Complement deficiencies limit CD20 monoclonal antibody treatment efficacy in CLL.

Monoclonal antibodies (MAbs) form a central part of chronic lymphocytic leukaemia (CLL) treatment. We therefore evaluated whether complement defects in CLL patients reduced the induction of complement-dependent cytotoxicity (CDC) by using anti-CD20 MAbs rituximab (RTX) and ofatumumab (OFA). Ofatumumab elicited higher CDC levels than RTX in all CLL samples examined, particularly in poor prognosis cohorts (11q- and 17p-). Serum sample analyses revealed that 38.1% of patients were deficient in one or more complement components, correlating with reduced CDC responses. Although a proportion of patients with deficient complement levels initially induced high levels of CDC, on secondary challenge CDC activity in sera was significantly reduced, compared with that in normal human serum (NHS; P<0.01; n=52). In addition, a high CLL cell number contributed to rapid complement exhaustion. Supplementing CLL serum with NHS or individual complement components, particularly C2, restored CDC on secondary challenge to NHS levels (P<0.0001; n=9). In vivo studies revealed that complement components were exhausted in CLL patient sera post RTX treatment, correlating with an inability to elicit CDC. Supplementing MAb treatment with fresh-frozen plasma may therefore maintain CDC levels in CLL patients with a complement deficiency or high white blood cell count. This study has important implications for CLL patients receiving anti-CD20 MAb therapy.

Murine models for chronic lymphocytic leukaemia.

CLL (chronic lymphocytic leukaemia) is characterized by the clonal outgrowth of B-lymphocytes with the distinctive phenotype: CD19(hi)CD5(+)CD23(+)IgM(lo). These malignant B-cells accumulate in the PB (peripheral blood) and lymphoid organs, and are generally arrested at the G(0)/G(1)-phase of cell cycle and display a resistance to apoptosis. To date, most of the CLL research has been carried out using PB samples obtained from patients with established CLL, which have proved instrumental in characterizing the disease. However, while CLL cells appear to have a defect in apoptosis in vivo, they rapidly undergo apoptosis ex vivo, suggesting that CLL cells are dependent on microenvironmental signals to enhance cell survival. One approach used to define the cellular and molecular events that govern CLL has been the development of murine models that replicate the human disease. As well as providing a deeper understanding of the potential triggers for CLL, these models provide preclinical in vivo systems to test novel therapies. The focus of the present review will be to highlight the recent advances in the development of mouse models for CLL.

Notch is constitutively active in Theileria-transformed B cells and can be further stimulated by the filarial nematode-secreted product, ES-62.

Theileria parva-infected B cells express Jagged-1 and activate Notch signalling in a parasite-dependent manner. ES-62, a filarial nematode-secreted phosphorylcholine-containing glycoprotein, is able to further stimulate Notch-mediated signalling in parasitized cells. Notch is also activated to a similar extent by addition of exogenous IL-10, and this occurs prior to any increase in proliferation in T. parva-infected B cells.

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.

Allelic exclusion and differentiation by protein kinase C-mediated signals in immature thymocytes.

Pre-T cell receptor (preTCR)-derived signals mediate the transition of thymocytes from the CD4(-) CD8(-) double-negative (DN) to CD4(+) CD8(+) double-positive stage of T lymphocyte development. This progression, termed beta-selection, is limited to thymocytes that have generated a functional TCR-beta chain able to associate with pTalpha to form the preTCR complex. Formation of the preTCR complex not only induces differentiation, survival, and proliferation of DN thymocytes; it also inhibits further TCR-beta gene rearrangement through an ill-defined process known as allelic exclusion. The signaling pathways controlling this critical developmental checkpoint have not been characterized. Here we demonstrate that formation of the preTCR complex leads to the activation of protein kinase C (PKC), and that activation of PKC is necessary for the differentiation and expansion of DN thymocytes. Importantly, we also show that allelic exclusion at the TCR-beta gene loci is enforced by PKC-mediated signals. These results define PKC as a central mediator of both differentiation and allelic exclusion during thymocyte development.

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.

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.

In vivo detection of intracellular signaling pathways in developing thymocytes.

Information regarding the intracellular signaling processes that occur during the development of T cells has largely been obtained with the use of transgenic mouse models, which although providing invaluable information are time consuming and costly. To this end, we have developed a novel system that facilitates the in vivo analysis of signal transduction pathways during T-lymphocyte development. This approach uses reporter-plasmids for the detection of intracellular signals mediated by the mitogen-activated protein kinase or cyclic AMP-dependent protein kinase. Reporter-plasmids are transfected into thymocytes in fetal thymic organ culture by accelerated DNA/particle bombardment (gene gun), and the activation of a signaling pathway is determined in the form of a standard luciferase assay. Importantly, this powerful technique preserves the structural integrity of the thymus, and will provide an invaluable tool to study how thymocytes respond to normal environmental stimuli encountered during differentiation within the thymic milieu. Thus, this method allows for the monitoring of signals that occur in a biological time frame, such as during differentiation, and within the natural environment of differentiating cells.

Extracellular signal-regulated kinase (ERK) activation by the pre-T cell receptor in developing thymocytes in vivo.

The first checkpoint in T cell development occurs between the CD4(-)CD8(-) and CD4(+)CD8(+) stages and is associated with formation of the pre-T cell receptor (TCR). The signaling mechanisms that drive this progression remain largely unknown. Here, we show that extracellular signal-regulated kinases (ERKs)-1/2 are activated upon engagement of the pre-TCR. Using a novel experimental system, we demonstrate that expression of the pre-TCR by developing thymocytes induces ERK-1/2 activation within the thymus. In addition, the activation of this pre-TCR signaling cascade is mediated through Lck. These findings directly link pre-TCR complex formation with specific downstream signaling components in vivo.

Natural killer cell development and function precede alpha beta T cell differentiation in mouse fetal thymic ontogeny.

Natural killer (NK) cells mediate MHC-unrestricted cytolysis of virus-infected cells and tumor cells. In the adult mouse, NK cells are bone marrow-derived lymphocytes that mature predominantly in extrathymic locations but have also been suggested to share a common intrathymic progenitor with T lymphocytes. However, mature NK cells are thought to be absent in mouse fetal ontogeny. We report the existence of thymocytes with a mature NK cell phenotype (NK1.1+/CD117-) as early as day 13 of gestation, approximately 3 days before the appearance of CD4+/CD8+ cells in T lymphocyte development. These mature fetal thymic NK cells express genes associated with NK cell effector function and, when freshly isolated, display MHC-unrestricted cytolytic activity in vitro. Moreover, the capacity of fetal thymic NK cells for sustained growth both in vitro and in vivo, in addition to their close phenotypic resemblance to early precursor thymocytes, confounds previous assessments of NK lineage precursor function. Thus, mature NK cells may have been inadvertently included in previous attempts to identify multipotent and bipotent precursor thymocytes. These results provide the first evidence of functional NK lymphocytes in mouse fetal ontogeny and demonstrate that NK cell maturation precedes alpha beta T cell development in the fetal thymus.

Changes following community living skills training: a controlled study.

Fifty-seven adults with mild to moderate learning disability served as participants in a community living skills training programme. Twenty-nine were trained using in vivo techniques, 13 were taught using classroom techniques and 15 acted as a no treatment control group. Assessments of community living skills and adaptive behaviour found those receiving in vivo training performed significantly better than the other groups. Eventual placement in the community reflected this superiority.

Upregulation of cAMP-specific PDE-4 activity following ligation of the TCR complex on thymocytes is blocked by selective inhibitors of protein kinase C and tyrosyl kinases.

We have previously shown that the major cAMP phosphodiesterase (PDE) isoforms present in murine thymocytes are the cGMP-stimulated PDE activity (PDE-2) and the cAMP-specific PDE activity (PDE-4), and that these isoforms are differentially regulated following ligation of the TCR (Michie, A.M., Lobban, M. D., Mueller, T., Harnett, M. M., and Houslay, M.D. [1996] Cell. Signalling 8, 97-110). We show here that the anti-CD3-stimulated elevation in PDE-4 activity in murine thymocytes is dependent on protein tyrosine kinase and protein kinase C (PKC)-mediated signals as the TCR-coupled increase in PDE-4 activity can be abrogated by both the tyrosine kinase inhibitor, genistein, and the PKC selective inhibitors chelerythrine and staurosporine. Moreover, the PKC-activating phorbol ester, phorbol-12-myristate, 13-acetate (PMA) caused an increase in PDE-4 activity, similar to that observed in cells challenged with anti-CD3 monoclonal antibodies and which was not additive with cochallenge using anti-CD3 antibodies. Both the PMA- and the anti-CD3 antibody-mediated increases in PDE-4 activity were blocked by treatment with either cycloheximide or actinomycin D. Despite the upregulation of PDE-4 activity consequent to TCR ligation, intracellular cAMP levels increased on challenge of thymocytes with anti-CD3 antibody, indicating that adenylate cyclase activity was also increased by TCR ligation. It is suggested that the anti-CD3-mediated increase in PDE-4 activity was owing to a rapid PKC-dependent induction of PDE-4 activity following crosslinking of the TCR complex. This identifies "crosstalk" occurring between the PKA and PKC signaling pathways initiated by ligation of the antigen receptor in murine thymocytes. That both adenylate cyclase and PDE-4 activities were increased may indicate the presence of compartmentalized cAMP responses present in these cells.

Early intrathymic precursor cells acquire a CD4(low) phenotype.

C4Dlow cells are a population of lymphoid lineage-restricted progenitor cells representing the earliest precursors present in the adult thymus. Paradoxically, thymic progenitors with a similar phenotype in fetal mice and adult RAG-2-deficient (RAG-2-/-) mice lack this characteristic low-level expression of CD4. We now show that radiation-induced differentiation of CD4+ CD8+ double positive thymocytes in RAG-2-/- mice results in the appearance of low levels of CD4 on thymocytes that are phenotypically identical to C4Dlow progenitor cells present in the normal adult thymus. This suggests that CD4 surface expression can be passively transferred from double positive cells to early progenitor thymocytes. Analysis of mixed bone marrow chimeras, reconstituted with hematopoietic stem cells from both CD4-/- (CD45.2) and CD4wt (CD45.1) congenic mice, revealed a CD4low phenotype on cells derived from CD4-/- bone marrow cells. Furthermore, these CD4-/- -derived "C4Dlow" progenitors were capable of reconstituting lymphocyte-depleted fetal thymi, with all thymocytes displaying a CD4-/- phenotype. This directly demonstrates that genetically CD4-deficient thymic progenitor cells can passively acquire a C4Dlow phenotype. Moreover, CD4 expression on C4Dlow progenitor thymocytes is sensitive to mild acid treatment, indicating that CD4 may not exist as an integral cell surface molecule on this thymocyte population. Our findings demonstrate that low-level CD4 surface expression can be passively acquired by intrathymic progenitor cells from the surrounding thymic microenvironment, suggesting that other cell surface molecules expressed at low levels may also result from an acquired phenotype.

Identification of a novel developmental stage marking lineage commitment of progenitor thymocytes.

Bipotent progenitors for T and natural killer (NK) lymphocytes are thought to exist among early precursor thymocytes. The identification and functional properties of such a progenitor population remain undefined. We report the identification of a novel developmental stage during fetal thymic ontogeny that delineates a population of T/NK-committed progenitors (NK1. 1(+)/CD117(+)/CD44(+)/CD25(-)). Thymocytes at this stage in development are phenotypically and functionally distinguishable from the pool of multipotent lymphoid-restricted (B, T, and NK) precursor thymocytes. Exposure of multipotent precursor thymocytes or fetal liver- derived hematopoietic progenitors to thymic stroma induces differentiation to the bipotent developmental stage. Continued exposure to a thymic microenvironment results in predominant commitment to the T cell lineage, whereas coculture with a bone marrow-derived stromal cell line results in the generation of mature NK cells. Thus, the restriction point to T and NK lymphocyte destinies from a multipotent progenitor stage is marked by a thymus-induced differentiation step.

The Chief Scientist reports... A controlled investigation of changes following a programme of community living, skills training and the validation of these changes through relocation.

Fifty-seven adults with mild to moderate learning disabilities served as subjects in a community living skills training project. Twenty-nine subjects were trained using in vivo techniques, 13 were taught using classroom techniques and 15 subjects acted as a no-treatment control group. The effects of the training programme were assessed using videotaped assessments rated by independent observers and the in vivo techniques were found to be significantly superior to the teaching and no-treatment control conditions. These effects were still evident at one year and two year follow-up assessments. Two years after the final follow-up, eventual relocation was examined and the in vivo group were found to have significantly more independent living circumstances than either of the two control groups.

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.

The consistency of reports about feelings and emotions from people with intellectual disability.

Sixty-seven subjects with mild or moderate intellectual disability were assessed on a variety of measures of emotion. All of the measures were self-report measures and all of the data is based on reports by the subjects' themselves. The battery included the Zung Self-Rating Anxiety Scale, the Zung Depression Inventory, the General Health Questionnaire and the Eysenck-Withers Personality Test. The results reveal an impressive amount of convergent validity in the subjects' emotional systems.