Batu Erman

Signaling by intrathymic cytokines, not T cell antigen receptors, specifies CD8 lineage choice and promotes the differentiation of cytotoxic-lineage T cells

Immature CD4+CD8+ (double-positive (DP)) thymocytes are signaled via T cell antigen receptors (TCRs) to undergo positive selection and become responsive to intrathymic cytokines such as interleukin 7 (IL-7). We report here that cytokine signaling is required for positively selected thymocytes to express the transcription factor Runx3, specify CD8 lineage choice and differentiate into cytotoxic-lineage T cells. In DP thymocytes genetically engineered to be cytokine responsive, IL-7 signaling induced TCR-unsignaled DP thymocytes to express Runx3 and to differentiate into mature CD8+ T cells, completely circumventing positive selection. We conclude that TCR-mediated positive selection converts DP cells into cytokine-responsive thymocytes, but it is subsequent signaling by intrathymic cytokines that specifies CD8 lineage choice and promotes differentiation into cytotoxic-lineage T cells.

Interleukin 7 signaling in dendritic cells regulates the homeostatic proliferation and niche size of CD4 + T cells

Interleukin 7 (IL-7) and T cell antigen receptor signals have been proposed to be the main drivers of homeostatic T cell proliferation. However, it is not known why CD4+ T cells undergo less-efficient homeostatic proliferation than CD8+ T cells do. Here we show that systemic IL-7 concentrations increased during lymphopenia because of diminished use of IL-7 but that IL-7 signaling on IL-7 receptor-α–positive (IL-7Rα+) dendritic cells (DCs) in lymphopenic settings paradoxically diminished the homeostatic proliferation of CD4+ T cells. This effect was mediated at least in part by IL-7-mediated downregulation of the expression of major histocompatibility complex class II on IL-7Rα+ DCs. Our results indicate that IL-7Rα+ DCs are regulators of the peripheral CD4+ T cell niche and that IL-7 signals in DCs prevent uncontrolled CD4+ T cell population expansion in vivo.Interleukin 7 (IL-7) and T cell receptor (TCR) signals have been proposed to be the primary drivers of homeostatic T cell proliferation. However, it is not known why CD4+ T cells undergo less efficient homeostatic proliferation than CD8+ T cells. Here we showed that systemic IL-7 concentrations rise during lymphopenia due to diminished IL-7 utilization, but that IL-7 signaling on IL-7Rα+ dendritic cells (DCs) in lymphopenic settings paradoxically diminishes CD4+ T cell homeostatic proliferation. This effect is mediated, at least in part, by IL-7-mediated downregulation of MHC class II expression on IL-7Rα+ DCs. These results implicate IL-7Rα+ DCs as regulators of the peripheral CD4+ T cell niche, and indicate that IL-7 signals in DCs prevent uncontrolled CD4+ T cell expansion in vivo.

In vitro evidence that cytokine receptor signals are required for differentiation of double positive thymocytes into functionally mature CD8+ T cells

CD4+8+ double positive (DP) thymocytes differentiate into CD4+ and CD8+ mature T cells in response to TCR signals. However, TCR signals that are initiated in DP thymocytes are unlikely to persist throughout all subsequent differentiation steps, suggesting that other signals must sustain thymocyte differentiation after TCR signaling has ceased. Using an in vitro experimental system, we now demonstrate that cytokine receptor signals, such as those transduced by IL-7 receptors, are required for differentiation of signaled DP thymocytes into functionally mature CD8+ T cells as they: (a) up-regulate Bcl-2 expression to maintain thymocyte viability; (b) enhance CD4 gene silencing; (c) promote functional maturation;and (d) up-regulate surface expression of glucose transporter molecules, which improve nutrient uptake and increase metabolic activity. IL-7Rs appear to be unique among cytokine receptors in maintaining the viability of newly generated CD4−8+ thymocytes, whereas several different cytokine receptors can provide the trophic/differentiative signals for subsequent CD8+ thymocyte differentiation and maturation. Thus, cytokine receptors provide both survival and trophic/differentiative signals with varying degrees of redundancy that are required for differentiation of signaled DP thymocytes into functionally mature CD8+ T cells.

IL-7 Receptor Signals Inhibit Expression of Transcription Factors TCF-1, LEF-1, and RORγt Impact on Thymocyte Development

Intrathymic T cell development depends on signals transduced by both T cell receptor and cytokine receptors. Early CD4−CD8− (double negative) thymocytes require interleukin (IL)-7 receptor (IL-7R) signals for survival and proliferation, but IL-7R signals are normally extinguished by the immature single positive (ISP) stage of thymocyte development. We now demonstrate that IL-7R signals inhibit expression of transcription factors TCF-1, LEF-1, and RORγt that are required for the ISP to double positive (DP) transition in the thymus. In addition, we demonstrate that IL-7R signals also inhibit TCF-1 and LEF-1 expression in mature peripheral T cells. Thus, the present work has identified several important downstream target genes of IL-7R signaling in T cells and thymocytes that provide a molecular mechanism for the inhibitory influence of IL-7R signaling on DP thymocyte development. We conclude that IL-7R signals down-regulate transcription factors required for the ISP to DP transition and so must be terminated by the ISP stage of thymocyte development.

Foxp3 Transcription Factor Is Proapoptotic and Lethal to Developing Regulatory T Cells unless Counterbalanced by Cytokine Survival Signals

Immune tolerance requires regulatory T (Treg) cells to prevent autoimmune disease, with the transcription factor Foxp3 functioning as the critical regulator of Treg cell development and function. We report here that Foxp3 was lethal to developing Treg cells in the thymus because it induced a unique proapoptotic protein signature (Puma⁺⁺⁺p-Bim⁺⁺p-JNK⁺⁺DUSP6⁻) and repressed expression of prosurvival Bcl-2 molecules. However, Foxp3 lethality was prevented by common gamma chain (γc)-dependent cytokine signals that were present in the thymus in limiting amounts sufficient to support only ∼1 million Treg cells. Consequently, most newly arising Treg cells in the thymus were deprived of this signal and underwent Foxp3-induced death, with Foxp3⁺CD25⁻ Treg precursor cells being the most susceptible. Thus, we identify Foxp3 as a proapoptotic protein that requires developing Treg cells to compete with one another for limiting amounts of γc-dependent survival signals in the thymus.

Oxidative Stress Is a Mediator for Increased Lipid Accumulation in a Newly Isolated Dunaliella salina Strain

Green algae offer sustainable, clean and eco-friendly energy resource. However, production efficiency needs to be improved. Increasing cellular lipid levels by nitrogen depletion is one of the most studied strategies. Despite this, the underlying physiological and biochemical mechanisms of this response have not been well defined. Algae species adapted to hypersaline conditions can be cultivated in salty waters which are not useful for agriculture or consumption. Due to their inherent extreme cultivation conditions, use of hypersaline algae species is better suited for avoiding culture contamination issues. In this study, we identified a new halophilic Dunaliella salina strain by using 18S ribosomal RNA gene sequencing. We found that growth and biomass productivities of this strain were directly related to nitrogen levels, as the highest biomass concentration under 0.05 mM or 5 mM nitrogen regimes were 495 mg/l and 1409 mg/l, respectively. We also confirmed that nitrogen limitation increased cellular lipid content up to 35% under 0.05 mM nitrogen concentration. In order to gain insight into the mechanisms of this phenomenon, we applied fluorometric, flow cytometric and spectrophotometric methods to measure oxidative stress and enzymatic defence mechanisms. Under nitrogen depleted cultivation conditions, we observed increased lipid peroxidation by measuring an important oxidative stress marker, malondialdehyde and enhanced activation of catalase, ascorbate peroxidase and superoxide dismutase antioxidant enzymes. These observations indicated that oxidative stress is accompanied by increased lipid content in the green alga. In addition, we also showed that at optimum cultivation conditions, inducing oxidative stress by application of exogenous H2O2 leads to increased cellular lipid content up to 44% when compared with non-treated control groups. Our results support that oxidative stress and lipid overproduction are linked. Importantly, these results also suggest that oxidative stress mediates lipid accumulation. Understanding such relationships may provide guidance for efficient production of algal biodiesels.

Cytokine signal transduction is suppressed in preselection double-positive thymocytes and restored by positive selection

Death by neglect requires that CD4+8+ double-positive (DP) thymocytes avoid cytokine-mediated survival signals, which is presumably why DP thymocytes normally extinguish IL-7R gene expression. We report that DP thymocytes before positive selection (preselection DP thymocytes) fail to transduce IL-7 signals even when they express high levels of transgenic IL-7R on their surface, because IL-7R signal transduction is actively suppressed in preselection DP thymocytes by suppressor of cytokine signaling (SOCS)–1. SOCS-1 is highly expressed in preselection DP thymocytes, but it is down-regulated by T cell receptor–mediated positive selection signals. Interestingly, we found that the uniquely small cell volume of DP thymocytes is largely the result of absent IL-7 signaling in preselection DP thymocytes. We also report that, contrary to current concepts, preselection DP thymocytes express high levels of endogenously encoded IL-4Rs. However, their ability to transduce cytokine signals is similarly suppressed by SOCS-1. Thus, despite high surface expression of transgenic or endogenous cytokine receptors, cytokine signal transduction is actively suppressed in preselection DP thymocytes until it is restored by positive selection.

ETS-core binding factor: A common composite motif in antigen receptor gene enhancers

ABSTRACT A tripartite domain of the murine immunoglobulin μ heavy-chain enhancer contains the μA and μB elements that bind ETS proteins and the μE3 element that binds leucine zipper-containing basic helix-loop-helix (bHLH-zip) factors. Analysis of the corresponding region of the human μ enhancer revealed high conservation of the μA and μB motifs but a striking absence of the μE3 element. Instead of bHLH-zip proteins, we found that the human enhancer bound core binding factor (CBF) between the μA and μB elements; CBF binding was shown to be a common feature of both murine and human enhancers. Furthermore, mutant enhancers that bound prototypic bHLH-zip proteins but not CBF did not activate transcription in B cells, and conversely, CBF transactivated the murine enhancer in nonlymphoid cells. Taking these data together with the earlier analysis of T-cell-specific enhancers, we propose that ETS-CBF is a common composite element in the regulation of antigen receptor genes. In addition, these studies identify the first B-cell target of CBF, a protein that has been implicated in the development of childhood pre-B-cell leukemias.

Conditional deletion of cytokine receptor chains reveals that IL-7 and IL-15 specify CD8 cytotoxic lineage fate in the thymus

Two cytokines that signal through the common gamma chain receptor, IL-7 and IL-15, are responsible for the differentiation of developing thymocytes into CD8+ cytotoxic lineage T cells in mice.

Transcriptional Activation by ETS and Leucine Zipper-Containing Basic Helix-Loop-Helix Proteins

ABSTRACT The immunoglobulin μ heavy-chain gene enhancer contains closely juxtaposed binding sites for ETS and leucine zipper-containing basic helix-loop-helix (bHLH-zip) proteins. To understand the μ enhancer function, we have investigated transcription activation by the combination of ETS and bHLH-zip proteins. The bHLH-zip protein TFE3, but not USF, cooperated with the ETS domain proteins PU.1 and Ets-1 to activate a tripartite domain of this enhancer. Deletion mutants were used to identify the domains of the proteins involved. Both TFE3 and USF enhanced Ets-1 DNA binding in vitro by relieving the influence of an autoinhibitory domain in Ets-1 by direct protein-protein associations. Several regions of Ets-1 were found to be necessary, whereas the bHLH-zip domain was sufficient for this effect. Our studies define novel interactions between ETS and bHLH-zip proteins that may regulate combinatorial transcription activation by these protein families.