NK-cell function is regulated by the integration of signals received from activating and inhibitory receptors. Here we show that a novel immune receptor, T-cell Ig and mucin-containing domain-3 (Tim-3), is expressed on resting human NK cells and is up-regulated on activation. The NK92 NK-cell line engineered to overexpress Tim-3 showed a marked increase in IFN-␥ production in the presence of soluble rhGal-9 or Raji tumor cells engineered to express Gal-9. The Tim-3 ؉ population of low-dose IL-12/IL-18-activated primary NK cells significantly increased IFN-␥ production in response to soluble rhGal-9, Gal-9 presented by cell lines, and primary acute myelogenous leukemia (AML) targets that endogenously express Gal-9. This effect is highly specific as Tim-3 Ab blockade significantly decreased IFN-␥ production, and Tim-3 cross-linking induced ERK activation and degradation of IB␣. Exposure to Gal-9-expressing target cells had little effect on CD107a degranulation. Reconstituted NK cells obtained from patients after hematopoietic cell transplantation had diminished expression of Tim-3 compared with paired donors. This observation correlates with the known IFN-␥ defect seen early posttransplantation. In conclusion, we show that Tim-3 functions as a human NK-cell coreceptor to enhance IFN-␥ production, which has important implications for control of infectious disease and cancer. (Blood. 2012;119(13): 3064-3072) IntroductionHuman NK cells are lymphocytes that develop from hematopoietic progenitor cells in the BM and secondary lymphoid tissues. 1 Peripheral blood (PB) NK cells are phenotypically defined as expressing the surface receptor CD56 (neural cell adhesion molecule [NCAM]) and lacking expression of CD3. 2 They mediate their function through the exocytosis of lytic granules that contain perforin and granzymes, 3 the expression of death receptor ligands, 4 the expression of FcR␥III, which mediates Ab-dependent cellmediated cytotoxicity, 5 and the secretion of cytokines and chemokines. 6 As a result, NK cells take part in both the innate and adaptive immune systems and play important roles in the control of viral infections, pregnancy, tumor immunosurveillance, and hematopoietic cell transplantation (HCT). 7,8 The ability of NK cells to differentiate normal healthy cells (self) from virally infected or malignantly transformed cells (nonself) is regulated by a sophisticated repertoire of cell-surface receptors that control their activation, proliferation, and effector functions. [9][10][11] Recently, a novel receptor, T-cell Ig and mucincontaining domain-3 (Tim-3), has been described to have various roles in immune regulation and is highly expressed on NK cells in mice and humans. [12][13][14][15][16][17] Tim-3 is a type I membrane glycoprotein that was first identified as a cell marker of terminally differentiated CD4 ϩ Th1 cells. 18 Galectin-9 (Gal-9), a 40-kDa S-type ␤-galactoside binding lectin, is a known ligand for Tim-3 and is highly expressed in tissues of the immune system, such as the BM, lymph nodes, thymu...

Using a novel qEBV assay, we demonstrated that young adults with primary EBV infection rapidly cleared virus from blood but not from the oropharynx. High oral concentrations of EBV in asymptomatic persons who have resumed normal activities support the concept that infectious mononucleosis is most likely acquired by kissing.

Protein-protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein-protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying crosslinked peptides from single LC/MS/MS datasets, without using specialized crosslinkers or custom-written software. To achieve this, each peptide in the pair of crosslinked peptides was considered to be “post-translationally” modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of crosslinked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich datasets for crosslinked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native crosslinks in proteins. The pipeline was validated by characterizing the known interactions in chemically crosslinked CYP2E1-b5 complex. Utility of this method in identifying native crosslinks was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.

The transcription factor forkhead box M1 (FOXM1) is a validated oncoprotein in solid cancers, but its role in malignant plasma cell tumors such as multiple myeloma (MM) is unknown. We analyzed publicly available MM datasets and found that overexpression of FOXM1 prognosticates inferior outcome in a subset (~15%) of newly diagnosed cases, particularly patients with high-risk disease based on global gene expression changes. Follow-up studies using human myeloma cell lines (HMCLs) as the principal experimental model system demonstrated that enforced expression of FOXM1 increased growth, survival and clonogenicity of myeloma cells, whereas knockdown of FOXM1 abolished these features. In agreement with that, constitutive up-regulation of FOXM1 promoted HMCL xenografts in laboratory mice, whereas inducible knockdown of FOXM1 led to growth inhibition. Expression of cyclin dependent kinase 6 (CDK6) and NIMA-related kinase 2 (NEK2) was co-regulated with FOXM1 in both HMCLs and myeloma patient samples, suggesting interaction of these 3 genes in a genetic network that may lend itself to targeting with small-drug inhibitors for new approaches to myeloma therapy and prevention. These results establish FOXM1 as high-risk myeloma gene and provide support for the design and testing of FOXM1-targeted therapies specifically for the FOXM1High subset of myeloma.

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