Elliott M. Faller

Interleukin-7 receptor expression on CD8 T-cells is downregulated by the HIV Tat protein.

We have previously shown decreased expression of the interleukin (IL)-7 receptor α-chain (CD127) on CD8 T-cells in HIV-infected patients and an apparent recovery of this receptor in those receiving antiretroviral therapy with sustained viral suppression. Here, we demonstrate that the HIV Tat protein specifically downregulates cell surface expression of CD127 on human CD8 T-cells in a dose- and time-dependent manner. The effects of Tat on CD127 expression could be blocked with anti-Tat monoclonal antibodies or by preincubating Tat with heparin. Tat had no effect on the expression of other cell surface proteins examined, including CD132, or on cell viability over 72 hours. Further, CD127 expression was not altered by other HIV proteins, including gp160 or Nef. Preincubation of purified CD8 T-cells with Tat protein inhibited CD8 T-cell proliferation and perforin synthesis after stimulation with IL-7. Because IL-7 signaling is essential for optimal CD8 T-cell proliferation and function, the downregulation of CD127 and apparent inhibition of cytotoxic activity by Tat may play an important role in HIV-induced immune dysregulation and impaired cell-mediated immunity.

Soluble HIV Tat Protein Removes the IL-7 Receptor α-Chain from the Surface of Resting CD8 T Cells and Targets It for Degradation

IL-7 signaling is essential to CD8 T cell development, activation, and homeostasis. We have previously shown decreased expression of the IL-7R α-chain (CD127) on CD8 T cells in HIV+ patients and that this downregulation is mediated at least in part by the HIV Tat protein. We show in this study that CD127 has a prolonged t1/2 in resting CD8 T cells and continuously recycles on and off the cell membrane. We also demonstrate soluble Tat protein significantly decreases the t1/2 of CD127. Soluble Tat is taken up from the medium and accumulates in CD8 T cells with a peak of 6 h. Once inside the cell, Tat exits the endosomes during their normal acidification and enters the cytosol. Tat then translocates to the inner leaflet of the cell membrane, where it binds directly to the cytoplasmic tail of CD127, inducing receptor aggregation and internalization through a process dependent on microtubules. Tat appears to then target CD127 for degradation via the proteasome. By removing CD127 from the cell surface, the HIV Tat protein is thus able to reduce IL-7 signaling and impair CD8 T cell proliferation and function.

IL-7 downregulates IL-7Rα expression in human CD8 T cells by two independent mechanisms.

Interleukin (IL)‐7 is an essential nonredundant cytokine, and throughout the lifespan of a T‐cell signaling via the IL‐7 receptor influences cell survival, proliferation and differentiation. It is therefore no surprise that expression of the IL‐7 receptor alpha‐chain (CD127) is tightly regulated. We have previously shown that IL‐7 downregulates expression of CD127 at the cell surface and now elucidate the kinetics of that suppression and demonstrate that IL‐7 downregulates CD127 transcripts and surface protein in primary human CD8 T cells by two separate pathways. We show that IL‐7 induces the initial reduction in cell‐surface CD127 protein independent of transcriptional suppression, which is delayed by 40–60 min. Although IL‐7‐mediated downregulation of CD127 transcripts is dependent on Janus kinase (JAK)/STAT5, the early downregulation of surface CD127 protein is independent of JAK activity. The data further illustrate that low levels of IL‐7 induce smaller and transient decreases in CD127 transcripts and surface protein, whereas higher concentrations induce more profound and sustained suppression. Such flexibility in receptor expression likely allows for fine‐tuned immune responses in human CD8 T cells in different microenvironments and in response to different immunological challenges.

IL-7 and the HIV Tat protein act synergistically to down-regulate CD127 expression on CD8 T cells.

IL-7 signaling is essential for optimal CD8 T cell function, homeostasis and establishment of memory. We have previously shown decreased expression of the IL-7 receptor alpha-chain (CD127) on CD8 T cells from HIV-infected patients with active viral replication. We have also shown that soluble HIV Tat protein specifically down-regulates CD127 on the surface of CD8 T cells and impairs cell proliferation and cytolytic potential following stimulation with IL-7 in vitro. We now show that soluble HIV Tat protein and IL-7 at near physiologic concentrations act synergistically to suppress CD127 expression. While soluble HIV Tat protein and IL-7 both independently reduce CD127 expression on the surface of CD8 T cells, Tat concentrations of 10 microg ml(-1) and IL-7 concentrations of 500 pg ml(-1) are required in vitro to have an appreciable effect. However, where 0.5 microg ml(-1) of Tat has no effect on CD127 expression and 200 pg ml(-1) of IL-7 decreases CD127 by only 14%, these two together at these same concentrations induce a 35% reduction in CD127 expression after 24 h. Inhibition of Janus kinase (JAK) completely blocks IL-7s ability to down-regulate CD127 on the surface of CD8 T cells and also abolishes synergy with Tat. Interestingly, while Tat acts synergistically with IL-7 to reduce CD127 expression, it antagonizes IL-7-induced cell proliferation and Ki-67 expression and has no effect on IL-7-mediated signal transducer and activator of transcription 5 (STAT5) phosphorylation or expression of the anti-apoptotic gene Bcl-2. Thus, by affecting different IL-7 signal transduction pathways, HIV Tat protein is able to impair both CD8 T cell activation and proliferation without inducing apoptosis.

Modulation of Microtubule Dynamics by the Microtubule-Associated Protein 1a

Structural microtubule‐associated proteins (MAPs) interact with microtubules to regulate the various dynamic stages of microtubules. The purpose of this study was to measure the impact of myc‐tagged MAP1a fragments on microtubule dynamic phases in vivo. Cells from an epithelial kidney cell line (LLCPK1) that had been permanently transfected with human green fluorescent protein (GFP)‐α‐tubulin were transiently transfected with myc‐tagged MAP1a fragments. Cells expressing MAP1a fragments were used to make direct observations of microtubule dynamics in living cells using fluorescence microscopy. All truncated MAP1a heavy chain fragments that contained the microtubule‐binding domain were shown to associate with microtubules. Truncated fragments containing different regions of the projection domain of MAP1a demonstrated variations in their impact on microtubule dynamic events by promoting growth or inhibition of shortening phases. In contrast to MAP1a, MAP2c bundled microtubules and resulted in a complete arrest of microtubule motility. Results from the present study suggest that MAP1a promotes slow, stable growth of microtubules. This type of growth may be important in the maintenance and restructuring of adult neurons.

Jak/STAT and PI3K signaling pathways have both common and distinct roles in IL-7-mediated activities in human CD8+ T cells

IL‐7 plays an important role in T cell survival, function, and memory cell development, yet the role of cytokine signaling pathways in these processes has not been fully elucidated. Moreover, the underlying mechanisms for the observed impairment of IL‐7 activity in diseases, such as HIV infection, breast cancer, and autoimmunity, are not well understood. It was therefore hypothesized that IL‐7‐induced signaling molecules could be linked with distinct IL‐7‐associated activities. To address this, the activation and functional associations of IL‐7‐induced signaling pathways, specifically antigen‐independent activities that are relevant to T cell homeostasis, were examined. Low concentrations of IL‐7 (100 pg/ml) are capable of activating the Jak‐STAT and PI3K signaling pathways, whereas higher concentrations (500–1000 pg/ml) were required to induce Bcl‐2 production and glucose uptake. Even higher concentrations of IL‐7 (10,000 pg/ml) were needed to induce cell proliferation and intracellular accumulation of perforin. Inhibition of Jak activation reduced IL‐7‐induced Bcl‐2 and perforin production, whereas inhibition of Jak/STAT or PI3K pathways reduced glucose uptake and proliferation. This study suggests a complex control of IL‐7‐associated activities in the absence of antigen stimulation. These data may provide insights into mechanisms of impaired IL‐7 signaling and function in disease and could be relevant for the study of IL‐7‐based immunotherapeutics. Specifically, this study has linked STAT5 and PI3K activation to shared and distinct IL‐7‐associated activities in human CD8+ T cells.

IL-7 induces clathrin-mediated endocytosis of CD127 and subsequent degradation by the proteasome in primary human CD8 T cells.

Interleukin‐7 (IL‐7), a key immunoregulatory cytokine, plays an essential role in peripheral T‐cell homeostasis and function. Signaling via the IL‐7 receptor is tightly regulated and we and others have shown IL‐7 provides negative feedback on its own signaling by downregulating expression of the IL‐7 receptor alpha‐chain (CD127) through both suppression of CD127 gene transcription and by internalization of existing CD127 proteins from the cell membrane. We show here for the first time in primary human CD8 T cells that upon stimulation with IL‐7, CD127 is internalized through clathrin‐coated pits, a process dependent on both lipid‐raft formation and the activity of dynamin. As visualized by confocal microscopy, CD127 shows increased co‐localization with clathrin within 5 min of IL‐7 stimulation and within 15–30 min is seen in multiple intracellular punctae co‐localizing with the early endosomal marker EEA1. By 2 h after addition of IL‐7, CD127 staining associates with the late endosomal marker RAB7 and with the proteasomal 20S subunit. By inducing receptor internalization and translocation from early endosomes to the proteasome, IL‐7 directly influences its receptor density on the cell surface and thus regulates the intensity of its own signaling cascades. Given the important role IL‐7 plays in T‐cell development, homeostasis and function, deciphering how expression of its receptor is controlled on the cell surface is essential in understanding how T‐cell activity can be regulated in different microenvironments and in response to different pathogens.

MAP1a associated light chain 3 increases microtubule stability by suppressing microtubule dynamics

Microtubule associated proteins (MAP) have been shown to play a role in microtubule stability in axons and dendrites, in determining neuronal shape and in regulating the balance between rigidity and plasticity in neuronal processes. MAP1a is the most abundant MAP in the adult brain, localized in axons and dendrites of neurons. MAP1a associates with three light chain molecules (LC1, LC2, LC3) that have been shown to bind microtubules independent of heavy chain molecules. In the present study we investigate the role of MAP1a associated light chain molecules in stabilizing microtubules and altering microtubule dynamics in vivo. All three light chain molecules co-localized with microtubules by fluorescence microscopy and bound taxol stabilized microtubules in an in vitro binding assay. LC-microtubule binding was associated with increased microtubule stability as shown by co localization of LC molecules with detyrosinated microtubules and increased amounts of detyrosinated tubulin in whole cell extracts. Both LC1 and LC2 binding to microtubules reorganized microtubules into wavy bundles that were resistant to nocodazole induced drug depolymerization. In contrast, LC3 bound microtubules were not resistant to nocodazole and the microtubule network of LC3 expressing cells was similar to media controls. Although LC3 bound microtubules were not resistant to drug induced depolymerization, in vivo measurement of microtubule dynamics shows that LC3 stabilizes microtubule networks by decreasing microtubule dynamicity and promoting growth over shortening events.

Suppressor of cytokine signaling (SOCS) proteins are induced by IL-7 and target surface CD127 protein for degradation in human CD8 T cells.

Given the essential role interleukin (IL)-7 plays in T-cell survival, homeostasis and function, it is no surprise expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. We have previously shown IL-7 binding to its receptor on the surface of CD8 T cells leads to both suppression of CD127 gene transcription and loss of existing CD127 protein from the cell membrane. Indeed upon binding IL-7, CD127 is rapidly internalized into early endosomes where phosphorylation by JAK targets the receptor for degradation. We now show that IL-7 induces the expression of suppressor of cytokine signaling (SOCS) proteins CIS, SOCS1 and SOCS2 through the JAK/STAT-5 pathway and that CIS and SOCS2 specifically interact with CD127 in early endosomes and direct the receptor complex to the proteasome for degradation. These results illustrate how expression of the IL-7 receptor and thus IL-7 signaling is modulated in human CD8 T cells by a negative feedback mechanism dependent on members of the SOCS family of proteins.

IL-7 receptor recovery on CD8 T-cells isolated from HIV+ patients is inhibited by the HIV Tat protein.

Expression of the IL-7 receptor α-chain (CD127) is decreased on CD8 T-cells in HIV infected patients and partially recovers in those receiving antiretroviral therapy with sustained viral suppression. We have shown that soluble HIV Tat protein down regulates CD127 expression on CD8 T-cells isolated from healthy HIV-negative individuals. Tat is taken up by CD8 T-cells via endocytosis, exits the endosome and then translocates to the inner leaflet of the cell membrane where it binds to the cytoplasmic tail of CD127 inducing receptor internalization and degradation by the proteasome. This down regulation of CD127 by Tat results in impaired CD8 T-cell function. Interestingly, suppression of CD127 by Tat is reversible and requires the continual presence of Tat in the culture media. We thus questioned whether the low IL-7 receptor expression evident on CD8 T-cells in HIV+ patients was similarly reversible and if suppression of the receptor could be maintained ex vivo by Tat protein alone. We show here that when CD8 T-cells isolated from HIV+ patients are incubated alone in fresh medium, low CD127 expression on the cell surface recovers to normal levels. This recovery of CD127, however, is completely inhibited by the addition of HIV Tat protein to the culture media. This study then provides evidence that soluble factor(s) are responsible for low CD127 expression on circulating CD8 T-cells in HIV+ individuals and further implicates Tat in suppressing this receptor essential to CD8 T-cell proliferation and function.