Human cytomegalovirus glycoprotein UL16 causes intracellular sequestration of NKG2D ligands, protecting against natural killer cell cytotoxicity.

The activating receptor, NKG2D, is expressed on a variety of immune effector cells and recognizes divergent families of major histocompatibility complex (MHC) class I-related ligands, including the MIC and ULBP proteins. Infection, stress, or transformation can induce NKG2D ligand expression, resulting in effector cell activation and killing of the ligand-expressing target cell. The human cytomegalovirus (HCMV) membrane glycoprotein, UL16, binds to three of the five known ligands for human NKG2D. UL16 is retained in the endoplasmic reticulum and cis-Golgi apparatus of cells and causes MICB to be similarly retained and stabilized within cells. Coexpression of UL16 markedly reduces cell surface levels of MICB, ULBP1, and ULBP2, and decreases susceptibility to natural killer cell-mediated cytotoxicity. Domain swapping experiments demonstrate that the transmembrane and cytoplasmic domains of UL16 are important for intracellular retention of UL16, whereas the ectodomain of UL16 participates in down-regulation of NKG2D ligands. The intracellular sequestration of NKG2D ligands by UL16 represents a novel HCMV immune evasion mechanism to add to the well-documented viral strategies directed against antigen presentation by classical MHC molecules.

Interleukin 18 is a primary mediator of the inflammation associated with dextran sulphate sodium induced colitis: blocking interleukin 18 attenuates intestinal damage.

BACKGROUND AND AIMS: Persistent inflammation observed in inflammatory bowel disease may be the consequence of an increased or aberrant immune response to normal gut constituents or an overall immune dysregulation and imbalance. Cytokines play an important role in immune regulation and interleukin 18 (IL-18) is one such cytokine that has emerged as being instrumental in driving CD4+ T cell responses towards a Th1-type. IL-18 can also directly mediate inflammation, moderate interleukin 1 activity, and can act on cell types other than T cells. It has been reported recently that IL-18 mRNA and protein are upregulated in gut tissue from IBD patients. The aim of this study was to understand more about the role of IL-18 in contributing to the pathology of IBD and to assess whether blocking IL-18 activity may be of therapeutic benefit as a treatment regimen for IBD. METHODS: Mice with dextran sulphate sodium (DSS) induced colitis were treated with recombinant IL-18 binding protein (IL-18bp.Fc), a soluble protein that blocks IL-18 bioactivity. Histopathological analysis was performed and RNA from the large intestine was analysed using the RNase protection assay and gene arrays. RESULTS: IL-18 RNA levels increased very early in the colon during DSS colitis. Treatment of mice with IL-18bp.Fc inhibited IBD associated weight loss and significantly inhibited the intestinal inflammation induced by DSS. IL-18bp.Fc treatment also attenuated mRNA upregulation of multiple proinflammatory cytokine genes, chemokine genes, and matrix metalloprotease genes in the large intestine that are commonly elevated during IBD. CONCLUSIONS: IL-18bp treatment attenuated inflammation during DSS induced colitis in mice. Neutralising IL-18 activity may therefore be of benefit for ameliorating the inflammation associated with human intestinal diseases.

Differentiation of NK1.1+, Ly49+ NK cells from flt3+ multipotent marrow progenitor cells.

To delineate factors involved in NK cell development, we established an in vitro system in which lineage marker (Lin)-, c-kit+, Sca2+ bone marrow cells differentiate into lytic NK1.1+ but Ly49- cells upon culture in IL-7, stem cell factor (SCF), and flt3 ligand (flt3L), followed by IL-15 alone. A comparison of the ability of IL-7, SCF, and flt3L to generate IL-15-responsive precursors suggested that NK progenitors express the receptor for flt3L. In support of this, when Lin-, c-kit+, flt3+ or Lin-, c-kit+, flt3- progenitors were utilized, 3-fold more NK cells arose from the flt3+ than from the flt3- progenitors. Furthermore, NK cells that arose from flt3- progenitors showed an immature NK1.1dim, CD2-, c-kit+ phenotype as compared with the more mature NK1.1bright, CD2+/-, c-kit- phenotype displayed by NK cells derived from flt3+ progenitors. Both progenitors, however, gave rise to NK cells that were Ly49 negative. To test the hypothesis that additional marrow-derived signals are necessary for Ly49 expression on developing NK cells, flt3+ progenitors were grown in IL-7, SCF, and flt3L followed by culture with IL-15 and a marrow-derived stromal cell line. Expression of Ly49 molecules, including those of which the MHC class I ligands were expressed on the stromal or progenitor cells, as well as others of which the known ligands were absent, was induced within 6-13 days. Thus, we have established an in vitro system in which Ly49 expression on developing NK cells can be analyzed and possibly experimentally manipulated.

Cutting edge: expression of functional CD94/NKG2A inhibitory receptors on fetal NK1.1+Ly-49- cells: a possible mechanism of tolerance during NK cell development.

Fetal liver- and thymus-derived NK1.1+ cells do not express known Ly-49 receptors. Despite the absence of Ly-49 inhibitory receptors, fetal and neonatal NK1.1+Ly-49- cells can distinguish between class Ihigh and class Ilow target cells, suggesting the existence of other class I-specific inhibitory receptors. We demonstrate that fetal NK1. 1+Ly-49- cell lysates contain CD94 protein and that a significant proportion of fetal NK cells are bound by Qa1b tetramers. Fetal and adult NK cells efficiently lyse lymphoblasts from Kb-/-Db-/- mice. Qa1b-specific peptides Qdm and HLA-CW4 leader peptide specifically inhibited the lysis of these blasts by adult and fetal NK cells. Qdm peptide also inhibited the lysis of Qa1b-transfected human 721.221 cells by fetal NK cells. Taken together, these results suggest that the CD94/NKG2A receptor complex is the major known inhibitory receptor for class I (Qa1b) molecules on developing fetal NK cells.

Natural killer cell differentiation: insights from knockout and transgenic mouse models and in vitro systems.

In the last few years, the routine development of knockout and transgenic mice and the ease with which rare progenitor populations can be isolated from hematopoietic organs and cultured in vitro has facilitated significant advances in understanding the lineage and development of natural killer (NK) cells. Fluorescence-activated cell sorter analyses have identified a common lymphoid progenitor capable of giving rise to NK, T, and B cells, confirming the lymphoid origin of NK cells. Knockout and transgenic mouse models have pointed to an absolutely critical role for signals sent through the interleukin (IL)-2/15 receptor beta (CD122) chain and common gamma (gamma c) chain for NK development. Such signals are likely relayed inside the cell by the tyrosine kinase Jak3, which associates with gamma c. Recently developed IL-15 and IL-15 receptor alpha knockout mice have pinpointed IL-15 as the mediator of this signal. Other mouse models have indicated an unexpected role for flt3 ligand in early NK-cell development as well as minor roles for stem cell factor and IL-7 in expanding NK-cell progenitor numbers. Finally, in vitro culture systems have proven useful in identifying the point in NK development at which each of these signals is critical.

Development of self-recognition systems in natural killer cells.

Differentiation of NK cells is bone marrow dependent. Although all the factors necessary for NK differentiation are yet to be fully characterized, IL-15 has emerged as the most likely candidate that drives terminal differentiation of NK cells. Other cytokines are needed for the expansion and maintenance of the progenitor population. Although the in vivo role for IL-15 cannot be established without the generation of either IL-15 or IL-15R alpha deficient mice, in vitro data suggests that it is responsible for the generation of lytic, NK1.1+ cells from immature progenitors. So far, it has not been possible to obtain Ly-49+ cells from marrow or fetal-derived progenitor cells in vitro. Stromal cells along with cytokines may be necessary to induce expression of Ly-49 on NK1.1+ cells. Expression of the NK receptors seems to be a sequential process with expression of IL-2/15R beta on progenitor cells occurring first followed by the expression of NK1.1 and then probably Ly-49. The same sequence seems to hold true in vivo as well, Ly-49 surface expression on splenic NK1.1+ cells is first detected 4-6 days after birth, and the frequency of cells expressing Ly-49 receptors reaches adult levels by days 20-24. Despite the lack of expression of Ly-49 receptors by fetal NK1.1+ as well as bone marrow derived NK1.1+ cells, they are able to distinguish between MHC class Ihi and class Ilo targets. This suggests that these NK1.1+Ly-49- cells express non-Ly-49 class I receptors. Efforts in the future need to be focused on elements responsible for the expression of Ly49 on these NK1.1+ cells in order to establish an in vitro system in which establishment of the Ly-49 repertoire can be studied.

Generation of lytic natural killer 1.1+, Ly-49- cells from multipotential murine bone marrow progenitors in a stroma-free culture: definition of cytokine requirements and developmental intermediates.

We have developed a stroma-free culture system in which mouse marrow or thymus cells, known to be enriched for lymphoid progenitors, can be driven to generate natural killer (NK) cells. Culture of lineage marker (Lin)-, c-kit+, Sca2+, interleukin (IL)-2/15Rbeta (CD122)- marrow cells in IL-6, IL-7, stem cell factor (SCF), and flt3 ligand (flt3-L) for 5-6 d followed by IL-15 alone for an additional 4-5 d expanded the starting population 30-40-fold and gave rise to a virtually pure population of NK1.1+, CD3- cells. Preculture in IL-6, IL-7, SCF, and flt3-L was necessary for inducing IL-15 responsiveness in the progenitors because the cells failed to significantly expand when cultured in IL-15 alone from the outset. Although culture of the sorted progenitors in IL-6, IL-7, SCF, and flt3-L for the entire 9-11-d culture period caused significant expansion, no lytic NK1.1+ cells were generated if IL-15 was not added, demonstrating a critical role for IL-15 in NK differentiation. Thus, two distinct populations of NK progenitors, IL-15 unresponsive and IL-15 responsive, have been defined. Similar results were obtained with Lin-, CD44+, CD25-, c-kit+ lymphoid progenitors obtained from adult thymus. The NK cells generated by this protocol lysed the NK-sensitive target YAC-1 and expressed markers of mature NK cells with the notable absence of Ly-49 major histocompatibility complex (MHC) receptors. However, despite the apparent lack of these inhibitory MHC receptors, the NK cells generated could distinguish MHC class I+ from class I- syngeneic targets, suggesting the existence of novel class I receptors.

Phenotypic and functional characterization of long-lived NK cell lines of different maturational status obtained from mouse fetal liver.

Culture of day 14 mouse fetal liver (FL) cells in high dose IL-2, together with appropriate combinations of IL-4 and PMA, resulted in the generation of cell lines, termed FL-A lines, that were phenotypically and functionally indistinguishable from cultured adult splenic NK cell populations with the single important exception that no Ly49-expressing cells were present. By contrast, when FL cells were cultured in low-dose IL-2 alone, a second population of slow-growing NK-like cells, termed FL-B cells, emerged. These cells expressed the NK markers asialoGM1, 10A7, 2B4, and Fc gammaRII/III but differed from FL-A and splenic NK cells in expressing IL-2R alpha and stem cell factor receptor (SCFR) but no B220. Most lines derived in this manner had minimal or no cytolytic activity and only very low levels of NK1.1. However, they could secrete substantial quantities of several lymphokines including IL-3, granulocyte-macrophage (GM)-CSF, TNF-alpha, and, most surprisingly, IL-2. A minority of FL-B lines, typified by line 903, displayed marked cytolytic activity, moderate levels of NK1.1, reduced production of IL-2, and the capacity for accelerated growth in high-dose IL-2. FL-B lines generally expressed mRNA for CD3gamma but not for other CD3 chains, whereas FL-A and fetal thymic (FT) NK lines often expressed mRNA for all four CD3 chains. Despite many similarities to pro-T cells, FL-B cells showed no capacity to differentiate into mature T cells. Taken together, our results suggest that NK lines of different maturity can be obtained from fetal liver, with FL-B lines being the most immature, FL-A lines the most mature, and lines such as FL-B 903 representing an intermediate state of differentiation.

Fetal and neonatal NK1.1+ Ly-49- cells can distinguish between major histocompatibility complex class I(hi) and class I(lo) target cells: evidence for a Ly-49-independent negative signaling receptor.

Natural killer (NK) cell function is regulated by both positive and negative signaling receptors. In adult splenic NK cells, negative signaling has been shown to be mediated by the Ly-49 family receptors. NK1.1- 2B4+ CD3- cells that are phenotypically and functionally similar to adult splenic NK cells can be derived from murine fetal liver and thymus. These cells do not express any known Ly-49 molecules on their surface nor do they contain the known Ly-49 transcripts. Surface expression of Ly-49 molecules is first detected on splenic NK1.1+ cells 4-6 days after birth. Despite the absence of these negative signaling receptors, fetal and neonatal Ly-49- NK cells lyse TAP-/- , major histocompatibility complex (MHC) class I(lo) but not TAP+/+, MHC class I(hi) target cells. This suggests that fetal and neonatal NK cells express negative signaling receptors other than Ly-49 molecules.