Faculty Bibliography

The Ca(2+)-activated K(+) channel KCa3.1 is required for Ca(2+) influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2beta (PI3K-C2beta) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2beta by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2beta siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2beta in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca(2+) influx, whereas silencing of PI3K-C2beta inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2beta colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation

CD4(+)CD25(+)Foxp3(+) natural regulatory T cells (T reg cells) maintain self-tolerance and suppress autoimmune diseases such as type 1 diabetes and inflammatory bowel disease (IBD). In addition to their effects on T cells, T reg cells are essential for maintaining normal numbers of dendritic cells (DCs): when T reg cells are depleted, there is a compensatory Flt3-dependent increase in DCs. However, little is known about how T reg cell homeostasis is maintained in vivo. We demonstrate the existence of a feedback regulatory loop between DCs and T reg cells. We find that loss of DCs leads to a loss of T reg cells, and that the remaining T reg cells exhibit decreased Foxp3 expression. The DC-dependent loss in T reg cells leads to an increase in the number of T cells producing inflammatory cytokines, such as interferon gamma and interleukin 17. Conversely, increasing the number of DCs leads to increased T reg cell division and accumulation by a mechanism that requires major histocompatibility complex II expression on DCs. The increase in T reg cells induced by DC expansion is sufficient to prevent type 1 autoimmune diabetes and IBD, which suggests that interference with this feedback loop will create new opportunities for immune-based therapies.

We have investigated the function of endogenous galectin-3 in T cells. Galectin-3-deficient (gal3(-/-)) CD4(+) T cells secreted more IFN-gamma and IL-4 than gal3(+/+)CD4(+) T cells after T-cell receptor (TCR) engagement. Galectin-3 was recruited to the cytoplasmic side of the immunological synapse (IS) in activated T cells. In T cells stimulated on supported lipid bilayers, galectin-3 was primarily located at the peripheral supramolecular activation cluster (pSMAC). Gal3(+/+) T cells formed central SMAC on lipid bilayers less effectively and adhered to antigen-presenting cells less firmly than gal3(-/-) T cells, suggesting that galectin-3 destabilizes the IS. Galectin-3 expression was associated with lower levels of early signaling events and phosphotyrosine signals at the pSMAC. Additional data suggest that galectin-3 potentiates down-regulation of TCR in T cells. By yeast two-hybrid screening, we identified as a galectin-3-binding partner, Alix, which is known to be involved in protein transport and regulation of cell surface expression of certain receptors. Co-immunoprecipitation confirmed galectin-3-Alix association and immunofluorescence analysis demonstrated the translocation of Alix to the IS in activated T cells. We conclude that galectin-3 is an inhibitory regulator of T-cell activation and functions intracellularly by promoting TCR down-regulation, possibly through modulating Alix's function at the IS.

Cytotoxic lymphocytes kill target cells by releasing the content of secretory lysosomes at the immune synapse. To understand the dynamics and control of cytotoxic immune synapses, we imaged human primary, live natural killer cells on lipid bilayers carrying ligands of activation receptors. Formation of an organized synapse was dependent on the presence of the beta2 integrin ligand ICAM-1. Ligands of coactivation receptors 2B4 and NKG2D segregated into central and peripheral regions, respectively. Lysosomal protein LAMP-1 that was exocytosed during degranulation accumulated in a large and spatially stable cluster, which overlapped with a site of membrane internalization. Lysosomal compartments reached the plasma membrane at focal points adjacent to centrally accumulated LAMP-1. Imaging of fixed cells revealed that perforin-containing granules were juxtaposed to an intracellular compartment where exocytosed LAMP-1 was retrieved. Thus, cytotoxic immune synapses include a central region of bidirectional vesicular traffic, which is controlled by integrin signaling.

The concept of an immunological synapse goes back to the early 1980s with the discovery of the relationship between T-cell antigen receptor mediated Ca(2+) signaling, adhesion, and directed secretion. However, this concept did not gain traction until images were published starting in 1998 that revealed a specific molecular pattern in the interface between T cells and model antigen-presenting cells or supported planar bilayers. The dominant pattern, a ring of adhesion molecules surrounding a central cluster of antigen receptors, was observed in both model systems. Analysis of the origins of this pattern over the past 10 years has presented a solution for a difficult problem in lymphocyte biology-how a highly motile cell can suddenly stop when it encounters a signal delivered by just a few antigenic ligands on the surface of another cell without disabling the sensory machinery of the motile cell. The T lymphocyte actively assembles the immunological synapse pattern following a modular design with roots in actin-myosin-based motility

T-cell acute lymphoblastic leukaemia (T-ALL) is a blood malignancy afflicting mainly children and adolescents. T-ALL patients present at diagnosis with increased white cell counts and hepatosplenomegaly, and are at an increased risk of central nervous system (CNS) relapse. For that reason, T-ALL patients usually receive cranial irradiation in addition to intensified intrathecal chemotherapy. The marked increase in survival is thought to be worth the considerable side-effects associated with this therapy. Such complications include secondary tumours, neurocognitive deficits, endocrine disorders and growth impairment. Little is known about the mechanism of leukaemic cell infiltration of the CNS, despite its clinical importance. Here we show, using T-ALL animal modelling and gene-expression profiling, that the chemokine receptor CCR7 (ref. 5) is the essential adhesion signal required for the targeting of leukaemic T-cells into the CNS. Ccr7 gene expression is controlled by the activity of the T-ALL oncogene Notch1 and is expressed in human tumours carrying Notch1-activating mutations. Silencing of either CCR7 or its chemokine ligand CCL19 (ref. 6) in an animal model of T-ALL specifically inhibits CNS infiltration. Furthermore, murine CNS-targeting by human T-ALL cells depends on their ability to express CCR7. These studies identify a single chemokine-receptor interaction as a CNS 'entry' signal, and open the way for future pharmacological targeting. Targeted inhibition of CNS involvement in T-ALL could potentially decrease the intensity of CNS-targeted therapy, thus reducing its associated short- and long-term complications

Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane

The interaction between a T cell and an antigen-presenting cell (APC) can trigger a signaling response that leads to T cell activation. Prior studies have shown that ligation of the T cell receptor (TCR) triggers a signaling cascade that proceeds through the coalescence of TCR and various signaling molecules (e.g., the kinase Lck and adaptor protein LAT [linker for T cell activation]) into microdomains on the plasma membrane. In this study, we investigated another ligand-receptor interaction (CD58-CD2) that facilities T cell activation using a model system consisting of Jurkat T cells interacting with a planar lipid bilayer that mimics an APC. We show that the binding of CD58 to CD2, in the absence of TCR activation, also induces signaling through the actin-dependent coalescence of signaling molecules (including TCR-zeta chain, Lck, and LAT) into microdomains. When simultaneously activated, TCR and CD2 initially colocalize in small microdomains but then partition into separate zones; this spatial segregation may enable the two receptors to enhance signaling synergistically. Our results show that two structurally distinct receptors both induce a rapid spatial reorganization of molecules in the plasma membrane, suggesting a model for how local increases in the concentration of signaling molecules can trigger T cell signaling.

Immunological synapses are initiated by signaling in discrete T cell antigen receptor microclusters and are important for the differentiation and effector functions of T cells. Synapse formation involves the orchestrated movement of microclusters toward the center of the contact area with the antigen-presenting cell. Microcluster movement is associated with centripetal actin flow, but the function of motor proteins is unknown. Here we show that myosin IIA was necessary for complete assembly and movement of T cell antigen receptor microclusters. In the absence of myosin IIA or its ATPase activity, T cell signaling was interrupted 'downstream' of the kinase Lck and the synapse was destabilized. Thus, T cell antigen receptor signaling and the subsequent formation of immunological synapses are active processes dependent on myosin IIA.