was critical for generating T cell memory. Up-regulation of Bcl-2 in the absence of IL-7 signaling was impaired after activation in vivo. Homeostatic proliferation of memory cells was also partially dependent on IL-7. These results point to IL-7 as a pivotal cytokine in T cell homeostasis.     

Intrathymic IL-7: the where, when, and why of IL-7 signaling during T cell development

The thymus is the birthplace of all T lineage cells. But the thymus is also a cradle as it provides the environment for further maturation and differentiation of immature thymocytes. While many factors contribute to make the thymus a unique place for T cell development, here we review the essential role of intrathymic interleukin-7 (IL-7). In the absence of IL-7 signaling, survival, proliferation and differentiation of immature thymocytes are all severely impaired. Consequently, IL-7 is critical to nurture and guide T precursor cells through the diverse steps of thymic maturation. Interestingly, even as IL-7 signaling is such a critical factor, IL-7 signaling must be also actively suppressed during specific stages of T cell differentiation. These contradictory observations are puzzling but can be satisfactorily explained when understanding the developmental context of IL-7 signaling. In this regard, here we will discuss the spatiotemporal expression of intrathymic IL-7 and address the stage-specific effects of IL-7 signaling in developing thymocytes. Specifically, we will review other facets of intrathymic IL-7 beyond its role as a pro-survival factor and so clarify and reaffirm the unique role of IL-7 as a prime factor in T cell development and differentiation.     

B cell early response gene expression coupled to B cell receptor,CD40 and interleukin-4 receptor co-stimulation: evidence for a role of the egr-2/krox20 transcription factor in B cell proliferation

B lymphocytes are activated following antigen stimulation of the B cell receptor but require co-stimulation with accessory molecules provided by interleukin (IL)-4/CD40 ligand for cell cycle progression and proliferation. By analyzing a panel of 11 early response genes induced by cross-linking of surface immunoglobulin, we show that CD40 signaling alone induces only 2 genes, c-myc together with an anonymous gene, 3L3, and that these are distinct from the set of genes induced in response to IL-4. Co-stimulation with the proliferative combination of anti-μ, IL-4 + CD40 signaling led to a fourfold enhancement of egr-2/krox20 expression over that seen with anti-μ alone. Egr-2 expression/activity was selectively inhibited by the immunosuppressive drug cyclosporin A, and antisense oligonucleotide blockade of Egr-2 activity elicited a dose-dependent inhibition of B cell proliferation. Taken together, these observations show that the early gene regulatory programs coupled to different surface receptors on B cells are largely distinct from each other, but that certain genes, exemplified by egr-2, may represent a point of convergence in the integration of different signaling pathways into the B cell proliferative response.     

Chicken IL-26 regulates immune responses through the JAK/STAT and NF-κB signaling pathways

Chicken interleukin 26 (ChIL-26), a member of the IL-10 family, is expressed in T cells and can induce expression of proinflammatory cytokines. We examined the response of signal transduction pathways to ChIL-26 stimulation in the chicken T (CU91), macrophage (HD11), and fibroblast (OU2) cell lines. ChIL-26 activated JAK2 and TYK2 phosphorylation, as well as activation of STAT1, STAT3, and SHP2 via tyrosine/serine residues. We also showed that ChIL-26 activates the phosphorylation of NF-κB1, TAK1, and MyD88 kinase, which are key regulators of NF-κB signaling pathways. Moreover, ChIL-26 stimulation upregulated mRNA expression of chemokines (CCL4, CCL20, and CXCL14), Th1 (IFN-α, IFN-β, IFN-γ, IL-1β, and IL-6), Th2 (IL-4 and IL-10), and Th17 (IL-12p40, IL-17A, and IL-17F), and the Treg cytokines (TGF-β4); additionally, it increased Th1 and Th17 protein levels and nitric oxide production but did not affect cell proliferation. Together, these results suggest that ChIL-26-induced activation of chemokines, Th1, Th2, and, Th17, and the Treg cytokines is mediated through JAK/STAT and NF-κB signaling pathways in chicken T, macrophage, and fibroblast cell lines. These results indicate a key role for ChIL-26-induced polarization of the immune response and could reveal new therapeutic approaches for use in combination with molecules that activate T and macrophage cells via activation JAK/STAT and NF-κB signaling pathways.     

Human lung innate immune response to Bacillus anthracis spore infection

Bacillus anthracis, the causative agent of inhalational anthrax, enters a host through the pulmonary system before dissemination. We have previously shown that human alveolar macrophages participate in the initial innate immune response to B. anthracis spores through cell signal-mediated cytokine release. We proposed that the lung epithelia also participate in the innate immune response to this pathogen, and we have developed a human lung slice model to study this process. Exposure of our model to B. anthracis (Sterne) spores rapidly activated the mitogen-activated protein kinase signaling pathways ERK, p38, and JNK. In addition, an RNase protection assay showed induction of mRNA of several cytokines and chemokines. This finding was reflected at the translational level by protein peak increases of 3-, 25-, 9-, 34-, and 5-fold for interleukin-6 (IL-6), tumor necrosis factor alpha, IL-8, macrophage inflammatory protein 1alpha/beta, and monocyte chemoattractant protein 1, respectively, as determined by an enzyme-linked immunosorbent assay. Inhibition of individual pathways by UO126, SP600125, and SB0203580 decreased induction of chemokines and cytokines by spores, but this depended on the pathways inhibited and the cytokines and chemokines induced. Combining all three inhibitors reduced induction of all cytokines and chemokines tested to background levels. An immunohistochemistry analysis of IL-6 and IL-8 revealed that alveolar epithelial cells and macrophages and a few interstitial cells are the source of the cytokines and chemokines. Taken together, these data showed the activation of the pulmonary epithelium in response to B. anthracis spore exposure. Thus, the lung epithelia actively participate in the innate immune response to B. anthracis infection through cell signal-mediated elaboration of cytokines and chemokines.     

Interelationship between CD3 and CD28 pathways in a murine T cell thymoma

It is well known that the CD28 costimulatory signal is important to complement T cell receptor (TCR)/CD3-initiated T cell activation, but the mechanism by which these two distinct signaling pathways are integrated is not clearly understood. In our laboratory, we dispose of a murine T cell hybridoma transfected with human CD28 molecule which is able to produce IL-2 in response to stimulation, suggesting that the signal transduction machinery coupled to the CD28 molecule is capable of triggering effector functions. Nevertheless, the action of three immunosuppressive agents previously shown in our model, suggested an interaction between the CD3 and CD28 pathways. We confirmed here this hypothesis by transfecting the cDNA of the human CD28 molecule in the BW5147 thymoma which lacks CD3 surface expression. Stimulation of the human CD28 did not lead to IL-2 secretion while the restoration of the TCR/CD3 complex re-established the functionality of this costimulatory molecule. These data demonstrate that the IL-2 production induced by the CD28 activation pathway is dependent of the TCR/CD3 complex cell surface expression and suggest the formation of a functional membrane complex between the CD3 and CD28 molecules. The molecular basis of the functional dependence of CD28 signaling on the TCR/CD3 complex is presently unknown. Nonetheless, we showed that some early events induced by CD28 stimulation, such as PI3-kinase association, are independent of the TCR/CD3 complex expression.     

An IL-2 receptor beta subdomain that controls Bcl-X(L) expression and cell survival

IL-2 binding to its high-affinity receptor regulates signaling events that control both lymphocyte cell survival and cell cycle progression. Although many studies have examined the mechanisms by which IL-2 regulates cell growth, few studies have dissected the pathways involved in promoting cell survival or the coupling of these pathways to the receptor. In the present study, using the pre-B cell line Baf-B03 transfected with a truncated form of the IL-2 receptor (IL-2R) beta chain, we demonstrate that IL-2-dependent cell survival requires only the N-terminal 350 amino acids of the IL-2Rbeta chain. IL-2-dependent survival of cells expressing the truncated receptor correlates with increases in receptor-associated phosphatidylinositol 3-kinase (PI3K) activity and expression of Bcl-X(L), but not with changes in c-Myc expression or proliferation. Inhibition of the PI3K pathway in these cells, but not in cells expressing the wild-type receptor, has a marked effect on the capacity of IL-2 to prevent cell death and diminishes the Bcl-X(L) response. The requirement for IL-2-induced PI3K activity in suppressing the onset of apoptotic cell death is discussed.