Alison M. Michie

Obligatory Role for Cooperative Signaling by Pre-TCR and Notch during Thymocyte Differentiation

The first checkpoint during T cell development, known as β selection, requires the successful rearrangement of the TCR-β gene locus. Notch signaling has been implicated in various stages during T lymphopoiesis. However, it is unclear whether Notch receptor-ligand interactions are necessary during β selection. Here, we show that pre-TCR signaling concurrent with Notch receptor and Delta-like-1 ligand interactions are required for the survival, proliferation, and differentiation of mouse CD4−CD8− thymocytes to the CD4+CD8+ stage. Furthermore, we address the minimal signaling requirements underlying β selection and show a hierarchical positioning of key proximal signaling molecules. Collectively, our results demonstrate an essential role for Notch receptor-ligand interactions in enabling the autonomous signaling capacity of the pre-TCR complex.

Regulation of thymocyte differentiation: pre-TCR signals and β-selection

The specificity of the adaptive immune response is, in part, dependent on the clonal expression of the mature T cell receptor (TCR) on T lymphocytes. One mechanism regulating the clonality of the TCR occurs at the level of TCR-beta gene rearrangements during lymphocyte development. Expression of a nascent TCR-beta chain together with pre-Talpha (pTalpha) and CD3 molecules to form the pre-TCR complex, represents a critical checkpoint in T cell differentiation known as beta-selection. Indeed, failure to generate a functionally rearranged TCR-beta chain at this stage of development results in apoptosis. Signals derived from the pre-TCR complex trigger a maturation program within developing thymocytes that includes: rescue from apoptosis; inhibition of further DNA recombination at the TCR-beta gene locus (allowing for the clonality of antigen receptor expression; allelic exclusion); and induction of proliferation and differentiation. The signaling mechanisms that control this developmental program remain largely undefined. Here, we discuss recent evidence investigating the molecular mechanisms that regulate thymocyte differentiation downstream of pre-TCR formation.

Branching out to gain control: how the pre-TCR is linked to multiple functions

How is signaling specificity achieved by the pre-TCR during selection of T-cell fate? Like the TCR, this receptor controls many functions, and recent studies define which pathways couple the pre-TCR to the molecular events controlling survival, proliferation, allelic exclusion at the TCRbeta locus, and further differentiation.

Duration and Strength of Extracellular Signal-Regulated Kinase Signals Are Altered During Positive Versus Negative Thymocyte Selection

During thymocyte development, high-affinity/avidity TCR engagement leads to the induction of negative selection and apoptosis, while lower TCR affinity-avidity interactions lead to positive selection and survival. To elucidate how these extracellular interactions are translated into intracellular signals that distinguish between positive and negative selection, we developed a culture system in which naive double-positive thymocytes were either induced to differentiate along the CD8+ lineage pathway or were triggered for clonal deletion. Using this system, we show that sustained low level activation of extracellular signal-regulated kinases (ERKs) promotes positive selection, whereas strong but transient ERK activation is coupled with negatively selecting stimuli. Importantly, similar ERK activation profiles were demonstrated during positive selection for strong agonist ligands presented at low concentrations or weak agonist ligands. This is consistent with the affinity/avidity model and a role for strong or weak agonists during positive selection. Surprisingly, the addition of a pharmacological inhibitor which blocks ERK activation prevented the induction of negative selection. These data suggest that the duration and strength of the TCR signal is involved in discriminating between positive and negative selection.

Clonal Characterization of a Bipotent T Cell and NK Cell Progenitor in the Mouse Fetal Thymus

We recently described a population of fetal thymocytes with a CD117+NK1.1+CD90lowCD25− phenotype, which were shown to contain committed T cell and NK cell progenitors. However, the characterization of a single cell with a restricted T and NK cell precursor potential was lacking. Here, using an in vitro model for T and NK cell differentiation, we provide conclusive evidence demonstrating the existence of a clonal lineage-restricted T and NK cell progenitor. These results establish that fetal thymocytes with a CD117+NK1.1+CD90lowCD25− phenotype represent bipotent T and NK cell progenitors.

Rapid regulation of PDE-2 and PDE-4 cyclic AMP phosphodiesterase activity following ligation of the T cell antigen receptor on thymocytes: analysis using the selective inhibitors erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) and rolipram

The PDE2, cyclic GMP-stimulated, and the PDE4, cyclic AMP-specific enzymes provide the major, detectable cyclic AMP phosphodiesterase activities in murine thymocytes. In the absence of the cyclic GMP, PDE4 activity predominated (approximately 80% total) but in the presence of low (10 microM) cyclic GMP concentrations, PDE2 activity constituted the major PDE activity in thymocytes (approximately 80% total). The PDE4 selective inhibitor rolipram dose-dependently inhibited thymocyte PDE4 activity (IC50 approximately 65 nM). PDE2 was dose-dependently activated (EC50 approximately 1 microM) by cyclic GMP and inhibited by erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) (IC50 approximately 4 microM). EHNA was shown to serve as a selective inhibitor of PDE-2 activity as assessed from studies using separated PDE1, PDE2, PDE3 and PDE4 species from hepatocytes as well as human PDE2 and PDE4 enzymes. EHNA completely ablated the ability of cyclic GMP to activate PDE2 activity, whilst having a much smaller inhibitory effect on the unstimulated PDE2 activity. EHNA exhibited normal Michaelian kinetics of inhibition for the cyclic GMP-stimulated PDE2 activity with Hill plots near unity. Apparent negative co-operative effect were seen in the absence of cyclic GMP with Hill coefficients of approximately 0.3 for inhibition of PDE2 activity. Within 5 min of challenge of thymocytes with the lectin phytohaemagglutinin (PHA) there was a transient decrease (approximately 83%) in PDE-4 activity and in PDE2 activity (approximately 40%). Both anti-TCR antibodies also caused an initial reduction in the PDE4 activity which was followed by a sustained and profound increase in activity. In contrast to that observed with PHA, anti-TCR/CD3 antisera had little effect on PDE2 activity. It is suggested that, dependent upon the intracellular concentrations of cyclic GMP, thymocyte cyclic AMP metabolism can be expected to switch from being under the predominant control of PDE4 activity to that determined predominantly by PDE2 activity. These activities may be rapidly and differentially regulated following ligation of different cell surface receptors.

Death-associated protein kinase (DAPK) and signal transduction: regulation in cancer

Death‐associated protein kinase (DAPK) is a pro‐apoptotic serine/threonine protein kinase that is dysregulated in a wide variety of cancers. The mechanism by which this occurs has largely been attributed to promoter hypermethylation, which results in gene silencing. However, recent studies indicate that DAPK expression can be detected in some cancers, but its function is still repressed, suggesting that DAPK activity can be subverted at a post‐translational level in cancer cells. This review will focus on recent data describing potential mechanisms that may alter the expression, regulation or function of DAPK.

Dasatinib inhibits B cell receptor signalling in chronic lymphocytic leukaemia but novel combination approaches are required to overcome additional pro-survival microenvironmental signals

As antigenic stimulation of the B cell antigen receptor (BCR) is key to chronic lymphocytic leukaemia (CLL) pathogenesis, targeting dysregulated kinases involved in BCR signalling is an attractive therapeutic approach. We studied the effects of the Src/c‐Abl tyrosine kinase inhibitor dasatinib on BCR signal transduction in CLL cells. Treatment of CLL cells with 100 nmol/l dasatinib induced apoptosis by an average reduction in viability of 33·7% at 48 h, with dasatinib sensitivity correlating with inhibition of SykY348 phosphorylation. Dasatinib inhibited calcium flux, phosphatidylinositol‐3‐kinase and mitogen‐activated protein kinase activation following BCR crosslinking, and blocked the Mcl‐1‐dependent increase in CLL cell survival on prolonged BCR stimulation. However, the pro‐apoptotic effect of dasatinib was abrogated by stromal cell contact alone or in the presence of CD154 and interleukin (IL)‐4 (CD154L/IL‐4 system). Whilst dasatinib retained the ability to sensitize CLL cells in stromal co‐culture to both fludarabine and chlorambucil, the addition of CD154 and IL‐4 rendered cells resistant to these drug combinations. We demonstrate that the HSP90 inhibitor 17‐DMAG exhibited synergy with dasatinib in vitro, and moreover, induced apoptosis of CLL cells in the CD154L/IL‐4 system. Our data provide evidence that dasatinib would be most clinically effective in combination with agents able to target antigen‐independent microenvironmental signals.

Megakaryocytes assemble podosomes that degrade matrix and protrude through basement membrane

Megakaryocytes give rise to platelets via extension of proplatelet arms, which are released through the vascular sinusoids into the bloodstream. Megakaryocytes and their precursors undergo varying interactions with the extracellular environment in the bone marrow during their maturation and positioning in the vascular niche. We demonstrate that podosomes are abundant in primary murine megakaryocytes adherent on multiple extracellular matrix substrates, including native basement membrane. Megakaryocyte podosome lifetime and density, but not podosome size, are dependent on the type of matrix, with podosome lifetime dramatically increased on collagen fibers compared with fibrinogen. Podosome stability and dynamics depend on actin cytoskeletal dynamics but not matrix metalloproteases. However, podosomes degrade matrix and appear to be important for megakaryocytes to extend protrusions across a native basement membrane. We thus demonstrate for the first time a fundamental requirement for podosomes in megakaryocyte process extension across a basement membrane, and our results suggest that podosomes may have a role in proplatelet arm extension or penetration of basement membrane.

Subversion of Protein Kinase Cα Signaling in Hematopoietic Progenitor Cells Results in the Generation of a B-Cell Chronic Lymphocytic Leukemia–Like Population In vivo

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived mature B cells with the distinctive phenotype CD19(hi) CD5+ CD23+ IgM(lo), which are refractory to apoptosis. An increased level of apoptosis has been observed on treatment of human B-CLL cells with protein kinase C (PKC) inhibitors, suggesting that this family of protein kinases mediate survival signals within B-CLL cells. Therefore, to investigate the ability of individual PKC isoforms to transform developing B cells, we stably expressed plasmids encoding PKC mutants in fetal liver-derived hematopoietic progenitor cells (HPC) from wild-type mice and then cultured them in B-cell generation systems in vitro and in vivo. Surprisingly, we noted that expression of a plasmid-encoding dominant-negative PKC alpha (PKC alpha-KR) in HPCs and subsequent culture both in vitro and in vivo resulted in the generation of a population of cells that displayed an enhanced proliferative capacity over untransfected cells and phenotypically resemble human B-CLL cells. In the absence of growth factors and stroma, these B-CLL-like cells undergo cell cycle arrest and, consistent with their ability to escape growth factor withdrawal-induced apoptosis, exhibited elevated levels of Bcl-2 expression. These studies therefore identify a unique oncogenic trigger for the development of a B-CLL-like disease resulting from the subversion of PKC alpha signaling. Our findings uncover novel avenues not only for the study of the induction of leukemic B cells but also for the development of therapeutic drugs to combat PKC alpha-regulated transformation events.