MR angiogenesis imaging with Robo4- vs. alphaVbeta3-targeted nanoparticles in a B16/F10 mouse melanoma model.

The primary objective of this study was to utilize MR molecular imaging to compare the 3-dimensional spatial distribution of Robo4 and α(V)ß(3)-integrin as biosignatures of angiogenesis, in a rapidly growing, syngeneic tumor. B16-F10 melanoma-bearing mice were imaged with magnetic resonance (MR; 3.0 T) 11 d postimplantation before and after intravenous administration of either Robo4- or α(V)ß(3)-targeted paramagnetic nanoparticles. The percentage of MR signal-enhanced voxels throughout the tumor volume was low and increased in animals receiving α(V)ß(3)- and Robo4-targeted nanoparticles. Neovascular signal enhancement was predominantly associated with the tumor periphery (i.e., outer 50% of volume). Microscopic examination of tumors coexposed to the Robo4- and α(V)ß(3)-targeted nanoparticles corroborated the MR angiogenesis mapping results and further revealed that Robo4 expression generally colocalized with α(V)ß(3)-integrin. Robo4- and α(V)ß(3)-targeted nanoparticles were compared to irrelevant or nontargeted control groups in all modalities. These results suggest that α(V)ß(3)-integrin and Robo4 are useful biomarkers for noninvasive MR molecular imaging in syngeneic mouse tumors, but α(V)ß(3)-integrin expression was more detectable by MR at 3.0 T than Robo4. Noninvasive, neovascular assessments of the MR signal of Robo4, particularly combined with α(V)ß(3)-integrin expression, may help define tumor character prior to and following cancer therapy.

A novel molecular interaction for the adhesion of follicular CD4 T cells to follicular DC.

Nectins and Nectin-like molecules (Necl) play a critical role in cell polarity within epithelia and in the nervous and reproductive systems. Recently, immune receptors specific for Nectins/Necl have been described. Since the expression and distribution of Nectins/Necl is often subverted during tumorigenesis, it has been suggested that the immune system may use these receptors to recognize and eliminate tumors. Here we describe a novel immunoreceptor, Washington University Cell Adhesion Molecule, which is expressed on human follicular B helper T cells (TFH) and binds a Nectin/Necl family member, the poliovirus receptor (PVR), under both static and flow conditions. Furthermore, we demonstrate that PVR is abundantly expressed by follicular DC (FDC) within the germinal center. These results reveal a novel molecular interaction that mediates adhesion of TFH to FDC and provide the first evidence that immune receptors for Nectins/Necl may be involved the generation of T cell-dependent antibody responses.

Glycoprotein structural genomics: solving the glycosylation problem.

Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the "glycosylation problem" can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis.

Digital-to-biological converter for on-demand production of biologics.

Manufacturing processes for biological molecules in the research laboratory have failed to keep pace with the rapid advances in automization and parellelization. We report the development of a digital-to-biological converter for fully automated, versatile and demand-based production of functional biologics starting from DNA sequence information. Specifically, DNA templates, RNA molecules, proteins and viral particles were produced in an automated fashion from digitally transmitted DNA sequences without human intervention.

The LLT1 receptor induces IFN-gamma production by human natural killer cells.

Natural killer cell functions are regulated by signals through activating and inhibitory receptors. These receptors belong to the immunoglobulin superfamily or the lectin superfamily. We have previously identified a lectin-like transcript, LLT1, expressed in human NK cells. In the present study, we have generated a monoclonal antibody, L9.7, that specifically binds LLT1 receptor and studied the functional role of LLT1 in human NK cells. Binding of mAb L9.7 to surface LLT1 induced IFN-gamma production, but did not modulate cytotoxicity by YT cells, a human NK cell line. We further demonstrate that in resting NK cells as well as in IL-2 activated NK cells LLT1 induced IFN-gamma production, but not cytotoxicity. Excess amounts of L9.7 mAb failed to increase natural or antibody-dependent cell-mediated cytolytic activity, whereas minimal amounts achieved maximal production of IFN-gamma by YT and activated NK cells. These findings further support the separation of signaling pathways that regulate cytotoxicity and IFN-gamma production in resting as well as activated NK cells.

DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors.

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1-deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell-mediated cytotoxicity caused by the paucity of other NK cell-activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

The tumor suppressor TSLC1/NECL-2 triggers NK-cell and CD8+ T-cell responses through the cell-surface receptor CRTAM.

The tumor suppressor in lung cancer-1 (TSLC1) gene is frequently silenced in human lung carcinomas, and its expression suppresses tumorigenesis in nude mice. TSLC1 encodes a cell-surface protein called Necl-2 that belongs to the Nectin and Nectin-like (Necl) family of molecules. Necl-2 mediates epithelial cell junctions by homotypic contacts and/or heterotypic interactions with other Nectins and Necls. Thus, it inhibits tumorigenesis by ensuring that epithelial cells grow in organized layers. Here, we demonstrate that natural killer (NK) cells and CD8+ T cells recognize Necl-2 through a receptor known as class I-restricted T-cell-associated molecule (CRTAM), which is expressed only on activated cells. CRTAM-Necl-2 interactions promote cytotoxicity of NK cells and interferon gamma (IFN-gamma) secretion of CD8+ T cells in vitro as well as NK cell-mediated rejection of tumors expressing Necl-2 in vivo. These results provide evidence for an additional mechanism of tumor suppression mediated by TSLC1 that involves cytotoxic lymphocytes. Furthermore, they reveal Necl-2 as one of the molecular targets that allows the immunosurveillance network to distinguish tumor cells from normal cells.

Adhesion of human T cells to antigen-presenting cells through SIRPbeta2-CD47 interaction costimulates T-cell proliferation.

Signal-regulatory proteins (SIRPs) are transmembrane glycoproteins belonging to the immunoglobulin (Ig) superfamily that are expressed in the immune and central nervous systems. SIRPalpha binds CD47 and inhibits the function of macrophages, dendritic cells, and granulocytes, whereas SIRPbeta1 is an orphan receptor that activates the same cell types. A recently identified third member of the SIRP family, SIRPbeta2, is as yet uncharacterized in terms of expression, specificity, and function. Here, we show that SIRPbeta2 is expressed on T cells and activated natural killer (NK) cells and, like SIRPalpha, binds CD47, mediating cell-cell adhesion. Consequently, engagement of SIRPbeta2 on T cells by CD47 on antigen-presenting cells results in enhanced antigen-specific T-cell proliferation.

Molecular and functional characterization of a CS1 (CRACC) splice variant expressed in human NK cells that does not contain immunoreceptor tyrosine-based switch motifs.

CS1 (CRACC, novel Ly9) is a novel member of the CD2 family expressed on natural killer (NK), T and stimulated B cells. Although the cytoplasmic domain of CS1 contains immunoreceptor tyrosine-based switch motifs (ITSM), which enables to recruite signaling lymphocyte activation molecule (SLAM)-associated protein (SAP/SH2D1A), it activates NK cells in the absence of a functional SAP. CS1 is a self ligand and homophilic interaction of CS1 regulates NK cell cytolytic activity. Here we have identified a novel splice variant of CS1 (CS1-S), which lacks ITSM. Human NK cells express mRNA for both wild-type CS1 (CS1-L) and CS1-S and their expression level remained steady upon various stimulations. To determine the function of each isoform, cDNA for CS1-L and CS1-S were transfected into the rat NK cell line RNK-16 and functionally tested using redirected cytotoxicity assays and calcium flux experiments. CS1-L was able to mediate redirected cytotoxicity of P815 target cells in the presence of monoclonal antibody against CS1 and a rise in intracellular calcium within RNK-16 cells, suggesting that CS1-L is an activating receptor, whereas CS1-S showed no effects. Interestingly, SAP associated with unstimulated CS1-L and dissociated upon pervanadate stimulation. These results indicate that CS1-L and CS1-S may differentially regulate human NK cell functions.