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Effects of KMT2A/MLL1 and Msk1 knockdown on HoxA gene expression. a Western analysis of KMT2A/MLL1 abundance in mouse embryo fibroblasts (MEFs), either mock-transfected (Con) or KMT2A/MLL1 knockdown (KD). RNA polymerase II (Pol II) was used as a loading control and to calculate the extent of KMT2A/MLL1 knockdown (37% of control). Full-length blots are given in Additional file 1. b RT-qPCR expression analysis of a selection of KMT2 genes in knockdown cells using actin as a control and with mock-transfected cells normalised to 1.0 (n = 3, T test, *p < 0.05; **p < 0.01; ***p < 0.001). The KMT2E protein has no methyltransferase activity (see text). c Western analysis of Msk1 and Msk2 in mock-transfected (Con) and Msk1 knockdown (KD) MEFs. Actin was used as a loading control and to calculate the extent of Msk1 knockdown (1% of control). A representative western (1 out of 3) is presented. Full-length blots are given in Additional file 1. d RT-qPCR expression analysis of Msk1 and 2 in Msk1 knockdown cells. Transcripts were analysed using actin as a control, with the level of transcripts in mock-transfected cells normalised to 1.0 (n = 3, T test, *p < 0.05; **p < 0.01; ***p < 0.001). e Relative gene expression levels in control (n = 3), KMT2A/MLL1 knockdown (n = 4) and Msk1 knockdown (n = 3) cells are indicated by colour intensity from 2.7-fold down-regulated (green) to 2.7-fold up-regulated (red). 3912 genes showed significant changes in expression following KMT2A/MLL1 and/or MSK1 knockdown and these were clustered into five groups by SOTA analysis; the number of genes in each cluster is indicated. f Venn diagrams showing the numbers and relationship of genes down-regulated (upper panel) or up-regulated (lower panel), by knockdown of KMT2A/MLL1 and/or Msk1. The corresponding SOTA cluster is indicated in brackets
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| 28.1 |
We used expression analysis on high-density microarrays to identify genes whose expression was sensitive to knockdown of KMT2A/MLL1 or Msk1. Using a fold change cut-off of 2 and false discovery rate <10%, 3912 different genes changed expression in response to either KMT2A/MLL1 or Msk1 knockdown. Undirected cluster analysis (SOTA) separated the responding genes into five groups, as shown in Fig. 2e. The up- and down-regulation of selected genes was validated by RT-qPCR (Additional file 2).
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| 32 |
Strikingly, all 758 genes that were significantly down-regulated in response to the loss of KMT2A/MLL1 [cluster 3 genes] were also down-regulated in response to the loss of Msk1 (Fig. 2f, upper panel). Thus, all genes whose ongoing transcriptional activity is dependent on KMT2A/MLL1 (i.e. that are down-regulated by KMT2A KD) are also dependent on Msk1. The overlap of responding genes was much greater than expected by chance (Chi-squared test with Yates correction, two-tailed p value <0.0001), indicating a functional relationship between the two enzymes. This 100% overlap is not attributable to down-regulation of Msk1 in KMT2A/MLL1 KD cells or vice versa. Expression of KMT2A/MLL1 in Msk1 knockdown cells was not significantly altered, whereas the expression of Msk1 showed a modest up-regulation in response to the loss of KMT2A/MLL1 (Additional file 3).
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| 27.42 |
Likewise, of the genes up-regulated in response to KMT2A/MLL1 KD, a majority (448, 62%) were also up-regulated in response to Msk1 KD (Fig. 2f, lower panel). However, these overlapping, up-regulated genes represent only a minority (19%) of the 2331 genes up-regulated in total by Msk1 KD (i.e. genes whose ongoing activity is repressed by Msk1). It seems that Msk1 alone, acting either directly or indirectly through phosphorylation of regulators such as NFkB or CREB [39, 41], is frequently involved in transcriptional repression.
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study
| 28.12 |
Ontology analysis of significant genes was consistent with KMT2A/MLL1 and Msk1 activating a wide range of gene targets (Additional file 4). Genes down-regulated by KMT2A/MLL1 and Msk1 KD were modestly (1.5–1.9-fold), though significantly enriched in general terms such as glycoprotein and extracellular region activity. In contrast, genes down-regulated by KD of Msk1 alone were enriched selectively in cell cycle terms (130 out of 543 genes), with high enrichments (3.5–9.8-fold). The gene set whose activity depends on Msk1 alone differs fundamentally from that requiring both Msk1 and KMT2A/MLL1. These very different gene sets may account for the different degrees of knockdown tolerated by the cells, almost complete for Msk1 and ~40% for KMT2A/MLL1 (Fig. 2c).
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other
| 29.98 |
In order to understand better the functional relationship between KMT2A/MLL1 and Msk1, we examined their interaction at the HoxA4 and HoxA5 loci, near the centre of the cluster and amongst the most severely down-regulated genes in both knockdowns. Using ChIP-PCR with antibodies to phosphorylated Msk1, using three primer pairs spanning the gene, we found that Msk1 is normally enriched over the HoxA4 TSS (Fig. 3c, upper left panel). This enrichment is lost when KMT2A/MLL1 is knocked down (Fig. 3c, upper left panel), suggesting that Msk1 must interact with the MLL1 complex to be recruited to these sites. In contrast, whilst a peak of KMT2A/MLL1 was detected over the HoxA4 TSS, it did not change significantly upon Msk1 knockdown (Fig. 3c, upper right panel), indicating that Msk1 is not essential for the recruitment of the MLL1 complex. Exactly the same response to KMT2A/MLL1 and Msk1 KD was detected across the HoxA5 locus (Fig. 3c, lower panels). It is interesting to note that the peaks of KMT2A/MLL1 and Msk1 are located at the TSS of HoxA5, but upstream for HoxA4; so, their co-location is not simple because they both are located at TSS (Fig. 3c).
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other
| 28.34 |
To define how KMT2A/MLL1 and Msk1 interact to regulate H3 modification levels at MLL1 complex target loci, we characterised key histone modifications at HoxA4 and HoxA5 in the context of KMT2A/MLL1 and Msk1 knockdown (Fig. 4a). Both H3K4me3 and H3K9acS10ph were enriched specifically at the TSS (left and middle panels). H3K4me3 fell to baseline levels, both upon KMT2A/MLL1 knockdown as expected and also in Msk1 knockdown cells (Fig. 4a, left panels). Likewise, the TSS-associated mark H3K9acS10ph was reduced both in Msk1 knockdown cells as expected and in KMT2A/MLL1 knockdown cells (Fig. 4a, middle panels). In contrast, the abundance of the silencing modification H3K27me3, used as a control, did not change upon knockdown of either protein (Fig. 4a, right panels). These results show that the KMT2A/MLL1 complex is required for recruitment of Msk1 to the HoxA4 and HoxA5 loci, and consistent with the proposition that Msk1-catalysed H3S10 phosphorylation facilitates the KMT2A/MLL1 complexes methyltransferase activity at these TSSs. However, the peaks of H3K4me3 and H3K9acS10ph at the HoxA4 TSS (Fig. 4a) do not coincide with the peaks of KMT2A and Msk1 at the same locus (Fig. 3c). Thus, whilst KMT2A and Msk1 are clearly necessary for the deposition of these histone modifications, the amount of enzyme at particular locations is not the sole determinant of their levels.Fig. 4Effect of KMT2A/MLL1 or Msk1 knockdown on H3K4me3, H3K9acS10ph and H3K27me3 distribution across HoxA4 and HoxA5. a Effect of KMT2A/MLL1 or Msk1 knockdown on H3K4me3, H3K9acS10ph and H3K27me3 levels at HoxA4 (upper panels) and HoxA5 (lower panels). Histograms present the modification abundance at these sites as a bound/unbound ratio, where 1.0 indicates there is no enrichment (n = 1 or 2, T test, *p < 0.05; **p < 0.01). b Proposed model of how the KMT2A/MLL1 and Msk1 interaction within the KMT2A/MLL1 complex facilitates H3K4 methylation at transcription start sites (TSSs). Msk1-catalysed H3S10 phosphorylation directly enhances KMT2A/MLL1-catalysed H3K4 methylation and, potentially, improves access of the complex to condensed chromatin by reversing chromatin compaction
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clinical case
| 28.1 |
We examined the effects of KMT2A/MLL1 and Msk1 knockdown on expression of genes within the HoxA cluster, important and well-described KMT2A/MLL1 target genes. Knockdown of KMT2A/MLL1 resulted in significant down-regulation of multiple HoxA genes, particularly those nearer the centre of the cluster (Fig. 3a). Msk1 knockdown generated a pattern of HoxA gene expression remarkably similar to that in KMT2A/MLL1 knockdown cells, with genes nearer the centre of the cluster, HoxA3–6 and HoxA10, showing the greatest down-regulation (Fig. 3b).Fig. 3HoxA gene expression and the distribution of KMT2A/MLL1 and Msk1 change upon KMT2A/MLL1 or Msk1 knockdown. a RT-qPCR expression analysis of HoxA genes in KMT2A knockdown cells, compared to mock-transfected control cells normalised to 1.0 (n = 3, T test, *p < 0.05; **p < 0.01; ***p < 0.001). b RT-qPCR expression analysis of changes in expression of HoxA genes in Msk1 knockdown cells (n = 3, T test, *p < 0.05; **p < 0.01; ***p < 0.001). c Effect of KMT2A/MLL1 knockdown on Msk1 binding (upper panels) or Msk1 knockdown on KMT2A binding (lower panels) across the HoxA4 gene (left hand panels) and HoxA5 gene (right hand panels). Binding was normalised to mock-transfected cells (n = 3, T test, *p < 0.05; **p < 0.01; ***p < 0.001). Gene maps for HoxA4 and HoxA5 show exons (boxes), TSS (upper arrow) and the locations of primers 1–3 (lower bars)
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| 28.05 |
HoxA gene expression and the distribution of KMT2A/MLL1 and Msk1 change upon KMT2A/MLL1 or Msk1 knockdown. a RT-qPCR expression analysis of HoxA genes in KMT2A knockdown cells, compared to mock-transfected control cells normalised to 1.0 (n = 3, T test, *p < 0.05; **p < 0.01; ***p < 0.001). b RT-qPCR expression analysis of changes in expression of HoxA genes in Msk1 knockdown cells (n = 3, T test, *p < 0.05; **p < 0.01; ***p < 0.001). c Effect of KMT2A/MLL1 knockdown on Msk1 binding (upper panels) or Msk1 knockdown on KMT2A binding (lower panels) across the HoxA4 gene (left hand panels) and HoxA5 gene (right hand panels). Binding was normalised to mock-transfected cells (n = 3, T test, *p < 0.05; **p < 0.01; ***p < 0.001). Gene maps for HoxA4 and HoxA5 show exons (boxes), TSS (upper arrow) and the locations of primers 1–3 (lower bars)
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| 30 |
Effect of KMT2A/MLL1 or Msk1 knockdown on H3K4me3, H3K9acS10ph and H3K27me3 distribution across HoxA4 and HoxA5. a Effect of KMT2A/MLL1 or Msk1 knockdown on H3K4me3, H3K9acS10ph and H3K27me3 levels at HoxA4 (upper panels) and HoxA5 (lower panels). Histograms present the modification abundance at these sites as a bound/unbound ratio, where 1.0 indicates there is no enrichment (n = 1 or 2, T test, *p < 0.05; **p < 0.01). b Proposed model of how the KMT2A/MLL1 and Msk1 interaction within the KMT2A/MLL1 complex facilitates H3K4 methylation at transcription start sites (TSSs). Msk1-catalysed H3S10 phosphorylation directly enhances KMT2A/MLL1-catalysed H3K4 methylation and, potentially, improves access of the complex to condensed chromatin by reversing chromatin compaction
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study
| 29.25 |
The KMT2A/MLL1 is necessary for regulation of genes with key roles in differentiation and development [1, 2] [42–44]. It has multiple domains that mediate binding to both proteins and DNA, and allow assembly of the multi-protein complex that regulates gene expression. KMT2A/MLL1 has a SET domain that, within the complex, confers lysine methyltransferase activity specific for histone H3 lysine 4 (H3K4), and is essential for gene regulation [3, 19]. The various proteins that form, or associate with, the core MLL1 complex regulate both its ability to methylate H3K4 and its ability to locate and gain access to its target genes, but the mechanisms that underpin these processes are not yet understood [1, 7]. Here, we show that the kinase Msk1, part of the MAPK signalling pathway, physically interacts with the MLL1 complex, is essential for the regulation of KMT2A/MLL1 target genes and, at HoxA4 and HoxA5, that this is via regulation of H3K4 methylation.
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clinical case
| 27.27 |
H3 serines 10 and 28 are both adjacent to lysine residues whose various modifications contribute to long- and short-term control of gene expression. H3K9 acetylation is associated with active gene promoters whilst its methylation is associated with silent, condensed chromatin (including centric heterochromatin) . H3K9me-mediated chromatin condensation is brought about largely through selective binding of heterochromatin protein 1 (HP1) [49, 50]. Msk1 phosphorylation of H3S10 appears to act in this process as a ‘phospho-switch’ that can block HP1 binding to H3K9me3, and even displace pre-bound HP1 [51, 52], thereby preventing or reversing gene silencing. This has been characterised on the mouse mammary tumour virus (MMTV) promoter, where Msk1 recruitment, along with the progesterone receptor (PR) and the extracellular signal-regulated kinase (Erk), leads to phosphorylation of H3S10 at the promoter, displacement of HP1 and recruitment of RNA polymerase II and the activator protein Brg1 . We propose that Msk1 facilitates binding of the KMT2A/MLL1 complex to some target genes by H3S10 phosphorylation and consequent diminution of HP1-mediated chromatin condensation (Fig. 4b).
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study
| 27.83 |
Methylation of H3K27 is necessary for gene silencing by the Polycomb repression complex PRC2 and is put in place, and bound, by a component of the complex, the methyltransferase Ezh2 . Genes can be protected against PRC2-mediated silencing by H3K27 acetylation , and by modification of other H3 lysines [46, 57–59], but phosphorylation of H3 at serine 28, catalysed by MSK1/2, is known to displace PRC2, presumably by disrupting binding to the adjacent, methylated residue [60, 61]. This is consistent with the increase in H3K28ph abundance observed at the promoter regions of genes that are transcriptionally activated when quiescent 3T3 cells are stimulated back into growth . H3S28 phosphorylation allows Polycomb-silenced genes to be reactivated without the need to demethylate H3K27 and may play a role in allowing the KMT2A/MLL1 complex to access such genes.
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| 28.61 |
In light of these results, the association of Msk1 with the MLL1 complex confers the potential ability to reverse both HP1-mediated and Polycomb-mediated chromatin silencing, as a prelude to selective gene activation. Whatever the detailed mechanisms may prove to be, the close physical and functional association between the KMT2A/MLL1 complex and Msk1 indicates novel routes by which the complex can implement its role as a transcriptional activator and developmental regulator.
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other
| 32.75 |
The histone kinase Msk1 physically associates in vivo with the KMT2A/MLL1 complex in human and mouse cells and is essential for the regulation of key developmental target genes. This represents a direct functional link between the MAPK cell signalling pathway and KMT2A-/MLL1-mediated gene regulation. At least, part of this regulatory role is accomplished through histone modification, specifically methylation of H3K4 and phosphorylation of H3S10 and possibly H3S28.
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study
| 26.9 |
Four lines of evidence demonstrate the physical association of Msk1 with the KMT2A/MLL1 complex. First, KMT2A/MLL1 and Msk1 co-immunoprecipitate from extracts of several different cell types. Second, the two proteins co-localise across HoxA genes (known targets). Third, knockdown of Msk1 diminishes methylation of H3K4 at MLL1 complex target sites. Likewise, KMT2A/MLL1 knockdown diminishes H3S10 phosphorylation at the same sites, suggesting that, in the absence of the KMT2A/MLL1 complex, Msk1 is not appropriately targeted. This was confirmed by showing that KMT2A/MLL1 knockdown prevented Msk1 recruitment to HoxA4 and HoxA5 genes, whilst Msk1 knockdown did not disrupt KMT2A/MLL1 binding. Finally, our finding that expression of 81% of KMT2A/MLL1-regulated genes are influenced by Msk1 knockdown, shows that the functional interaction between Msk1 and the KMT2A/MLL1 complex in vivo is very frequent. Because our knockdown experiments targeting Msk1 left its close homologue Msk2 unaffected, we conclude that the effects we observe are mediated by Msk1 alone.
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| 28.56 |
Msk1 is known to phosphorylate histone H3 at serines 10 and 28, as well as key regulatory proteins such as cAMP response element binding (CREB) and NFκB . All of these substrates can, potentially, alter gene expression. The choice of which substrate, or even which lysine, to phosphorylate is likely to be context dependent [45, 46]. The catalytic properties of Msk1, previous data and the present results suggest that the enzyme regulates the KMT2A/MLL1 complex in one or both of two general ways, namely by enhancing SET domain-catalysed H3K4 methylation and/or by allowing the complex to access condensed chromatin. We have shown previously that SET domain methyltransferase activity is enhanced by the acetylation and phosphorylation of the H3 substrate at lysines 9 and 14 and serine 10, respectively . The most effective catalytic improvement was provided by the fully modified substrate (H3K9acS10phK14ac) . We have now shown that recruitment of Msk1 to KMT2A/MLL1 responsive genes and consequent increases in H3S10 phosphorylation leads to increased H3K4 methylation and up-regulation of transcription. In light of these data, we propose that (in addition to its other activities) Msk1-mediated phosphorylation of H3S10 directly stimulates the ability of the KMT2A/MLL1 SET domain to methylate H3K4 in vivo. It may be that the recruitment of the lysine acetyltransferase CBP by the KMT2A/MLL1 complex also contributes to enhancement of H3K4 methylation through enhanced acetylation of H3K9 and K14. This model is shown in Fig. 4b.
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clinical case
| 27.05 |
Human lymphoblastoid cells (LCLs) with a normal karyotype were generated by M. Rowe (Uni. Birmingham) and grown in RPMI 1640, 100 µg/ml streptomycin, 100 U/ml penicillin and 10% FBS (Foetal bovine serum, Invitrogen) at 37 °C, 5% CO2. Human embryonic kidney cells (HEKs) were cultured in DMEM, 100 µg/ml streptomycin, 100 U/ml penicillin and 10% FBS at 37 °C, 5% CO2. Mouse embryonic fibroblasts (MEFs) were generated in-house, via isolation from Balb/c wild-type mouse embryos between day 12 and 15 of gestation, and cultured in DMEM, 100 µg/ml streptomycin, 100 U/ml penicillin, 1/100 MEM non-essential amino acids, 1/1000 2-mercaptoethanol and 10% FBS at 37 °C, 5% CO2. Only cells between passage 2 and 4 were used. Drosophila SL2 (Schneider line-2) cells were cultured in Schneider’s medium supplemented with 8% FBS and 1/100 penicillin/streptomycin, and incubated at 26 °C.
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| 30.28 |
RNA was isolated using RNeasy Minikit (Quaigen), and reverse transcription/cDNA amplification performed in one step using QuantiTech™ Sybr® Green Mix (Qiagen), according to the manufacturer’s instructions. Reactions used commercial primers (Qiagen: Msk1 QT00141554, actin QT01336772, HoxA1 QT00248322, HoxA2 QT0062812, HoxA3 QT00138264, HoxA4 QT00174986, HoxA5 QT00098819, HoxA6 QT00140742, HoxA7 QT00168707, HoxA9 QT00108885, HoxA10 QT00240212, HoxA11 QT00250404, HoxA13 QT0027642, MLL1 QT00240954, MLL2 QT01075620, MLL3 QT00310527, MLL5 QT00279426), with three analyses performed for each sample. RT-qPCR was performed on a 7900 HT machine (Applied Biosystems).
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| 33.5 |
Cells were washed and incubated in lysis buffer [20 mM Tris–HCl pH 7.4, 100 mM EDTA, 10 mM NaCl, 1% Triton X-100, 1 mM β-glycerophosphate, 1 mM EGTA, 5 mM sodium pyrophosphate, ‘Complete’ protease inhibitor (Roche)] for 30 min on ice. Insoluble material was removed by centrifugation and protein concentration determined by Coomassie Plus protein assay (Thermo Scientific). Proteins were separated by gel electrophoresis and stained with silver nitrate or blotted for western analysis. Primary antibody binding was detected by fluorescently tagged secondary antibodies (Licor) and gel loading normalised using anti-actin or anti-Pol II antibodies (Abcam).
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| 33.1 |
Antibody for H3K4me3 (R612) was generated in-house [63, 64]. Other antibodies were obtained commercially: H3K9acS10p [Abcam ab12181]; H3K27me3 [Millipore 07-449]; phosphorylated Msk1, this antibody is specific for Msk1 phosphorylated at S360, one of the last residues to become phosphorylated and detects active Msk1 [Abcam ab81294]; KMT2A [Millipore 05-764]; Msk2 [R&D systems MAB2310]; actin [Abcam ab1801]; RNA Pol II [Abcam ab5408]; NFκB [Abcam ab7970-1]; FLAG [Sigma F1804].
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| 28.3 |
Immunoprecipitation of formaldehyde cross-linked chromatin (X-ChIP) in MEFs was carried out essentially as described [67, 68]. Briefly, MEFs were cross-linked with 1% para-formaldehyde in medium for 10 min at RT. The reaction was quenched by the addition of 200 mM glycine before washing the cells twice with ice-cold PBS. Cells were resuspended in lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8, complete inhibitor cocktail(Roche)] to a concentration of 105 cells in 500 μl and sonicated at high power with 30 s ON/OFF for 7 cycles (Bioruptor, Diagenode). The chromatin was diluted 1:2 with dilution buffer [1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8, 150 mM NaCl, complete inhibitor cocktail (Roche)]. Protein G Dynabeads (Thermo Fischer) were prepared resuspended in citrate–phosphate buffer (0.1 M citric acid, 0.1 M Na2HPO4) with 0.5% BSA. Beads were coated with antibodies for 2 h at 4 °C. The beads were washed twice, added to the chromatin solution and incubated for 4 h at 4 °C. The beads were washed twice with low-salt buffer (1% Triton X-100, 0.1% SDS, 2 mM EDTA, 150 mM NaCl, 20 mM Tris–HCl, pH 8) and twice with high-salt buffer (1% Triton X-100, 0.1% SDS, 2 mM EDTA, 100 mM NaCl, 20 mM Tris–HCl, pH 8). The bound material was eluted with Elution buffer (100 mM NaHCO3, 1% SDS decross-linked, and the DNA extracted with QIAquick PCR purification kit (QIAGEN). Primer pairs used for analysing ChIP DNA by qPCR are listed in Additional file 5.
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| 36.1 |
Co-immunoprecipitation experiments were carried out as described before, using 1 × 108 cells per precipitation and in the presence of 50 µg/ml ethidium bromide, in order to release chromatin bound proteins from DNA [69–72]. Cells were lysed in NP40 buffer [1% NP40, 10% glycerol, 50 mM Tris pH 7.5, 0.1% sodium azide, 150 mM NaCl, complete proteinase inhibitor (Roche)] for 20 min on ice before sonication (high power, 10 s, Bioruptor Diagenode). The lysate was cleared by centrifugation (10 min, 17000g, 4 °C) and pre-cleaned by adding protein A Sepharose beads for 30 min at 4 °C. The cleaned lysate was incubated overnight with 15 µg of antibody at 4 °C on a rotating wheel and then with 40 µl beads for 3 h at RT. The beads were washed three times with high-salt buffer (1% NP40, 10% glycerol, 50 mM Tris pH 7.5, 0.1% sodium azide, 200 mM NaCl). The beads were analysed on a SDS gel and western analysis, together with a no-antibody control and 10% input samples.
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other
| 34 |
HEK293 cells were split the day before transfection and 4 × 106 cells seeded into 10 cm plates in complete medium. The next day, a mix of 15 μg vector DNA (pMSCV-FLAG-MLL1, R. Slany, University Erlangen), 1000 μl OptiMEM (Gibco) and 60 μl of polyethyleneimine (1 mg/ml, Polyscience Inc.) per plate was incubated for 15 min at RT prior to addition to the plates. The cells were cultured at 37 °C, 5% CO2 for 3 days and harvested, washed with TBS and resuspended in lysis buffer (1× TBS, 1 mM EDTA, 1% TritonX-100). Cells were incubated on ice for 30 min prior to centrifuging (15 min, 13,000 rpm, 4 °C), addition of 50 μl anti-FLAG M2 magnetic beads (Sigma) and incubation for 2 h at RT with rotation. Beads were washed with TBS and FLAG-MLL1 was eluted using 5 μg of 3× FLAG peptide (Sigma) and diluted in 500 μl TBS. The beads were incubated twice with 250 μl elution for 30 min, 4 °C with rotation and the supernatants pooled.
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other
| 29.16 |
shRNA vectors were purchased from Origene (Msk1: TR504795; MLL1: TR517798) and transfected into MEFs by electroporation (GenePulser XCell, Bio-Rad). MEFs were brought into a single cell suspension by trypsination and washed with ice-cold PBS. 4 × 106 cells per transfection were resuspended in 400 μl electroporation buffer (10 mM Hepes, pH 7.5, 135 mM KCl, 2 mM MgCl2, 5 mM EGTA, 25% FBS) and transferred to a 4 mm electroporation cuvette (Bio-Rad). 20 μg of vector DNA was added to the cells immediately before transfection and incubated on ice for 1 min. Cells were transfected with one pulse (300 V, 600 μF, 1000 Ω) and allowed to recover (RT, 1 min), before addition of 10 ml pre-warmed MEF medium and incubation at 37 °C, 5% CO2 for 6 h. Non-transfected cells were removed by puromycin treatment (10 μg/ml, 14 h). After 20 h, cells were harvested. The KD efficiency is determined by quantitation of Wester blots using the corresponding loading controls for normalisation.
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other
| 36.03 |
Expression analysis was performed on MEFs transfected with knockdown shRNA vectors, using NimbleGen arrays (100718_MM9_EXP_HX12) containing 12 individual 135 k arrays with 3 probes per sequence for 42,576 sequences. 10 μg of RNA per array was isolated (RNeasy, Qiagen) and reverse transcribed using the cDNA synthesis system (NimbleGen). cDNA samples were labelled with the NimbleGen One-Colour DNA labelling kit and subsequently hybridised on the array, washed and scanned with the MS 200 Microarray Scanner (NimbleGen). Four replicate transfections were carried out for each knockdown and control but one control and one MSK1 KD replicate were eliminated as outliers. Data were extracted in DEVA (Nimblegen), processed with R (background correction and normalisation) and analysed with MeV (clustering, statistical analysis and visualisation). Statistical significance of co-regulated genes was analysed by 2 × 2 contingency tables and Chi-squared analysis with Yates correction (GraphPad, www.graphpad.com). Microarray data have been deposited in GEO (www.ncbi.nlm.nih.gov/geo), accession number GSE89141.
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other
| 33.12 |
Influenza A virus is a very common respiratory virus causing 3–5 million cases of severe disease each year worldwide1. Symptoms include fever, muscle pain, sore throat and coughing. Most people recover from the infection but between 250.000–500.000 humans succumb to the disease annually1. Existing antibody based vaccines target the surface proteins HA and NA, which is problematic due to their high degree of antigenic variation2. Point mutations (antigenic drift) and genetic reassortment (antigenic shift) change these surface glycoproteins, so that existing antibodies rapidly become of limited protective value. Due to this, ongoing global surveillance and typically the production of a new vaccine combination is required for each winter season. Not only is this an expensive process, but if the predictions turn out to be wrong, then the consequences may be devastating. Further, this vaccine strategy does not work in relation to new pandemic strains, since the antigenic composition is stochastic and unpredictable. Consequently, there is a need for the development of a new vaccine strategy.
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study
| 31 |
In contrast to antibody mediated immunity, CD8+ T-cell immunity also targets the conserved internal proteins of influenza. Several studies, in both mice and man, have identified CD8+ T cells to be of major importance for clearance of influenza virus infections, and CD8+ memory T cells can remain for at least a couple of years after the infection is cleared34567. As CD8+ T cells target infected cells, not cell-free virus, T cells from influenza primed mice do not prevent infection but causes earlier clearance from the lungs and hence protects the mice from otherwise lethal disease8. T-cell mediated immunity can therefore provide a basic protection against influenza A, important in certain times of need, such as during a pandemic, when the majority of the population lacks antibody mediated protection. If we can induce long lasting CD8+ T-cell immunity, the population would also be protected from severe disease and the deadly consequences of seasonal flu. We have previously described a replication-deficient adenovirus vector encoding the influenza A nucleoprotein (AdNP) with the capacity to induce a long-standing CD8+ memory T-cell response against influenza infection9. In the same study, we also demonstrated that the protection, induced by the vaccine, is exerted predominantly by CD8+ T cells. Adenovirus serotype 5 used in this study has been shown to induce high quality CD8+ memory T-cell when used as a vaccine vector10. The fact that adenovirus naturally infects the airways and therefore may induce homing of immune cells to the lungs also speaks in its favor for usage as a vector in an influenza vaccine. Several studies have used adenovirus vectors encoding influenza genes HA, M1 or NP to vaccinate against influenza with varying success111213. Even though the internal proteins are highly conserved between influenza A strains, variations do exist which could cause the virus to escape CD8+ T-cell mediated immunity. To circumvent this problem, we propose a vaccine-cocktail, targeting several of the internal proteins of the influenza virus, creating a vaccine able to protect against challenge with a broad variety of strains. PB1 is highly conserved8 and has previously been sparsely studied as a vaccine target. A DNA vaccine expressing PB1 has shown promise of protective capacity. However, immunization had to be performed trice to boost the immune response, and mice were only challenged with low doses14. One limitation in this context could be a low intrinsic immunogenicity of PB1. However, we have recently found that CD8+ T-cell responses directed towards weak antigens may be markedly augmented by expressing the antigen linked to the invariant chain (Ii); thus tethering of the antigen to Ii increases the number of epitope/MHC class I complexes on the cell surface and probably through this mechanism accelerates, augments and prolongs the antigen-specific CD8+ T-cell responses particularly against epitopes of low/intermediate immunogenicity1516. The aim of this study is therefore to investigate the protective capacities of a replication deficient adenovirus encoding PB1 with and without Ii chain (AdIiPB1 and AdPB1, respectively). H-2b mice vaccinated both locally and systemically with AdIiPB1 were significantly protected up to 60 days post vaccination compared to non-vaccinated mice. However, despite high numbers of virus-specific CD8+ T cells in the circulation, the protection never matched that following AdNP vaccination. Part of the reason for this appears to be a less stable surface expression of the major PB1 epitope (PB1703), leading to a reduced capacity of PB1-specific cells to kill target cells expressing this epitope compared to the killing of NP366 expressing cells by NP366-specific CD8+ T cells. Consequently, a vaccine targeting PB1 is not sufficient as a stand-alone vaccine, even when tethered to Ii, but PB1 may be included in a combination with other internal genes of influenza.
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study
| 28.1 |
Adenovirus vaccines encoding PB1 with or without invariant chain (AdPB1 or AdIiPB1) were used to vaccinate mice s.c. in the foot pad; an adenovector expressing invariant chain combined with an irrelevant antigen (the glycoprotein of lymphocytic choriomeningitis virus, AdIiGP) was used as control. Fourteen days after vaccination, and following ex vivo stimulation with PB1703 peptide, numbers of antigen-specific, IFN-γ+ CD8+ T cells from the spleen were enumerated through intracellular cytokine staining and flow cytometry. A significantly higher numbers of PB1-specific CD8+ T cells were detected in animals vaccinated with the construct expressing PB1 tethered to invariant chain, AdIiPB1, compared to the untethered construct (Fig. 1A+B). Very few PB1-specific CD8+ T cells were detected in mice vaccinated with AdPB1, and, notably, tethering to invariant chain did not influence the antigen-specificity of the elicited CD8+ T-cell response. In mice vaccinated with AdIiPB1, the response peaked between day 14 and 17 after vaccination (Fig. 2A), and decreased until day 20, where after the number of cells remained stable up to 30 days post vaccination. Despite the high numbers of PB1 specific cells elicited in AdIiPB1 vaccinated mice, we found little or no protection when vaccinated mice were challenged after 30 days with homologous influenza virus PR8, neither 3 nor 5 days post challenge (Fig. 2B).
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| 29.56 |
Previous studies have shown that i.n. vaccination increases the number of antigen specific CD8+ T cells in the lungs and can therefore provide better protection against a respiratory infection17. Even though we observed no protection from s.c. vaccination with neither AdIiPB1 nor AdPB1, we hypothesized that due to the high number of antigen specific CD8+ T cells in the spleen induced by AdIiPB1, AdIiPB1 could still induce significant protection if the primed cells were effectively recruited to the lungs. To test this theory, C57BL/6 mice were vaccinated systemically (s.c.), locally (i.n.) or through both routes (s.c. + i.n.) and then challenged with PR8 virus 30 days later. Viral titers were measured in the lungs 3 and 5 days post challenge.
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| 34.38 |
Mice that were vaccinated both locally and systemically tended to have lower viral titers in their lungs compared to unvaccinated controls on day 3 post infection, and on day 5 after challenge a significant reduction was found (Fig. 3A). Unlike most experiments (cf. Fig. 2B), even s.c. vaccinated mice had significantly reduced viral titers on day 5. Mice vaccinated exclusively by the i.n. route were the only mice to have significantly lower lung virus levels compared to unvaccinated mice on day 3 after challenge, and, notably, this difference disappeared when similar mice were analyzed 2 days later; this pattern suggests that the effect of local vaccination per se wanes with time after challenge.
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other
| 32.75 |
Parallel to the measurements of viral loads, the infection-induced weight loss was recorded after challenge and until the animals were sacrificed (Fig. 3B). Mice vaccinated through both routes displayed less morbidity 3–5 days post challenge compared to mice vaccinated s.c. or unvaccinated mice. Even though the protection of mice vaccinated only i.n. seemed to fade with time, these mice were still clinically protected, displaying a weight loss on days 3–5 post challenge marginally bigger than that of mice vaccinated both locally and systemically. Nevertheless, if both viral loads and weight loss are considered, the overall impression was that combined vaccination provided the most consistent protection.
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| 33.16 |
Knowing that combined local and systemic vaccination could reduce the viral titer in challenged mice, we wanted to investigate how the different vaccination routes impacted the locations of CD8+ T cells following viral challenge. C57BL/6 mice were vaccinated i.n., s.c, or s.c and i.n. with AdIiPB1. 17 (Fig. 4A) and 30 days (Fig. 4B) after vaccination, PB1-specific IFNγ+ CD8+ T cells in the spleen and MLNs were enumerated after peptide stimulation in half of the mice through intracellular staining and flow cytometry. The other half was challenged with PR8 virus and PB1-specific CD8+ T cells from the spleen, MLNs and BAL were enumerated after peptide stimulation through flow cytometry 5 days post challenge. As seen in Fig. 3, high numbers of IFNγ+ CD8+ T cells were detected in the spleen before challenge of mice vaccinated s.c. + i.n. or s.c., and these numbers were markedly reduced 5 days after challenge. In contrast, the number of PB1-specific cells in the MLN increased in all groups post challenge. There was no difference in the number of cells in the MLN between the groups 5 days post challenge. This was found both for mice challenged 17 as well as 30 days post vaccination. Notably, numbers of IFNγ+ CD8+ T cells in the BAL 5 days post challenge were consistently higher in the mice vaccinated via both routes compared to a single route.
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| 30 |
Next, we wanted to examine if the capacity of AdIiPB1 to induce protective CD8+ T cells lasted into the memory phase. To this end, C57BL/6 mice were vaccinated s.c., i.n. or both s.c. and i.n. and challenged with PR8 virus 60 days later. Analysis of viral titers in the lungs 5 days post challenge revealed a small, but statistically significant difference between several of the vaccinated groups and unvaccinated controls (Fig. 5B), and the lowest titers were found in mice subjected to the combined vaccination. This pattern was repeated 7 days post challenge. Regarding the virus-induced morbidity, we again followed the weight loss. Similar to what was found on day 30 post vaccination, mice vaccinated both systemically and locally lost significantly less weight compared to mice vaccinated s.c. or non-vaccinated mice (Fig. 5C). Analysis of CD8+ T cells isolated from the spleen, MLN and BAL (Fig. 5A) showed similar patterns as on day 17 and 30 (Fig. 4A,B) post vaccination and are also present in almost comparable numbers indicating a good recall response.
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| 30.39 |
To investigate the morbidity and mortality after challenge of mice vaccinated with AdIiPB1 both i.n. as well as s.c., groups of mice were followed until 21 days after challenge. C57BL/6 mice were vaccinated s.c., i.n. or by both routes, and 60 days later all mice were challenged with a lethal dose of PR8. Mice were weighed daily (Fig. 6A) and survival (Fig. 6B) was recorded. Neither the mice in the control group nor the mice vaccinated s.c. survived past day 8. 25% of the mice vaccinated both s.c. and i.n. survived the infection and displayed no weight loss 21 days post challenge. In the group of mice vaccinated only i.n., a very small proportion survived, and these mice had not regained as much weight 21 days post challenge as had the mice subjected to combined vaccination.
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| 32.7 |
The antiviral protection presented above (and associated with PB1vaccination) appear not to match that previously published for NP vaccination9, suggesting that the induced PB1 specific CD8+ T-cell response provide markedly inferior protection compared to that induced by NP vaccination. To confirm this impression, groups of mice were vaccinated i.n. plus s.c. with one of the following adenovector constructs: AdIiPB1, AdNP or AdIiGP for control. Thirty days later these mice and a group of naïve controls were challenged i.n. with PR8 and 5 days later virus titers in the lungs were determined. As can be seen in Fig. 7, NP vaccination provided substantially better protection than vaccination against PB1, which still induced significant protection compared to naïve controls. Sham vaccination with an irrelevant vector did not induce any protection compared to naïve controls.
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| 33.3 |
As the first step in this analysis, we tested if the observed difference should reflect an intrinsic difference in the immunogenicity of the involved vaccines. To this end, we performed a dose/response experiment where groups of mice were vaccinated in the s.c. with one of the doses, 106, 107 and 108 PFU, of one of the following adenovectors: AdPB1, AdIiPB1, AdNP and AdIiNP. Fourteen days later numbers of antigen-specific CD8 T cells in the spleen were enumerated. We found (Fig. 8A) that over a 100-fold dose range, three of the constructs (AdIiPB1, AdNP and AdIiNP) consistently induced substantial and almost equal numbers of antigen-specific CD8+ effector T cells (between 1–3 × 106 antigen-specific cells), while even the highest dose of the fourth constructs (AdPB1) elicited very few cells (<105/spleen). Besides underscoring the strong immunogenicity of the AdIiPB1 vector, these results confirmed that NP396-specific cells could be very effectively elicited even without tethering to Ii, whereas a PB1703-specific response of the same magnitude critically required tethering of this protein to Ii.
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| 32.53 |
Interestingly, PB1-specific T cells do not seem to be nearly as efficient in controlling influenza replication as NP-specific cells. There may be several reasons for that. First, vaccine-driven NP-specific CD8+ T cells tend to accumulate in MLN and BAL to a higher degree than similarly stimulated PB1-specific T cells, at least early in the memory phase. The underlying mechanism for this is not clear, but following primary influenza infection the expansion of NP-specific cells is prolonged and contraction delayed. This is due to continued presentation of NP by a subset of lymph node DCs that are CD103−CD11bhigh and this programs these T cells for a robust recall response22. If antigen presentation is similarly biased after adenovector immunization, which is known to cause sustained local antigen presentation23, this may lead to a preferential accumulation of NP-specific cells in the airways compared to PB1-specific cells. Given that a more diverse range of APCs present NP24 local accumulation is very likely to increase further following flu challenge. This difference and in particular migratory CD103+ DCs have been found to induce an early and efficient presentation of NP in the MLN that favors the expansion of CD8+ T cells with this specificity above others25. Additionally, we find that under similar conditions, PB1-specific T cells are less efficient in recognizing and eliminating their relevant target cells in vivo, probably because of a lower stability of the targeted peptide/MHC combination. Since low peptide/MHC complex stability will tend to reduce epitope expression also on infected target cells, this, combined with the reduced numbers of PB1-specific CD8+ T cells present locally, might explain why PB1-specific cells provide less efficient protection compared to NP-specific CD8+ T cells.
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study
| 29.81 |
What are the implications for human vaccination against influenza A? First, our results underscore that a potent systemic response does not guarantee efficient protection; not surprising localization, but also fine specificity of the involved cells matter more. Second, even though our results are based on analysis of only two dominant epitopes in a single inbred mouse strain, these and other results2224 suggest that relevant local expression of PB1 in the lungs is limited compared to that of NP. If this observation can be extrapolated to humans, local accumulation as well as the efficiency of PB1-specific effector CD8+ T cells would be less substantial also in human patients.
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other
| 32.75 |
To further study the difference between PB1 and NP vaccination, we directly compared the numbers of PB1703 and NP366 -specific CD8+ T cells found in different organs (spleen, MLN and BAL) 30 days after s.c + i.n. vaccination with either AdIiPB1 or AdNP. Interestingly, in this situation AdIiPB1 vaccinated mice had more antigen-specific cells in the spleen compared to NP vaccinated mice, whereas similar numbers were found in MLNs, and more antigen-specific cells were recovered from the BAL of AdNP vaccinated animals (Fig. 8B). Thus, NP-specific cells seemed to accumulate near the airways to a higher degree than PB1- specific cells.
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other
| 32.97 |
Next, we made a head-to-head comparison of the quality of adenovirus vector elicited PB1703- and NP366-specific CD8+ T cells using several parameters as end-points for our analysis. To evaluate effector cell status/functionality, PB1- and NP-specific CD8+ T cells were optimally stimulated with peptide for 5 hours, and the fraction of IFNγ producing cells also degranulating or co-producing TNF-α or IL-2 was determined. As can be seen in Fig. 9A, a similar composition regarding functional subsets was observed irrespective of the antigenic specificity of the CD8+ T cells.
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other
| 31.28 |
Furthermore, we wanted to compare the capacity of PB1- and NP-specific cells to recognize their relevant targets in vivo. As a functional parameter to evaluate T-cell recognition in vivo, we performed standard in vivo cytotoxicity assays in which the cytolytic capacity is determined by the ability to find and kill injected peptide loaded spleen cells18. Thirty days after vaccination with AdIiPB1 or AdNP, these mice and naïve controls received a 1:1 mixed population of spleen cells loaded with PB1703 or NP366 under similar conditions. Sixteen hours after cell transfer, the fraction of injected cells recovered in the host spleens was determined by flow cytometry. As can be seen in Fig. 9B, levels of in vivo killing did not match the frequencies of relevant antigen-specific CD8+ T cells in each type of host, as a significantly lower killing of the injected cells was observed in AdIiPB1 vaccinated mice. Since the frequency of relevant effector cells in spleen is higher in AdIiPB1 vaccinated mice compared to AdNP vaccinated mice, and the quality of the effector cells apparently does not differ, this result suggests that there is reduced in vivo recognition of the relevant target cells in AdIiPB1 vaccinated mice.
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other
| 33.78 |
Seeking an explanation to this conundrum, we compared the functional avidity of PB1 and NP specific CD8+ T cells, i.e. the ability of these antigen specific cells to sense peptide presentation. To this end, we determined the relative frequencies of IFN-γ producing CD8+ T cells induced by ex vivo incubation in the presence of decreasing concentrations of either peptide. Interestingly, the threshold for activation of the PB1703- and NP366-specific CD8+ T-cell subset was similar under these conditions (data not shown). Therefore, in order to be able to explain the results of the in vivo cytotoxic analysis, we hypothesized that the peptide/MHC class I complexes were less stable with the PB1 peptide and that with time, this would significantly limit the triggering of PB1-specific CD8+ T cells, thus explaining the observed difference in a long-term assay like the in vivo cytotoxic test. To evaluate this possibility, an experiment similar to that just described above was set up with the notable difference that instead of peptide in solution, washed naïve splenocytes loaded with decreasing concentrations of either peptide were used for stimulation of IFNγ production. Either immediately or 6 hours later, splenocytes from vaccinated mice were added and intracellular staining was performed after 5 hours of co-incubation according to standard procedures. A comparison of the results obtained under these conditions (Fig. 9C) revealed a clear difference in the presentation of the two peptides, as PB1703-specific CD8+ T cells required a much higher loading concentration of peptide to be fully stimulated when the addition of responder T cells was delayed. From this, we conclude that the functional avidities of PB1703 and NP366-specific CD8+ T cells are very similar, but the PB1703 peptide is less stably expressed on the cell surface than the NP366 peptide.
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other
| 34.56 |
Thus, the protection from AdIiPB1 vaccination is not complete. However, compared to real life human infection, the dose of influenza used for challenge in our animal model is probably much higher than what humans are normally exposed to. Furthermore, PB1 induced immunity may matter, if immunization against this antigen is combined with other adenovirus encoded flu antigens. Adding PB1 will then increase the breadth of the induced T-cell response and might contribute significantly to protection if some of the other responses fail due to mutations in the involved epitopes. Hence, PB1 could still represent a relevant vaccine target to consider in a future flu vaccine.
|
study
| 29.44 |
Female C57BL/6 mice (H-2b), 6–8 weeks old, were obtained from Taconic Farms (Ry, Denmark) and housed in a specific pathogen–free facility. Upon arrival, all mice were allowed to acclimatize for ≥1 week at the facility before being used in experiments. Experiments were conducted in accordance with national Danish guidelines (Amendment # 1306 of November 23, 2007) regarding animal experiments as approved by the Danish Animal Experiments Inspectorate, Ministry of Justice, permission numbers 2015-15-0201-00623, and the mice were housed in an AAALAC accredited facility in accordance with good animal practice as defined by FELASA.
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| 35.53 |
Influenza remains one of the most widespread human infections despite annual vaccination programs. The virus continuously changes through antigenic drift and shift creating new seasonal and pandemic strains. As previously mentioned, the current vaccines are unable to provide heterosubtypic protection and there is a need for development of a new vaccine strategy against influenza A infections. This was especially highlighted in the recent H1N1 2009 pandemic as well as in the increased number of cases and deaths of the highly pathogenic avian influenza in humans, H5N1, in the last year19. Here, we have investigated an adenovirus encoding the internal influenza protein PB1 as a potential vaccine candidate. PB1 is highly conserved and expressed by all influenza A viruses making it a good target for a broad protection8. AdPB1 does not at first glance seem to show much promise, but by adding invariant chain to the antigen, we observe a very marked increase in the number of PB1-specific CD8+ T cells induced. These results confirm previous studies, where the addition of invariant chain has been found to increase CD8+ T cell responses. This is especially true for weaker antigens containing subdominant epitopes1520. The mechanism behind this phenomenon is, however, not yet fully elucidated as Ii normally is involved in MHC II presentation, but this does not appear to play a role in this case.
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| 29.56 |
Even though AdIiPB1 could induce a high number of PB1-specific CD8+ T cells, we did not detect significant protection when AdIiPB1 was used in s.c. vaccination. In our previous study with AdNP, we saw an increase in the number of CD8+ T cells in the lungs and MLN when vaccinating both s.c. and i.n. As with AdNP, combined local and systemic vaccination with AdIiPB1 resulted in higher numbers of CD8+ T cells in the MLN and BAL, and this correlated with a reduction in viral titers in the lungs 5 and 7 days post infection. Mice vaccinated only i.n. had higher mortality, lost more weight and eventually had slightly higher viral titers compared to mice vaccinated both i.n. and s.c. Based on these findings and on our previous experience with AdNP vaccination, we speculate that a good systemic priming of T cells is important for a more prolonged T-cell response, which explain why these mice are the last to lose weight. A similar mechanism has been discussed concerning tuberculosis infection, where a combination of i.n. and s.c. immunization gives the benefit of both a good early and a late response21.
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| 30.95 |
A replication-deficient E1-deleted Adenovirus serotype 5 vector with a nonfunctional E3 gene, expressing the PB1 gene from influenza strain A/Puerto Rico/8/34 unlinked or linked to invariant chain (designated AdPB1 or AdliPB1) was produced as described previously26. Adenoviral particles were purified using standard methods, aliquoted, and frozen at −80 °C in 10% glycerol. Insert was verified by sequencing (data not shown). Infectivity of the adenovirus stocks was determined using Adeno-X Rapid Titer Kit (Clontech Laboratories, Mountain View, CA). The production of other adenovectors used here has previously been described9.
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other
| 30.28 |
Vaccination was given subcutaneous (s.c.) and/or intranasal (i.n.). Mice s.c. vaccinated were briefly anesthetized with isoflurane and injected in the right foot pad with 2 × 107 particle forming units (PFU) in 30 μl of PBS. Mice vaccinated i.n were first anaesthetized by intraperitoneal (i.p.) injection with avertin (2,2,2 tribromoethanol in 2-methyl-2-butanol, 250 mg/kg) and then vaccinated with 2 × 107 PFU in 30 μl of PBS in the nostrils.
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| 34.6 |
All antibodies were purchased from Nordic Biosite. The following flourochrome-conjugated monoclonal rat anti-mouse antibodies were used for surface and intracellular cytokine staining: PerCP-Cy5.5 conjugated α-CD8 (clone 53-6.7), FITC conjugated α-CD44 (clone IM7), APC conjugated α-IFNγ (clone XMG1.2), APC/Cy7 conjugated CD44 (clone IM7), PE conjugated IL-2 (clone JES6-5H4), PE/Cy7 conjugated TNFα (clone MP6-XY22) and AlexaFlour488 conjugated CD107a (clone 1D4B).
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| 29.47 |
Lungs were homogenized using sterilized sand and a mortar and pistil, 1% FBS in PBS was added to obtain a 10% weight/volume suspension. Samples were spun down at 600 G for 15 min at 4 °C, and the supernatant was transferred to a new tube and kept on ice until use.
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| 35.8 |
Challenge was performed with diluted A/Puerto Rico/8/34 (PR8) influenza virus. The lethal dose was determined, and 1–3 LD50 were used for all challenge. Mice to be challenged were first anaesthetized by i.p. injection with avertin (2,2,2 tribromoethanol in 2-methyl-2-butanol, 250 mg/kg) and subsequently infected i.n. with 30 μl of appropriately diluted influenza virus. After influenza infection, animals were weighed daily and euthanized by cervical dislocation if the weight loss exceeded 25% or the experiment was terminated 21 days post challenge.
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other
| 38.12 |
To obtain single cells from spleens and mediastinal lymph node (MLN), these organs were aseptically isolated and pressed through a fine steel mesh. For brochoalveolar lavage (BAL) sampling, mice were first anaesthetized using avertin and exsanguinated in order to reduce the risk of lymphocyte contamination from the blood. Next, the trachea was exposed and a small incision was made. A venflon was inserted into the incision and the lungs were flushed 3 times with ice-cold Hanks BSS medium. BAL samples were pooled within groups in order to obtain enough cells for analysis. All samples were centrifuged and re-suspended in Hanks BSS. Cells were then counted on an automated cell counter, Countess (Invitrogen). This was followed by centrifugation and resuspension in RPMI 1640 cell culture medium containing 10% FCS supplemented with 2-ME, l-glutamine, and penicillin-streptomycin. For enumeration of Ag-specific T cells, splenocytes were incubated at 37 °C and 5% CO2 for 5 h in the presence of 1 μg/ml PB1703 peptide (SSYRRPVGI) or NP366 peptide (ASNENMETM), 50 IU/ml IL-2, and 3 μM monensin; cells incubated without peptide or with irrelevant peptide was included for control. Cells were then stained for surface markers. Subsequent to surface staining, the cells were washed, permeabilized, and stained for intracellular cytokines10. Samples were analyzed using a LSR II (BD Biosciences), and data analysis was conducted using FlowJo v7.6.5 software (TreeStar); see Supplementary Figure 1 for our gating strategy.
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other
| 35.1 |
Madin-Darby Canine Kidney epithelial (MDCK) cells were used for influenza plaque assay and grown in complete medium. 4.5 × 104 MDCK cells in 100 μl medium were grown in 96-well plates overnight. For the plaque assay, 10-fold dilutions of the lung suspensions were prepared using an influenza growth medium containing DMEM 1965 medium with 2 mM L-glutamine, 200 IU/ml penicillin, 50 μg/ml streptomycin, 0.2% BSA, 1% sodium-pyruvate and 5 units/ml TPCK Trypsin. MDCK-cells were first washed twice with PBS and then incubated with 50 μl of virus dilution for 2 hour at 37 °C, 5% CO2. Samples were then removed, and an overlay medium containing 2x minimum essential medium (MEM) eagle supplemented with 0.4% BSA, 10% NaHCO3, 2% Streptomycin, 2% penicillin and 5 units/ml TPCK trypsin mixed 1:1 with 1.8% methylcellulose was added to the cells. Cells were incubated for 48 hour at 37 °C, 5% CO2. Then overlay was removed and wells were washed 2x with PBS. Cells were fixated with 4% formaldehyde in PBS for 30 min, RT. After fixation cells were washed twice with PBS and permeabilized with warm 0.5% Triton-X in Hanks balanced salt solution medium for 10 min, RT. Cells were subsequently washed twice with PBS. Next, cells were incubated with primary α-influenza nucleocapsid A mAb (Nordic Biosite) diluted 1:1500 in 10% FBS in PBS for 1 hour at 37 °C, 5% CO2. Antibody was removed and cells washed 5x. This was followed by incubation with a secondary goat α-mouse HRP conjugated mAb (Dako) diluted 1:500 in 10% FBS in PBS for 1 hour at 37 °C, 5% CO2. After secondary antibody incubation cells were washed 5x with PBS. 200 μl substrate solution containing 3 mg/ml 3-amino-9-ethylcarbazole and 0.07% H2O2 and 5 mM citrate phosphate buffer pH5 was added to wells for 30 minutes, RT. Substrate was removed and cells were washed once with PBS before counting. All samples were run in duplicates. Plaque forming units per g lung tissue were calculated accordingly:
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| 39.78 |
Splenocytes from naive B6.SJL mice (CD45.1 positive) were pulsed with NP366 or PB1703 (10 μg/spleen) for 30 minutes at 37 °C. After incubation with peptide, the cells were washed and stained with 0.2 μM CFSE (NP366) or 2 μM (PB1703). Following another washing step, the labeled cells were mixed in a 1:1 ratio, and 2 × 107 cells were injected i.v. into vaccinated C57BL/6 mice (CD45.1 negative); 16 h later, recipient splenocytes were isolated, and target cells were identified using flow cytometry by the expression of CD45.1 and CFSE staining. The percentage of killing was calculated using the following equation: 100−[(percentage of Influenza peptide-labeled cells in infected mice/percentage of cells labeled with irrelevant peptide in infected mice)/(percentage of Influenza peptide-labeled cells in uninfected mice/percentage of cells labeled with irrelevant peptide in uninfected mice) ×100] as described in ref. 18.
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| 42.34 |
The 5G community has already started investigating solutions that can lead to the 1000× challenge. These solutions include among others, enhanced massive Multiple-Input Multiple-Output (MIMO), carrier aggregation, higher-order modulation schemes such as 64-Quadrature Amplitude Modulation (QAM) or 256-QAM, cloud computing services and advanced network architecture modifications. At the same time, the adoption of LTE from different applications gains ground, as it is a technology that approaches the Shannon limit and can contribute significantly to solving the network capacity challenge.
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| 27.61 |
Recently, key players of the mobile world have proposed standards to the 3rd Generation Partnership Project (3GPP), which allow LTE operation in the unlicensed spectrum. To this end, 3GPP announced the operation of LTE Licensed-Assisted Access (LTE LAA) , as an enhancement within 3GPP LTE Release 13. LTE LAA will allow operators to use a secondary cell operating in the unlicensed spectrum, alongside the primary cell operating in the licensed band they own. The carrier aggregation that has been introduced in 3GPP LTE Release 10 will be used to enable this feature. There are two predominant proposals for LTE LAA. According to the first one, the secondary cell will operate in the unlicensed spectrum for supplemental Downlink (DL) traffic only, while Uplink (UL) traffic will be transmitted over the operator’s licensed spectrum. In the second proposal, the secondary cell operating in the unlicensed spectrum can be used for both DL and UL LTE traffic.
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| 27.95 |
Analysis was performed using Graphpad Prism software v6.01. Statistical comparison of two groups was done using the Mann-Whitney test (two-tailed). Data from all experiments with more than two groups were first compared using a one-way ANOVA test, and, only if groups were found to differ significantly, this analysis was followed by pair-wise comparisons using Mann-Whitney rank sum test. A significant difference between two groups was acknowledged if p < 0.05. Statistical significance was marked with an asterix (*).
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| 36.1 |
Over the past few years, the technological growth combined with the proliferation of wireless devices such as sensors, smartphones, laptops and wearable technology has changed the way that information is exchanged. The number of interconnected devices and the number of Heterogeneous Networks (HetNets) increase rapidly. The development and the consolidation of wireless sensor networks has further contributed to the increase of the wireless traffic, as often they consist of hundreds to thousands of wireless sensor nodes. The first-generation Internet has evolved into the Internet of Everything, where massive amounts of information are exchanged between devices using different types of mainstream and well-established wireless technologies such as LTE, Wi-Fi, IEEE 802.15.4 and Bluetooth. Recently, the sub-gigahertz bands have been extensively exploited by wireless technologies that offer wide ranging communications, such as LORA, SIGFOX and 802.11ah. Moreover, high frequency bands such as mmWave are also being used for multi-gigabit speeds (IEEE 802.11ad). According to Qualcomm, the amount of wireless traffic is expected to further increase by a factor of 1000 by 2020 . Additionally, Cisco’s latest forecast expects that the traffic from wireless and mobile devices will exceed the overall wired traffic by 2019 . Based on these predictions, the wireless network capacity will soon become a bottleneck for the massive growth of wireless traffic.
|
study
| 30.33 |
On the other hand, leading wireless stakeholders other than mobile operators are taking the first steps towards exploitation of LTE in the unlicensed spectrum as a standalone wireless solution complementary to Wi-Fi. To this end, they formed the MulteFire Alliance . Their target is to decouple LTE from the operators, so it can be deployed by Internet service providers (ISPs) enterprises, building owners, cable companies, etc.
|
study
| 27.83 |
Figure 1 indicates how LTE in the unlicensed spectrum could be deployed next to the current wireless infrastructure, where an LTE-U small cell could be either an LTE LAA small cell, controlled by a mobile operator, or a small cell operating solely in the unlicensed spectrum without mobile operator control.
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other
| 32.22 |
LTE is a technology that has been initially designed to operate in the licensed spectrum. Hence, it assumes that it can exclusively use the whole assigned spectrum, and therefore, it does not incorporate any techniques for harmonious coexistence with other possible co-located technologies. It is clear that introducing LTE in the unlicensed spectrum as is will cause significant coexistence issues with other well-established technologies such as Wi-Fi, IEEE 802.15.4 or Bluetooth. This means that LTE will have a negative impact on the performance of traditional unlicensed technologies in terms of throughput, latency and other Quality of Service (QoS) guarantees , affecting their applications such as wireless (sensor) networks, Device-to-Device (D2D) and Machine-to-Machine (M2M) communications. To this end, research has focused on the design and evaluation of coexistence techniques for LTE, in order to enable fair spectrum sharing with other technologies operating in the unlicensed spectrum, and in particular with Wi-Fi. On the other hand, much less attention has been paid to cooperation techniques between the two technologies. The networks that participate in a cooperation scheme are able to exchange information directly or indirectly (via a third-party entity) in order to improve the efficiency of spectrum usage in a fair way.
|
other
| 28.47 |
In this article, we distinguish two different classes of cooperation between LTE and Wi-Fi, and for each class, we propose and analyze potential cooperation techniques that can be applied. For each cooperation technique, we analyze the advantages and disadvantages regarding the design and deployment complexity, the flexibility and the efficiency they could offer. The proposed techniques can contribute to the open discussion regarding the standardization process of the LTE operation in the unlicensed spectrum. The main contribution of this work is summarized as follows:Classification of techniques that can be applied on co-located LTE and Wi-Fi networksDetailed analysis of the current state of the art regarding LTE in the unlicensed spectrum and Wi-Fi covering: ‒Analysis of the standard LTE and Wi-Fi protocols‒The regional regulations for the unlicensed spectrum‒The impact of LTE on Wi-Fi without applying any coexistence technique‒The current approaches for coexistence between LTE and Wi-FiAnalysis of the different concepts of cooperation between LTE and Wi-Fi and potential techniques that can be applied for realizing each conceptComparison and feasibility of the different presented concepts
|
review
| 26.48 |
Detailed analysis of the current state of the art regarding LTE in the unlicensed spectrum and Wi-Fi covering: ‒Analysis of the standard LTE and Wi-Fi protocols‒The regional regulations for the unlicensed spectrum‒The impact of LTE on Wi-Fi without applying any coexistence technique‒The current approaches for coexistence between LTE and Wi-Fi
|
study
| 35.3 |
The remainder of the article is organized as follows. Section 2 presents a classification of the techniques that can be applied when LTE and Wi-Fi networks are co-located and operate in the same (unlicensed) frequency band. Section 3 discusses the current state of the art for LTE operation in the unlicensed spectrum. Next, in Section 4, we analytically present the concept of direct cooperation between LTE and Wi-Fi via in-band energy level patterns and showcase possible cooperation techniques. Section 5 presents the concept of cooperation between LTE and Wi-Fi using indirect communication through a third-party entity and describes possible cooperation techniques. In Section 6, we compare the proposed concepts and techniques. Finally, in Section 7, we conclude the paper and discuss plans for future work.
|
other
| 30.47 |
The other two categories classify two co-located networks based on whether the applied technique aims to provide coexistence or cooperation between the networks. Coexistence and cooperation are two terms that differ significantly. With the term coexistence, we refer to methodologies that enable peaceful operation of a wireless technology next to another. The technologies must respect each other, as well as the regional regulations and seek equal opportunities to access the wireless medium, under the condition that there is no exchange of any information between different technologies. On the other hand, the cooperation term refers to methodologies that seek collaboration among the technologies towards harmonious coexistence and optimal spectrum usage by exchanging information. The cooperation between different technologies is in line with the 5G vision, where all of the available wireless technologies will act towards enhancing the user experience.
|
other
| 31.81 |
As can be seen from Figure 2, coexistence techniques include the 3GPP LTE LAA mechanism and other techniques that are described later in Section 3. Although coexistence techniques between LTE operating in the unlicensed spectrum and Wi-Fi have been studied widely, the literature, to the best of our knowledge, consists of only a limited number of studies focusing on cooperation techniques among the two technologies. This paper targets covering this gap by classifying the possible cooperation schemes and proposing potential techniques that can be applied in each category. The different cooperation techniques can be classified into the following two big categories, based on the way that the participating networks communicate with each other:Direct cooperation via in-band energy level patternsIndirect cooperation via a third-party entity
|
other
| 33.5 |
This section presents a taxonomy of techniques that can be applied when an LTE network is co-located with a Wi-Fi network and both networks operate in the same frequency band. This taxonomy is presented in Figure 2. As can be seen, co-located LTE and Wi-Fi networks can be classified into three big categories depending on the techniques that are applied between them.
|
other
| 35.56 |
In the first category, the networks operate next to each other in the way they were initially designed, without any technique that improves the symbiosis between them. This is the worst type of co-location scenario as LTE transmissions result in a severe impact on Wi-Fi .
|
other
| 30.5 |
Upon a transmission, LTE uses the multi-user versions of the Orthogonal Frequency Division Multiplexing (OFDM) digital modulation scheme, called Orthogonal Frequency Division Multiple Access (OFDMA) for the DL and Single-Carrier Frequency Division Multiple Access (SC-FDMA) for the UL . The available spectrum is divided into subcarriers, and each subcarrier occupies 15-kHz of bandwidth. The time domain is organized in timeslots of 0.5 ms in duration. One timeslot corresponds to seven OFDM symbols when the normal Cyclic Prefix (CP) is used and six OFDM symbols when the extended CP is used. Combining the subcarriers and timeslots, LTE defines the Resource Block (RB). The RB is a unit of transmission resource and consists of one slot in the time domain and 12 subcarriers in the frequency domain. An LTE radio frame has a duration of 10 ms and consists of 10 sub-frames, each of which lasts 1 ms corresponding to two slots. Figure 3 shows the structure of resource blocks in the frequency-time domain and how they are scheduled for different User Equipment (UE).
|
other
| 32.94 |
LTE sends user traffic in the DL and in the UL using the Physical Downlink Shared Channel (PDSCH) and the Physical Uplink Shared Channel (PUSCH), respectively. In addition to user traffic, several RB are allocated for control traffic in dedicated channels. Such control traffic includes among others the transmission of synchronization signals and reference signals.
|
review
| 26.53 |
The first category includes techniques that make use of one or multiple in-band energy patterns in order to perform technology identification, inform about their actions or achieve synchronization between multiple networks that participate in the cooperation scheme.
|
review
| 27.1 |
This section briefly describes the mechanisms that LTE and Wi-Fi use to transmit in the way that they were initially designed. The analysis of the core differences among these mechanisms will give us the insight to understand in depth the reasons why LTE cannot operate next to Wi-Fi without appropriate coexistence and cooperation mechanisms. Moreover, it will be used as a basis for the subsequent description of the different cooperation protocols.
|
other
| 33.72 |
CCA consists of two functions named Carrier Sense (CS) and Energy Detection (ED). The CS function refers to the ability of the receiver to detect and decode a received Wi-Fi preamble. According to the specifications, if the power of the detected signal is higher or equal to −82 dBm for the 20-MHz bandwidth, then CCA reports the channel as busy for the timeslot that is indicated in the frame’s Physical Layer Convergence Protocol (PLCP) length field . This field contains either the time in µs that is required for the Medium Access Control (MAC) Protocol Data Unit (MPDU) payload transmission or the number of octets carried in the frame MPDU payload, which is used to compute the time required for the MPDU transmission.
|
other
| 30.86 |
If the received signal cannot be decoded, the ED function is used. The ED function refers to the ability of the receiver to detect the energy level in the operating channel based on non-Wi-Fi signals that are sensed, introducing interference, or based on corrupted Wi-Fi transmissions that cannot be decoded. If the energy level is higher than −62 dBm for a 20-MHz bandwidth, then CCA reports the channel as busy. ED senses the channel every time slot to estimate the corresponding energy level .
|
other
| 31.64 |
LTE is a scheduled technology designed to operate in the licensed spectrum. Therefore, it does not require sensing the medium before transmission. The scheduling in LTE is performed by the LTE base station named the evolved NodeB (eNB) on a subframe basis. That means that every 1 ms, the assignment of the subframes to the active UEs can change.
|
other
| 29.47 |
Wi-Fi uses the Distributed Coordination Function (DCF) as the fundamental mechanism to access the medium and is designed to be asynchronous and decentralized . Additionally, it uses the OFDM digital modulation scheme that divides the spectrum into multiple OFDM subcarriers spanning 20 MHz or multiple spectral portions of 20 MHz. In order to sense and gain access to the medium, Wi-Fi uses the Carrier Sensing Multiple Access with Collision Avoidance (CSMA/CA) mechanism before a packet transmission. According to this contention-based protocol, a Wi-Fi node first has to listen to the shared medium to determine if there are other ongoing transmissions. This procedure is known as Clear Channel Assessment (CCA). The node senses the channel for a DCF Inter-Frame Space (DIFS) duration. Only when the channel is estimated as idle is the node permitted to transmit.
|
other
| 30.52 |
In case the channel is sensed as idle for a DCF Inter-Frame Spacing (DIFS) period, then the Wi-Fi node can transmit. Otherwise and also prior to attempting to transmit again immediately after a successful transmission, the node has to postpone its transmission and wait for a free DIFS plus a random backoff time to avoid packet collisions. After a transmission, the node waits for an acknowledgment during a Short Inter-Frame Space (SIFS) period. If the acknowledgment is not received after this timeout period, the node schedules a retransmission after a new exponential backoff period and until the maximum number of retransmissions is reached. Figure 4 illustrates the CSMA/CA algorithm described above.
|
other
| 30.16 |
In order to enable fair coexistence among LTE and Wi-Fi, research has been focusing on designing coexistence techniques that will allow LTE to operate in the unlicensed spectrum, respecting the different regional regulations. Concurrently, these techniques aim to fairly share the medium with other well-established technologies like Wi-Fi. For instance, the European Telecommunications Standards Institute (ETSI) defines requirements that should be fulfilled by each technology that operates in the unlicensed spectrum. These requirements among others include:CCA before transmission together with timing requirements for each CCA phaseMaximum antenna gainTransmission power limitations
|
other
| 26.75 |
The previous section has described the different operational methods for LTE and Wi-Fi. It is clear that introducing LTE into the unlicensed spectrum, in the way it was originally designed, will have a significant impact on the performance (throughput, latency, packet loss, spectral efficiency) of a co-located Wi-Fi network. As LTE can schedule traffic without sensing the medium for ongoing transmissions, it can interfere with Wi-Fi within the overlapping spectrum. Hence, the CCA mechanism of Wi-Fi, and more specifically the ED function, will force Wi-Fi to backoff. This impact can become even higher by consecutive LTE transmissions. Then, LTE will either seriously degrade the signal quality of Wi-Fi due to collisions (if the LTE signal power is below the ED threshold, but still high enough to interfere with the Wi-Fi transmissions), or lead to Wi-Fi starvation, as it will be forced to backoff continuously.
|
other
| 29.2 |
Several studies have evaluated the impact of traditional LTE on Wi-Fi, when both technologies operate in the same frequency band without any coexistence mechanism being applied. In our previous work , we studied the impact of LTE operating in the unlicensed spectrum on Wi-Fi using Off The Shelf (OTS) hardware equipment . The experiments were performed on the LTE and Wi-Fi infrastructure of the W-iLab2 testbed at imec . Three different levels of LTE signal have been examined, representing different possible levels of LTE impact on Wi-Fi. The results show that the Wi-Fi performance, in terms of throughput and latency, can be significantly affected by LTE.
|
other
| 27.64 |
Other approaches evaluate the Wi-Fi performance degradation based on simulations and mathematical models. In , the authors evaluate the performance of both LTE and Wi-Fi when both technologies operate in a shared band. All studies come to the same conclusion, namely that LTE causes a serious impact on Wi-Fi, when both operate in the same band without any coexistence mechanism among them and no medium sensing mechanism enabled at the LTE side.
|
other
| 28.83 |
Towards a coexistence technique that respects the regional regulations, 3GPP announced the LTE LAA standards in Release 13, including the description of a Listen Before Talk (LBT) procedure (also known as CCA) . Initially, LTE LAA is scheduled to operate within the 5-GHz unlicensed spectrum and for DL traffic only, but in a later phase, it is expected to be extended to the 2.4-GHz unlicensed band, as well as for both DL and UL traffic. Initially, an eNB will be able to activate and deactivate a secondary cell operating in the unlicensed spectrum. Through this cell, only data traffic (via the PDSCH) can be sent, while the LTE control signals and the UL traffic (PUSCH) will be transmitted via the licensed anchor. The eNB must perform the LBT procedure and sense the channel prior to a transmission in the unlicensed spectrum. When the channel is sensed as busy, the eNB must defer its transmission by performing an exponential backoff. If the channel is sensed to be idle, it performs a transmission burst with a duration from 2–10 ms, depending on the channel access priority class. The authors in analytically describe the LTE LAA procedure. They provide an overview of the LAA mechanism including the motivation and use cases where it can be applied. Additionally, they present a coexistence evaluation methodology and results, which have been contributed by 3GPP. Figure 5 shows the LTE LAA and Wi-Fi coexistence in the same channel in the unlicensed spectrum.
|
other
| 32.1 |
In regions such as the U.S., China and South Korea, where an LBT procedure is not required by the local regulations, different types of coexistence techniques can be applied. Carrier Sensing Adaptive Transmission (CSAT) , proposed by Qualcomm, is a technique that can enable coexistence among LTE and Wi-Fi based on minor modifications of the 3GPP LTE Release 10/11/12 Carrier Aggregation protocols . CSAT introduces the use of duty cycle periods and divides the time into LTE “ON” and LTE “OFF” slots. During the LTE “OFF” period, also known as the “mute” period, LTE remains silent, giving the opportunity to other coexistent networks, such as Wi-Fi, to transmit. During the LTE “ON” period, LTE accesses the channel without sensing it before a transmission. Moreover, CSAT allows short transmission gaps during the LTE “ON” period to allow for latency sensitive applications, such as VoIP in co-located networks. In CSAT, the eNB senses the medium for a time period ranging from tens of ms up to 100 ms and according to the observed channel utilization (based on the estimated number of Wi-Fi Access Points (APs)) defines the duration of the LTE “ON” and LTE “OFF” periods . Figure 6 depicts the CSAT duty cycle periods.
|
other
| 32.53 |
Coexistence between LTE and Wi-Fi in the unlicensed spectrum has attracted the attention of the mobile and the research community. There are several proposed mechanisms, trying to achieve fair coexistence between the two technologies. These mechanisms are evaluated on the success of providing the desired fairness.
|
other
| 30.22 |
In , the authors discuss a preliminary design of a semi-distributed LTE in the unlicensed spectrum scheme, where the eNB senses the carrier before a transmission in a similar way to Wi-Fi. They use a method that exploits the LTE Almost Blank Subframes (ABS). The ABS were initially designed to enhance Inter-Cell Interference Coordination (eICIC) as part of 3GPP LTE Release 10 . The proposed method evaluates different duty cycles, different distances between an eNB and an AP and different numbers of cells. Different ABS patterns are also studied.
|
other
| 27.98 |
In , the authors propose an LTE subframe design consisting of three phases named the data transmission phase, the mute phase and the sensing and reservation phase. Based on this subframe design, they proposed three schemes to enable coexistence among LTE and Wi-Fi. The first scheme assumes a fixed mute duration. The second scheme uses a randomized mute duration. The third scheme introduces a random backoff counter during the mute period. The three proposed schemes are evaluated through simulations. The results show that the scheme that uses a random mute duration offers better overall throughput performance, while the scheme that uses a random backoff counter results in smaller throughput difference between LTE and Wi-Fi.
|
other
| 27.08 |
The authors in describe an analytical framework for interference characterization of Wi-Fi and LTE. Initially, a first model is described for single LTE and single Wi-Fi AP separated by a specific distance. The results show that Wi-Fi performance is significantly decreased compared to LTE for which the degradation is minimal. They observe that the conventional perception of the inverse proportion of throughput to inter-AP distance is not valid for LTE-Wi-Fi co-channel deployment. A second model with many LTE and Wi-Fi systems is also described. The results show that the overall system throughput first increases and then decreases with growing density. Finally, in order to increase the individual Radio Access Technology (RAT) and system throughput, random channel assignment, intra- and inter-RAT channel coordination are considered. In the intra- and inter-RAT channel coordination schemes, the channel is allocated at an AP as a graph multi-coloring problem. The results show 3.5–5× gains in system capacity. In this technique, the networks do not exchange specific information in order to optimize the offered QoS, but different frequencies are assigned to them in order to avoid overlapping frequencies between co-located networks.
|
study
| 28 |
In , the authors use Q-Learning techniques to achieve the desired coexistence. In , they propose a Q-Learning-based dynamic duty cycle selection mechanism for configuring LTE transmission gaps. LTE LAA and Wi-Fi performance using a fixed transmission gap is evaluated and then compared with the proposed Q-Learning mechanism. Simulation results show that the proposed scheme enhances the overall capacity performance. The authors in propose a Q-Learning mechanism for advanced learning of the activity within the unlicensed band resulting in efficient coexistence between LTE LAA and Wi-Fi. As a next step, the coexistence is further enhanced through a double Q-Learning method that takes into account both discontinuous transmission and transmit power control of LTE to improve both LTE and Wi-Fi performance.
|
study
| 27.1 |
Coexistence of LTE and Wi-Fi when LTE uses an LBT procedure is studied in . The authors in propose an adaptive LBT protocol for LTE LAA. This protocol enhances the coexistence with Wi-Fi and increases the overall system performance. The protocol consists of two different mechanisms named on-off adaptation for channel occupancy time and short-long adaptation for idle time. The first mechanism is responsible for adapting the channel occupancy time of LTE based on the load of the network, while the second one adapts the idle period based on the Contention Window (CW) duration of Wi-Fi. The authors in propose an LBT mechanism for LTE LAA that aims to share the medium in a fair way towards the increase of the overall system performance. This work includes both a mathematical analysis and a validation via simulation of the proposed LBT scheme. The results show that a proper selection of LAA channel occupancy and the backoff counter can increase the performance of Wi-Fi. In , the authors study the coexistence among LTE LAA and Wi-Fi using the LBT category four-channel access scheme. The behavior of LAA eNB is modeled as a Markov chain, and the obtained throughput is adopted as the performance metric. The proposed LBT scheme uses an adaptive CW size for LTE LAA. According to the results, the proposed scheme outperforms the fixed CW size. In , the authors examine how LTE cells in the unlicensed spectrum from different operators can adjust their CW in order to tune the LBT algorithm and provide coexistence both with Wi-Fi and among themselves in an altruistic way. The interaction of LTE cells in the unlicensed spectrum is studied using a coalition formation game framework, which is based on the Shapley value.
|
other
| 29.27 |
In , the authors present an analytical model for evaluating the performance of coexistence between LTE and Wi-Fi. This model has been used to obtain baseline performance measures. The results of the model have been partially validated via experimental evaluation using Universal Software Radio Peripheral (USRP) platforms. Moreover, the authors propose an inter-network coordination with logically centralized radio resource management across LTE and Wi-Fi as a solution to improve coexistence.
|
other
| 31.44 |
The authors in propose two non-coordinated and two coordinated network management approaches to enable coexistence. Regarding the non-coordinated techniques, the first one proposes eNB to perform LBT on different channels and to switch to a different channel after a transmission, while the second proposes LTE to offer transmission opportunities of variable duration to Wi-Fi after a transmission based on the occupancy of the medium. Concerning the coordinated methodologies, the first one proposes a Network Function Virtualization (NFV) interconnection to combine the Wi-Fi network and the service provider of LTE in the unlicensed spectrum. This way, channel selection and seamless transfer of resources between the two technologies can be enabled, using the in-the-cloud control of distributed APs. The second method proposes the management of coexistence using the X2 interface among the eNBs. The eNBs can exchange information and schedule ABS in different subframes, thus giving more opportunities to any Wi-Fi network that is located potentially within their proximity. In the aforementioned schemes, the different RATs are under the control of the same mobile operator. The coordination between the wireless technologies targets the enhancement of the overall QoS that the operator offers (e.g., perform load balancing via frequency coordination).
|
other
| 28.69 |
Finally, the authors in provide a detailed survey of the coexistence of LTE and Wi-Fi on 5 GHz with the corresponding deployment scenarios. They provide a detailed description of the coexistence-related features of LTE and Wi-Fi, the coexistence challenges, the differences in performance between the two different technologies and co-channel interference. They extensively discuss the proposed coexistence mechanisms between LTE and Wi-Fi in the current literature. Furthermore, the survey discusses the concept of the scenario-oriented coexistence, in which coexistence-related problems are solved according to different deployment scenarios.
|
other
| 29.03 |
Although the coexistence between LTE operating in the unlicensed spectrum and Wi-Fi is being investigated extensively, little attention is given to studies that investigate cooperation scenarios among the two technologies. As has been discussed in Section 2, in this paper, we distinguish two different types of cooperation between LTE in the unlicensed spectrum and Wi-Fi. Furthermore, for each solution, we propose and describe different techniques that can lead to efficient and fair spectrum use.
|
other
| 33.97 |
This section describes cooperation techniques between co-located LTE and Wi-Fi networks that operate in the same frequency band, using in-band pattern recognition in order to enhance the spectral efficiency of the coexisting networks. A cooperation scheme that uses in-band pattern recognition can be applied, when the co-located networks do not have the ability to communicate between each other (e.g., via a coordinator) in order explicitly to express their requirements. The in-band pattern recognition methodology allows direct cooperation between different wireless technologies, as it can be used for technology identification and inter-RAT synchronization. Moreover, a wireless technology can use one or more in-band special patterns in order to inform other technologies about different actions that it performs. Upon the recognition of such a pattern, a wireless network will be able to adapt its behavior towards an increased performance (e.g., higher throughput) and more advanced spectrum usage. Figure 7 depicts an example of an in-band pattern recognition. In this example, a predefined energy level pattern is transmitted by the LTE eNB. This pattern is used for the identification of the LTE network by a Wi-Fi AP.
|
other
| 37 |
For a technique of such a nature, the complexity of the design and the implementation is relatively low, as only small modifications of the current protocols of each wireless technology are required in order to transmit and interpret such energy level patterns. Nevertheless, the low complexity of the methodologies implies also a limited flexibility, meaning that upon sensing a co-located wireless technology, each network takes some predefined actions that can contribute to more efficient spectrum sharing and/or performs readjustment and tuning of existing coexistence techniques.
|
other
| 31.5 |
One negative aspect of the CSAT algorithm, as described in Section 3.4.2, is that it requires a very long sensing period ranging from 20 ms up to 100 ms, in order to observe the activity in the medium and decide the LTE “ON” and LTE “OFF” periods. Furthermore, at the end of an LTE “ON” period or at the end of a Wi-Fi opportunity slot during the LTE “ON” period, LTE starts transmitting without sensing the medium for ongoing Wi-Fi transmissions. This results in several collisions among LTE and Wi-Fi. These drawbacks can be eliminated by the use of an energy level pattern periodically transmitted by the eNB. Such a pattern can be sensed by Wi-Fi and other LTE networks in order to achieve inter- and intra-technology synchronization and to adjust their behavior.
|
other
| 32.7 |
In the proposed methodology, we define three different energy level patterns that can be used for different purposes. These patterns are defined as follows:Synchronization pattern that enables inter- and intra-technology synchronization and that is transmitted by the first activated networkLTE identification pattern that is transmitted by the eNB of a newly-activated LTE network in order to inform the rest of the networks about its presenceWi-Fi identification pattern that is transmitted by the AP of a newly-activated Wi-Fi network in order to inform the rest of the networks about its presence
|
study
| 26.7 |
Additionally, we define a new time frame as is depicted in Figure 8. This time frame starts with a period TSYNC dedicated to transmission and reception of the synchronization pattern. Then, the main part of the frame is called TTX and is divided into slots for LTE and Wi-Fi traffic. The last part of the frame is called TIDENT, and it is used for LTE and Wi-Fi pattern transmission, in order to identify any new networks and adjust the LTE and Wi-Fi slots for the next time frame. Initially, when a new network is activated, it must sense the medium for a period of time equal to a frame in order to discover potential synchronization patterns. If such a pattern does not exist, then the network starts periodically transmitting a synchronization pattern signal to enable inter- and intra-technology synchronization. On the other hand, if the new network senses a synchronization pattern, then it does not initialize a periodic synchronization pattern signal transmission, but it keeps sensing the medium to identify the next synchronization pattern that is expected at the beginning of the next frame. At the moment that two sequentially synchronization patterns are sensed, then the new network can be in synchronization with the rest of the networks. The length of a frame can be stable over time or can vary based on the number of cooperating networks and the amount of transmitted traffic. In the case that a variable frame size is used, then the new network must sense the medium to discover a potential synchronization pattern for a time period that is equal to the maximum frame length.
|
other
| 32.66 |
From the moment that the newly-activated network is in synchronization with the rest of the networks, it must transmit a corresponding LTE or Wi-Fi identification pattern during the next TIDENT period. This way, the rest of the networks will be notified for the new LTE or Wi-Fi network. The newly-activated network can identify the LTE and Wi-Fi slots that have already been created by sensing the medium during a TTX period. The network that is in charge of transmitting the synchronization pattern transmits its identification pattern only after it senses the first identification pattern from another network during the same TIDENT period. If an LTE or Wi-Fi identification pattern is sensed during the TIDENT, then the LTE and Wi-Fi slots during the TTX period are readjusted. This readjustment is done based on the number and the type of the co-located networks. The creation of the new slots will be decided based on the same predefined mechanisms for both LTE and Wi-Fi networks. Moreover, the length of the LTE and Wi-Fi slots will be decided based on the common scheduling mechanism that is used by LTE and Wi-Fi. Furthermore, during a time slot, the eNBs and the APs will measure the channel utilization in order to further adapt the slots for the next frame.
|
other
| 31.17 |
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