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High affinity soluble ILT2 receptor:a potent inhibitor of CD8^(+)T cell activation

Protein & Cell 2010年第12期1卷 1118-1127页

作者： Ruth K.Moysey Yi Li Samantha J.Paston Emma E.Baston Malkit S.Sami Brian J.Cameron Jessie Gavarret Penio Todorov Annelise Vuidepot Steven M.Dunn Nicholas J.Pumphrey Katherine J.Adams Fang Yuan Rebecca E.Dennis Deborah H.Sutton Andy D.Johnson Joanna E.Brewer Rebecca Ashfield Nikolai M.Lissin Bent K.Jakobsen immunocore limited 57c Milton ParkAbingdonOxonOX144RXUK Current address:Oxford Nanopore Technologies UK Current address:Geneva Merck Serono S.A. Switzerland Current address:Adaptimmune limited UK

Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin superfamily receptor ILT2(synonyms:LIR1,MIR7,CD85j),we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex(MHC)class I *** in a dimeric form,either by chemical cross-linking with bivalent polyethylene glycol(PEG)derivatives or as a genetic fusion with human IgG Fc-fragment,the mutants exhibited a further increase in ligand-binding strength due to the avidity effect,with resident half-times(t1/2)on the surface of MHC I-positive cells of many *** novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors(TCRs).In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8^(+)cytotoxic T lymphocytes(CTLs)in the presence of their target cells,with subnanomolar potency and in a dose-dependent *** a selective inhibitor of CD8^(+)CTL responses,the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

关键词： CD8^(+)T cells cellular activation autoimmunity cell surface molecules binding affinity phage display

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A Multifunctional Lentiviral-Based Gene Knockdown with Concurrent Rescue that Controls for Off-Target Effects of RNAi

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Genomics, Proteomics & Bioinformatics 2010年第4期8卷 238-245页

作者： Yunfeng Feng Linghu Nie Meghna Das Thakur Qin Su Zhenfen Chi Yongliang Zhao Gregory D. Longmore Departments of Medicine and Cell Biology Washington University St. Louis MO 63110 USA Key Laboratory of Genome Sciences and Information Cancer Biology and Genetics Group Beijing Institute of Genomics Chinese Academy of Sciences Beijing 100029 China immunocore limited Abingdon Oxon OX14 4RX UK

The efficient, stable delivery of siRNA into cells, and the appropriate controls for non-specific off-target effects of siRNA are major limitations to functional studies using siRNA technology. To overcome these drawbacks, we have developed a single lentiviral vector that can concurrently deplete endogenous gene expression while expressing an epitope-tagged siRNA-resistant target gene in the same cell. To demonstrate the functional utility of this system, we performed RNAi-depleted α-actinin-1 （α-ACTN1） expression in human T cells. α-ACTN1 RNAi resulted in inhibited chemotaxis to SDF-1α, but it can be completely rescued by concurrent expression of RNAi-resistant α-ACTN1 （rr-α-ACTN1） in the same cell. The presence of a GFP tag on rr-α-ACTN1 allowed for detection of appropriate subcellular localization of rr-α-ACTN1. This system provides not only an internal control for RNAi off-target effects, but also the potential tool for rapid structure-function analyses and gene therapy.

关键词： lentivirus RNAi shRNA α-actinin-1 chemotaxis

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Regulation of U6 Promoter Activity by Transcriptional Interference in Viral Vector-Based RNAi

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Genomics, Proteomics & Bioinformatics 2010年第3期8卷 170-179页

作者： Linghu Nie Meghna Das Thakur Yumei Wang Qin Su Yongliang Zhao Yunfeng Feng Key Laboratory of Genome Sciences and Information Cancer Biology and Genetics Group Beijing Institute of Genomics Chinese Acadamy of Sciences Beijing 100029 China Departments of Medicine and Cell Biology Washington University St. Louis MO 63110 USA immunocore limited Abingdon Oxon OX14 4RX UK.

The direct negative impact of the transcriptional activity of one component on the second one in c/s is referred to as transcriptional interference （TI）. U6 is a type III RNA polymerase III promoter commonly used for driving small hairpin RNA （shRNA） expression in vector-based RNAi. In the design and construction of viral vectors, multiple transcription units may be arranged in close proximity in a space-limited vector. Determining if U6 promoter activity can be affected by TI is critical for the expression of target shRNA in gene therapy or loss-of-function studies. In this research, we designed and implemented a modified retroviral system where shRNA and exogenous gene expressions were driven by two independent transcriptional units. We arranged U6 promoter driving .shRNA expression and UbiC promoter in two promoter arrangements. In primary macrophages, we found U6 promoter activity was inhibited by UbiC promoter when in the divergent arrangement but not in tandem. In contrast, PKG promoter had no such negative impact. Instead of enhancing U6 promoter activity, CMV enhancer had significant negative impact on U6 promoter activity in the presence of UbiC promoter. Our results indicate that U6 promoter activity can be affected by TI in a proximal promoter-specific and arrange- ment-dependent manner.

关键词： transcriptional interference U6 promoter shRNA retrovirus

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Identification of host miRNAs that may limit human rhinovirus replication

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World Journal of Biological Chemistry 2014年第4期5卷 437-456页

作者： Victor Paky Bondanese Ana Francisco-Garcia Nicole Bedke Donna E Davies Tilman Sanchez-Elsner LGL Lab Ex LIO ENS Lyon Site Monod 69364 Lyon France Academic Unit of Clinical and Experimental Sciences University of Southampton Faculty of Medicine University Hospital Southampton Southampton SO16 6YD United Kingdom immunocore limited Milton Park Abingdon Oxon OX14 4RY United Kingdom

AIM: To test whether the replication of human rhinovirus(HRV) is regulated by micro RNAs in human bronchial epithelial ***: For the present study, the human cell line BEAS-2B(derived from normal human bronchial epithelial cells) was adopted. DICER knock-down, by si RNA transfection in BEAS-2B cells, was performed in order to inhibit micro RNA maturation globally. Alternatively, antisense oligonucleotides(anti-mi Rs) were transfectedto inhibit the activity of specific micro RNAs. Cells were infected with HRV-1B. Viral replication was assessed by measuring the genomic viral RNA by reverse transcription quantitative polymerase chain reaction(RT-q PCR). Association between micro RNA-induced-silencing-complex and viral RNA was detected by Ago2 co-immunoprecipitation followed by RT-q PCR. Targetscan v.6 was used to predict micro RNA target sites on several HRV ***: Here, we show that micro RNAs affect replication of HRV-1B. DICER knock-down significantly reduced the expression of mature micro RNAs in a bronchial epithelial cell line(BEAS-2B) and in turn, increased the synthesis of HRV-1B RNA. Additionally, HRV-1B RNA co-immunoprecipitated with argonaute 2 protein, an important effector for micro RNA activity suggesting that micro RNAs bind to viral RNA during infection. In order to identify specific micro RNAs involved in this interaction, we employed bioinformatics analysis, and selected a group of micro RNAs that have been reported to be under-expressed in asthmatic bronchial epithelial cells and were predicted to target different strains of rhinoviruses(HRV-1B,-16,-14,-27). Our results suggest that, out of this group of micro RNAs, mi R-128 and mi R-155 contribute to the innate defense against HRV-1B: transfection of specific anti-mi Rs increased viral replication, as anticipated ***: Taken together, our results suggest that pathological changes in micro RNA expression, as already reported for asthma or chronic obstructive pulmona

关键词： Human Rhinovirus Micro RNAs Mi R-155 Si RNA Lentiviral transduction Antiviral innate immunity

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