Spatial and Chemical Surface Guidance of NK Cell Cytotoxic Activity

Studying how different signaling pathways spatially integrate in cells requires selective manipulation and control of different transmembrane ligand–receptor pairs at the same time. This work explores a novel method for precisely arranging two arbitrarily chosen ligands on a micron-scale two-dimensional pattern. The approach is based on lithographic patterning of Au and TiO₂ films, followed by their selective functionalization with Ni–nitrilotriacetic acid–histidine and biotin–avidin chemistries, respectively. The selectivity of chemical and biological functionalizations is demonstrated by X-ray photoelectron spectroscopy and immunofluorescence imaging, respectively. This approach is applied to produce the first type of bifunctional surfaces with controllably positioned ligands for activating the receptors of natural killer (NK) immune cells. NK cells were used as a model system to demonstrate the potency of the surface in guiding site-selective cell attachment and activation. Upon applying the suitable ligand or ligand combination, the surfaces guided the appropriate single- or bifunctional attachment and activation. These encouraging results demonstrate the effectiveness of the system as an experimental platform aimed at the comprehensive understanding of the immunological synapse. The great simplicity, modularity, and specificity of this approach make it applicable for a myriad of combinations of other biomolecules and applications, turning it into the “Swiss knife” of biointerfaces

Synergistic Activity of Anticancer Polyphenols Embedded in Amphiphilic Dendrimer Nanoparticles

Dendritic polymer nanoparticles (NPs) are promising vehicles for drug delivery. Most dendrimer polymer NPs, however, exhibit positive surface charge which make them, in many instances, cytotoxic. We constructed noncationic, amphiphilic dendrimer NPs embedding curcumin and resveratrol, natural polyphenols exhibiting anticancer properties. The curcumin/resveratrol/dendrimer NPs both effectively shielded the embedded polyphenols and facilitated their slow release and, notably, targeted cancer cells. The experimental data trace the cancer cell toxicity of the curcumin/resveratrol/dendrimer NPs to impairment of mitochondrial functions, specifically giving rise to enhanced intracellular calcium release, inhibition of cytochrome c oxidase enzyme activity, decreased mitochondrial membrane potential, and mitochondrial membrane perturbation. Importantly, synergism between the dendrimer-NP-embedded curcumin and resveratrol was observed, as more pronounced cancer cell death and mitochondrial disruption were induced by the curcumin/resveratrol/dendrimer NPs as compared to either the freely dissolved polyphenols or amphiphilic dendrimer NPs incorporating curcumin or resveratrol separately. This work suggests that amphiphilic dendrimer NPs encapsulating curcumin and resveratrol may constitute a promising anticancer therapeutic platform

Targeting Natural Killer Cell Reactivity by Employing Antibody to NKp46: Implications for Type 1 Diabetes

<div><p>Natural killer (NK) cells belong to the innate lymphoid cells. Their cytotoxic activity is regulated by the delicate balance between activating and inhibitory signals. NKp46 is a member of the primary activating receptors of NK cells. We previously reported that the NKp46 receptor is involved in the development of type 1 diabetes (T1D). Subsequently, we hypothesized that blocking this receptor could prevent or hinder disease development. To address this goal, we developed monoclonal antibodies for murine NKp46. One mAb, named NCR1.15, recognizes the mouse homologue protein of NKp46, named Ncr1, and was able to down-regulate the surface expression of NKp46 on primary murine NK cells following antibody injection <i>in vivo</i>. Additionally, NCR1.15 treatments were able to down-regulate cytotoxic activity mediated by NKp46, but not by other NK receptors. To test our primary assumption, we examined T1D development in two models, non-obese diabetic mice and low-dose streptozotocin. Our results show a significantly lower incidence of diabetic mice in the NCR1.15-treated group compared to control groups. This study directly demonstrates the involvement of NKp46 in T1D development and suggests a novel treatment strategy for early insulitis.</p></div

Prolonged multiple treatments with NCR1.15 down-regulates the surface expression of NKp46.

<p>Mice were injected i.p. 8 doses of PBS, 100 μg of NCR1.15 or cIgG1,κ every other day; 3 days after the last inoculation mice were sacrificed for further analysis. (A) C57BL/6 WT PBMCs and splenocytes were harvested and stained to detect the levels of CD3^-NK1.1⁺ NK cells. Data from one representative of three independent experiments are shown. (B) PBMCs drained from <i>NCR1</i>^<i>gfp/+</i> mice were analyzed for GFP expression following the prolonged repeated treatments. (C, D) Membrane associated NKp46 and NKG2D levels were analyzed on gated CD3ε^-NK1.1⁺ NK cells. (E) <i>NCR1</i> transcript levels from treated mice splenocytes were assessed by qRT-PCR. Data from one representative of two independent experiments are shown. * <i>p</i><0.05, ** <i>p</i><0.01 by Student's unpaired <i>t</i>-test. Bars, ±SD.</p

Single dose treatment with NCR1.15 inhibits NKp46-mediated activity on NK cells.

<p>(A) 3 days following i.p. injection of NCR1.15, isotype control or PBS purified splenic NK cells were co-incubated for 2–4 hours with YAC-1 target cells in the presence of anti-CD107a antibody. CD107a surface levels on NK cells were assessed by FACS. (B) Labeled YAC-1 and RMA cells were injected into the tail vein of 5 NCR1.15-, c-IgG1 κ- or PBS-treated mice. Cells in the lungs were quantified 3–4 hours following the injection using FACS analysis. Data from one of two independent experiments are shown. (C) 3 days following mice i.p. injection c-IgG1 κ-treated <i>UBC-GFP</i> and NCR1.15-treated C57BL/6 WT purified splenic NK cells were co-incubated with PD1.6 target cells in 1:1:1 ratio (E₁:E₂:T) with the presence of anti-CD107a antibody for 3–4 hours. CD107a surface levels on NK cells were assessed by FACS. (D) Purified splenic NK cells harvested from control- or NCR1.15-treated mice 3 days after the treatment were co-incubated with Ba/F3-Rae1ε target cells in 1:1 E:T ratio for 3–4 hours. CD107a surface expression levels were analyzed using FACS. Data from one representative of three independent experiments are shown. ** <i>p</i><0.01 by Student's unpaired <i>t</i>-test. Bars, ±SD.</p

Single dose treatment with NCR1.15 down-regulates the surface expression of NKp46 on NK cells, but not the NKp46 transcript or spleen/blood NK levels.

<p>3 days following i.p. injection of NCR1.15, isotype control or PBS splenocytes (A, B) and PBMCs (B) were stained and analyzed for CD3^-NK1.1⁺ NK cells using flow cytometry. Data from one representative of five independent experiments are shown. The membrane associated NKp46 (C, D) and NKG2D (C) on NK cells were stained and analyzed using flow cytometry. (E) Representative confocal images of purified splenic NK cells stained for extra- and intracellular expression of the NKp46. (F) qRT-PCR of NKp46 transcripts in splenocytes harvested from treated mice. (G) GFP intensity (GeoMFI) expressed by NK cells from <i>NCR1</i>^<i>gfp/+</i> following the various treatments. Data from one representative of three independent experiments are shown. ** <i>p</i><0.01 by Student's unpaired <i>t</i>-test. Bars, ±SD.</p

Treatment with NCR1.15 reduces NKp46-mediated NK activity on β-cells and inhibits T1D development.

<p>(A) 8 weeks after the first treatment NOD PBMCs drained from mice within the indicated groups were stained and analyzed for NK levels. NK cell levels were normalized to mock (PBS) treatment. (B) Representative histogram of membrane associated NKp46 levels on gated CD3ε-CD49b+ NK cells from the indicated groups. (C) Normalized GeoMFI of membrane associated NKp46 on gated NK cells, ** p<0.01 by Student's unpaired t-test. GeoMFI values were normalized to mock (PBS) treatment. (D) T1D development in NOD female mice in the indicated groups. (n = 14–15). Animals were considered diabetic when blood glucose was ≥250mg/dl. * p<0.05 Kaplan-M log-rank (Mental-Cox). Bars, ±SD.</p

Short term treatment with NCR1.15 down-regulates NKp46-mediated activity on pancreatic β-cells and inhibits LD-STZ induced diabetes.

<p>(A) Purified splenic NK cells from treated cells were co-incubated for 2–4 hours with purified C57BL/6 β-cells in the presence of anti-CD107a antibody. CD107a surface levels on NK cells were assessed by FACS. * <i>p</i><0.05 by Student's unpaired <i>t</i>-test. (B) LD-STZ induced diabetes development in C57BL/6 mice following 50μg injections at days-2 and 5. (<i>n</i> = 8). Animals were considered diabetic when blood glucose was ≥300mg/dl. *** <i>p</i><0.005 Kaplan-M log-rank (Mental-Cox). (C) Body weight of mice in the indicated groups following the LD-STZ administration; body weight baselines at day 0 of the control and NCR1.15 groups were 19.2±0.86, 19.8±1 grams, respectively. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.005 by Student's unpaired <i>t</i>-test. Bars, ±SD. (D) Representative H&E sections of the pancreas from sex&age-matched C57BL/6 mice treated with NCR1.15 or mock-treated (IgG1, κ) in LD-STZ induced diabetes model (magnification X40). (E) Summary of insulitis score over the days calculated as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118936#pone.0118936.ref037" target="_blank">37</a>]. Score is quantified from 0 (low) to 4 (most severe).</p

image_5_The Ebola-Glycoprotein Modulates the Function of Natural Killer Cells.JPEG

<p>The Ebola virus (EBOV) uses evasion mechanisms that directly interfere with host T-cell antiviral responses. By steric shielding of human leukocyte antigen class-1, the Ebola glycoprotein (GP) blocks interaction with T-cell receptors (TCRs), thus rendering T cells unable to attack virus-infected cells. It is likely that this mechanism could promote increased natural killer (NK) cell activity against GP-expressing cells by preventing the engagement of NK inhibitory receptors; however, we found that primary human NK cells were less reactive to GP-expressing HEK293T cells. This was manifested as reduced cytokine secretion, a reduction in NK degranulation, and decreased lysis of GP-expressing target cells. We also demonstrated reduced recognition of GP-expressing cells by recombinant NKG2D and NKp30 receptors. In accordance, we showed a reduced monoclonal antibody-based staining of NKG2D and NKp30 ligands on GP-expressing target cells. Trypsin digestion of the membrane-associated GP led to a recovery of the recognition of membrane-associated NKG2D and NKp30 ligands. We further showed that membrane-associated GP did not shield recognition by KIR2DL receptors; in accordance, GP expression by target cells significantly perturbed signal transduction through activating, but not through inhibitory, receptors. Our results suggest a novel evasion mechanism employed by the EBOV to specifically avoid the NK cell immune response.</p

image_3_The Ebola-Glycoprotein Modulates the Function of Natural Killer Cells.JPEG

<p>The Ebola virus (EBOV) uses evasion mechanisms that directly interfere with host T-cell antiviral responses. By steric shielding of human leukocyte antigen class-1, the Ebola glycoprotein (GP) blocks interaction with T-cell receptors (TCRs), thus rendering T cells unable to attack virus-infected cells. It is likely that this mechanism could promote increased natural killer (NK) cell activity against GP-expressing cells by preventing the engagement of NK inhibitory receptors; however, we found that primary human NK cells were less reactive to GP-expressing HEK293T cells. This was manifested as reduced cytokine secretion, a reduction in NK degranulation, and decreased lysis of GP-expressing target cells. We also demonstrated reduced recognition of GP-expressing cells by recombinant NKG2D and NKp30 receptors. In accordance, we showed a reduced monoclonal antibody-based staining of NKG2D and NKp30 ligands on GP-expressing target cells. Trypsin digestion of the membrane-associated GP led to a recovery of the recognition of membrane-associated NKG2D and NKp30 ligands. We further showed that membrane-associated GP did not shield recognition by KIR2DL receptors; in accordance, GP expression by target cells significantly perturbed signal transduction through activating, but not through inhibitory, receptors. Our results suggest a novel evasion mechanism employed by the EBOV to specifically avoid the NK cell immune response.</p