Denis R. Alexander

Membrane phosphatidylserine distribution as a non-apoptotic signalling mechanism in lymphocytes

Phosphatidylserine (PS) exposure is normally associated with apoptosis and the removal of dying cells. We observed that PS is exposed constitutively at high levels on T lymphocytes that express low levels of the transmembrane tyrosine phosphatase CD45RB. CD45 was shown to be a negative regulator of PS translocation in response to various signals, including activation of the ATP receptor P2X7. Changes in PS distribution were shown to modulate several membrane activities: Ca2+ and Na+ uptake through the P2X7 cation channel itself; P2X7-stimulated shedding of the homing receptor CD62L; and reversal of activity of the multidrug transporter P-glycoprotein. The data identify a role for PS distribution changes in signal transduction, rapidly modulating the activities of several membrane proteins. This seems to be an all-or-none effect, coordinating the activity of most or all the molecules of a target protein in each cell. The data also suggest a new approach to circumventing multidrug resistance.

CD45 tyrosine phosphatase-activated p59fyn couples the T cell antigen receptor to pathways of diacylglycerol production, protein kinase C activation and calcium influx.

The role of the CD45 phosphotyrosine phosphatase in coupling the T cell antigen receptor complex (TCR) to intracellular signals was investigated. CD45‐ HPB‐ALL T cells were transfected with cDNA encoding the CD45RA+B+C‐ isoform. The tyrosine kinase activity of p59fyn was found to be 65% less in CD45‐ cells than in CD45+ cells, whereas p56lck kinase activity was comparable in both sub‐clones. In CD45‐ cells the TCR was uncoupled from protein tyrosine phosphorylation, phospholipase C gamma 1 regulation, inositol phosphate production, calcium signals, diacylglycerol production and protein kinase C activation. Restoration of TCR coupling to all these pathways correlated with the increased p59fyn activity observed in CD45‐transfected cells. Co‐aggregation of CD4‐ or CD8‐p56lck kinase with the TCR in CD45‐ cells restored TCR‐induced protein tyrosine phosphorylation, phospholipase C gamma 1 regulation and calcium signals. Receptor‐mediated calcium signals were largely due (60–90%) to Ca2+ influx, and only a minor component (10–40%) was caused by Ca2+ release from intracellular stores. Maximal CD3‐mediated Ca2+ influx occurred at CD3 mAb concentrations at which inositol phosphate production was non‐detectable. These results indicate that CD45‐regulated p59fyn plays a critical role in coupling the TCR to specific intracellular signalling pathways and that CD4‐ or CD8‐p56lck can only restore signal transduction coupling in CD45‐ cells when brought into close association with the TCR.

Protein tyrosine phosphatases in T-cell development, apoptosis and signalling

The study of phosphatases was viewed as a rather esoteric subject for immunologists until eight years ago, when the discovery that CD45 is a protein tyrosine phosphatase (PTPase) began to make the topic respectable. Now, as reviewed by Julie Frearson and Denis Alexander, PTPases are increasingly being shown to play key roles in the molecular physiology of haematopoietic cells and some have been shown to regulate critical events in T-cell development and signalling.

Cutting Edge: TREM-Like Transcript-1, a Platelet Immunoreceptor Tyrosine-Based Inhibition Motif Encoding Costimulatory Immunoreceptor that Enhances, Rather than Inhibits, Calcium Signaling via SHP-2

To date, immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been shown to mediate inhibitory properties. We report a novel triggering receptor expressed on myeloid cells (TREM) family member, TREM-like transcript-1 (TLT1), which differs from the activating members because its cytoplasmic tail contains two ITIMs at Y245 and Y281. A TLT1 splice variant (TLT1sp) encodes a different cytoplasmic tail lacking ITIMs. Both isoforms are expressed in resting platelet α-granules, which are up-regulated to the cell surface following activation. TLT1 recruited Src homology 2 domain-containing tyrosine phosphatase (SHP)-2 to the “classical” ITIM (Y281) but not the “nonclassical” ITIM (Y245). In contrast to previously characterized ITIM receptors, TLT1 enhanced, rather than inhibited, FcεRI-mediated calcium signaling in rat basophilic leukemia cells, a property dependent on the SHP-2 recruiting classical Y281 ITIM. Therefore, TLT1 represents a new costimulatory ITIM immunoreceptor and is the second ITIM-bearing receptor to be identified in platelets after platelet endothelial cell adhesion molecule-1.

DNA Damage–Induced Bcl-xL Deamidation Is Mediated by NHE-1 Antiport Regulated Intracellular pH

The pro-survival protein Bcl-xL is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage–induced Bcl-xL deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-xL deamidation remains unknown and its functional consequences unclear. We show here that rBcl-xL deamidation generates an iso-Asp52/iso-Asp66 species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-xL deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage–induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-xL deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-xL deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy.

CTLA-4 suppresses proximal TCR signaling in resting human CD4(+) T cells by inhibiting ZAP-70 Tyr(319) phosphorylation: a potential role for tyrosine phosphatases.

The balance between positive and negative signals plays a key role in determining T cell function. CTL-associated Ag-4 is a surface receptor that can inhibit T cell responses induced upon stimulation of the TCR and its CD28 coreceptor. Little is known regarding the signaling mechanisms elicited by CTLA-4. In this study we analyzed CTLA-4-mediated inhibition of TCR signaling in primary resting human CD4+ T cells displaying low, but detectable, CTLA-4 cell surface expression. CTLA-4 coligation with the TCR resulted in reduced downstream protein tyrosine phosphorylation of signaling effectors and a striking inhibition of extracellular signal-regulated kinase 1/2 activation. Analysis of proximal TCR signaling revealed that TCR ζ-chain phosphorylation and subsequent ζ-associated protein of 70 kDa (ZAP-70) tyrosine kinase recruitment were not significantly affected by CTLA-4 engagement. However, the association of p56lck with ZAP-70 was inhibited following CTLA-4 ligation, correlating with reduced actions of p56lck in the ZAP-70 immunocomplex. Moreover, CTLA-4 ligation caused the selective inhibition of CD3-mediated phosphorylation of the positive regulatory ZAP-70 Y319 site. In addition, we demonstrate protein tyrosine phosphatase activity associated with the phosphorylated CTLA-4 cytoplasmic tail. The major phosphatase activity was attributed to Src homology protein 2 domain-containing tyrosine phosphatase 1, a protein tyrosine phosphatase that has been shown to be a negative regulator of multiple signaling pathways in hemopoietic cells. Collectively, our findings suggest that CTLA-4 can act early during the immune response to regulate the threshold of T cell activation.

Inhibition of the Bcl-xL Deamidation Pathway in Myeloproliferative Disorders

BACKGROUND The myeloproliferative disorders are clonal disorders with frequent somatic gain-of-function alterations affecting tyrosine kinases. In these diseases, there is an increase in DNA damage and a risk of progression to acute leukemia. The molecular mechanisms in myeloproliferative disorders that prevent apoptosis induced by damaged DNA are obscure. METHODS We searched for abnormalities of the proapoptotic Bcl-x(L) deamidation pathway in primary cells from patients with chronic myeloid leukemia (CML) or polycythemia vera, myeloproliferative disorders associated with the BCR-ABL fusion kinase and the Janus tyrosine kinase 2 (JAK2) V617F mutation, respectively. RESULTS The Bcl-x(L) deamidation pathway was inhibited in myeloid cells, but not T cells, in patients with CML or polycythemia vera. DNA damage did not increase levels of the amiloride-sensitive sodium-hydrogen exchanger isoform 1 (NHE-1), intracellular pH, Bcl-x(L) deamidation, and apoptosis. Inhibition of the pathway was reversed by enforced alkalinization or overexpression of NHE-1, leading to a restoration of apoptosis. In patients with CML, the pathway was blocked in CD34+ progenitor cells and mature myeloid cells. Imatinib or JAK2 inhibitors reversed inhibition of the pathway in cells from patients with CML and polycythemia vera, respectively, but not in cells from a patient with resistance to imatinib because of a mutation in the BCR-ABL kinase domain. CONCLUSIONS BCR-ABL and mutant JAK2 inhibit the Bcl-x(L) deamidation pathway and the apoptotic response to DNA damage in primary cells from patients with CML or polycythemia vera.

Alefacept selectively promotes NK cell-mediated deletion of CD45R0+ human T cells.

Modulation of the immune response using immunoglobulin fusion proteins has shown great promise for clinical immunotherapy of autoimmune diseases. Alefacept is an immunoglobulin fusion protein composed of the first extracellular domain of human LFA‐3 fused to the hinge, CH2 and CH3 domains of human IgG1. Alefacept has previously been reported to inhibit T cell proliferation. Here, we analyzed the effects of alefacept on lymphocytes in vitro and characterized the role of autologous NK cells in its mechanism of action. Alefacept, but not a CH2 binding mutant of Alefacept, inhibited CD3‐induced T cell proliferation only in the presence of live NK cells, consistent with an important role for FcγR engagement. Alefacept caused preferential depletion of CD69+CD45R0+CD25+ T cell subsets. Cytotoxicity assays revealed that alefacept, but not the CH2 binding mutant, induced NK cell‐mediated death of activated T cells and sorting into CD45R0+ and CD45RA+ subpopulations showed that lymphocyte deletion occurred preferentially in the CD45R0+ subset. Activated CD45R0+ cells expressed higher levels of CD2 than CD45R0– cells, providing a possible explanation for the selective targeting of this subset. Our results suggest that selective targeting of CD45R0+ T cells by NK cells represents a potential therapeutic mechanism of action of alefacept.

Vav -promoter regulated oncogenic fusion protein NPM-ALK in transgenic mice causes B-cell lymphomas with hyperactive Jun kinase

Anaplastic large-cell lymphoma is associated with a chromosomal translocation generating an oncogenic fusion protein: the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). We have generated several independent lines of human NPM-ALK transgenic mice using the haematopoietic cell-specific Vav promoter. Lymphomas develop in two transgenic lines in which the Vav promoter regulates NPM-ALK expression. The transgenic line with higher copy number displays an early-onset phenotype in which all mice succumb to aggressive lymph node tumours with intestinal involvement, whereas the second line displays late-onset tumour development in the spleen and/or liver. Lymphomas from both lines are phenotypically distinct and display B-lineage characteristics with aberrant coexpression of myeloid markers. The NPM-ALK kinase is active in primary tumour tissue and forms a multimeric complex with tyrosine-phosphorylated proteins, that is, Shc. Jun and ERK kinase activities in tumours are elevated by up to 30-fold and fivefold, respectively, in comparison with sIgM-stimulated primary B cells. The new transgenic models provide a system for investigating the oncogenic events mediated by NPM-ALK in situ and a physiologically relevant context for developing tyrosine kinase inhibitor therapies of potential use in the clinic.

Development of T-leukaemias in CD45 tyrosine phosphatase-deficient mutant lck mice

The CD45 tyrosine phosphatase lowers T‐cell antigen receptor signalling thresholds by its positive actions on p56lck tyrosine kinase function. We now show that mice expressing active lckF505 at non‐oncogenic levels develop aggressive thymic lymphomas on a CD45−/− background. CD45 suppresses the tumorigenic potential of the kinase by dephosphorylation of the Tyr394 autophosphorylation site. In CD45−/− thymocytes the kinase is switched to a hyperactive oncogenic state, resulting in increased resistance to apoptosis. Transformation occurs in early CD4−CD8− thymocytes during the process of TCR‐β chain rearrangement by a recombinase‐independent mechanism. Our findings represent the first example in which a tyrosine phosphatase in situ prevents the oncogenic actions of a Src family tyrosine kinase.