Natural Killer Cell Inhibitory Receptors Block Actin Cytoskeleton-dependent Recruitment of 2B4 (CD244) to Lipid Rafts

A dynamic balance of positive and negative signals regulates target cell lysis by natural killer (NK) cells upon engagement of a variety of different activation receptors and of inhibitory receptors that recruit the tyrosine phosphatase SHP-1. However, the step at which activation signals are blocked by SHP-1 is not known. We have been using activation receptor 2B4 (CD244) to study the influence of inhibitory receptors on NK cell activation. Engagement of inhibitory receptors by HLA class I on target cells blocks phosphorylation of 2B4, placing the inhibitory step at the level, or upstream of 2B4 phosphorylation. Here we show that phosphorylated 2B4, after engagement with either antibodies or target cells that express the 2B4 ligand, is found exclusively in a detergent-resistant membrane fraction that contains lipid rafts. Integrity of lipid rafts was essential for phosphorylation and activating function of 2B4. Coengagement of inhibitory receptors blocked 2B4 phosphorylation and 2B4 association with detergent-resistant membranes, indicating that inhibitory receptors function upstream of raft-dependent signals. Recruitment of 2B4 into detergent-resistant membrane fractions and 2B4 phosphorylation were dependent on actin polymerization. Blocking actin cytoskeleton-dependent raft recruitment of different receptors may be a general mechanism by which inhibitory receptors control NK cell activation

Distinct Role of Rab27a in Granule Movement at the Plasma Membrane and in the Cytosol of NK Cells

Protocols were developed to automate image analysis and to track the movement of thousands of vesicular compartments in live cells. Algorithms were used to discriminate among different types of movement (e.g. random, caged, and directed). We applied these tools to investigate the steady-state distribution and movement of lytic granules (LG) in live natural killer (NK) cells by high-speed 3-dimensional (3D) spinning disc confocal and 2-dimensional total internal reflection fluorescence microscopy. Both mouse NK cells and a human NK cell line deficient in the small GTPase Rab27a were examined. The unbiased analysis of large datasets led to the following observations and conclusions. The majority of LG in the cytosol and at the plasma membrane of unstimulated NK cells are mobile. The use of inhibitors indicated that movement in the cytosol required microtubules but not actin, whereas movement at the plasma membrane required both. Rab27a deficiency resulted in fewer LG, and in a reduced fraction of mobile LG, at the plasma membrane. In contrast, loss of Rab27a increased the fraction of mobile LG and the extent of their movement in the cytosol. Therefore, in addition to its documented role in LG delivery to the plasma membrane, Rab27a may restrict LG movement in the cytosol

Recruitment of Activation Receptors at Inhibitory NK Cell Immune Synapses

Natural killer (NK) cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR) family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses

Vav1 Phosphorylation Is Induced by β2 Integrin Engagement on Natural Killer Cells Upstream of Actin Cytoskeleton and Lipid Raft Reorganization

The guanine nucleotide exchange factor Vav1 regulates actin polymerization and contributes to cytotoxicity by natural killer (NK) cells. An open question is how Vav1 becomes activated and what receptor can signal upstream of actin cytoskeleton rearrangement upon NK cell contact with target cells. Using transfected insect cells that express ligands of human NK cell receptors, we show that engagement of the β2 integrin LFA-1 on NK cells by intercellular adhesion molecule (ICAM)-1 led to a tyrosine phosphorylation of Vav1 that was not sensitive to cholesterol depletion and to inhibition of actin polymerization. Vav1 phosphorylation was blocked by an inhibitor of Src-family kinases, and correlated with activation of its downstream effector PAK. Binding of activation receptor 2B4 to its ligand CD48 was not sufficient for Vav1 phosphorylation. However, coengagement of 2B4 with LFA-1 resulted in an enhancement of Vav1 phosphorylation that was sensitive to cholesterol depletion and to inhibition of actin polymerization. Vav1 was recruited to a detergent-resistant membrane (DRM) fraction only when 2B4 and LFA-1 were coengaged, but not after LFA-1 engagement. Therefore, binding of LFA-1 to ICAM-1 on target cells may initiate an early signaling cascade in NK cells through activation of Vav1, leading to cytoskeleton reorganization and amplification of signals from other activation receptors

Killer cell inhibitory receptors specific for HLA-C and HLA-B identified by direct binding and by functional transfer

AbstractAn inhibitory signal is delivered to natural killer (NK) cells and a subset of cytotoxic T cells upon recognition of HLA class 1 molecules on target cells. We demonstrate that soluble forms of killer cell inhibitory receptors (KIR) bind directly and specifically to HLA-C alleles on transfected cells. Furthermore, transfer of individual KIR Into NK clones reconstituted recognition of HLA-C on target cells, leading to inhibition of lysis. Using such functional reconstitution, a related KIR that confers specificity for some HLA-B alleles was also identified. These KIR share conserved tyrosine phosphorylation motifs in their cytoplasmic tails. Thus, a single receptor in NK cells provides both specificity for HLA class I on target cells and the Inhibitory signal that prevents lysis

Synergistic Signals for Natural Cytotoxicity Are Required to Overcome Inhibition by c-Cbl Ubiquitin Ligase

SummaryNatural killer (NK) cell cytotoxicity toward target cells depends on synergistic coactivation by NK cell receptors such as NKG2D and 2B4. How synergy occurs is not known. Synergistic phosphorylation of phospholipase PLC-γ2, Ca2+ mobilization, and degranulation triggered by NKG2D and 2B4 coengagement were blocked by Vav1 siRNA knockdown, but enhanced by knockdown of c-Cbl. c-Cbl inhibited Vav1-dependent signals, given that c-Cbl knockdown did not rescue the Vav1 defect. Moreover, c-Cbl knockdown and Vav1 overexpression each circumvented the necessity for synergy because NKG2D or 2B4 alone became sufficient for activation. Thus, synergy requires not strict complementation but, rather, strong Vav1 signals to overcome inhibition by c-Cbl. Inhibition of NK cell cytotoxicity by CD94-NKG2A binding to HLA-E on target cells was dominant over synergistic activation, even after c-Cbl knockdown. Therefore, NK cell activation by synergizing receptors is regulated at the level of Vav1 by a hierarchy of inhibitory mechanisms

Higher measured than modeled ozone production at increased NOx levels in the Colorado Front Range

Abstract. Chemical models must correctly calculate the ozone formation rate, P(O3), to accurately predict ozone levels and to test mitigation strategies. However, air quality models can have large uncertainties in P(O3) calculations, which can create uncertainties in ozone forecasts, especially during the summertime when P(O3) is high. One way to test mechanisms is to compare modeled P(O3) to direct measurements. During summer 2014, the Measurement of Ozone Production Sensor (MOPS) directly measured net P(O3) in Golden, CO, approximately 25 km west of Denver along the Colorado Front Range. Net P(O3) was compared to rates calculated by a photochemical box model that was constrained by measurements of other chemical species and that used a lumped chemical mechanism and a more explicit one. Median observed P(O3) was up to a factor of 2 higher than that modeled during early morning hours when nitric oxide (NO) levels were high and was similar to modeled P(O3) for the rest of the day. While all interferences and offsets in this new method are not fully understood, simulations of these possible uncertainties cannot explain the observed P(O3) behavior. Modeled and measured P(O3) and peroxy radical (HO2 and RO2) discrepancies observed here are similar to those presented in prior studies. While a missing atmospheric organic peroxy radical source from volatile organic compounds co-emitted with NO could be one plausible solution to the P(O3) discrepancy, such a source has not been identified and does not fully explain the peroxy radical model–data mismatch. If the MOPS accurately depicts atmospheric P(O3), then these results would imply that P(O3) in Golden, CO, would be NOx-sensitive for more of the day than what is calculated by models, extending the NOx-sensitive P(O3) regime from the afternoon further into the morning. These results could affect ozone reduction strategies for the region surrounding Golden and possibly other areas that do not comply with national ozone regulations. Thus, it is important to continue the development of this direct ozone measurement technique to understand P(O3), especially under high-NOx regimes