An Historical Overview: The Discovery of How NK Cells Can Kill Enemies, Recruit Defense Troops, and More

Natural killer (NK) cells were originally defined as effector lymphocytes of innate immunity characterized by the unique ability of killing tumor and virally infected cells without any prior priming and expansion of specific clones. The \u201cmissing-self\u201d theory, proposed by Klas Karre, the seminal discovery of the first prototypic HLA class I-specific inhibitory receptors, and, later, of the Natural Cytotoxicity Receptors (NCRs) by Alessandro Moretta, provided the bases to understand the puzzling behavior of NK cells. Actually, those discoveries proved crucial also for many of the achievements that, along the years, have contributed to the modern view of these cells. Indeed, NK cells, besides killing susceptible targets, are now known to functionally interact with different immune cells, sense pathogens using TLR, adapt their responses to the local environment, and, even, mount a sort of immunological memory. In this review, we will specifically focus on the main activating NK receptors and on their crucial role in the ever-increasing number of functions assigned to NK cells and other innate lymphoid cells (ILCs)

UniPR1331, a small molecule targeting Eph/ephrin interaction, prolongs survival in glioblastoma and potentiates the effect of antiangiogenic therapy in mice

Glioblastoma multiforme (GBM) is the most malignant brain tumor, showing high resistance to standard therapeutic approaches that combine surgery, radiotherapy, and chemotherapy. As opposed to healthy tissues, EphA2 has been found highly expressed in specimens of glioblastoma, and increased expression of EphA2 has been shown to correlate with poor survival rates. Accordingly, agents blocking Eph receptor activity could represent a new therapeutic approach. Herein, we demonstrate that UniPR1331, a pan Eph receptor antagonist, possesses significant in vivo anti-angiogenic and anti-vasculogenic properties which lead to a significant anti-tumor activity in xenograft and orthotopic models of GBM. UniPR1331 halved the final volume of tumors when tested in xenografts (p<0.01) and enhanced the disease-free survival of treated animals in the orthotopic models of GBM both by using U87MG cells (40 vs 24 days of control, p<0.05) or TPC8 cells (52 vs 16 days, p<0.01). Further, the association of UniPR1331 with the anti-VEGF antibody Bevacizumab significantly increased the efficacy of both monotherapies in all tested models. Overall, our data promote UniPR1331 as a novel tool for tackling GBM

Amyotrophic Lateral Sclerosis-Linked Mutant VAPB Inclusions Do Not Interfere with Protein Degradation Pathways or Intracellular Transport in a Cultured Cell Model

<div><p>VAPB is a ubiquitously expressed, ER-resident adaptor protein involved in interorganellar lipid exchange, membrane contact site formation, and membrane trafficking. Its mutant form, P56S-VAPB, which has been linked to a dominantly inherited form of Amyotrophic Lateral Sclerosis (ALS8), generates intracellular inclusions consisting in restructured ER domains whose role in ALS pathogenesis has not been elucidated. P56S-VAPB is less stable than the wild-type protein and, at variance with most pathological aggregates, its inclusions are cleared by the proteasome. Based on studies with cultured cells overexpressing the mutant protein, it has been suggested that VAPB inclusions may exert a pathogenic effect either by sequestering the wild-type protein and other interactors (loss-of-function by a dominant negative effect) or by a more general proteotoxic action (gain-of-function). To investigate P56S-VAPB degradation and the effect of the inclusions on proteostasis and on ER-to-plasma membrane protein transport in a more physiological setting, we used stable HeLa and NSC34 Tet-Off cell lines inducibly expressing moderate levels of P56S-VAPB. Under basal conditions, P56S-VAPB degradation was mediated exclusively by the proteasome in both cell lines, however, it could be targeted also by starvation-stimulated autophagy. To assess possible proteasome impairment, the HeLa cell line was transiently transfected with the ERAD (ER Associated Degradation) substrate CD3δ, while autophagic flow was investigated in cells either starved or treated with an autophagy-stimulating drug. Secretory pathway functionality was evaluated by analyzing the transport of transfected Vesicular Stomatitis Virus Glycoprotein (VSVG). P56S-VAPB expression had no effect either on the degradation of CD3δ or on the levels of autophagic markers, or on the rate of transport of VSVG to the cell surface. We conclude that P56S-VAPB inclusions expressed at moderate levels do not interfere with protein degradation pathways or protein transport, suggesting that the dominant inheritance of the mutant gene may be due mainly to haploinsufficiency.</p></div

The response of pigs inoculated with a thymidine kinase-negative (TK) pseudorabies virus to challenge infection with virulent virus

12 Large-White-Landrace piglets were subdivided in four groups of 3 and housed in separate units. The piglets of three groups were inoculated with the 86/27V 6C2 thymidine kinase negative (TK-) mutant of pseudorabies virus (PRV), by different routes. A second inoculation with the same mutant was given to the pigs 21 days later. The animals of a fourth group were left as uninoculated controls. 21 days following the second inoculation with the TK- mutant all pigs were challenge infected with the virulent PRV. On post challenge day (PCD) 30 all pigs were killed and samples for virus detection and histology were taken from several organs. The inoculated TK- mutant of PRV did not induce any ill effects in the pigs except a transient febrile reaction in some animals. Virus was recovered from nasal swabbings from one pig 2 days after the first inoculation of the mutant. After challenge exposure with virulent PRV, the TK- mutant-inoculated pigs were apparently protected, whereas the control pigs all were severely affected and recovered very slowly over 3 weeks. Virus was isolated from the nasal swabbings from the TK- mutant-inoculated pigs on PCDs 2 and 4, whereas the nasal swabbings from the control piglets were all positive for virus from PCD 2 through PCD 10. DNA analysis of the virus recovered showed a pattern identical to that of the virulent PRV. Histologic lesions were found in the respiratory and the central nervous systems, however, the lesions in the TK- mutant-inoculated pigs were much milder compared to those registered for the control pigs. Virus was not isolated from any of the tissue samples that were tested, but viral DNA with sequences typical of PRV genome was detected by PCR in all samples of trigeminal ganglia from either the TK- mutant-inoculated pigs or from the controls

Transport of VSVG to the cell surface occurs normally in cells expressing P56S-VAPB inclusions.

<p><b>A:</b> HeLa-TetOff cells, induced (−Dox) or not induced (+Dox) to express <i>myc</i>-P56S-VAPB, were transfected with VSVG-EGFP at 39.3°C. After 24 h, cells were shifted to 32°C. At the indicated times, the cells were chilled and incubated with anti-lumenal domain of VSVG under non-permeabilizing conditions (red). The cells were then permeabilized and stained with anti-VAPB antibodies (blue in merge panel - see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113416#s2" target="_blank">Methods</a>). Total VSVG (intracellular+surface) was revealed by GFP fluorescence (green). Maximum intensity projections of z-stacks are shown. The acquisition parameters were the same in all images. Scale bar, 10 µm. <b>B:</b> Time course (means ± SD) of VSVG surface labeling normalized to total EGFP fluorescence. Significant differences between induced or non-induced samples were not detected by Student's t-test.</p

P56S-VAPB inclusions in a model motoneuronal cell line are degraded by the proteasome.

<p><b>A:</b> Immunofluorescence analysis of NSC34 Tet-Off cells induced to express <i>myc</i>-wt-VAPB (left) or <i>myc</i>-P56S-VAPB (right). The upper panel shows anti-<i>myc</i> immunofluorescence, the lower one the superposition of <i>myc</i> staining with phase contrast. The inset of the upper left panel shows a 2 fold enlargement of the boxed area, and illustrates the web-like distribution of wt VAPB typical of an ER protein. Scale bar: 15 µm. <b>B:</b> Degradation of P56S-VAPB stably expressed in NSC34 cells. Induced cells were supplemented with Dox; 3 h thereafter the cells were either left untreated or treated with MG132 (MG) or Bafilomycin (Baf) for 7 h. Control (Ctl) cells were grown in the presence of Dox. Equal aliquots of each sample were loaded. The lower panel shows Ponceau staining of the blotted gel region; the positions of the 25 and 37 kDa size marker are indicated. The vertical white line indicates removal of irrelevant lanes form the image. The levels of P56S-VAPB, as percentage of values in untreated cells at 3 h after Dox addition, are indicated below the lanes. p62 immunoblotting was performed to check the efficacy of bafilomycin to inhibit autophagy (upper). <b>C:</b> Confocal analysis (single sections are shown) of P56S-VAPB inclusions stained with anti-<i>myc</i> antibody (red) at 3 h after Dox addition (left) and 7 h later in the presence or absence of the indicated drugs. Nuclei were stained with DAPI. The number and size of the inclusions decreased in the absence of drugs or in the presence of Bafilomycin, but remained similar to the 3 h cells when MG132 was present. Scale bar, 10 µm.</p

Close relationship between P56S-VAPB inclusions and the Golgi Complex.

<p>Induced HeLa Tet-Off cells were doubly immunostained with anti-<i>myc</i> antibodies, to reveal P56S-VAPB, and antibodies against the Golgi proteins GM130 or giantin, as indicated. Nuclei, stained with DAPI, are shown in the merge panel. Shown are maximum intensity projections of z-stacks. Scale bars: upper row, 10 µm; middle and lower row 5 µm.</p

Transport of VSVG to the Golgi Complex occurs normally in cells expressing P56S-VAPB inclusions.

<p><b>A:</b> HeLa-TetOff cells, induced (−Dox, right) or not induced (+Dox, left) to express <i>myc</i>-P56S-VAPB, were transfected with VSVG-EGFP at 39.3°C. After 24 h, one coverslip of each sample was fixed (0 min), while the others were shifted to 32°C and fixed after incubation for the indicated times. Cells were stained with anti-Giantin (red) and anti-<i>myc</i> (blue) antibodies. Maximum intensity projections of z-stacks are shown. The cell boundaries at the 30 min time point are indicated by the white line in the merge panel. Acquisition parameters were the same in all images. Scale bar, 10 µm. <b>B:</b> Time course (means ± SD) of VSVG transport through the Golgi. Significant differences between induced or non-induced samples were not detected by Student's t-test.</p

P56S-VAPB is degraded by the proteasome and by activated, but not basal, autophagy.

<p><b>A:</b> Immunoblotting analysis of degradation of P56S-VAPB in the presence or absence of proteasome or autophagy inhibitors. 3 h after the inhibition of transcription of the P56S-VAPB transgene by addition of Dox to the media (lanes 2 and 7), cells were either left untreated (lanes 3 and 8), treated with the autophagy inhibitor Bafilomycin (Baf) or with the proteasome inhibitors MG132 (MG) or Lactacystin (Lact) for 6–7 h, as indicated. Control (Ctl) cells were grown in the presence of Dox. Equal aliquots of each sample were loaded (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113416#s2" target="_blank">Methods</a>). The lower panel shows Ponceau staining of the blotted gel region, as loading control. The vertical white line (here and in panel D) juxtaposes lanes deriving from the same blot exposure. The position of the 25 kDa size marker is indicated. <b>B:</b> Quantification (means from 2–5 experiments +SEM) of P56S-VAPB remaining at 10 h after Dox addition in the presence or absence of drugs, as indicated, compared to levels measured at 3 h *: p = 0.013 and 0.025 for MG132 and lactacystin treated samples <i>vs</i> untreated by Student's t test. respectively. The difference between 3-MA or bafilomycin-treated samples and untreated was non-significant (ns). <b>C:</b> Equal amounts of protein of the samples of lanes 3 and 4 of panel A were analyzed for p62 by immunoblotting, to control for inhibition of autophagy by bafilomycin. Actin was probed as loading control. <b>D:</b> Effect of starvation on clearance of P56S-VAPB. 3 h after addition of Dox to the media (lane 2), cells were either left untreated (lane 3), or treated with bafilomycin (Baf) or MG132 (MG), as indicated, for 6 h; the samples of lanes 6–8 were also starved during the incubation with or without the drugs. Control (Ctl) cells were cultured in presence of Dox. Ponceau staining of the blotted region is shown in the lower panel. <b>E:</b> Quantification of three experiments (means +S.E.M.) of P56S-VAPB remaining 9 h after Dox addition under the indicated conditions compared to levels measured before drug treatment and/or starvation at 3 h after Dox addition. *: p = 0.036 by Student's t test; ns, non significant.</p