Louise Swainson

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.

IL-7Rα Gene Expression Is Inversely Correlated with Cell Cycle Progression in IL-7-Stimulated T Lymphocytes

IL-7 plays a major role in T lymphocyte homeostasis and has been proposed as an immune adjuvant for lymphopenic patients. This prospect is based, at least in part, on the short-term expansion of peripheral T cells in rIL7-treated mice and primates. Nevertheless, in vivo, following initial increases in T cell proliferation and numbers, lymphocytes return to a quiescent state. As the bases for this cell cycle exit have not yet been elucidated, it is important to assess the long-term biological effects of IL-7 on quiescent human T lymphocyte subsets. In this study, we find that IL-7-stimulated CD4+ naive lymphocytes enter into cell cycle with significantly delayed kinetics as compared with the memory population. Importantly though, these lymphocytes exit from the cell cycle despite the continuous replenishment of rIL-7. This response is distinct in memory and naive CD4+ lymphocytes with memory cells starting to exit from cycle by day 10 vs day 18 for naive cells. Return to quiescence is associated with a cessation in IL-7R signaling as demonstrated by an abrogation of STAT-5 phosphorylation, despite an up-regulation of surface IL-7Rα. Indeed, an initial 10-fold decrease in IL-7Rα mRNA levels is followed by increased IL-7Rα expression in naive as well as memory T cells, with kinetics paralleling cell cycle exit. Altogether, our data demonstrate 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. Thus, IL-7 therapy should be designed to allow optimal responsiveness of naive and memory T cell subsets.

Glut1-mediated glucose transport regulates HIV infection

Cell cycle entry is commonly considered to positively regulate HIV-1 infection of CD4 T cells, raising the question as to how quiescent lymphocytes, representing a large portion of the viral reservoir, are infected in vivo. Factors such as the homeostatic cytokine IL-7 have been shown to render quiescent T cells permissive to HIV-1 infection, presumably by transiently stimulating their entry into the cell cycle. However, we show here that at physiological oxygen (O2) levels (2–5% O2 tension in lymphoid organs), IL-7 stimulation generates an environment permissive to HIV-1 infection, despite a significantly attenuated level of cell cycle entry. We identify the IL-7–induced increase in Glut1 expression, resulting in augmented glucose uptake, as a key factor in rendering these T lymphocytes susceptible to HIV-1 infection. HIV-1 infection of human T cells is abrogated either by impairment of Glut1 signal transduction or by siRNA-mediated Glut1 down-regulation. Consistent with this, we show that the susceptibility of human thymocyte subsets to HIV-1 infection correlates with Glut1 expression; single-round infection is markedly higher in the Glut1-expressing double-positive thymocyte population than in any of the Glut1-negative subsets. Thus, our studies reveal the Glut1-mediated metabolic pathway as a critical regulator of HIV-1 infection in human CD4 T cells and thymocytes.

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

BackgroundWe 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.ResultsAs 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 (H1RBD or H2RBD). 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 H1RBD as well as H2RBD, 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.ConclusionTransfection 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.

Molecular and cellular basis of T cell lineage commitment

The thymus forms as an alymphoid thymic primordium with T cell differentiation requiring the seeding of this anlage. This review will focus on the characteristics of the hematopoietic progenitors which colonize the thymus and their subsequent commitment/differentiation, both in mice and men. Within the thymus, the interplay between Notch1 and IL-7 signals is crucial for the orchestration of T cell development, but the precise requirements for these factors in murine and human thympoeisis are not synonymous. Recent advances in our understanding of the mechanisms regulating precursor entry and their maintenance in the thymus will also be presented.

Lentiviral Transduction of Immune Cells

Gene 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.

HTLV envelopes and their receptor GLUT1, the ubiquitous glucose transporter: a new vision on HTLV infection?

We identified the ubiquitous glucose transporter GLUT1 as a receptor for Deltaretroviruses HTLV-1 and HTLV-2 envelopes (Env), mediating viral binding and entry. Here, we review the context and key observations that led us to this finding: functional modules of HTLV SU are similar to those of Gammaretrovirus Env which use multimembrane-spanning nutrient transporters as receptors; the HTLV Env receptor is an early marker of T lymphocyte activation; and HTLV Env inhibits glucose transport. We review several molecular, viral, cellular and physiological aspects of HTLV infection in relation to the in vivo and in vitro properties of GLUT1. Also, we examine the implications of HTLV-1 Env-GLUT1 interactions and altered glucose transport on the two major HTLV-1-induced diseases, adult T cell leukemia (ATL) and neurodegenerative tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM). Complementary to the classical models of disease progression, we propose new schemes that emphasize the potential metabolic alterations caused in different cellular compartments. Finally, we review the potential use of HTLV Env-derived constructs as tools for labeling GLUT1 in vivo and inhibiting GLUT1 transport in tumor cells.

In Vivo and Ex Vivo Gene Transfer in Thymocytes and Thymocyte Precursors

The thymus provides a specialized environment allowing the differentiation of T lymphocytes from bone marrow-derived progenitor cells. We and others have demonstrated that gene transfer into distinct thymocyte populations can be obtained, both in vivo and ex vivo, using lentiviral vectors. Here, we describe techniques for intrathymic lentiviral transduction in mice, using a surgical approach wherein the thoracic cavity is exposed as well as a significantly less invasive strategy wherein virions are directly injected through the skin. Moreover, thymocyte differentiation from murine and human progenitors is now feasible in vitro, under conditions wherein the Notch and IL-7 signaling pathways are activated. We describe methods allowing transduction of murine and human progenitors and their subsequent differentiation into more mature thymocytes. Conditions for lentiviral gene transfer into more differentiated human thymocyte subsets are also presented. Optimization of technologies for HIV-based gene transfer into murine and human thymocyte progenitors will advance strategies aimed at modulating T-cell differentiation and function in-vivo; approaches potentially targeting patients with genetic and acquired immunodeficiencies as well as immune-sensitive tumors. Furthermore, this technology will foster the progression of basic research aimed at elucidating molecular aspects of T-cell differentiation in mice and humans.