CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4+CD8+ thymocytes, and B cell maturation.

The CD45 transmembrane glycoprotein has been shown to be a protein phosphotyrosine phosphatase and to be important in signal transduction in T and B lymphocytes. We have employed gene targeting to create a strain of transgenic mice that completely lacks expression of all isoforms of CD45. The spleens from CD45-null mice contain approximately twice the number of B cells and one fifth the number of T cells found in normal controls. The increase in B cell numbers is due to the specific expansion of two B cell subpopulations that express high levels of immunoglobulin (IgM) staining. T cell development is significantly inhibited in CD45-null animals at two distinct stages. The efficiency of the development of CD4-CD8- thymocytes into CD4+ CD8+ thymocytes is reduced by twofold, subsequently the frequency of successful maturation of the double positive population into mature, single positive thymocytes is reduced by a further four- to fivefold. In addition, we demonstrate that CD45-null thymocytes are severely impaired in their apoptotic response to cross-linking signals via T cell receptor (TCR) in fetal thymic organ culture. In contrast, apoptosis can be induced normally in CD45-null thymocytes by non-TCR-mediated signals. Since both positive and negative selection require signals through the TCR complex, these findings suggest that CD45 is an important regulator of signal transduction via the TCR complex at multiple stages of T cell development. CD45 is absolutely required for the transmission of mitogenic signals via IgM and IgD. By contrast, CD45-null B cells proliferate as well as wild-type cells to CD40-mediated signals. The proliferation of B cells in response to CD38 cross-linking is significantly reduced but not abolished by the CD45-null mutation. We conclude that CD45 is not required at any stage during the generation of mature peripheral B cells, however its loss reveals a previously unrecognized role for CD45 in the regulation of certain subpopulations of B cells.

The role of intracellular signalling pathways regulating thymocyte and leukemic T cell apoptosis.

Apoptosis plays a critical role during T cell development, both in the generation of functionally competent T cells in the thymus and the regulation of peripheral T cell populations. The fate of any T cell, whether it is developing in the thymus, or functioning in the peripheral immune system, is dependent on T cell receptor (TCR) specificity for antigens presented by MHC molecules and on the consequences of TCR-generated intracellular signalling pathways which lead to activation, anergy or apoptosis. This review describes data that have elucidated the way in which these highly regulated TCR-derived signalling pathways lead to such diverse final outcomes in thymocytes. Contributions to the induction of apoptosis in thymocytes by signalling pathways and receptors such as Fas, CD45 and CD28 are summarized, particularly with regard to the analysis of relevant transgenic mice. Developments concerning regulation of apoptosis by bcl-2 family members and the possible effectors of apoptosis, proteases, are assessed. Finally, this information is contrasted with the relatively scarce data on signalling pathways in thymic-derived T-ALL cells together with potential explanations of how transformation might occur by perturbation of apoptotic mechanisms. Precise understanding of these pathways may lead to the development of novel therapeutic reagents.

Defective depletion of CD45-null thymocytes by the Staphylococcus aureus enterotoxin B superantigen.

The development of a normal T-cell repertoire is critically dependent on the negative and positive selection events which occur at the CD4+CD8+ (double positive, DP) stage of thymic development. Depending on the avidity of the T-cell antigen receptor (TCR) for peptides presented within the thymus, DP thymocytes are either positively selected for maturation to CD4+/CD8+ single positive cells or are depleted by apoptosis. The addition of superantigen to thymocytes within foetal thymic organ culture (FTOC) mimics the negative selection signal of potentially autoreactive thymocytes and induces the responding population of thymocytes to apoptose. Here we present evidence that the transmembrane phosphotyrosine phosphatase CD45 critically regulates TCR-induced signals in thymic differentiation and present data to show defective depletion of CD45-null transgenic TCR-Vbeta8 DP thymocytes in FTOC by the Staphylococcus aureus Enterotoxin B (SEB) superantigen.

Preclinical development of CAT-354, an IL-13 neutralizing antibody, for the treatment of severe uncontrolled asthma.

BACKGROUND AND PURPOSE: IL-13 is a pleiotropic Th2 cytokine considered likely to play a pivotal role in asthma. Here we describe the preclinical in vitro and in vivo characterization of CAT-354, an IL-13-neutralizing IgG4 monoclonal antibody (mAb), currently in clinical development. EXPERIMENTAL APPROACH: In vitro the potency, specificity and species selectivity of CAT-354 was assayed in TF-1 cells, human umbilical vein endothelial cells and HDLM-2 cells. The ability of CAT-354 to modulate disease-relevant mechanisms was tested in human cells measuring bronchial smooth muscle calcium flux induced by histamine, eotaxin generation by normal lung fibroblasts, CD23 upregulation in peripheral blood mononuclear cells and IgE production by B cells. In vivo CAT-354 was tested on human IL-13-induced air pouch inflammation in mice, ovalbumin-sensitization and challenge in IL-13 humanized mice and antigen challenge in cynomolgus monkeys. KEY RESULTS: CAT-354 has a 165 pM affinity for human IL-13 and functionally neutralized human, human variant associated with asthma and atopy (R130Q) and cynomolgus monkey, but not mouse, IL-13. CAT-354 did not neutralize human IL-4. In vitro CAT-354 functionally inhibited IL-13-induced eotaxin production, an analogue of smooth muscle airways hyperresponsiveness, CD23 upregulation and IgE production. In vivo in humanized mouse and cynomolgus monkey antigen challenge models CAT-354 inhibited airways hyperresponsiveness and bronchoalveolar lavage eosinophilia. CONCLUSIONS AND IMPLICATIONS: CAT-354 is a potent and selective IL-13-neutralizing IgG4 mAb. The preclinical data presented here support the trialling of this mAb in patients with moderate to severe uncontrolled asthma.

Regulation of T-cell-receptor-stimulated bivalent-cation entry in Jurkat E6 cells: role of protein kinase C.

Stimulation of Jurkat E6 cells with anti-CD3 antibody results in a characteristic rise in [Ca2+]i which is due to both the release of Ca2+ from intracellular stores and the entry of external Ca2+. Individual components of the [Ca2+]i increase were investigated by measuring intracellular Ca2+ release in the absence of external Ca2+ and determining influx of bivalent cations by following the entry of Mn2+. The increase in [Ca2+]i induced by anti-CD3 antibody in the presence or absence of extracellular Ca2+ could be inhibited by the non-selective kinase inhibitor staurosporine, which also inhibits anti-CD3-stimulated phospholipase C activity. Staurosporine also inhibits the influx of bivalent cations induced by anti-CD3 antibody, but not that induced by depletion of intracellular Ca2+ stores using thapsigargin. The effect of staurosporine was compared with that of Ro 31-8425, a potent and selective inhibitor of protein kinase C (PKC). Ro 31-8425, at concentrations up to 10 microM, has no inhibitory effect on the anti-CD3 antibody-induced [Ca2+]i increase or phospholipase C activity. These studies are consistent with the concept that augmentation of [Ca2+]i by stimulated T-cell receptors requires activation of a kinase, probably a tyrosine kinase such as p56lck, ZAP-70 or p59fyn, and is independent of PKC. Phorbol esters inhibit the anti-CD3-stimulated [Ca2+]i increase and phospholipase C activity, showing that this can be negatively regulated by PKC. A small potentiation of the anti-CD3 antibody-induced [Ca2+]i rise in the presence of extracellular Ca2+ was detected in the presence of Ro 31-8425; this suggests that T-cell-receptor ligation can also limit the increase in [Ca2+]i via PKC activation.

Phosphatidylinositol 4,5-bisphosphate hydrolysis accompanies T cell receptor-induced apoptosis of murine thymocytes within the thymus.

Regulation of the development of thymocytes into mature T cells within the thymus is now known to involve antigen-induced deletion, by apoptosis, of potentially autoreactive thymocytes, and it can be mimicked either by stimulating the T cell receptor (TcR) complex by monoclonal antibody (mAb) or by ionophore-induced elevation of cytosolic [Ca2+]. To identify signaling pathways employed by the TcR complex of immature thymocytes, we examined the effects of anti-CD3 and anti-TcR beta constant (c) region mAb, staphylococcal enterotoxin B (SEB) and pharmacological agents on the generation of inositol phosphates through hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] both in cultured fetal mouse thymic lobes and in the CD4+CD8+ immature thymocyte cell line, TM10G. Stimulation of the TcR complex with anti-CD3 mAb provoked an accumulation of inositol phosphates diagnostic of the occurrence of receptor-stimulated phosphoinositidase C (PLC) activation. Anti-TcRC beta mAb and SEB provoked smaller but similar responses. The PLC activation evoked by anti-CD3 mAb was suppressed by inhibitors of receptor tyrosine kinases and was unmodified by protein kinase C activation or elevation of cytosolic [Ca2+]. It thus appears that apoptosis triggered by TcR stimulation is associated with PLC activation by a receptor-regulated tyrosine kinase. Treatment of thymic lobes or TM10G cells with fluoroaluminate provoked apoptosis of a wider range of thymocyte subtypes and such stimulation also provoked an accumulation of inositol phosphates. The responses to fluoroaluminate were not prevented by inhibitors of tyrosine kinases, suggesting that unidentified GTP-binding proteins which couple to PLC activation may also be capable of initiating apoptosis by a route independent of the TcR. These results, when considered alongside previous studies of mature T cells, indicate that stimulation of immature thymocytes or of mature T cells through their TcR complex activates the PLC-catalyzed PtdIns(4,5)P2 hydrolysis signaling pathway, and thus that this signaling pathway may be implicated both in provoking apoptosis in immature T cells and in initiating proliferation in mature T cells.

Phosphatidylserine-mediated adhesion of T-cells to endothelial cells.

Phosphatidylserine was exposed on the surface of human umbilical endothelial cells (ECV304) a few minutes after adding thrombin in vitro, as monitored by prothrombinase assays with and without annexin V. Jurkat T cells adhered to the thrombin-treated cells. The adhesion was inhibited by annexin V, indicating that it was mediated by exposed phosphatidylserine on the endothelial cells.

Intracellular signaling events during positive and negative selection of CD4+CD8+ thymocytes in vitro.

We have used in vitro models of thymocyte positive and negative selection in conjunction with selective inhibitors of the TCR-mediated signaling cascade to investigate the intracellular signaling events that mediate these processes. We report that Ro 31.8425, a potent and selective inhibitor of protein kinase C, which blocks the activation of mature T cells in a dose-dependent fashion, has no effect on either positive or negative selection of CD4+8+ thymocytes. In contrast, cyclosporin A fails to prevent negative selection, but inhibits positive selection through a direct effect on developing thymocytes, rather than through the perturbation of stromal cell support. Thus, our data suggest that positive and negative selection may operate via distinct intracellular signaling pathways.

Aberrant TCR-mediated signaling in CD45-null thymocytes involves dysfunctional regulation of Lck, Fyn, TCR-zeta, and ZAP-70.

CD45 is a transmembrane phosphotyrosine phosphatase expressed on all nucleated hemopoietic cells. Targeting of CD45 exon 9 has generated a mouse line completely lacking CD45 expression (CD45-null) in which there are severe abnormalities in T cell development. Defects in TCR-mediated signals underlying these abnormalities have now been investigated using CD45-null T cells. No T cell proliferation was detected in response to a CD3 mAb. In thymocytes the p56(lck) and p59(fyn) tyrosine kinases were hyperphosphorylated, and p56(lck) was in its inactive conformation. Both basal and TCR-stimulated tyrosine phosphorylation of TCR-zeta and CD3-epsilon were much reduced, and TCR stimulation induced an abnormal p18 phosphoisomer of TCR-zeta previously noted in T cells stimulated by altered peptide ligands. These defects were associated with the failure of ZAP-70 kinase recruitment to the TCR-zeta chain. TCR coupling to the tyrosine phosphorylation of several proteins, including HS1 and p120(cbl), was also much reduced. However, TCR-induced signaling was not ablated, and significant inositol phosphate and calcium signals were observed in CD45-null thymocytes. Our molecular analysis suggests that the threshold for TCR signal transduction is greatly increased in CD45-null T cells, thus explaining the profound defects in thymic development.

Inositol lipids and phosphates in the regulation of the growth and differentiation of haemopoietic and other cells.

Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis is an important signalling reaction involved in the responses of cells to some, but not all, stimuli that promote cell proliferation. Active agents in this regard include antigens activating T and B lymphocytes, angiotensin (employing a receptor encoded by the mas oncogene), bombesin and platelet-derived growth factor PDGF). However, accumulating evidence suggests that inositol lipids and phosphates also have other roles in the regulation of cell growth and differentiation. Growth factor receptors that encode tyrosine kinases (such as that for PDGF) activate a kinase that synthesises phosphatidylinositol 3-phosphate, a novel lipid, and loss of this kinase-activating function abolishes growth-promoting activity. Human interleukin-4, a lymphokine that activates B lymphocytes, appears to employ phosphatidylinositol 4,5-bisphosphate hydrolysis as a brief initial signal that is followed by a sustained rise in cyclic adenosine monophosphate (cAMP): both signals are needed for the successful induction of the surface antigen CD23. Moreover, the same inositol lipid signalling pathway as is employed by antigen-stimulated mature T lymphocytes to provoke proliferation may be redeployed in immature T cells to trigger their elimination when they encounter self-antigens. Finally, studies of HL60 promyelocytic cells have shown that these cells contain high concentrations of inositol 3,4,5,6-tetrakisphosphate, 1,3,4,5,6-pentakisphosphate and hexakisphosphate, three inositol polyphosphates that are probably formed independently of inositol lipid metabolism. When these cells are induced to differentiate either towards neutrophils (in the presence of dimethylsulphoxide) or macrophages (in phorbol myristate acetate), cessation of growth and acquisition of differentiated characteristics are accompanied by large and different changes in the concentrations of these inositol phosphates that may be characteristic of these two pathways of differentiation.

Inositol lipids and phosphates in the proliferation and differentiation of lymphocytes and myeloid cells.

It is established that receptor-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate is an essential signalling reaction in the responses of many haemopoietic cells to stimuli: examples include platelet activation, antigen-driven initiation of cell proliferation in mature B and T lymphocytes and histamine release by mast cells, and chemotaxis and oxygen radical generation by neutrophils. However, the roles of inositol lipids and phosphates in the development of haemopoietic and immune cells are less well understood. This paper discusses three such situations: the sequential employment of phosphatidylinositol 4,5-bisphosphate hydrolysis and cyclic AMP accumulation as two signals essential to the action of the B lymphocyte-stimulatory cytokine interleukin 4; the involvement of antigen receptor-triggered inositol lipid hydrolysis in apoptotic elimination of immature anti-self T lymphocytes in the fetal mouse thymus; and the possible role of changes in the levels of abundant inositol polyphosphates in the differentiation of HL-60 promyelocytic cells and of normal human myeloid blast cells.