Naomi Taylor

The ubiquitous glucose transporter GLUT-1 is a receptor for HTLV.

The human T cell leukemia virus (HTLV) is associated with leukemia and neurological syndromes. The physiopathological effects of HTLV envelopes are unclear and the identity of the receptor, present on all vertebrate cell lines, has been elusive. We show that the receptor binding domains of both HTLV-1 and -2 envelope glycoproteins inhibit glucose transport by interacting with GLUT-1, the ubiquitous vertebrate glucose transporter. Receptor binding and HTLV envelope-driven infection are selectively inhibited when glucose transport or GLUT-1 expression are blocked by cytochalasin B or siRNAs, respectively. Furthermore, ectopic expression of GLUT-1, but not the related transporter GLUT-3, restores HTLV infection abrogated by either GLUT-1 siRNAs or interfering HTLV envelope glycoproteins. Therefore, GLUT-1 is a receptor for HTLV. Perturbations in glucose metabolism resulting from interactions of HTLV envelope glycoproteins with GLUT-1 are likely to contribute to HTLV-associated disorders.

Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites.

Helminth parasitic infections are a major global health and social burden. The host defence against helminths such as Nippostrongylus brasiliensis is orchestrated by type 2 cell-mediated immunity. Induction of type 2 cytokines, including interleukins (IL) IL-4 and IL-13, induce goblet cell hyperplasia with mucus production, ultimately resulting in worm expulsion. However, the mechanisms underlying the initiation of type 2 responses remain incompletely understood. Here we show that tuft cells, a rare epithelial cell type in the steady-state intestinal epithelium, are responsible for initiating type 2 responses to parasites by a cytokine-mediated cellular relay. Tuft cells have a Th2-related gene expression signature and we demonstrate that they undergo a rapid and extensive IL-4Rα-dependent amplification following infection with helminth parasites, owing to direct differentiation of epithelial crypt progenitor cells. We find that the Pou2f3 gene is essential for tuft cell specification. Pou2f3−/− mice lack intestinal tuft cells and have defective mucosal type 2 responses to helminth infection; goblet cell hyperplasia is abrogated and worm expulsion is compromised. Notably, IL-4Rα signalling is sufficient to induce expansion of the tuft cell lineage, and ectopic stimulation of this signalling cascade obviates the need for tuft cells in the epithelial cell remodelling of the intestine. Moreover, tuft cells secrete IL-25, thereby regulating type 2 immune responses. Our data reveal a novel function of intestinal epithelial tuft cells and demonstrate a cellular relay required for initiating mucosal type 2 immunity to helminth infection.

Erythrocyte Glut1 triggers dehydroascorbic acid uptake in mammals unable to synthesize vitamin C.

Of all cells, human erythrocytes express the highest level of the Glut1 glucose transporter. However, the regulation and function of Glut1 during erythropoiesis are not known. Here, we report that glucose transport actually decreases during human erythropoiesis despite a >3-log increase in Glut1 transcripts. In contrast, Glut1-mediated transport of L-dehydroascorbic acid (DHA), an oxidized form of ascorbic acid (AA), is dramatically enhanced. We identified stomatin, an integral erythrocyte membrane protein, as regulating the switch from glucose to DHA transport. Notably though, we found that erythrocyte Glut1 and associated DHA uptake are unique traits of humans and the few other mammals that have lost the ability to synthesize AA from glucose. Accordingly, we show that mice, a species capable of synthesizing AA, express Glut4 but not Glut1 in mature erythrocytes. Thus, erythrocyte-specific coexpression of Glut1 with stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize vitamin C.

Insulin-like growth factor induces the survival and proliferation of myeloma cells through an interleukin-6-independent transduction pathway

Multiple myeloma (MM) is a B‐cell neoplasia that is associated with an increased level of bone resorption. One important mediator of bone remodelling, insulin‐like growth factor (IGF‐I), has been shown to stimulate the proliferation of human myeloma cells. However, the mechanisms of action of IGF‐I in these cells have not been determined. Using interleukin (IL)‐6‐dependent myeloma cell lines, we show IGF‐I to be as potent a survival and proliferation factor as IL‐6. We demonstrated that IGF‐I functions independently of the IL‐6 transducer gp130 and that these two cytokines have additive effects. Moreover, inhibition of the IGF‐I pathway did not modulate the proliferative effect of IL‐6. Accordingly, we found that IL‐6 and IGF‐I activated distinct downstream signalling molecules: IL‐6 activated STAT3 phosphorylation, whereas IGF‐I treatment resulted in the phosphorylation of IRS‐1. Interestingly, these signalling pathways appear to converge as both cytokines activated the ras/MAPK pathway. Thus, IGF‐I acts as a potent survival and proliferation factor for myeloma cells by stimulating an IL‐6‐independent signalling cascade. These data, together with the finding that, in vivo, IGF‐I is normally expressed in close proximity to myeloma cells within the bone matrix, strongly suggest a role for this cytokine in the pathophysiology of multiple myeloma.

ZAP‐70 kinase regulates HIV cell‐to‐cell spread and virological synapse formation

HIV efficiently spreads in lymphocytes, likely through virological synapses (VSs). These cell–cell junctions share some characteristics with immunological synapses, but cellular proteins required for their constitution remain poorly characterized. We have examined here the role of ZAP‐70, a key kinase regulating T‐cell activation and immunological synapse formation, in HIV replication. In lymphocytes deficient for ZAP‐70, or expressing a kinase‐dead mutant of the protein, HIV replication was strikingly delayed. We have characterized further this replication defect. ZAP‐70 was dispensable for the early steps of viral cycle, from entry to expression of viral proteins. However, in the absence of ZAP‐70, intracellular Gag localization was impaired. ZAP‐70 was required in infected donor cells for efficient cell‐to‐cell HIV transmission to recipients and for formation of VSs. These results bring novel insights into the links that exist between T‐cell activation and HIV spread, and suggest that HIV usurps components of the immunological synapse machinery to ensure its own spread through cell‐to‐cell contacts.

IL-7 differentially regulates cell cycle progression and HIV-1-based vector infection in neonatal and adult CD4+ T cells

Differences in the immunological reactivity of umbilical cord (UC) and adult peripheral blood (APB) T cells are poorly understood. Here, we show that IL-7, a cytokine involved in lymphoid homeostasis, has distinct regulatory effects on APB and UC lymphocytes. Neither naive nor memory APB CD4+ cells proliferated in response to IL-7, whereas naive UC CD4+ lymphocytes underwent multiple divisions. Nevertheless, both naive and memory IL-7-treated APB T cells progressed into the G1b phase of the cell cycle, albeit at higher levels in the latter subset. The IL-7-treated memory CD4+ lymphocyte population was significantly more susceptible to infection with an HIV-1-derived vector than dividing CD4+ UC lymphocytes. However, activation through the T cell receptor rendered UC lymphocytes fully susceptible to HIV-1-based vector infection. These data unveil differences between UC and APB CD4+ T cells with regard to IL-7-mediated cell cycle progression and HIV-1-based vector infectivity. This evidence indicates that IL-7 differentially regulates lymphoid homeostasis in adults and neonates.

Isolation and functional characterization of human erythroblasts at distinct stages: implications for understanding of normal and disordered erythropoiesis in vivo

Terminal erythroid differentiation starts from morphologically recognizable proerythroblasts that proliferate and differentiate to generate red cells. Although this process has been extensively studied in mice, its characterization in humans is limited. By examining the dynamic changes of expression of membrane proteins during in vitro human terminal erythroid differentiation, we identified band 3 and α4 integrin as optimal surface markers for isolating 5 morphologically distinct populations at successive developmental stages. Functional analysis revealed that these purified cell populations have distinct mitotic capacity. Use of band 3 and α4 integrin enabled us to isolate erythroblasts at specific developmental stages from primary human bone marrow. The ratio of erythroblasts at successive stages followed the predicted 1:2:4:8:16 pattern. In contrast, bone marrows from myelodysplastic syndrome patients exhibited altered terminal erythroid differentiation profiles. Thus, our findings not only provide new insights into the genesis of the red cell membrane during human terminal erythroid differentiation but also offer a means of isolating and quantifying each developmental stage during terminal erythropoiesis in vivo. Our findings should facilitate a comprehensive cellular and molecular characterization of each specific developmental stage of human erythroblasts and should provide a powerful means of identifying stage-specific defects in diseases associated with pathological erythropoiesis.

Lentivirus-mediated gene transfer in primary T cells is enhanced by a central DNA flap.

Retroviral vectors have become the primary tool for gene delivery into hematopoietic cells, including T lymphocytes. Lentiviral vectors offer an advantage over Moloney murine leukemia virus (MuLV) vectors because of their ability to translocate across an intact nuclear membrane and integrate into the genome of nonproliferating cells. We have recently demonstrated that a central strand displacement event, controlled by the central polypurine tract (cPPT) and the central termination sequence (CTS), results in the formation of a central DNA flap which acts as a cis-determinant of HIV-1 genome nuclear import. Here, we show that insertion of this DNA determinant in a classical lentiviral vector resulted in a significantly higher level of transduction in activated T cells (51 ± 12.7% versus 15 ± 1.4%). CD4+ and CD8+ T cells were transduced at equivalent levels. Importantly, freshly isolated T cells stimulated only during the 12-h transduction period could be efficiently transduced with this new flap-containing lentiviral vector, but not with the parental lentiviral vector nor an MuLV vector. Transgene expression in the flap-containing lentiviral vector, under the control of either an internal cytomegalovirus or the elongation factor-1 alpha (EF1α) promoter, was significant and expression remained elevated in resting T cells. Thus, this system allows stable expression of transgenes in T lymphocytes following a short ex vivo transduction protocol.

Defective expression of p56lck in an infant with severe combined immunodeficiency.

Severe combined immune deficiency (SCID) is a heterogeneous disorder characterized by profound defects in cellular and humoral immunity. We report here an infant with clinical and laboratory features of SCID and selective CD4 lymphopenia and lack of CD28 expression on CD8(+) T cells. T cells from this patient showed poor blastogenic responses to various mitogens and IL-2. Other T cell antigen receptor- induced responses, including upregulation of CD69, were similarly inhibited. However, more proximal T cell antigen receptor signaling events, such as anti-CD3 induced protein tyrosine phosphorylation, phosphorylation of mitogen-associated protein kinase, and calcium mobilization were intact. Although p59fyn and ZAP-70 protein tyrosine kinases were expressed at normal levels, a marked decrease in the level of p56lck was noted. Furthermore, this decrease was associated with the presence of an alternatively spliced lck transcript lacking the exon 7 kinase encoding domain. These data suggest that a deficiency in p56lck expression can produce a SCID phenotype in humans.

Homeostasis of naive and memory CD4+ T cells: IL-2 and IL-7 differentially regulate the balance between proliferation and Fas-mediated apoptosis.

Cytokines play a crucial role in the maintenance of polyclonal naive and memory T cell populations. It has previously been shown that ex vivo, the IL-7 cytokine induces the proliferation of naive recent thymic emigrants (RTE) isolated from umbilical cord blood but not mature adult-derived naive and memory human CD4+ T cells. We find that the combination of IL-2 and IL-7 strongly promotes the proliferation of RTE, whereas adult CD4+ T cells remain relatively unresponsive. Immunological activity is controlled by a balance between proliferation and apoptotic cell death. However, the relative contributions of IL-2 and IL-7 in regulating these processes in the absence of MHC/peptide signals are not known. Following exposure to either IL-2 or IL-7 alone, RTE, as well as mature naive and memory CD4+ T cells, are rendered only minimally sensitive to Fas-mediated cell death. However, in the presence of the two cytokines, Fas engagement results in a high level of caspase-dependent apoptosis in both RTE as well as naive adult CD4+ T cells. In contrast, equivalently treated memory CD4+ T cells are significantly less sensitive to Fas-induced cell death. The increased susceptibility of RTE and naive CD4+ T cells to Fas-induced apoptosis correlates with a significantly higher IL-2/IL-7-induced Fas expression on these T cell subsets than on memory CD4+ T cells. Thus, IL-2 and IL-7 regulate homeostasis by modulating the equilibrium between proliferation and apoptotic cell death in RTE and mature naive and memory T cell subsets.