David C. Scherer

A role for Wnt signalling in self-renewal of haematopoietic stem cells

Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated β-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.

Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation.

The eukaryotic transcription factor NF-kappa B plays a central role in the induced expression of human immunodeficiency virus type 1 and in many aspects of the genetic program mediating normal T-cell activation and growth. The nuclear activity of NF-kappa B is tightly regulated from the cytoplasmic compartment by an inhibitory subunit called I kappa B alpha. This cytoplasmic inhibitor is rapidly phosphorylated and degraded in response to a diverse set of NF-kappa B-inducing agents, including T-cell mitogens, proinflammatory cytokines, and viral transactivators such as the Tax protein of human T-cell leukemia virus type 1. To explore these I kappa B alpha-dependent mechanisms for NF-kappa B induction, we identified novel mutants of I kappa B alpha that uncouple its inhibitory and signal-transducing functions in human T lymphocytes. Specifically, removal of the N-terminal 36 amino acids of I kappa B alpha failed to disrupt its ability to form latent complexes with NF-kappa B in the cytoplasm. However, this deletion mutation prevented the induced phosphorylation, degradative loss, and functional release of I kappa B alpha from NF-kappa B in Tax-expressing cells. Alanine substitutions introduced at two serine residues positioned within this N-terminal regulatory region of I kappa B alpha also yielded constitutive repressors that escaped from Tax-induced turnover and that potently inhibited immune activation pathways for NF-kappa B induction, including those initiated from antigen and cytokine receptors. In contrast, introduction of a phosphoserine mimetic at these sites rectified this functional defect, a finding consistent with a causal linkage between the phosphorylation status and proteolytic stability of this cytoplasmic inhibitor. Together, these in vivo studies define a critical signal response domain in I kappa B alpha that coordinately controls the biologic activities of I kappa B alpha and NF-kappa B in response to viral and immune stimuli.

The natural killer T-cell ligand α-galactosylceramide prevents autoimmune diabetes in non-obese diabetic mice

Diabetes in non-obese diabetic (NOD) mice is mediated by pathogenic T-helper type 1 (Th1) cells that arise because of a deficiency in regulatory or suppressor T cells. Vα14–Jα15 natural killer T (NKT) cells recognize lipid antigens presented by the major histocompatibility complex class I-like protein CD1d (refs. 3,4). We have previously shown that in vivo activation of Vα14 NKT cells by α-galactosylceramide (α-GalCer) and CD1d potentiates Th2-mediated adaptive immune responses. Here we show that α-GalCer prevents development of diabetes in wild-type but not CD1d-deficient NOD mice. Disease prevention correlated with the ability of α-GalCer to suppress interferon-γ but not interleukin-4 production by NKT cells, to increase serum immunoglobulin E levels, and to promote the generation of islet autoantigen-specific Th2 cells. Because α-GalCer recognition by NKT cells is conserved among mice and humans, these findings indicate that α-GalCer might be useful for therapeutic intervention in human diseases characterized by Th1-mediated pathology such as Type 1 diabetes.

Cell-fate conversion of lymphoid-committed progenitors by instructive actions of cytokines

The primary role of cytokines in haemato-lymphopoiesis is thought to be the regulation of cell growth and survival. But the instructive action of cytokines in haematopoiesis has not been well addressed. Here we show that a clonogenic common lymphoid progenitor, a bone marrow-resident cell that gives rise exclusively to lymphocytes (T, B and natural killer cells), can be redirected to the myeloid lineage by stimulation through exogenously expressed interleukin (IL)-2 and GM-CSF (granulocyte/macrophage colony-stimulating factor) receptors. Analysis of mutants of the β-chain of the IL-2 receptor revealed that the granulocyte- and monocyte-differentiation signals are triggered by different cytoplasmic domains, showing that the signalling pathway(s) responsible for these unique developmental outcomes are separable. Finally, we show that the endogenous myelo-monocytic cytokine receptors for GM-CSF and macrophage colony-stimulating factor (M-CSF) are expressed at low to moderate levels on the more primitive haematopoietic stem cells, are absent on common lymphoid progenitors, and are upregulated after myeloid lineage induction by IL-2. We conclude that cytokine signalling can regulate cell-fate decisions and propose that a critical step in lymphoid commitment is downregulation of cytokine receptors that drive myeloid cell development.

Inactivation of IkappaBbeta by the tax protein of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of NF-kappaB.

In resting T lymphocytes, the transcription factor NF-kappaB is sequestered in the cytoplasm via interactions with members of the I kappa B family of inhibitors, including IkappaBalpha and IkappaBbeta. During normal T-cell activation, IkappaBalpha is rapidly phosphorylated, ubiquitinated, and degraded by the 26S proteasome, thus permitting the release of functional NF-kappaB. In contrast to its transient pattern of nuclear induction during an immune response, NF-kappaB is constitutively activated in cells expressing the Tax transforming protein of human T-cell leukemia virus type I (HTLV-1). Recent studies indicate that HTLV-1 Tax targets IkappaBalpha to the ubiquitin-proteasome pathway. However, it remains unclear how this viral protein induces a persistent rather than transient NF-kappaB response. In this report, we provide evidence that in addition to acting on IkappaBalpha, Tax stimulates the turnover Of IkappaBbeta via a related targeting mechanism. Like IkappaBalpha, Tax-mediated breakdown of IkappaBbeta in transfected T lymphocytes is blocked either by cell-permeable proteasome inhibitors or by mutation Of IkappaBbeta at two serine residues present within its N-terminal region. Despite the dual specificity of HTLV-1 Tax for IkappaBalpha and IkappaBbeta at the protein level, Tax selectively stimulates NF-kappaB-directed transcription of the IkappaBalpha gene. Consequently, IkappaBbeta protein expression is chronically downregulated in HTLV-1-infected T lymphocytes. These findings with IkappaBbeta provide a potential mechanism for the constitutive activation of NF-kappaB in Tax-expressing cells.

Lipid antigen presentation in the immune system : lessons learned from CD1d knockout mice

Summary: CD I molecules represent a distinct lineage of antigen‐presenting molecules chat are evolutionarily related to the classical major histocompatility complex (MHC) dass I and class II molecules, Unlike the classical MHC products that bind peptides, GDI molecules have evolved Co bind lipids and glycolipids, Murine and human CD Id molecules can present glycolipid antigens such as α‐galactosylceramide (α‐GalCer) to CD 1d‐restricced natural killer (NK) T cells. Using CD 1d knockout mice we demonstrated chat CDI d expression is required for the development of NK T cells. These animals were also deficient in the rapid production of inter‐leukin‐4 and intcrferon‐γ in response to stimulation by anti‐CD3 antibodies. Despite these defects, CD Id knockout animals were able to generate strong T‐helper type 1 (TH1) and TH2 responses. Spleen cells from these animals neither proliferated nor produced cytokines in response to stimulation by α‐GalCer, Repeated injection of α‐GalCer into wild‐type but not CD 1 d mutant mice was able to clear metastatic tumors. We further showed that α‐GalCer can inhibit disease in diabetes‐prone non‐obese diabetic mice. Collectively, these findings with CD ld knockout animals indicate a critical role for CD 1 d‐dependent T cells in various disease conditions, and suggest that α‐GalCer may be useful for therapeutic intervention in these diseases.

Lineage infidelity in myeloid cells with TCR gene rearrangement: A latent developmental potential of proT cells revealed by ectopic cytokine receptor signaling

The most immature lymphoid-committed progenitors in both the bone marrow (common lymphoid progenitor) and thymus (proT1) maintain a latent granulocyte/macrophage (G/M) differentiation potential that can be initiated by signals emanating from exogenously expressed IL-2 receptors. In this study, we investigate at which developmental stage thymocytes lose this G/M differentiation potential. We demonstrate that the next maturational stage after proT1 cells (proT2), but not preT (TN3) cells, can convert cell fate from lymphoid to myeloid in response to ectopic IL-2 receptor signaling in human IL-2Rβ transgenic mice. It is significant that approximately 10% of clonogenic G/M colonies derived from proT cells of IL-2Rβ transgenic mice have DJ rearrangement specifically at the Dβ1 but not Dβ2 segment in the TCRβ locus. No TCR gene rearrangement is observed in G/M cells from nontransgenic mice, suggesting that the G/M cells we observe in this system were truly lymphoid-committed before stimulation with IL-2. In addition, Dβ1 and Dβ2 DJ rearrangement of the TCRβ gene may be differentially regulated and thus serve as markers for distinct proT cell maturational stages.

Lymphocyte development from hematopoietic stem cells.

The recent application of new techniques, such as multi-color cell sorting and the production of transgenic and gene-knockout mice, has contributed to a better understanding of lymphocyte development from hematopoietic stem cells. Now that we can purify progenitors at different maturational stages during lymphocyte development, the challenge is to understand the processes that govern each developmental stage transition.

Corepression of RelA and c-Rel Inhibits Immunoglobulin κ Gene Transcription and Rearrangement in Precursor B Lymphocytes

Multiple members of the NF-kappa B/Rel protein family are induced during B cell differentiation and have been implicated in transcriptional activation of the immunoglobulin kappa (Ig kappa) locus. Despite these findings, normal numbers of Ig kappa + B lymphocytes are produced by mice bearing targeted mutations in individual NF-kappa B/Rel genes. In the present study, precursor B lymphocytes were engineered to express a trans-dominant form of I kappa B alpha that simultaneously impairs the c-Rel and RelA transactivating subunits of NF-kappa B. This dual block in NF-kappa B/Rel signaling led to potent inhibition of germline Ig kappa transcription and rearrangement, whereas recombinase activity was unaffected. These findings suggest that c-Rel and RelA serve compensatory functional roles in the developmental mechanisms that govern Ig kappa gene assembly.

Transcription Factor NF-κB Regulates Inducible Oct-2 Gene Expression in Precursor B Lymphocytes

The POU transcription factors Oct-1 and Oct-2 regulate the activity of octamer-dependent promoters, including those that direct transcription from rearranged immunoglobulin genes. Unlike Oct-1, which is constitutively expressed in many cell types, Oct-2 expression is restricted primarily to B lymphocytes and can be induced in precursor B cells by stimulation with bacterial lipopolysaccharide (LPS). However, the precise factors that mediate this induction mechanism remain unknown. In the present study, we monitored Oct-2 expression in cells arrested for the activation of NF-κB, an LPS-responsive member of the Rel transcription factor family. Despite stimulation with LPS, disruption of the NF-κB signaling pathway in precursor B cells led to the loss of inducible Oct-2 DNA binding activity in vitro and the suppression of Oct-2-directed transcription in vivo. This biochemical defect correlated with a specific block to Oct-2 gene expression at the level of transcription, whereas the expression of Oct-1 was unaffected. The finding that Oct-2 is under NF-κB control highlights an important cross-talk mechanism involving two distinct transcription factor families that regulate B lymphocyte function.