KIR2DL4 is an unusual killer cell immunoglobulin-like receptor (KIR) family member in terms of its structure, expression, cellular localization, and signaling properties. The most conserved KIR in ...evolution, it is referred to as a framework KIR gene and is expressed by all natural killer (NK) cells and a subset of T cells. Although it has a long cytoplasmic tail that is typical of inhibitory KIR, engagement of this receptor results in the activation of NK cells, not for cytotoxicity, but for cytokine and chemokine secretion. Unlike all other KIRs, which are expressed on the surface of NK cells, KIR2DL4 resides in endosomes. It signals from this intracellular site for a proinflammatory and proangiogenic response, using a novel endosomal signaling pathway that involves the serine/threonine kinases DNA-PKcs and Akt. The only known ligand of KIR2DL4 is HLA-G. Soluble HLA-G accumulates in KIR2DL4(+) endosomes. Unlike classical HLA molecules that serve as ligands for other KIR family members, in healthy individuals, HLA-G expression is restricted to the fetal trophoblast cells that invade the maternal decidua during early pregnancy. Since NK cells constitute the predominant lymphocyte subset at this site, the proinflammatory/proangiogenic outcome of the interaction between KIR2DL4 and soluble HLA-G supports a role for KIR2DL4 in the extensive remodeling of the maternal vasculature during the early weeks of pregnancy.
Interleukin-2 (IL-2) regulates lymphocyte function by signaling through heterodimerization of the IL-2Rβ and γc receptor subunits. IL-2 is of considerable therapeutic interest, but harnessing its ...actions in a controllable manner remains a challenge. Previously, we have engineered an IL-2 “superkine” with enhanced affinity for IL-2Rβ. Here, we describe next-generation IL-2 variants that function as “receptor signaling clamps.” They retained high affinity for IL-2Rβ, inhibiting binding of endogenous IL-2, but their interaction with γc was weakened, attenuating IL-2Rβ-γc heterodimerization. These IL-2 analogs acted as partial agonists and differentially affected lymphocytes poised at distinct activation thresholds. Moreover, one variant, H9-RETR, antagonized IL-2 and IL-15 better than blocking antibodies against IL-2Rα or IL-2Rβ. Furthermore, this mutein prolonged survival in a model of graft-versus-host disease and blocked spontaneous proliferation of smoldering adult T cell leukemia (ATL) T cells. This receptor-clamping approach might be a general mechanism-based strategy for engineering cytokine partial agonists for therapeutic immunomodulation.
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•Partial IL-2 agonists with altered signaling amplitudes have been created•A cell’s activation state influences its response to particular partial agonists•One IL-2 mutant, H9-RETR, blocked actions of IL-2 and IL-15, with therapeutic utility•Our approach should be broadly applicable to many other cytokines
There is resurging interest in controlling the actions of interleukin-2. Leonard, Garcia, and colleagues generated partial agonists of IL-2 with enhanced binding to IL-2Rβ but attenuated γc interaction. These reagents yielded a spectrum of signaling amplitudes and biological effects, including the mutein H9-RETR that blocked IL-2- and IL-15-mediated proliferation and cytotoxicity, with therapeutic utility.
Thermoelectric performance of the p-type semiconductor bornite, Cu5FeS4, is greatly enhanced through chemical substitution. Nonstoichiometric materials in which the Cu:Fe ratio and overall ...cation-vacancy content were adjusted are reported, and a figure of merit, ZT = 0.79, is achieved at temperatures as low as 550 K in Cu4.972Fe0.968S4. All materials were synthesized mechanochemically and characterized by powder X-ray diffraction, differential scanning calorimetry (DSC), and thermal and electrical transport property measurements. Single-phase behavior is retained in copper deficient phases, Cu5–x FeS4, for vacancy levels up to x = 0.1, while in materials Cu5+y Fe1–y S4, in which the Cu:Fe ratio is varied while maintaining full occupancy of cation sites, single-phase behavior persists for y ≤ 0.08. Adjusting the Cu:Fe ratio at a constant cation-vacancy level of 0.06 in Cu4.94+z Fe1–z S4, leads to single phases for z ≤ 0.04. DSC measurements indicate the temperature of the intermediate- (2a) to high-temperature (a) phase transition shows a more marked dependence on the Cu:Fe ratio than the lower temperature 4a to 2a transition. The thermoelectric power factor increases almost linearly with increasing Cu(II) content. The maximum figures of merit are obtained for materials with Cu(II) contents in the range 0.10–0.15 (corresponding to 2.0–2.8% Cu(II)), which simultaneously contain ca. 1% of cation vacancies.
Dominant inhibitory receptors for HLA class I (HLA-I) endow NK cells with high intrinsic responsiveness, a process termed licensing or education, but hinder their ability to kill HLA-I
tumor cells. ...Cancer immunotherapy with adoptive transfer of NK cells must overcome inhibitory signals by such receptors to promote elimination of HLA-I
tumor cells. As proof of concept, we show here that a chimeric antigen receptor (CAR) can be engineered to overcome inhibition by receptors for HLA-I and to promote lysis of HLA-I
tumor cells by CAR-NK cells. The design of this NK-tailored CAR (NK-CAR) relied on the potent NK cell activation induced by the synergistic combination of NK receptors CD28H (CD28 homolog,
) and 2B4 (CD244,
). An NK-CAR consisting of the single-chain fragment variable (scFv) of a CD19 antibody, the CD28H transmembrane domain, and the fusion of CD28H, 2B4, and TCRζ signaling domains was compared to a third-generation T-cell CAR with a CD28-41BB-TCRζ signaling domain. The NK-CAR delivered stronger activation signals to NK cells and induced more robust tumor cell lysis. Furthermore, such CAR-NK cells could overcome inhibition by HLA-E or HLA-C expressed on tumor cells. Therefore, engineering of CAR-NK cells that could override inhibition by HLA-I in patients undergoing cancer immunotherapy is feasible. This approach offers an attractive alternative to more complex strategies, such as genetic editing of inhibitory receptors in CAR-NK cells or treatment of patients with a combination of CAR-NK cells and checkpoint blockade with antibodies to inhibitory receptors. A significant benefit of inhibition-resistant NK-CARs is that NK cell inhibition would be overcome only during contact with targeted tumor cells and that HLA-I on healthy cells would continue to maintain NK cell responsiveness through licensing.
Freshly isolated, resting natural killer (NK) cells are generally less lytic against target cells than in vitro interleukin 2 (IL-2)-activated NK cells. To investigate the basis for this difference, ...the contribution of several receptors to activation of human NK cells was examined. Target-cell lysis by IL-2-activated NK cells in a redirected, antibody-dependent cytotoxicity assay was triggered by a number of receptors. In contrast, cytotoxicity by resting NK cells was induced only by CD16, and not by NKp46, NKG2D, 2B4 (CD244), DNAM-1 (CD226), or CD2. Calcium flux in resting NK cells was induced with antibodies to CD16 and, to a weaker extent, antibodies to NKp46 and 2B4. Although NKp46 did not enhance CD16-mediated calcium flux, it synergized with all other receptors. 2B4 synergized with 3 other receptors, NKG2D and DNAM-1 each synergized with 2 other receptors, and CD2 synergized with NKp46 only. Resting NK cells were induced to secrete tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ), and to kill target cells by engagement of specific, pair-wise combinations of receptors. Therefore, natural cytotoxicity by resting NK cells is induced only by mutual costimulation of nonactivating receptors. These results reveal distinct and specific patterns of synergy among receptors on resting NK cells.
Natural killer (NK) cells possess potent perforin‐ and interferon‐γ‐dependent effector functions that are tightly regulated. Inhibitory receptors for major histocompatibility complex class I display ...variegated expression among NK cells, which confers specificity to individual NK cells. Specificity is also provided by engagement of an array of NK cell activation receptors. Target cells may express ligands for a multitude of activation receptors, many of which signal through different pathways. How inhibitory receptors intersect different signaling cascades is not fully understood. This review focuses on advances in understanding how activation receptors cooperate to induce cytotoxicity in resting NK cells. The role of activating receptors in determining specificity and providing redundancy of target cell recognition is discussed. Using Drosophila insect cells as targets, we have examined the contribution of individual receptors. Interestingly, the strength of activation is not determined simply by additive effects of parallel activation pathways. Combinations of signals from different receptors can have different outcomes: synergy, no enhancement over individual signals, or additive effects. Cytotoxicity requires combined signals for granule polarization and degranulation. The integrin leukocyte function‐associated antigen‐1 contributes a signal for polarization but not for degranulation. Conversely, CD16 alone or in synergistic combinations, such as NKG2D and 2B4, signals for phospholipase‐C‐γ‐ and phosphatidylinositol‐3‐kinase‐dependent degranulation.
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 β2 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 anti-leukemia activity of NK cells helps prevent relapse during hematopoietic stem cell transplantation (HSCT) in leukemia patients. However, the factors that determine the sensitivity or ...resistance of leukemia cells in the context of NK-mediated cytotoxicity are not well-established. Here, we performed a genome-wide CRISPR screen in the human chronic-myelogenous-leukemia (CML) cell line K562 to identify genes that regulate the vulnerability of leukemia cells to killing by primary human NK cells. The distribution of guide RNAs (gRNAs) in K562 cells that survived co-incubation with NK cells showed that loss of
, which encodes the ligand of the natural cytotoxicity receptor NKp30, protected K562 cells from killing. In contrast, loss of genes that regulate the antigen-presentation and interferon-γ-signaling pathways increased the vulnerability of K562 cells. The addition of IFN-γ neutralizing antibody increased the susceptibility of K562 cells to NK-mediated killing. Upregulation of MHC class I on K562 cells after co-incubation with NK cells was dependent on
. Analysis of RNA-seq data from The Cancer Genome Atlas (TCGA) showed that low
expression in cancer tissues was associated with improved overall survival in acute myeloid leukemia (AML) and Kidney Renal Clear Cell Carcinoma (KIRC) patients. Our results, showing that the upregulation of MHC class I by NK-derived IFN-γ leads to resistance to NK cytotoxicity, suggest that targeting IFN-γ responses might be a promising approach to enhance NK cell anti-cancer efficacy.
ABSTRACT
Introduction: Ocular myasthenia gravis (OMG) is a common condition of the neuromuscular junction that may convert to generalized myasthenia gravis (GMG). Our aim in this study was to ...determine the conversion rate and predictive factors for generalization in OMG, in an Asian population. Methods: The investigation consisted of a retrospective study of OMG patients with a minimum 2 years of follow‐up. Results: Among 191 patients with OMG, 155 had the minimum 2‐year follow‐up. The conversion rate at median follow‐up (40.8 months) was 10.6% (95% confidence interval 7.9%–13.3%), and at the 2‐year follow‐up it was 7.7% (95% confidence interval 5.6%–9.8%). At baseline, the predictive factors for generalization were positive acetylcholine receptor antibodies (hazard ratio 3.71, P = 0.024), positive repetitive nerve stimulation (RNS) studies (hazard ratio 4.42, P = 0.003), and presence of radiologically presumed or pathologically confirmed thymoma (hazard ratio 3.10, P = 0.013). Discussion: The conversion rate of OMG to GMG in Asian patients is low, as predicted by presence of acetylcholine receptor antibodies, presence of thymoma, and positive RNS studies. Muscle Nerve 57: 756–760, 2018
Tetrahedrite, Cu12Sb4S13, is an abundant mineral with excellent thermoelectric properties owing to its low thermal conductivity. The electronic and structural origin of the intriguing physical ...properties of tetrahedrite, including its metal‐to‐semiconductor transition (MST), remains largely unknown. This work presents the first determination of the low‐temperature structure of tetrahedrite that accounts for its unique properties. Contrary to prior conjectures, the results show that the trigonal–planar copper cations remain in planar coordination below the MST. The atomic displacement parameters of the trigonal–planar copper cations, which have been linked to low thermal conductivity, increase by 200% above the MST. The phase transition is a consequence of the orbital degeneracy of the highest occupied 3d cluster orbitals of the copper clusters found in the cubic phase. This study reveals that a Jahn–Teller electronic instability leads to the formation of “molecular‐like” Cu57+ clusters and suppresses copper rattling vibrations due to the strengthening of direct copper–copper interactions. First principles calculations demonstrate that the structural phase transition opens a small band gap in the electronic density of states and eliminates the unstable phonon modes. These results provide insights on the interplay between phonon transport, electronic properties, and crystal structure in mixed‐valence compounds.
Direct copper–copper interactions in tetrahedrite result in the formation of pentameric Cu57+ clusters below the metal‐to‐semiconductor transition. The enhancement of copper–copper interactions in these “molecular‐like” clusters “locks” the trigonal–planar copper cations in place, suppressing the out‐of‐plane rattling vibrations to which low thermal conductivity is attributed.
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