1,239 research outputs found
Natural killer cell evolution: cellular and molecular studies on Xenopus
No full textThe presence of natural killer cells at lower evolutionary levels was investigated in the amphibian Xenopus laevis. Chromium release microcytotoxicity assays revealed that fresh splenocytes from early-thymectomised Xenopus displayed significant spontaneous cytotoxicity against allogeneic B(_3)B(_7) thymic tumor cell targets, unlike those from control Xenopus, suggesting that 'NK-like' activity is greater in thymectomised (T cell-deficient) animals. Addition of Concanavalin A- derived active supernatants to splenocytes from a thymectomised animal caused a significant increase in cytolytic activity, but had no effect on cells from a control animal. This finding of enhanced cytotoxicity was indicative of lymphokine-activated killing in Xenopus, and supported the concept that tumour cell lysis was mediated by NK - like cells. Attempts were made to enrich the splenocytes for natural killer cells through the selective depletion of other lymphocyte subsets, using the techniques of 'panning’ and 'magnetic bead' separation following monoclonal antibody labelling of cells. On comparison of the two techniques, it was found that both were able to deplete a splenocyte culture of B cells to the same extent, but that magnetic sorting produced far superior results for depletion of T cells. Optimum conditions for magnetic sorting were determined, and used to generate 'purified' populations which were tested for their cytolytic activity. Such preliminary investigations suggested that natural killer like activity in Xenopus is likely to be mediated by a 'non-T / non-B' lymphoid subset. Finally, preliminary work was undertaken into the development of 'phage display' technology for the generation of single chain Fy antibody fragments (ultimately against NK cell surface antigens). PGR amplification of the V(_H) and Kappa chains was attempted on RNA extracted (using various methods) from Carboxypeptidase Y-injected-, B(_3)B(_7)-injected-, and unimmunised mice. Following RNA extraction under optimum conditions. Kappa chains were successfully amplified from experimental spleens, but the heavy chains still require more development
Development of antibody technology to identify natural killer cell surface antigens in Xenopus Laevis
No full textNatural killer (NK)-like lymphocytes have recently been identified in thymectomised (Tx) Xenopus which are capable of spontaneous cytotoxicity towards the MHC- deficient, allogeneic thymus tumour cell line B(_3)B(_7). This Thesis describes attempts to raise antibodies to Xenopus NK cell surface antigens by phage display and hybridoma technology. The phage display technique was optimised for raising antibodies to novel, cellular antigens in a trial run using the Xenopus thymus tumour cell line B(_3)B(_7). Having isolated a phage antibody which was shown by flow cytometry to bind B(_3)B(_7) cells, the technique was then used to try and raise antibodies to Xenopus NK cells. Isolation of an NIC-specific phage antibody was not achieved but phage antibody XL-6 was raised, which bound an antigen on Xenopus lymphocytes. Phage antibody XL-6, and soluble scFv derived from this, were able to identify a putative mature T cell population in the thymus and may be specific for an amphibian homologue of the mammalian leukocyte common antigen CD45. Hybridoma technology was used to isolate three monoclonal antibodies, 1F8, 4D4 and 1G5, which were shown by flow cytometric analysis to identify a putative NK cell population in control and Tx Xenopus. Following immunomagnetic purification, 1F8- positive spleen cells from control and Tx animals were shown to kill the MHC- deficient tumour target B(_3)B(_7), confirming that this antibody was specific for Xenopus NK cells. Western blotting experiments showed that 1F8, 4D4 and 1G5 identified a doublet of protein bands at 72 and 74 kilodaltons in Xenopus gut lymphoid lysates. Initial attempts to isolate cDNA encoding a Xenopus NK cell surface antigen through immunoscreening a xenopus gut cDNA expression library with antibody 1G5 were unsuccessful as was an attempt to clone a Xenopus homologue of the mammalian NK receptor NKR-Pl by PGR
The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells
No full textSummaryNatural killer (NK) cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells) and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells
[Portion of America] [cartographic material].
No full textSection of a map of America with relief shown pictorially.; Title and author information taken from pencil notes on verso of map.; Rex Nan Kivell Collection Map NK 10432
In Vivo IFN-γ Secretion by NK Cells in Response to Salmonella Typhimurium Requires NLRC4 Inflammasomes
No full textabstract: Natural killer (NK) cells are a critical part of the innate immune defense against viral infections and for the control of tumors. Much less is known about how NK cells contribute to anti-bacterial immunity. NK cell-produced interferon gamma (IFN-γ) contributes to the control of early exponential replication of bacterial pathogens, however the regulation of these events remains poorly resolved. Using a mouse model of invasive Salmonellosis, here we report that the activation of the intracellular danger sensor NLRC4 by Salmonella-derived flagellin within CD11c[superscript +] cells regulates early IFN-γ secretion by NK cells through the provision of interleukin 18 (IL-18), independently of Toll-like receptor (TLR)-signaling. Although IL18-signalling deficient NK cells improved host protection during S. Typhimurium infection, this increased resistance was inferior to that provided by wild-type NK cells. These findings suggest that although NLRC4 inflammasome-driven secretion of IL18 serves as a potent activator of NK cell mediated IFN-γ secretion, IL18-independent NK cell-mediated mechanisms of IFN-γ secretion contribute to in vivo control of Salmonella replication.The article is published at http://journals.plos.org/plosone/article?id=10.1371/journal.pone.009741
HCMV vCXCL1 Binds Several Chemokine Receptors and Preferentially Attracts Neutrophils over NK Cells by Interacting with CXCR2
No full textSummaryHCMV is a highly sophisticated virus that has developed various mechanisms for immune evasion and viral dissemination throughout the body (partially mediated by neutrophils). NK cells play an important role in elimination of HCMV-infected cells. Both neutrophils and NK cells utilize similar sets of chemokine receptors to traffic, to and from, various organs. However, the mechanisms by which HCMV attracts neutrophils and not NK cells are largely unknown. Here, we show a unique viral protein, vCXCL1, which targets three chemokine receptors: CXCR1 and CXCR2 expressed on neutrophils and CXCR1 and CX3CR1 expressed on NK cells. Although vCXCL1 attracted both cell types, neutrophils migrated faster and more efficiently than NK cells through the binding of CXCR2. Therefore, we propose that HCMV has developed vCXCL1 to orchestrate its rapid systemic dissemination through preferential attraction of neutrophils and uses alternative mechanisms to counteract the later attraction of NK cells
HIV-1 Control by NK Cells via Reduced Interaction between KIR2DL2 and HLA-C∗12:02/C∗14:03
No full textSummaryNatural killer (NK) cells control viral infection in part through the interaction between killer cell immunoglobulin-like receptors (KIRs) and their human leukocyte antigen (HLA) ligands. We investigated 504 anti-retroviral (ART)-free Japanese patients chronically infected with HIV-1 and identified two KIR/HLA combinations, KIR2DL2/HLA-C∗12:02 and KIR2DL2/HLA-C∗14:03, that impact suppression of HIV-1 replication. KIR2DL2+ NK cells suppressed viral replication in HLA-C∗14:03+ or HLA-C∗12:02+ cells to a significantly greater extent than did KIR2DL2− NK cells in vitro. Functional analysis showed that the binding between HIV-1-derived peptide and HLA-C∗14:03 or HLA-C∗12:02 influenced KIR2DL2+ NK cell activity through reduced expression of the peptide-HLA (pHLA) complex on the cell surface (i.e., reduced KIR2DL2 ligand expression), rather than through reduced binding affinity of KIR2DL2 to the respective pHLA complexes. Thus, KIR2DL2/HLA-C∗12:02 and KIR2DL2/HLA-C∗14:03 compound genotypes have protective effects on control of HIV-1 through a mechanism involving KIR2DL2-mediated NK cell recognition of virus-infected cells, providing additional understanding of NK cells in HIV-1 infection
Failure of mycoplasma lipoprotein MALP-2 to induce NK cell activation through dendritic cell TLR2
No full textMacrophage-activating lipopeptide 2 (MALP-2), a mycoplasmal diacylated lipopeptide with palmitic acid moiety (Pam2), activates Toll-like receptor (TLR) 2 to induce inflammatory cytokines. TLR2 is known to mature myeloid dendritic cells (mDC) to drive mDC contact-mediated natural killer (NK) cell activation. Here we tested if MALP-2 activates NK cells through stimulation of TLR2 on mDC. Although synthetic MALP-2 with 6 or 14 amino acids (a.a.) stretch (designated as s and f) matured mDC to induce IL-6, IL-12p40 and TNF-α to a similar extent, they far less activated NK cells than Pam2CSK4, a positive control of 6 a.a.-containing diacyl lipopeptide. MALP-2s and f were TLR2/6 agonists and activate the MyD88 pathway similar to Pam2CSK4, but MALP-2s having the CGNNDE sequence acted on mDC TLR2 to barely induce external NK activation. Even the s form, with slightly high induction of IL-6 compared to the f form, barely induced in vivo growth retardation of NK-sensitive implant tumor. Pam2CSK4 and MALP-2 have the common lipid moiety but different peptides, which are crucial for NK cell activation. The results infer that MALP-2 is applicable to a cytokine inducer but not to an adjuvant for antitumor NK immunotherapy
Map of the world taken from an Arabian manuscript of Al Edrisi in the Bodleian Library [cartographic material] /
No full text"The author lived in the 12th Century. The manuscript is of the 15th NB The Arabian geographers represent the world as an egg floating in a Bason of water".; Facsimile.; Map of the world with relief shown pictorially.; Rex Nan Kivell Collection Map NK 4852
T and NK Cells in IL2RG-Deficient Patient 50 Years After Hematopoietic Stem Cell Transplantation
No full textThe first successful European hematopoietic stem cell transplantation (HSCT) was performed in 1968 as treatment in a newborn with IL2RG deficiency using an HLA-identical sibling donor. Because of declining naive T and natural killer (NK) cells, and persistent human papilloma virus (HPV)-induced warts, the patient received a peripheral stem cell boost at the age of 37 years. NK and T cells were assessed before and up to 14 years after the boost by flow cytometry. The boost induced renewed reconstitution of functional NK cells that were 14 years later enriched for CD56dimCD27+ NK cells. T-cell phenotype and T-cell receptor (TCR) repertoire were simultaneously analyzed by including TCR Vβ antibodies in the cytometry panel. Naive T-cell numbers with a diverse TCR Vβ repertoire were increased by the boost. Before and after the boost, clonal expansions with a homogeneous TIGIT and PD-1 phenotype were identified in the CD27− and/or CD28− memory population in the patient, but not in the donor. TRB sequencing was applied on sorted T-cell subsets from blood and on T cells from skin biopsies. Abundant circulating CD8 memory clonotypes with a chronic virus-associated CD57+KLRG1+CX3CR1+ phenotype were also present in warts, but not in healthy skin of the patient, suggesting a link with HPV. In conclusion, we demonstrate in this IL2RG-deficient patient functional NK cells, a diverse and lasting naive T-cell compartment, supported by a stem cell boost, and an oligoclonal memory compartment half a century after HSCT.Pattern Recognition and Bioinformatic
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