Fredrick G. Karnell

Notch1 Expression in Early Lymphopoiesis Influences B versus T Lineage Determination

Notch receptors regulate fate decisions in many cells. One outcome of Notch signaling is differentiation of bipotential precursors into one cell type versus another. To investigate consequences of Notch1 expression in hematolymphoid progenitors, mice were reconstituted with bone marrow (BM) transduced with retroviruses encoding a constitutively active form of Notch1. Although neither granulocyte or monocyte differentiation were appreciably affected, lymphopoiesis was dramatically altered. As early as 3 weeks following transplantation, mice receiving activated Notch1-transduced BM contained immature CD4+ CD8+ T cells in the BM and exhibited a simultaneous block in early B cell lymphopoiesis. These results suggest that Notch1 provides a key regulatory signal in determining T lymphoid versus B lymphoid lineage decisions, possibly by influencing lineage commitment from a common lymphoid progenitor cell.

Essential roles for ankyrin repeat and transactivation domains in induction of T-cell leukemia by notch1

ABSTRACT Notch receptors participate in a conserved signaling pathway that controls the development of diverse tissues and cell types, including lymphoid cells. Signaling is normally initiated through one or more ligand-mediated proteolytic cleavages that permit nuclear translocation of the intracellular portion of the Notch receptor (ICN), which then binds and activates transcription factors of the Su(H)/CBF1 family. Several mammalian Notch receptors are oncogenic when constitutively active, including Notch1, a gene initially identified based on its involvement in a (7;9) chromosomal translocation found in sporadic T-cell lymphoblastic leukemias and lymphomas (T-ALL). To investigate which portions of ICN1 contribute to transformation, we performed a structure-transformation analysis using a robust murine bone marrow reconstitution assay. Both the ankyrin repeat and C-terminal transactivation domains were required for T-cell leukemogenesis, whereas the N-terminal RAM domain and a C-terminal domain that includes a PEST sequence were nonessential. Induction of T-ALL correlated with the transactivation activity of each Notch1 polypeptide when fused to the DNA-binding domain of GAL4, with the exception of polypeptides deleted of the ankyrin repeats, which lacked transforming activity while retaining strong transactivation activity. Transforming polypeptides also demonstrated moderate to strong activation of the Su(H)/CBF1-sensitive HES-1 promoter, while polypeptides with weak or absent activity on this promoter failed to cause leukemia. These experiments define a minimal transforming region for Notch1 in T-cell progenitors and suggest that leukemogenic signaling involves recruitment of transcriptional coactivators to ICN1 nuclear complexes.

Deltex1 redirects lymphoid progenitors to the B cell lineage by antagonizing Notch1.

Notch1 signaling drives T cell development at the expense of B cell development from a common precursor, an effect that is dependent on a C-terminal Notch1 transcriptional activation domain. The function of Deltex1, initially identified as a positive modulator of Notch function in a genetic screen in Drosophila, is poorly understood. We now demonstrate that, in contrast to Notch1, enforced expression of Deltex1 in hematopoietic progenitors results in B cell development at the expense of T cell development in fetal thymic organ culture and in vivo. Consistent with these effects, Deltex1 antagonizes Notch1 signaling in transcriptional reporter assays by inhibiting coactivator recruitment. These data suggest that a balance of inductive Notch1 signals and inhibitory signals mediated through Deltex1 and other modulators regulate T-B lineage commitment.

Notch1 Regulates Maturation of CD4+ and CD8+ Thymocytes by Modulating TCR Signal Strength

Notch signaling regulates cell fate decisions in multiple lineages. We demonstrate in this report that retroviral expression of activated Notch1 in mouse thymocytes abrogates differentiation of immature CD4+CD8+ thymocytes into both CD4 and CD8 mature single-positive T cells. The ability of Notch1 to inhibit T cell development was observed in vitro and in vivo with both normal and TCR transgenic thymocytes. Notch1-mediated developmental arrest was dose dependent and was associated with impaired thymocyte responses to TCR stimulation. Notch1 also inhibited TCR-mediated signaling in Jurkat T cells. These data indicate that constitutively active Notch1 abrogates CD4+ and CD8+ maturation by interfering with TCR signal strength and provide an explanation for the physiological regulation of Notch expression during thymocyte development.

Tonic B cell antigen receptor signals supply an NF-κB substrate for prosurvival BLyS signaling

The survival of transitional and mature B cells requires both the B cell antigen receptor (BCR) and BLyS receptor 3 (BR3), which suggests that these receptors send signals that are nonredundant or that engage in crosstalk with each other. Here we show that BCR signaling induced production of the nonclassical transcription factor NF-κB pathway substrate p100, which is required for transmission of BR3 signals and thus B cell survival. The capacity for sustained p100 production emerged during transitional B cell differentiation, the stage at which BCR signals begin to mediate survival rather than negative selection. Our findings identify a molecular mechanism for the reliance of primary B cells on continuous BR3 and BCR signaling, as well as for the gradual resistance to negative selection that is acquired during B cell maturation.

Analysis of the Individual Contributions of Igα (CD79a)- and Igβ (CD79b)-Mediated Tonic Signaling for Bone Marrow B Cell Development and Peripheral B Cell Maturation

The individual contribution of Igα and Igβ for BCR-triggered fates is unclear. Prior evidence supports conflicting ideas concerning unique as well as redundant functions for these proteins in the context of BCR/pre-BCR signaling. Part of this ambiguity may reflect the recent appreciation that Igα and Igβ participate in both Ag-independent (tonic) and Ag-dependent signaling. The present study undertook defining the individual requirement for Igα and Igβ under conditions where only ligand-independent tonic signaling was operative. In this regard, we have constructed chimeric proteins containing one or two copies of the cytoplasmic domains of either Igα or Igβ and Igα/Igβ heterodimers with targeted Tyr→Phe modifications. The ability of these proteins to act as surrogate receptors and trigger early bone marrow and peripheral B cell maturation was tested in RAG2−/− primary pro-B cell lines and in gene transfer experiments in the μMT mouse model. We considered that the threshold for a functional activity mediated by the pre-BCR/BCR might only be reached when two functional copies of the Igα/Igβ ITAM domain are expressed together, and therefore the specificity conferred by these proteins can only be observed in these conditions. We found that the ligand-independent tonic signal is sufficient to drive development into mature follicular B cells and both Igα and Igβ chains supported formation of this population. In contrast, neither marginal zone nor B1 mature B cell subsets develop from bone marrow precursors under conditions where only tonic signals are generated.

Notch Ankyrin Repeat Domain Variation Influences Leukemogenesis and Myc Transactivation

Background The functional interchangeability of mammalian Notch receptors (Notch1-4) in normal and pathophysiologic contexts such as cancer is unsettled. We used complementary in vivo, cell-based and structural analyses to compare the abilities of activated Notch1-4 to support T cell development, induce T cell acute lymphoblastic leukemia/lymphoma (T-ALL), and maintain T-ALL cell growth and survival. Principal Findings We find that the activated intracellular domains of Notch1-4 (ICN1-4) all support T cell development in mice and thymic organ culture. However, unlike ICN1-3, ICN4 fails to induce T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) and is unable to rescue the growth of Notch1-dependent T-ALL cell lines. The ICN4 phenotype is mimicked by weak gain-of-function forms of Notch1, suggesting that it stems from a failure to transactivate one or more critical target genes above a necessary threshold. Experiments with chimeric receptors demonstrate that the Notch ankyrin repeat domains differ in their leukemogenic potential, and that this difference correlates with activation of Myc, a direct Notch target that has an important role in Notch-associated T-ALL. Conclusions/Significance We conclude that the leukemogenic potentials of Notch receptors vary, and that this functional difference stems in part from divergence among the highly conserved ankyrin repeats, which influence the transactivation of specific target genes involved in leukemogenesis.

The Role of Membrane Lipids in the Regulation of Immune Cell Activity

Engagement of immune receptors by antigen triggers processes that culminate in activation, cell proliferation, differentiation and effector functions. A growing body of evidence suggests that it is within specialized membrane regions called lipid rafts that the initiation and propagation of the signaling events taking place in immune cells occurs. These specialized membrane compartments are enriched in cholesterol and glycolipids and contain many lipid-modified signaling proteins such as GPI (glycosylphosphatidylinositol)- linked proteins, Src family tyrosine kinases and other adaptor proteins. By confining certain signaling molecules in membrane subdomains, lipid rafts have been proposed to function as platforms for the formation of multi-unit transduction complexes. Therefore, it has been suggested that the translocation of immune receptors into lipid rafts regulates immune cell activation. In lymphocytes it has been shown that immune receptors become lipid raft-associated upon antigen cross-linking, and signaling complexes are formed when additional components of the signaling pathways are recruited to lipid rafts. Furthermore, substantial evidence from a number of experiments shows that the integrity of lipid rafts is crucial for the initiation and maintenance of intracellular signals. Recent studies have also shown that the access and translocation of immune receptors to lipid rafts are developmentally regulated (immature versus mature cells, naïve versus effector) and that the cholesterol content plays an important role in maintaining plasma membrane heterogeneity and influencing a variety of cellular processes including signaling, adhesion, and permeability. The aim of this review is to examine the role membrane lipids play in regulating immune activation.