Phosphorylation of Collapsin Response Mediator Protein 2 on Tyr-479 Regulates CXCL12-induced T Lymphocyte Migration

In the central nervous system, collapsin response mediator protein 2 (CRMP2) is a transducer protein that supports the semaphorin-induced guidance of axons toward their cognate target. However, we previously showed that CRMP2 is also expressed in immune cells and plays a crucial role in T lymphocyte migration. Here we further investigated the molecular mechanisms underlying CRMP2 function in chemokine-directed T-cell motility. Examining Jurkat T-cells treated with the chemokine CXCL12, we found that 1) CXCL12 induces a dynamic re-localization of CRMP2 to uropod, the flexible structure of migrating lymphocyte, and increases its binding to the cytoskeletal protein vimentin; 2) CXCL12 decreases phosphorylation of the glycogen synthase kinase-3β-targeted residues CRMP2-Thr-509/514; and 3) tyrosine Tyr-479 is a new phosphorylation CRMP2 residue and a target for the Src-family kinase Yes. Moreover, phospho-Tyr-479 increased under CXCL12 signaling while phospho-Thr-509/514 decreased. The functional importance of this tyrosine phosphorylation was demonstrated by Y479F mutation that strongly reduced CXCL12-mediated T-cell polarization and motility as tested in a transmigration model and on neural tissue. We propose that differential phosphorylation by glycogen synthase kinase-3β and Yes modulates the contribution of CRMP2 to cytoskeletal reorganization during chemokine-directed T-cell migration. In addition to providing a novel mechanism for T lymphocyte motility, our findings reveal CRMP2 as a transducer of chemokine signaling.T lymphocyte migration is the basis of major immune functions such as responses to infection and inflammation, as well as normal recirculation through the lymphoid organs. Indeed, the role of T-cells depends strongly on their ability to travel between organs via the blood and lymph and to move rapidly within these tissues, by extravasation (1). This latter function is dependent on extracellular signals, among which chemokines play a major role.Chemokines form a superfamily of small proteins that orchestrate lymphocyte polarization and migration (2). These proteins exert their functions by binding specific seven-transmembrane-domain G-protein-coupled receptors on the T-cell surface (3). T-lymphocytes exposed to chemokines, in a soluble or surface-bound gradient, develop a polarized shape, extending at the front, an F-actin-rich lamellipodium, which constitutes the leading edge, and a trailing edge or uropod in which both the microtubule and vimentin networks are retracted during migration. Although F-actin has the well known function of producing the mechanical forces required to generate movement (4), the role of microtubules and vimentin in T-cell migration requires further investigation.Cytoskeletal remodeling is of key importance in migrating cells (5) and is one of the functions carried out by the chemokine stromal cell-derived factor-1α, also named CXCL12. In association with its cognate receptor CXCR4, CXCL12 is a potent chemoattractant for mature T-cells and monocytes (6). Following ligand recognition and binding, CXCR4 signaling starts with the activation of G proteins, followed by various signaling cascade effectors, including MAP² kinases, phosphoinositide 3-kinase, and phospholipase Cγ (7). Although this intracellular signaling cascade has not been completely elucidated, the Src family non-receptor tyrosine kinase Lck and the Syk kinase ZAP-70 have emerged as the main candidates for delivering the input signal following CXCR4 activation (8). Thus, tyrosine kinase activity appears as a central step in CXCR4-dependent chemotaxis.While searching for molecules involved in T-cell motility, we recently identified collapsin response mediator protein 2 (CRMP2) (9), a protein first described in the context of neuronal growth cone advance (10, 11). We demonstrated that CRMP2 regulated both T-cell polarization and spontaneous/chemokine-induced migration of T-lymphocytes. Moreover, CRMP2 was found at the uropod of motile T-cells and has the ability to bind cytoskeletal elements, including vimentin. A correlation between CRMP2 expression levels and cell migratory rates toward a chemokine gradient, including CXCL12, was demonstrated by overexpression and knockdown experiments in T-cells (9). In addition, we recently reported that, in mouse model of neuroinflammation, elevated CRMP2 expression in T lymphocytes correlated with their elevated migratory rates and their ability to target the central nervous system (12). The importance of CXCL12 in the central nervous system and its implication in the pathogenesis of central nervous system disorders, including neuroinflammatory diseases, are well documented (review in Ref. 13). Thus, the aim of the present study was to determine whether and how CRMP2 participates in the transduction pathway induced by CXCL12 on T lymphocytes.     

A role for the neuronal protein collapsin response mediator protein 2 in T lymphocyte polarization and migration

The semaphorin-signaling transducer collapsin response mediator protein 2 (CRMP2) has been identified in the nervous system where it mediates Sema3A-induced growth cone navigation. In the present study, we provide first evidence that CRMP2 is present in the immune system and plays a critical role in T lymphocyte function. CRMP2 redistribution at the uropod in polarized T cells, a structural support of lymphocyte motility, suggests that it may regulate T cell migration. This was evidenced in primary T cells by small-interfering RNA-mediated CRMP2 gene silencing and blocking Ab, as well as CRMP2 overexpression in Jurkat T cells tested in a chemokine- and semaphorin-mediated transmigration assay. Expression analysis in PBMC from healthy donors showed that CRMP2 is enhanced in cell subsets bearing the activation markers CD69+ and HLA-DR+. Heightened expression in T lymphocytes of patients suffering from neuroinflammatory disease with enhanced T cell-transmigrating activity points to a role for CRMP2 in pathogenesis. The elucidation of the signals and mechanisms that control this pathway will lead to a better understanding of T cell trafficking in physiological and pathological situations.     

The role of CTLs in persistent viral infection: cytolytic gene expression in CD8+ lymphocytes distinguishes between individuals with a high or low proviral load of human T cell lymphotropic virus type 1

The proviral load in human T cell lymphotropic virus type 1 (HTLV-1) infection is typically constant in each infected host, but varies by >1000-fold between hosts and is strongly correlated with the risk of HTLV-1-associated inflammatory disease. However, the factors that determine an individual's HTLV-1 proviral load remain uncertain. Experimental evidence from studies of host genetics, viral genetics, and lymphocyte function and theoretical considerations suggest that a major determinant of the equilibrium proviral load is the CD8+ T cell response to HTLV-1. In this study, we tested the hypothesis that the gene expression profile in circulating CD8+ and CD4+ lymphocytes distinguishes between individuals with a low proviral load of HTLV-1 and those with a high proviral load. We show that circulating CD8+ lymphocytes from individuals with a low HTLV-1 proviral load overexpressed a core group of nine genes with strong functional coherence: eight of the nine genes encode granzymes or other proteins involved in cell-mediated lysis or Ag recognition. We conclude that successful suppression of the HTLV-1 proviral load is associated with strong cytotoxic CD8+ lymphocyte activity in the peripheral blood.     

L-plastin regulates polarization and migration in chemokine-stimulated human T lymphocytes

Chemokines such as SDF-1α play a crucial role in orchestrating T lymphocyte polarity and migration via polymerization and reorganization of the F-actin cytoskeleton, but the role of actin-associated proteins in this process is not well characterized. In this study, we have investigated a role for L-plastin, a leukocyte-specific F-actin-bundling protein, in SDF-1α-stimulated human T lymphocyte polarization and migration. We found that L-plastin colocalized with F-actin at the leading edge of SDF-1α-stimulated T lymphocytes and was also phosphorylated at Ser(5), a site that when phosphorylated regulates the ability of L-plastin to bundle F-actin. L-plastin phosphorylation was sensitive to pharmacological inhibitors of protein kinase C (PKC), and several PKC isoforms colocalized with L-plastin at the leading edge of SDF-1α-stimulated lymphocytes. However, PKC ζ, an established regulator of cell polarity, was the only isoform that regulated L-plastin phosphorylation. Knockdown of L-plastin expression with small interfering RNAs demonstrated that this protein regulated the localization of F-actin at the leading edge of chemokine-stimulated cells and was also required for polarization, lamellipodia formation, and chemotaxis. Knockdown of L-plastin expression also impaired the Rac1 activation cycle and Akt phosphorylation in response to SDF-1α stimulation. Furthermore, L-plastin also regulated SDF-1α-mediated lymphocyte migration on the integrin ligand ICAM-1 by influencing velocity and persistence, but in a manner that was independent of LFA-1 integrin activation or adhesion. This study, therefore, demonstrates an important role for L-plastin and the signaling pathways that regulate its phosphorylation in response to chemokines and adds L-plastin to a growing list of proteins implicated in T lymphocyte polarity and migration.     

CCR7/CCL21 migration on fibronectin is mediated by phospholipase Cgamma1 and ERK1/2 in primary T lymphocytes

CCR7 binds to its cognate ligand, CCL21, to mediate the migration of circulating naive T lymphocytes to the lymph nodes. T lymphocytes can bind to fibronectin, a constituent of lymph nodes, via their β1 integrins, which is a primary mechanism of T lymphocyte migration; however, the signaling pathways involved are unclear. We report that rapid (within 2 min) and transient phosphorylation of ERK1/2 is required for T cell migration on fibronectin in response to CCL21. Conversely, prevention of ERK1/2 phosphorylation by inhibition of its kinase, MAPK/MEK, prevented T lymphocyte migration. Previous studies have suggested that phospholipase Cγ1 (PLCγ1) can mediate phosphorylation of ERK1/2, which is required for β1 integrin activation. Paradoxically, we found that inhibition of PLCγ1 phosphorylation by the general PLC inhibitor U73122 was associated with a delayed and reduced phosphorylation of ERK1/2 and reduced migration of T lymphocytes on fibronectin. To further characterize the relationship between ERK1/2 and PLCγ1, we reduced PLCγ1 levels by 85% using shRNA and observed a reduced phosphorylation of ERK1/2 and a significant loss of CCR7-mediated migration of T lymphocytes on fibronectin. In addition, we found that inhibition of ERK1/2 phosphorylation by U0126 resulted in a decreased phosphorylation of PLCγ1, suggesting a feedback loop between ERK1/2 and PLCγ1. Overall, these results suggest that the CCR7 signaling pathway leading to T lymphocyte migration on fibronectin is a β1 integrin-dependent pathway involving transient ERK1/2 phosphorylation, which is modulated by PLCγ1.     

Evidence that the lipid phosphatase SHIP-1 regulates T lymphocyte morphology and motility

SHIP-1 negatively regulates the PI3K pathway in hematopoietic cells and has an emerging role in T lymphocyte biology. PI3K and SHIP can regulate cell migration in leukocytes, particularly in neutrophils, although their role in T cell migration has been less clear. Therefore, we sought to explore the role of SHIP-1 in human CD4(+) T lymphocyte cell migration responses to chemoattractants using a lentiviral-mediated expression system and a short hairpin RNA approach. Silencing of SHIP-1 leads to increased basal phosphorylation of protein kinase B/Akt and its substrate GSK3β, as well as an increase in basal levels of polymerized actin, suggesting that SHIP-1 might regulate changes in the cytoskeleton. Accordingly, silencing of SHIP-1 led to loss of microvilli and ezrin/radixin/moesin phosphorylation, which could not be rescued by the PI3K inhibitor Ly294002. There were striking morphological changes, including a loss of microvilli projections, which mirrored changes in wild type cells after stimulation with the chemokine CXCL11. There was no defect in directional T cell migration toward CXCL11 in the SHIP-1-silenced cells but, importantly, there was a defect in the overall basal motility of SHIP-1 knockdown cells. Taken together, these results implicate SHIP-1 as a key regulator of basal PI3K signaling in human CD4(+) T lymphocytes with important phosphatase-independent actions, which together are key for maintaining normal morphology and basal motility.     

Lymphocyte migration through brain endothelial cell monolayers involves signaling through endothelial ICAM-1 via a rho-dependent pathway

Lymphocyte extravasation into the brain is mediated largely by the Ig superfamily molecule ICAM-1. Several lines of evidence indicate that at the tight vascular barriers of the central nervous system (CNS), endothelial cell (EC) ICAM-1 not only acts as a docking molecule for circulating lymphocytes, but is also involved in transducing signals to the EC. In this paper, we examine the signaling pathways in brain EC following Ab ligation of endothelial ICAM-1, which mimics adhesion of lymphocytes to CNS endothelia. ICAM-1 cross-linking results in a reorganization of the endothelial actin cytoskeleton to form stress fibers and activation of the small guanosine triphosphate (GTP)-binding protein Rho. ICAM-1-stimulated tyrosine phosphorylation of the actin-associated molecule cortactin and ICAM-1-mediated, Ag/IL-2-stimulated T lymphocyte migration through EC monolayers were inhibited following pretreatment of EC with cytochalasin D. Pretreatment of EC with C3 transferase, a specific inhibitor of Rho proteins, significantly inhibited the transmonolayer migration of T lymphocytes, endothelial Rho-GTP loading, and endothelial actin reorganization, without affecting either lymphocyte adhesion to EC or cortactin phosphorylation. These data show that brain vascular EC are actively involved in facilitating T lymphocyte migration through the tight blood-brain barrier of the CNS and that this process involves ICAM-1-stimulated rearrangement of the endothelial actin cytoskeleton and functional EC Rho proteins.     

Intermediate-affinity LFA-1 binds alpha-actinin-1 to control migration at the leading edge of the T cell

T lymphocytes use LFA-1 to migrate into lymph nodes and inflammatory sites. To investigate the mechanisms regulating this migration, we utilize mAbs selective for conformational epitopes as probes for active LFA-1. Expression of the KIM127 epitope, but not the 24 epitope, defines the extended conformation of LFA-1, which has intermediate affinity for ligand ICAM-1. A key finding is that KIM127-positive LFA-1 forms new adhesions at the T lymphocyte leading edge. This LFA-1 links to the cytoskeleton through alpha-actinin-1 and disruption at the level of integrin or actin results in loss of cell spreading and migratory speed due to a failure of attachment at the leading edge. The KIM127 pattern contrasts with high-affinity LFA-1 that expresses both 24 and KIM127 epitopes, is restricted to the mid-cell focal zone and controls ICAM-1 attachment. Identification of distinctive roles for intermediate- and high-affinity LFA-1 in T lymphocyte migration provides a biological function for two active conformations of this integrin for the first time.     

Interactions between brain endothelial cells and human T-cell leukemia virus type 1-infected lymphocytes: mechanisms of viral entry into the central nervous system

Human T-cell leukemia virus type 1 (HTLV-1) is associated with a variety of clinical manifestations, including tropical spastic paraparesis or HTLV-1-associated myelopathy (TSP/HAM). Viral detection in the central nervous system (CNS) of TSP/HAM patients demonstrates the ability of HTLV-1 to cross the blood-brain barrier (BBB). To investigate viral entry into the CNS, rat brain capillary endothelial cells were exposed to human lymphocytes chronically infected by HTLV-1 (MT2), to lymphocytes isolated from a seropositive patient, or to a control lymphoblastoid cell line (CEM). An enhanced adhesion to and migration through brain endothelial cells in vitro was observed with HTLV-1-infected lymphocytes. HTLV-1-infected lymphocytes also induced a twofold increase in the paracellular permeability of the endothelial monolayer. These effects were associated with an increased production of tumor necrosis factor alpha by HTLV-1-infected lymphocytes in the presence of brain endothelial cells. Ultrastructural analysis showed that contact between endothelial cells and HTLV-1-infected lymphocytes resulted in a massive and rapid budding of virions from lymphocytes, followed by their internalization into vesicles by brain endothelial cells and apparent release onto the basolateral side, suggesting that viral particles may cross the BBB using the transcytotic pathway. Our study also demonstrates that cell-cell fusion occurs between HTLV-1-infected lymphocytes and brain endothelial cells, with the latter being susceptible to transient HTLV-1 infection. These aspects may help us to understand the pathogenic mechanisms associated with neurological diseases induced by HTLV-1 infection.     

Astrocyte-specific expression of human T-cell lymphotropic virus type 1 (HTLV-1) Tax: induction of tumor necrosis factor alpha and susceptibility to lysis by CD8+ HTLV-1-specific cytotoxic T cells.

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with a chronic neurological disease termed HTLV-1-associated myelopathy/tropical spastic paraperesis (HAM/TSP). Although the pathogenesis of this disease remains to be elucidated, the evidence suggests that immunopathological mechanisms are involved. Since HTLV-1 tax mRNA was colocalized with glial acidic fibrillary protein, a marker for astrocytes, we developed an in vitro model to assess whether HTLV-1 infection activates astrocytes to secrete cytokines or present viral immunodominant epitopes to virus-specific T cells. Two human astrocytic glioma cell lines, U251 and U373, were transfected with the 3' portion of the HTLV-1 genome and with the HTLV-1 tax gene under astrocyte-specific promoter control. In this study, we report that Tax-expressing astrocytic glioma transfectants activate the expression of tumor necrosis factor alpha mRNA in vitro. Furthermore, these Tax-expressing glioma transfectants can serve as immunological targets for HTLV-1-specific cytotoxic T lymphocytes (CTL). We propose that these events could contribute to the neuropathology of HAM/TSP, since infected astrocytes can become a source for inflammatory cytokines upon HTLV-1 infection and serve as targets for HTLV-1-specific CTL, resulting in parenchymal damage by direct lysis and/or cytokine release.     

Induction of human T lymphocyte motility by interleukin 2

Interleukin 2 (IL 2) is known to have multiple immunoenhancing activities that are related to its ability to promote the proliferation and the expression of effector functions of human T lymphocytes. We investigated the potential of IL 2 to induce human T lymphocyte migration. Unstimulated T cells did not respond to IL 2, but T cells exposed to dextran or phytohemagglutinin did respond to IL 2 concentrations from 0.01 to 10.0 U/ml, with significantly increased migration. This activity could be specifically blocked with anti-Tac antibody. Analysis of T lymphocyte subsets revealed that OKT4+ but not OKT8+ lymphocytes responded to IL 2 in the chemotaxis assay. Checkerboard analysis demonstrated that the IL 2-induced chemoattractant activity was predominantly chemotactic rather than chemokinetic in nature. The activity of IL 2 was compared with that of another chemoattractant lymphokine, lymphocyte chemoattractant factor, which was found to stimulate lymphocyte migration without prior exposure to mitogen, and which was not inhibited by anti-Tac. Our data suggest that the lymphocyte migratory response to IL 2 is under the control of the inducible receptor recognized by anti-Tac in a manner similar to the proliferative response to IL 2, but differs from proliferation in its OKT4+ cell specificity.     

Lysis of human T cell leukemia virus infected T and B lymphoid cells by interleukin 2-activated killer cells

The human T cell leukemia (HTLV-1) retrovirus is the etiologic agent for adult T cell leukemia. Interleukin 2 (IL-2) activated killer (AK) cells have been shown to lyse freshly explanted tumor cells in vitro and have been used as a form of adoptive immunotherapy for the treatment of cancer. In this report, the ability of AK cells to lyse HTLV-1-infected targets was examined. Normal lymphocytes, when cultured in recombinant IL-2 for periods of 3 to 7 days, killed infected T and B cell lines. The precursor for these AK cells resided in the CD-16 antigen-positive subset (i.e., natural killer (NK) cells). Resting T cells, NK cells, or unfractionated lymphocytes did not lyse the infected targets. However, when isolated NK cells were incubated for 24 hr in IL-2, suboptimal cytolysis was induced whereas activation of NK cells with a four pulse of IL-2 was insufficient to generate effector cells. The results of performing cold target inhibition studies with Epstein-Barr virus-infected B cell lines and HTLV-1-infected T and B cell lines suggest that there are discrete subsets (i.e., clonotypic) in the AK population that preferentially lyse a given virally infected cell line. Thus to consider AK cells as true polyspecific killer cells may be inaccurate. Alternately AK cells may express a number of different receptors with variable affinities for the Epstein-Barr virus- and HTLV-1-infected cell lines. In addition, it was shown that HTLV-1-infected B cells are relatively resistant to AK cell-mediated lysis. These results clearly indicate that AK cells but not resting NK cells kill HTLV-1-infected cells.     

T lymphocytes and neutrophil granulocytes differ in regulatory signaling and migratory dynamics with regard to spontaneous locomotion and chemotaxis

Chemotactic migration of T lymphocytes and neutrophil granulocytes within a three-dimensional collagen matrix is distinct from spontaneous, matrix-induced migration concerning dynamic parameters and regulatory intracellular signaling. Both spontaneous T lymphocyte locomotion and stromal-cell-derived factor-1 (SDF-1)-induced chemotaxis-involved protein tyrosine kinase (PTK) activity, whereas only SDF-1-induced migration was protein kinase C (PKC) dependent. Spontaneous locomotion of neutrophil granulocytes was independent of PKC and PTK activity, but formyl-methionyl-leucyl-phenylalanine-induced migration involved PKC activity. In addition, the microtubule cytoskeleton was not changed after induction of chemotaxis in both cell types. T lymphocytes had a well-developed microtubule cytoskeleton with the microtubule organizing center located in the uropod, whereas neutrophil granulocytes revealed a clustered tubulin distribution at the leading edge of the migrating cell. Therefore, differences of the microtubule cytoskeleton might contribute to differences in locomotion between T lymphocytes and neutrophil granulocytes but not to differences between spontaneous locomotion and chemotaxis.     

Tax-inducible production of CC chemokine ligand 22 by human T cell leukemia virus type 1 (HTLV-1)-infected T cells promotes preferential transmission of HTLV-1 to CCR4-expressing CD4+ T cells

Adult T cell leukemia is a mature CD4+ T cell malignancy which predominantly expresses CCR4 and is etiologically associated with human T cell leukemia virus type 1 (HTLV-1). Because HTLV-1 transmission depends on close cell-cell contacts, HTLV-1-infected T cells may preferentially interact with CCR4+CD4+ T cells for efficient viral transmission. In terms of gene expression and protein secretion, we found a strong correlation between HTLV-1 Tax oncoprotein and CCL22, a CCR4 ligand, in HTLV-1-infected T cells. Transient Tax expression in an HTLV-1-negative T cell line activated the CCL22 promoter and induced CCL22. Additionally, tax gene knockdown by small interference RNA reduced CCL22 expression in the infected T cells. These findings indicate that CCL22 is a cellular target gene of Tax. In chemotaxis assays, the culture supernatants of HTLV-1-infected T cells selectively attracted CCR4+CD4+ T cells in PBMCs. This was blocked by pretreating the supernatants with anti-CCL22 Ab or PBMCs with a synthetic CCR4 antagonist. In coculture experiments, primary CCR4+CD4+ T cells significantly adhered to Tax-expressing cells. This adhesion was blocked by the CCR4 antagonist or pertussis toxin. Interestingly, CCR4 was redistributed to the contact region, and in some cases, this was accompanied by a polarized microtubule-organizing center, which is an indicator of virological synapse formation, in the infected T cells. Finally, anti-CCL22 Ab treatment also blocked HTLV-1 transmission to primary CD4+ T cells in coculture experiments with HTLV-1 producer cells. Thus, HTLV-1-infected T cells produce CCL22 through Tax and selectively interact with CCR4+CD4+ T cells, resulting in preferential transmission of HTLV-1 to CCR4+CD4+ T cells.     

Cross-talk between TCR and CCR7 signaling sets a temporal threshold for enhanced T lymphocyte migration

Lymphocyte homing to, and motility within, lymph nodes is regulated by the chemokine receptor CCR7 and its two ligands CCL19 and CCL21. There, lymphocytes are exposed to a number of extracellular stimuli that influence cellular functions and determine the cell fate. In this study, we assessed the effect of TCR engagement on CCR7-mediated cell migration. We found that long-term TCR triggering of freshly isolated human T cells through CD3/CD28 attenuated CCR7-driven chemotaxis, whereas short-term activation significantly enhanced CCR7-mediated, but not CXCR4-mediated, migration efficiency. Short-term activation most prominently enhanced the migratory response of naive T cells of both CD4 and CD8 subsets. We identified distinct roles for Src family kinases in modulating CCR7-mediated T cell migration. We provide evidence that Fyn, together with Ca(2+)-independent protein kinase C isoforms, kept the migratory response of naive T cells toward CCL21 at a low level. In nonactivated T cells, CCR7 triggering induced a Fyn-dependent phosphorylation of the inhibitory Tyr505 of Lck. Inhibiting Fyn in these nonactivated T cells prevented the negative regulation of Lck and facilitated high CCR7-driven T cell chemotaxis. Moreover, we found that the enhanced migration of short-term activated T cells was accompanied by a synergistic, Src-dependent activation of the adaptor molecule linker for activation of T cells. Collectively, we characterize a cross-talk between the TCR and CCR7 and provide mechanistic evidence that the activation status of T cells controls lymphocyte motility and sets a threshold for their migratory response.     

PI3Kgamma is the dominant isoform involved in migratory responses of human T lymphocytes: effects of ex vivo maintenance and limitations of non-viral delivery of siRNA

Use of mice in which individual PI3K isoforms have been deleted or mutated by gene targeting, has determined that PI3Kγ provides a key migratory signal for T lymphocyte migration. Since PI3Kγ can be a dispensable signal for directional migration of human T cells, we have adopted a pharmacological and siRNA strategy to assess the contribution of individual PI3K isoforms to chemokine-stimulated migration of human T cells. The broad spectrum PI3K isoform inhibitor Ly294002 inhibits CXCL12-stimulated migration of freshly isolated T lymphocytes. Use of second generation inhibitors that can discriminate between individual PI3K isoforms, revealed that PI3Kγ was the major contributor to CXCL12-induced migration and PI3K/Akt signaling (as assessed by S6 phosphorylation). Non-viral delivery of siRNA targeting class I (PI3Kγ), class II (PI3KC2 and PI3KC2β) and class III PI3Ks, followed by 3 days ex vivo culture, reduces the levels of isoform mRNA, but is insufficient to impact on cell migration responses. However, ex vivo maintenance of T cells alone, independently of siRNA treatment, resulted in the migratory response of T cells toward CXCL12 becoming insensitive to Ly294002. Remarkably, random migration remains sensitive to Ly294002. This study therefore, highlights that the migratory response of freshly isolated human T cells is dependent on PI3K signals that are provided predominantly by PI3Kγ. However, the role of PI3K in cell migration is context-dependent and diminishes during ex vivo maintenance.     

HTLV-2 Tax Immortalizes Human CD4+ Memory T Lymphocytes by Oncogenic Activation and Dysregulation of Autophagy

Human T cell leukemia virus type 1 and type 2 (HTLV-1 and -2) are two closely related retroviruses with the former causing adult T cell leukemia. HTLV-2 infection is prevalent among intravenous drug users, and the viral genome encodes the viral transactivator Tax, which is highly homologous to the transforming protein Tax from HTLV-1. However, the link between HTLV-2 infection and leukemia has not been established. In the present study, we evaluated the activity of HTLV-2 Tax in promoting aberrant proliferation of human CD4 T lymphocytes. Tax2 efficiently immortalized CD4⁺ memory T lymphocytes with a CD3/TCRαβ/CD4/CD25/CD45RO/CD69 immunophenotype, promoted constitutive activation of PI3K/Akt, IκB kinase/NF-κB, mitogen-activated protein kinase, and STAT3, and it also increased the level of Mcl-1. Disruption of these oncogenic pathways led to growth retardation and apoptotic cell death of the Tax2-established T cell lines. We further found that Tax2 induced autophagy by interacting with the autophagy molecule complex containing Beclin1 and PI3K class III to form the LC3⁺ autophagosome. Tax2-mediated autophagy promoted survival and proliferation of the immortalized T cells. The present study demonstrated the oncogenic properties of Tax2 in human T cells and also implicated Tax2 in serving as a molecular tool to generate distinct T cell subtype lines.