Isolated receptor binding domains of HTLV-1 and HTLV-2 envelopes bind Glut-1 on activated CD4+ and CD8+ T cells

<p>Abstract</p> <p>Background</p> <p>We previously identified the glucose transporter Glut-1, a member of the multimembrane-spanning facilitative nutrient transporter family, as a receptor for both HTLV-1 and HTLV-2. However, a recent report concluded that Glut-1 cannot serve as a receptor for HTLV-1 on CD4 T cells: This was based mainly on their inability to detect Glut-1 on this lymphocyte subset using the commercial antibody mAb1418. It was therefore of significant interest to thoroughly assess Glut-1 expression on CD4 and CD8 T cells, and its association with HTLV-1 and -2 envelope binding.</p> <p>Results</p> <p>As previously reported, ectopic expression of Glut-1 but not Glut-3 resulted in significantly augmented binding of tagged proteins harboring the receptor binding domains of either HTLV-1 or HTLV-2 envelope glycoproteins (H1_RBDor H2_RBD). Using antibodies raised against the carboxy-terminal peptide of Glut-1, we found that Glut-1 expression was significantly increased in both CD4 and CD8 cells following TCR stimulation. Corresponding increases in the binding of H1_RBDas well as H2_RBD, not detected on quiescent T cells, were observed following TCR engagement. Furthermore, increased Glut-1 expression was accompanied by a massive augmentation in glucose uptake in TCR-stimulated CD4 and CD8 lymphocytes. Finally, we determined that the apparent contradictory results obtained by Takenouchi et al were due to their monitoring of Glut-1 with a mAb that does not bind cells expressing endogenous Glut-1, including human erythrocytes that harbor 300,000 copies per cell.</p> <p>Conclusion</p> <p>Transfection of Glut-1 directly correlates with the capacities of HTLV-1 and HTLV-2 envelope-derived ligands to bind cells. Moreover, Glut-1 is induced by TCR engagement, resulting in massive increases in glucose uptake and binding of HTLV-1 and -2 envelopes to both CD4 and CD8 T lymphocytes. Therefore, Glut-1 is a primary binding receptor for HTLV-1 and HTLV-2 envelopes on activated CD4 as well as CD8 lymphocytes.</p

Critical Loss of the Balance between Th17 and T Regulatory Cell Populations in Pathogenic SIV Infection

Chronic immune activation and progression to AIDS are observed after SIV infection in macaques but not in natural host primate species. To better understand this dichotomy, we compared acute pathogenic SIV infection in pigtailed macaques (PTs) to non-pathogenic infection in African green monkeys (AGMs). SIVagm-infected PTs, but not SIVagm-infected AGMs, rapidly developed systemic immune activation, marked and selective depletion of IL-17-secreting (Th17) cells, and loss of the balance between Th17 and T regulatory (Treg) cells in blood, lymphoid organs, and mucosal tissue. The loss of Th17 cells was found to be predictive of systemic and sustained T cell activation. Collectively, these data indicate that loss of the Th17 to Treg balance is related to SIV disease progression

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Metabolism and IL-7-responsiveness of human thymocyte and T cell populations

La cytokine IL-7 joue un rôle majeur dans l'homéostasie des cellules T et a été proposée comme adjuvant immunitaire pour les patients lymphopéniques. Le potentiel thérapeutique de l'IL-7 nécessite une meilleure connaissance de ses effets à court et long terme sur les populations de cellules T humaines. Au cours de ma thèse, j'ai montré que l'IL-7 induit la survie des cellules T CD4+ naïves et mémoires alors que leur cinétique d'entrée en cycle cellulaire est différente. Un arrêt de phosphorylation de STAT-5, induisant ainsi un défaut de signalisation via l'IL-7R, est associé au retour à un stade quiescent malgré une augmentation de transcription et d'expression de l'IL-7R. Ceci permet une boucle de régulation de la réponse des cellules T à l'IL-7. La réponse immune est contrôlée par une régulation fine des processus de prolifération et de mort cellulaire. Nous avons alors étudié l'influence de l'IL-7 sur différentes sous-populations de cellules T en présence ou non d'IL-2. En présence d'IL-7, les cellules récemment sorties du thymus (RTE) prolifèrent plus que les cellules naïves et mémoires d'adultes. Cet effet est même augmenté en présence d'IL-2. De plus, l'IL-2 agit en synergie avec l'IL-7 en augmentant la susceptibilité des RTE et des cellules T naïves adultes à la mort cellulaire induite par Fas. Cependant, la sous-population mémoire reste moins sensible à cette apoptose induite par Fas. Par ces mécanismes, l'IL-2 et l'IL-7 régulent l'homéostasie des sous-populations T en modulant l'équilibre entre prolifération et mort cellulaire. L'IL-7 entraîne des effets divers sur les cellules T, mais ses mécanismes d'action sont peu connus. J'ai montré que les effets distincts de l'IL-7 sur la survie et la prolifération des RTE dépendent de la dose et de la durée d'administration. Sous ces conditions, différents intermédiaires de signalisation sont activés. La phosphorylation de STAT5 ne corrèle pas avec la prolifération. Au cours de l'entrée en cycle, l'IL-7 augmente l'expression du transporteur de glucose GLUT1 et la captation de glucose. En effet, l'expression de GLUT1 est régulée par la voie PI3K et l'inhibition de cette voie bloque la prolifération mais pas la survie. GLUT1 jouant un rôle important dans la réponse des RTE à l'IL-7, j'ai donc analysé plus précisément son impact sur la différenciation des précurseurs T. Chez l'homme, au cours de la thymopoïèse, j'ai identifié une population unique de thymocytes CD4+CD8+ en cycle, basée sur l'augmentation de l'expression de GLUT1 et de la captation de glucose. Ces cellules DP GLUT1+, contrairement aux cellules DP GLUT1-, expriment des niveaux élevés de CD4 et montrent un important chimiotactisme induit par CXCR4. De plus, ces cellules DP GLUT1+, sont intrinsèquement plus susceptibles à l'infection par le VIH-1, indépendamment des récepteurs CD4 et CXCR4. Ceci réside probablement dans leur forte activité métabolique et proliférative. Ces études soulignent l'importance non seulement du métabolisme du glucose dans la thymopoïèse mais aussi de la régulation de la réponse des cellules T à l'IL-7. Elles ont de plus des implications pour l'utilisation de l'IL-7 en clinique comme un adjuvant immunitaire pour les patients lymphopéniques.The IL-7 cytokine plays a major role in T lymphocyte homeostasis and has been proposed as an immune adjuvant for lymphopenic patients. The potential for IL-7 therapy necessitates an profound understanding of the short and long-term effects of this cytokine on ex vivo cultured human T cell subsets. During my PhD, I have demonstrated that IL-7 promotes the extended survival of both naive and memory CD4+ T cells, whereas cycling of these two subsets is distinct and transient. We established that return to quiescence is associated with a cessation in IL-7R signaling as demonstrated by an abrogation of STAT5 phosphorylation, despite an up-regulation of IL-7R transcription and surface expression; thereby indicating an inhibitory feedback mechanism limiting IL-7 responsiveness. Immunological activity is, however, controlled not just by the regulation of cell cycle and proliferation, but by compensatory processes inducing T cell death. We therefore endeavoured to elucidate how IL-7 may influence this delicate equilibrium in different T cell subsets, both alone and in conjunction with another major cytokine, IL-2. IL-7 induces greater proliferation of recent thymic emigrants (RTE) than mature adult-derived naive and memory human CD4+ T cells, and we found that this effect is enhanced by IL-2. IL-2 also synergises with IL-7 to provoke high levels of Fas-induced cell death in RTE and naïve adult T cells, while the memory subset remain significantly less sensitive to Fas-mediated apoptosis. In this manner, 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. IL-7 can mediate diverse effects on T cells, however, little is known about the molecular bases of these distinct effects on primary human T lymphocytes. I determined that the survival and proliferative effects of IL-7 on human RTE can be distinguished, on the basis of dose as well as duration of IL-7 administration. Distinct signaling intermediates are activated under conditions of IL-7-induced survival and proliferation: STAT5 phosphorylation does not correlate with proliferation whereas upregulation of the glucose transporter GLUT1 as well as increased glucose uptake are markers of IL-7-induced cell cycle entry. GLUT1 is regulated by PI3K and indeed, inhibiting PI3K activity abrogates IL-7-induced proliferation but not survival. Given the importance of GLUT1 in RTE responses to IL-7, I then went on to analyse the role of GLUT1 in T cell precursor differentiation. During human thymopoiesis, GLUT1 and glucose uptake are up-regulated after b-selection, and I identified a unique population of cycling CD4+CD8+DP thymocytes expressing GLUT1. The GLUT1 +DP cells express uniformly high levels of CD4, and show enhanced CXCR4-induced chemotaxis as compared to the GLUT1- DP subset. Moreover, GLUT1+DP cells are intrinsically more permissive to HIV-1 vector infection, independent of their high CD4 and CXCR4 receptor levels, likely due to their active metabolism and rapidly cycling status. These studies highlight the importance of glucose metabolism in thymopoiesis as well as the regulation of T cell responses to IL-7, and have major implications for the clinical use of IL-7 as an immunorestorative agent in lymphopenic patients.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Lentiviral transduction of immune cells.

International audienceGene transfer into mammalian cells has been of crucial importance for studies determining the role of specific genes in the differentiation and cell fate of various hematopoietic lineages. Until recently, the majority of these studies were performed in transformed cell lines due to difficulties in achieving levels of transfection of greater than 1-3% in primary hematopoietic cells. Vectors based on retrovirus and lentivirus backbones have revolutionized our ability to transfer genes into primary hematopoietic cells. These vectors have allowed extensive ex vivo and in vivo studies following introduction of a gene of interest and have been used clinically in individuals suffering from cancers, infections, and genetic diseases. Ex vivo lentiviral gene transfer can result in efficient transduction of progenitor cells (>80%) that can then be further differentiated into immune lineage cells including T, B, dendritic, or natural killer cells. Alternatively, differentiated immune cells can themselves be transduced ex vivo with lentiviral vectors. Here, we discuss optimization of technologies for human immunodeficiency virus (HIV)-based gene transfer into murine and human progenitor and immune cell lineages

Glucose transporter 1 expression identifies a population of cycling CD4(+)CD8(+) human thymocytes with high CXCR4-induced chemotaxis

GLUT1, the major glucose transporter in peripheral T lymphocytes, is induced upon T cell receptor activation. However, the role of GLUT1 during human thymocyte differentiation remains to be evaluated. Our identification of GLUT1 as the human T lymphotrophic virus (HTLV) receptor has enabled us to use tagged HTLV-receptor-binding domain fusion proteins to specifically monitor surface GLUT1 expression. Here, we identify a unique subset of CD4(+)CD8(+) double-positive (DP) thymocytes, based on their GLUT1 surface expression. Whereas these cells express variable levels of CD8, they express uniformly high levels of CD4. Glucose uptake was 7-fold higher in CD4(hi)DP thymocytes than in CD4(lo)DP thymocytes (P = 0.0002). Further analyses indicated that these GLUT1(+) thymocytes are early post-β-selection, as demonstrated by low levels of T cell receptor (TCR)αβ and CD3. This population of immature GLUT1(+)DP cells is rapidly cycling and can be further distinguished by specific expression of the transferrin receptor. Importantly, the CXCR4 chemokine receptor is expressed at 15-fold higher levels on GLUT1(+)DP thymocytes, as compared with the DP GLUT1(-) subset, and the former cells show enhanced chemotaxis to the CXCR4 ligand CXCL12. Thus, during human thymopoiesis, GLUT1 is up-regulated after β-selection, and these immature DP cells constitute a population with distinct metabolic and chemotactic properties

Colony-Stimulating Factor 1 Receptor Antagonists Sensitize Human Immunodeficiency Virus Type 1-Infected Macrophages to TRAIL-Mediated Killing

Strategies aimed at eliminating persistent viral reservoirs from HIV-1-infected individuals have focused on CD4(+) T-cell reservoirs. However, very little attention has been given to approaches that could promote elimination of tissue macrophage reservoirs. HIV-1 infection of macrophages induces phosphorylation of colony-stimulating factor 1 receptor (CSF-1R), which confers resistance to apoptotic pathways driven by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), thereby promoting viral persistence. In this study, we assessed whether CSF-1R antagonists (PLX647, PLX3397, and PLX5622) restored apoptotic sensitivity of HIV-1-infected macrophages in vitro. PLX647, PLX3397, and PLX5622 at clinically relevant concentrations blocked the activation of CSF-1R and reduced the viability of infected macrophages, as well as the extent of viral replication. Our data show that strategies targeting monocyte colony-stimulating factor (MCSF) signaling could be used to promote elimination of HIV-1-infected myeloid cells and to contribute to the elimination of persistent viral reservoirs. IMPORTANCE As the HIV/AIDS research field explores approaches to eliminate HIV-1 in individuals on suppressive antiviral therapy, those approaches will need to eliminate both CD4(+) T-cell and myeloid cell reservoirs. Most of the attention has focused on CD4(+) T-cell reservoirs, and scant attention has been paid to myeloid cell reservoirs. The distinct nature of the infection in myeloid cells versus CD4(+) T cells will likely dictate different approaches in order to achieve their elimination. For CD4(+) T cells, most strategies focus on promoting virus reactivation to promote immune-mediated clearance and/or elimination by viral cytopathicity. Macrophages resist viral cytopathic effects and CD8(+) T-cell killing. Therefore, we have explored clearance strategies that render macrophages sensitive to viral cytopathicity. This research helps inform the design of strategies to promote clearance of the macrophage reservoir in infected individuals on suppressive antiviral therapy