The Glycophosphatidylinositol Anchor of the MCMV Evasin, m157, Facilitates Optimal Cell Surface Expression and Ly49 Receptor Recognition.

The murine cytomegalovirus-encoded protein m157 is a cognate ligand for both inhibitory and activating receptors expressed by natural killer cells. Additionally, m157 is expressed on the surface of infected cells by a glycophosphatidylinositol (GPI) anchor. Although endogenous GPI-anchored proteins are known to be ligands for the NK cell receptor, NKG2D, the contribution of the GPI anchor for viral m157 ligand function is unknown. To determine whether the GPI anchor for m157 is dispensable for m157 function, we generated m157 variants expressed as transmembrane fusion proteins and tested cells expressing transmembrane m157 for the capacity to activate cognate Ly49 receptors. We found that the GPI anchor is required for high-level cell surface expression of m157, and that the transmembrane m157 ligand retains the capacity to activate reporter cells and NK cells expressing Ly49H, as well as Ly49I(129) reporter cells, but with reduced potency. Importantly, target cells expressing the transmembrane form of m157 were killed less efficiently and failed to mediate Ly49H receptor downregulation on fresh NK cells compared to targets expressing GPI-anchored m157. Taken together, these results show that the GPI anchor for m157 facilitates robust cell surface expression, and that NK cells are sensitive to the altered cell surface expression of this potent viral evasin.

Role of NK cell subsets in organ-specific murine melanoma metastasis.

Tumor metastasis plays a major role in the morbidity and mortality of cancer patients. Among solid tumors that undergo metastasis, there is often a predilection to metastasize to a particular organ with, for example, prostate cancer preferentially metastasizing to bones and colon cancer preferentially metastasizing to the liver. Although many factors are thought to be important in establishing permissiveness for metastasis, the reasons for organ-specific predilection of each tumor are not understood. Using a B16 murine melanoma model, we tested the hypothesis that organ-specific NK cell subsets play a critical role in organ-specific metastasis of this tumor. Melanoma cells, given intravenously, readily colonized the lungs but not the liver. NK cell depletion (either iatrogenically or by using genetically targeted mice) resulted in substantial hepatic metastasis. Analysis of NK cell subsets, defined by the differential expression of a combination of CD27 and CD11b, indicated a significant difference in the distribution of NK cell subsets in the lung and liver with the mature subset being dominant in the lung and the immature subset being dominant in the liver. Several experimental approaches, including adoptive transfer, clearly indicated that the immature hepatic NK cell subset, CD27+ CD11b-, was protective against liver metastasis; this subset mediated its protection by a perforin-dependent cytotoxic mechanism. In contrast, the more mature NK cell subsets were more efficient at reducing pulmonary tumor load. These data indicate that organ-specific immune responses may play a pivotal role in determining the permissiveness of a given organ for the establishment of a metastatic niche.

A dynamic flux in natural killer cell subsets as a function of the duration of alcohol ingestion.

BACKGROUND: Chronic ethanol (EtOH) consumption is associated with a wide variety of immune abnormalities including changes in T cells, B cells, dendritic cells, and natural killer (NK) cells. However, there is conflicting information as to the direction of such immune changes. The hypothesis that was tested in this report is that, for NK cells, the changes can vary as a function of the duration of alcohol ingestion. METHODS: Using the Meadows-Cook murine model of chronic alcohol ingestion, the changes in NK cell function and subset distribution were examined as a function of the duration of alcohol ingestion. RESULTS: Acute alcohol ingestion resulted in decreased number and cytotoxic function of NK cells with no effect on intracellular interferon gamma expression. These abnormalities normalized after 12 to 14 days of alcohol ingestion and there was an increase of NK cell number and cytotoxicity after 8 weeks of continued EtOH ingestion. Ten weeks of continued alcohol consumption results in a significant decrease in the Ly49H+ CD11b+ CD27- splenic NK cell subset; this difference continued to be significant at 30 weeks. CONCLUSIONS: This report may explain some of the conflicting data in the literature that examined NK cell activity in alcoholic patients. It is apparent that various abnormalities can be seen in NK cell activity and subset distribution with the flux being a function of the duration of alcohol ingestion. The demonstration of a decrease in the Ly49H+ subset (which is known to be involved in resisting murine cytomegalovirus infection) may explain the reported increase in susceptibility to some viral infections in chronic alcohol abuse. Another novel finding is that changes of some subsets of NK cells are not evident until at least 10 weeks of continued EtOH consumption.

Glycosylation contributes to variability in expression of murine cytomegalovirus m157 and enhances stability of interaction with the NK-cell receptor Ly49H.

NK cell-mediated resistance to murine cytomegalovirus (MCMV) is controlled by allelic Ly49 receptors, including activating Ly49H (C57BL/6 strain) and inhibitory Ly49I (129 strain), which specifically recognize MCMV m157, a glycosylphosphatidylinositol-linked protein with homology to MHC class I. Although the Ly49 receptors retain significant homology to classic carbohydrate-binding lectins, the role of glycosylation in ligand binding is unclear. Herein, we show that m157 is expressed in multiple, differentially N-glycosylated isoforms in m157-transduced or MCMV-infected cells. We used site-directed mutagenesis to express single and combinatorial asparagine (N)-to-glutamine (Q) mutations at N178, N187, N213, and N267 in myeloid and fibroblast cell lines. Progressive loss of N-linked glycans led to a significant reduction of total cellular m157 abundance, although all variably glycosylated m157 isoforms were expressed at the cell surface and retained the capacity to activate Ly49H(B6) and Ly49I(129) reporter cells and Ly49H(+) NK cells. However, the complete lack of N-linked glycans on m157 destabilized the m157-Ly49H interaction and prevented physical transfer of m157 to Ly49H-expressing cells. Thus, glycosylation on m157 enhances expression and binding to Ly49H, factors that may impact the interaction between NK cells and MCMV in vivo where receptor-ligand interactions are more limiting.

Assessing siRNA pharmacodynamics in a luciferase-expressing mouse.

A significant barrier to the successful general development of small-interfering RNA (siRNA) therapeutics is the ability to deliver them systemically to target organs and cell types. In this study, we have developed a mouse strain that will facilitate the evaluation of the efficacy of siRNA delivery strategies. This strain contains robust ubiquitous expression of firefly luciferase from germ line Cre-mediated recombination of the ROSA26-LSL-Luc allele. We show that luciferase is highly and uniformly expressed in all tissues examined. Using this mouse model, we describe a facile assay that enables the assessment of the pharmacodynamics of a systemically delivered siRNA formulation. These mice can also be used as universal donors, enabling the efficient and sensitive monitoring of cell trafficking or tissue transplantation. The primary advantage of this approach is that siRNA efficacy against a nonessential target can be easily evaluated in any tissue. This strain should generally enhance the ability to rapidly screen, compare and optimize various siRNA formulations for tissue-targeted or -enhanced systemic delivery in a preclinical development setting.

Assessment of natural killer (NK) and NKT cells in murine spleens and livers.

Natural killer (NK) cells part of innate immunity. NK cells have been assigned numerous functions, including the ability to serve as a bridge between innate and adaptive immunity. In evaluating NK cell function, two pathways need to be examined: their ability to kill certain tumors spontaneously and their ability to secrete cytokines, interferon-gamma (IFN-gamma), in particular. Although NK cells are distinct from T lymphocytes, a new lymphocyte subset, termed NKT cell, has been described. NKT cells express surface markers that are unique to NK cells (e.g., NK1.1) as well as markers that are unique to T cells (e.g., CD3). Most NKT cells recognize glycolipids and are thought to play an important immunoregulatory role. This chapter will detail the methodology needed for examination of NK and NKT cells in mice.