9th Meeting of The Society for Natural Immunity November 4-8, 2005 nk hawaii Poipu Beach, Kauai




Название9th Meeting of The Society for Natural Immunity November 4-8, 2005 nk hawaii Poipu Beach, Kauai
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9th Meeting of The Society for Natural Immunity

November 4-8, 2005

NK Hawaii -- Poipu Beach, Kauai




A001

Lectin-Like Transcript 1 (LLT1) is a ligand for CD161 receptor. Hatice Aldemir1, Virginie Prodhomme1, Marie-Jeanne Dumaurier1, Christelle Retiere2, Julie Cazareth1, Franck Bihl1, and Veronique M. Braud1. 1CNRS UMR6097, University of Nice-Sophia Antipolis, 660 Route des Lucioles, 06560 Valbonne, France, 2Etablissement Francais du Sang, 44011 Nantes cedex 1, France.



Human NK cells and subsets of T cells or NKT cells express the orphan C-type lectin receptor CD161 (NKR-P1A) of unknown function. In contrast to rodents that possess several NKR-P1 genes coding for either activating or inhibitory receptors, the nature of signals delivered by the single human NKR-P1A receptor is still to be clarified. We report that the Lectin-Like Transcript 1 (LLT1) molecule is a ligand for CD161 receptor. Engagement of CD161 on NK cells with LLT1 expressed on target cells inhibited NK cell-mediated cytotoxicity and IFN- secretion. Conversely, LLT1/CD161 interaction in the presence of a TCR signal enhanced IFN- production by T cells. These findings identify a novel ligand/receptor pair that differentially regulates NK and T cell functions.


A002

Synapses and nanotubes in innate immunity. Catarina R. Almeida, Bjorn Önfelt, Shlomo Nedvetzki, Stefanie Sowinski, Bebhinn Treanor and Daniel M Davis (with numerous collaborators). Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.

Micrometer-scale segregation of proteins across an intercellular contact is the hallmark characteristic of an immunological synapse (IS) and here, we report that the extent of segregation of ICAM-1 from MHC class I protein at the NK cell IS varies with the level of target cell expression of MHC class I protein. At NK cell synapses with target cells expressing low levels of MHC protein (i.e. 104/cell surface), ICAM-1 and MHC protein are largely co-localized, whereas at synapses involving target cells expressing high levels of MHC protein (105/cell surface), clusters of ICAM-1 and MHC protein are clearly segregated. In addition, with target cells expressing a low amount of HLA-C, a multi-focal patterning of MHC protein occurs at the NK cell IS whereas for higher levels of target cell expression, patterning of MHC protein was homogeneous, ring-shaped, or containing multiple exclusions. Thus, supramolecular patterning and segregation of proteins at an intercellular contact reflects protein expression levels and therefore, could be used generally to report such information between cells.

After disassembly of the IS, membrane tethers or nanotubes leave cells connected for some time. Here, we demonstrate the transport of vesicles and other cargo within membrane nanotubes that connect various immune cells. Some of these nanotubular connections contain both f-actin and tubulin while others contain only f-actin, with relative frequencies that depend on cell type. Lipid vesicles were observed to move within a subset of tubes in a step-wise and ATP-dependent manner. Thus, nanotubular connections between cells are more complex than simple ubiquitous membrane tethers and can traffic different cargoes in a controllable manner.

Finally, we report that after seven days of co-culture with autologous mature macrophages, NK cells proliferate, secrete IFN-, and increase expression of activating receptors NKG2D and NKp44. After co-culture, NK cells show increased cytotoxicity to susceptible target cells. This activation of NK cells is dependent on the intercellular contact with macrophages where ICAM-1, 2B4 and macrophage f-actin accumulate. Activation of macrophages by LPS induces transcription of ULBP1, 2 and 3, and renders macrophages directly susceptible to NK cell cytotoxicity via NKG2D recognition. At this cytolytic NK cell/macrophage immunological synapse, NK cell f-actin and macrophage ICAM-1 often accumulate within a peripheral ring at the synapse around a central cluster of NKG2D and activating adaptors DAP10 and CD3. Thus, macrophages can augment NK cell effector functions whereas LPS-activated macrophages are directly lysed; the different outcomes correlating with distinct NK cell/macrophage immune synapses.

A003

Dramatic alterations of NK cell subsets and function starting in Acute HIV-1 infection.

Galit Alter, Nickolas Teigen, and Marcus Altfeld. Massachusetts General Hospital, 149 13th Street, Room 6613, Charlestown MA 02129 USA.


NK cells are critical in the first line defense against viral infections. Here we characterized for the first time the phenotypic and functional evolution of the NK cell compartment by multiparameter flow-cytometry starting in acute HIV-1 infection. Acute HIV-1 infection was associated with elevated NK cell numbers, an expansion of CD3negCD56dimCD16pos NK cells and an early depletion of CD3negCD56brightCD16neg NK cells. Ongoing viral replication resulted in a secondary depletion of CD3negCD56dimCD16pos NK cells with a paralleled increase in functionally anergic CD3negCD56negCD16pos NK cells. While NK cell activity was elevated in acute HIV-1 infection following stimulation with MHC devoid target cells, maximal NK cell response to mitogens was already significantly reduced in acute infection. The increased NK cell activity to MHC devoid target cells early in infection was associated with increased expression of KIR on NK cells. During the first year of infection, NK cell numbers, function, perforin expression, and cytolytic activity following stimulation with MHC devoid target cells decreased to levels observed in chronically HIV-1 infected subjects. This was associated with the accumulation of functionally anergic CD3negCD56negCD16pos NK cells, expressing high levels of SHIP-1 and reduced levels of perforin. Taken together, these data demonstrate significant perturbations in NK cell distribution and function as early as acute HIV-1 infection, which may have important consequences on the subsequent immune control of opportunistic infections, as well as HIV-1 replication itself.

A004

Phosphorylation of a KIR by protein kinase C impacts on receptor expression on the surface of NK cells. Diana A. Alvarez Arias and Kerry S. Campbell, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA


The KIR3DL1 (3DL1) inhibitory receptor negatively regulates human NK cell activation by recruiting the SHP1 and SHP2 protein tyrosine phosphatases following phosphorylation of two cytoplasmic tyrosine residues. Remarkably, phosphoamino acid analysis of 3DL1 from 32P-orthophosphate-labelled primary NK and NK-92 cells revealed strong constitutive phosphorylation on serine (S) and weak phosphorylation on threonine (T). Truncation mapping in NK-92 cells and in vitro phosphorylation analysis identified S364 and S367 as casein kinase II phosphorylation sites and S394 and T386 as protein kinase C (PKC) sites. PKC stimulation with phorbol ester did not increase 3DL1 phosphorylation, indicating that the S/T phosphorylation is constitutive and stable. To determine whether serine phosphorylation plays a role in the receptor function or expression/turnover, we created NK cell lines containing S/T to alanine (A) mutants of 3DL1. None of these mutations affected the inhibitory capacity of 3DL1 in cytotoxicity assays. However, the 3DL1-S394A mutant exhibited an increased level of surface expression in NK-92 cells and an increased turnover rate relative to the wild type receptor. In contrast, mutation of S394 to a phosphomimetic residue, aspartic acid, decreased 3DL1 expression and turnover. Our results indicate that serine phosphorylation of 3DL1 by PKC regulates surface expression in NK cells.

Supported by NIH grants R01-CA083859 and CA-09035.

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