scholarly journals Human Cytomegalovirus UL40 Signal Peptide Regulates Cell Surface Expression of the NK Cell Ligands HLA-E and gpUL18

2012 ◽  
Vol 188 (6) ◽  
pp. 2794-2804 ◽  
Author(s):  
Virginie Prod’homme ◽  
Peter Tomasec ◽  
Charles Cunningham ◽  
Marius K. Lemberg ◽  
Richard J. Stanton ◽  
...  
Keyword(s):  
Cell Surface ◽  
Human Cytomegalovirus ◽  
Signal Peptide ◽  
Nk Cell ◽  
Surface Expression ◽  
Cell Surface Expression

Impact of β2 integrin deficiency on mouse natural killer cell development and function

Blood ◽  
2011 ◽  
Vol 117 (10) ◽  
pp. 2874-2882 ◽  
Author(s):  
Karine Crozat ◽  
Céline Eidenschenk ◽  
Baptiste N. Jaeger ◽  
Philippe Krebs ◽  
Sophie Guia ◽  
...  
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Surface Expression ◽  
Cell Maturation ◽  
Cell Surface Expression ◽  
Mcmv Infection ◽  
Β2 Integrin

Abstract Natural killer (NK) cells are innate immune cells that express members of the leukocyte β2 integrin family in humans and mice. These CD11/CD18 heterodimers play critical roles in leukocyte trafficking, immune synapse formation, and costimulation. The cell-surface expression of one of these integrins, CD11b/CD18, is also recognized as a major marker of mouse NK-cell maturation, but its function on NK cells has been largely ignored. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated a mouse carrying an A → T transverse mutation in the Itgb2 gene, resulting in a mutation that prevented the cell-surface expression of CD18 and its associated CD11a, CD11b, and CD11c proteins. We show that β2 integrin–deficient NK cells have a hyporesponsive phenotype in vitro, and present an alteration of their in vivo developmental program characterized by a selective accumulation of c-kit+ cells. NK-cell missing-self recognition was partially altered in vivo, whereas the early immune response to mouse cytomegalovirus (MCMV) infection occurred normally in CD18-deficient mice. Therefore, β2 integrins are required for optimal NK-cell maturation, but this deficiency is partial and can be bypassed during MCMV infection, highlighting the robustness of antiviral protective responses.

Down-Regulation of HLA-G1 Cell Surface Expression in Human Cytomegalovirus Infected Cells

2003 ◽  
Vol 50 (4) ◽  
pp. 328-333 ◽  
Author(s):  
Nathalie Pizzato ◽  
Barbara Garmy-Susini ◽  
Philippe Le Bouteiller ◽  
Francoise Lenfant
Keyword(s):  
Cell Surface ◽  
Human Cytomegalovirus ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Down Regulation ◽  
Infected Cells

scholarly journals HIV-1 Vpr up-regulates expression of ligands for the activating NKG2D receptor and promotes NK cell–mediated killing

Blood ◽  
2010 ◽  
Vol 115 (7) ◽  
pp. 1354-1363 ◽  
Author(s):  
Jonathan Richard ◽  
Sardar Sindhu ◽  
Tram N. Q. Pham ◽  
Jean-Philippe Belzile ◽  
Éric A. Cohen
Keyword(s):  
Dna Damage ◽  
Cell Surface ◽  
Nk Cell ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Nkg2d Ligands ◽  
Infected Cells ◽  
Nkg2d Receptor ◽  
Hiv 1

AbstractHIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor, including ULBP-1, -2, and -3, but not MICA or MICB, in infected cells both in vitro and in vivo. However, the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/stress-sensing ATR kinase and promotes G2 cell-cycle arrest, conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4+ T lymphocytes by a process that is Vpr dependent. Importantly, Vpr enhanced the susceptibility of HIV-1–infected cells to NK cell–mediated killing. Strikingly, Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell–mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells, suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall, these results indicate that Vpr is a key determinant responsible for HIV-1–induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1–induced CD4+ T-lymphocyte depletion but may also take part in HIV-1–induced NK-cell dysfunction.

scholarly journals The PAR2 signal peptide prevents premature receptor cleavage and activation

2019 ◽  
Author(s):  
Belinda Liu ◽  
Grace Lee ◽  
Jiejun Wu ◽  
Janise Deming ◽  
Chester Kuei ◽  
...  
Keyword(s):  
Cell Surface ◽  
Signal Peptide ◽  
Synthetic Peptide ◽  
Surface Expression ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Peptide Ligand ◽  
Protease Activated Receptors ◽  
N Terminus

AbstractUnlike closely related GPCRs, protease-activated receptors (PAR1, PAR2, PAR3, and PAR4) have a predicted signal peptide at their N-terminus, which is encoded by a separate exon, suggesting that the signal peptides of PARs may serve an important and unique function, specific for PARs. In this report, we show that the PAR2 signal peptide, when fused to the N-terminus of IgG-Fc, effectively induced IgG-Fc secretion into culture medium, thus behaving like a classical signal peptide. The presence of PAR2 signal peptide has a strong effect on PAR2 cell surface expression, as deletion of the signal peptide (PAR2ΔSP) led to dramatic reduction of the cell surface expression and decreased responses to trypsin or the synthetic peptide ligand (SLIGKV). However, further deletion of the tethered ligand region (SLIGKV) at the N-terminus rescued the cell surface receptor expression and the response to the synthetic peptide ligand, suggesting that the signal peptide of PAR2 may be involved in preventing PAR2 from intracellular protease activation before reaching the cell surface. Supporting this hypothesis, an Arg36Ala mutation on PAR2ΔSP, which disabled the trypsin activation site, increased the receptor cell surface expression and the response to ligand stimulation. Similar effects were observed when PAR2ΔSP expressing cells were treated with protease inhibitors. Our findings indicated that these is a role of the PAR2 signal peptide in preventing the premature activation of PAR2 from intracellular protease cleavage before reaching the cells surface. The same mechanism may also apply to PAR1, PAR3, and PAR4.

scholarly journals Promising Preliminary Activity of Optimized Affinity, CD38 CAR NK Cells Generated Using a Non-Viral Engineering Approach in Gene Edited Cord Blood Derived NK Cells for the Treatment of Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4793-4793
Author(s):  
Rohit Duggal ◽  
Sumit Sen Santara ◽  
Myra Gordon ◽  
Aoife Kilgallon ◽  
David Hermanson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Surface ◽  
Cord Blood ◽  
Nk Cells ◽  
Nk Cell ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Feeder Layer ◽  
Guide Rnas ◽  
Rnp Complex

Abstract CD38 is a multifunctional cell surface protein that is frequently overexpressed on malignant plasma cells as well as on immune suppressive cells within the tumor microenvironment and constitutes a validated immunotherapeutic target in the treatment of multiple myeloma (MM). At ONK Therapeutics we are developing a gene edited, cord blood-derived NK (CBNK) cell product targeting CD38 for treatment of patients with relapsed and/or refractory MM. The product will be generated using a workflow shown in Figure 1A. This involves starting with cord blood that is processed for NK expansion using a clinically validated, Epstein Barr Virus-transformed lymphoblastoid cell line (EBV-LCL) feeder layer. The NK cells would undergo genetic engineering that involves gene editing followed by a non-viral chimeric antigen receptor (CAR) introduction process mediated by the TcBuster (TcB) DNA transposon system (Biotechne). This is followed by a second round of expansion on the EBV-LCL feeder layer resulting in a characterized NK cell product that can then be cryopreserved. In order to develop protocols for optimizing the best transfection efficiencies using the Maxcyte ATx instrument, GFP mRNA (TriLink) was used for transfecting CBNK cells using different electroporation programs. High transfection efficiency was obtained using all programs (Figure 1B.), with the best from program NK4. Since the product employs an optimized affinity second generation anti CD38 CAR (Stikvoort et al., Hemasphere 2021) which could also target CD38 expressed on neighbouring activated NK cells, it is imperative to knock out (KO) the cell surface expression of CD38 on the CAR-NK cells. To achieve this we carried out CRISPR Cas9 based KO studies of CD38 (Figure 1C. left top), using guide RNAs targeting CD38 (Synthego) in the form of a ribonucleoprotein (RNP) complex with Cas9. CBNK cells were transfected using the Maxcyte ATx instrument and CD38 cell surface expression monitored. As shown in Figure 1C. (left top), complete CD38 KO was achieved 11 days post transfection. ONK Therapeutics is actively involved in targeting and downregulating the negative regulator of cytokine signalling, cytokine inducible SH2-containing protein (CIS), which is encoded by the CISH gene, as part of their CBNK products. It has been demonstrated that in addition to facilitating greater cytokine signalling, CISH KO also confers greater metabolic capacity to NK cells resulting in their increased persistence (Daher et al., Blood 2021). Therefore, ONK Therapeutics have evaluated CISH KO in CBNK cells (Figure 1C, top right) using the same scheme that was used for the CD38 KO. Guide RNAs in the form of a RNP complex with Cas9 (Synthego) were transfected into CBNK cells and intracellular CIS protein levels monitored over time. Almost complete KO was attained by 9 days post transfection. In order to dial in CISH KO as part of the product, we further carried out a simultaneous KO of CD38 and CISH, in addition to individual KO of CD38 or CISH (Fig 1C, bottom). Simultaneous multiplexing of the CD38 and CISH KOs resulted in efficient double KO (DKO) . The extent of knock down leading to KO in the DKO setting was very similar to that of individual gene KOs. We then introduced the anti CD38 CAR as part of a transposon that could be transposed by TcB transposase in CBNK cells. After DKO of CD38 and CISH in CBNK cells, the transposon DNA and mRNA for transposase were electroporated. CAR expression was detected 4-5 days post transposition (Figure 1D) with more than 50% of cells expressing the anti CD38 CAR. These CAR expressing CBNK cells were then tested for functionality in a co-culture kill assay against the CD38 positive MM cell line, RPMI8226, which was engineered to express firefly luciferase. In a 4 hour killing assay, robust killing of the RPMI8226 cells was achieved by the CAR-CBNK cells with an EC 50 ten times lower (more potent) than that of mock electroporation control CBNK cells. To our knowledge this is the first successful expression of an anti CD38 CAR in cord-derived NK cells, and with a double CD38/CISH KO, using non-viral CAR insertion approaches. Current work is focusing on designing and developing a manufacturing-ready workflow for this potential product and further examining the effects of CAR NK cell activity in a DKO setting where both KOs contribute to improved metabolism and potentially NK cell persistence, as well as exploring the added benefit of a DR5 TRAIL variant to enhance cytotoxicity. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Microbial Disease Spectrum Linked to a Novel IL-12Rβ1 N-Terminal Signal Peptide Stop-Gain Homozygous Mutation with Paradoxical Receptor Cell-Surface Expression

2017 ◽  
Vol 8 ◽  
Author(s):  
Thais Louvain de Souza ◽  
Regina C. de Souza Campos Fernandes ◽  
Juliana Azevedo da Silva ◽  
Vladimir Gomes Alves Júnior ◽  
Adelia Gomes Coelho ◽  
...  
Keyword(s):  
Cell Surface ◽  
Signal Peptide ◽  
Receptor Cell ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Homozygous Mutation ◽  
Disease Spectrum ◽  
Microbial Disease

scholarly journals Human cytomegalovirus suppresses Fas expression and function

2014 ◽  
Vol 95 (4) ◽  
pp. 933-939 ◽  
Author(s):  
Sepehr Seirafian ◽  
Virginie Prod’homme ◽  
Daniel Sugrue ◽  
James Davies ◽  
Ceri Fielding ◽  
...  
Keyword(s):  
Cell Surface ◽  
Human Cytomegalovirus ◽  
De Novo ◽  
Late Phase ◽  
Surface Expression ◽  
Productive Infection ◽  
Cell Surface Expression ◽  
Trail Receptor ◽  
Infected Cells ◽  
High Level

Human cytomegalovirus (HCMV) is known to evade extrinsic pro-apoptotic pathways not only by downregulating cell surface expression of the death receptors TNFR1, TRAIL receptor 1 (TNFRSF10A) and TRAIL receptor 2 (TNFRSF10B), but also by impeding downstream signalling events. Fas (CD95/APO-1/TNFRSF6) also plays a prominent role in apoptotic clearance of virus-infected cells, so its fate in HCMV-infected cells needs to be addressed. Here, we show that cell surface expression of Fas was suppressed in HCMV-infected fibroblasts from 24 h onwards through the late phase of productive infection, and was dependent on de novo virus-encoded gene expression but not virus DNA replication. Significant levels of the fully glycosylated (endoglycosidase-H-resistant) Fas were retained within HCMV-infected cells throughout the infection within intracellular membranous structures. HCMV infection provided cells with a high level of protection against Fas-mediated apoptosis. Downregulation of Fas was observed with HCMV strains AD169, FIX, Merlin and TB40.

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