Michael R. Daws

Pattern Recognition by TREM-2: Binding of Anionic Ligands

We recently described the cloning of murine triggering receptor expressed by myeloid cells (TREM) 2, a single Ig domain DNAX adaptor protein 12-associated receptor expressed by cells of the myeloid lineage. In this study, we describe the identification of ligands for TREM-2 on both bacteria and mammalian cells. First, by using a TREM-2A/IgG1-Fc fusion protein, we demonstrate specific binding to a number of Gram-negative and Gram-positive bacteria and to yeast. Furthermore, we show that fluorescently labeled Escherichia coli and Staphylococcus aureus bind specifically to TREM-2-transfected cells. The binding of TREM-2A/Ig fusion protein to E. coli can be inhibited by the bacterial products LPS, lipoteichoic acid, and peptidoglycan. Additionally, binding can be inhibited by a number of other anionic carbohydrate molecules, including dextran sulfate, suggesting that ligand recognition is based partly on charge. Using a sensitive reporter assay, we demonstrate activation of a TREM-2A/CD3ζ chimeric receptor by both bacteria and dextran sulfate. Finally, we demonstrate binding of TREM-2A/Ig fusion to a series of human astrocytoma lines but not to a variety of other cell lines. The binding to astrocytomas, like binding to bacteria, is inhibited by anionic bacterial products, suggesting either a similar charge-based ligand recognition method or overlapping binding sites for recognition of self- and pathogen-expressed ligands.

Cloning and characterization of a novel mouse myeloid DAP12-associated receptor family.

The presence of a negatively charged residue in the transmembrane domain of DAP12 precludes its cell surface expression in the absence of a partner receptor containing a positive charge in its transmembrane domain. We utilized this property of DAP12 to screen a BALB / c macrophage cDNA library for novel molecules that induce cell surface expression of DAP12. By this method, we cloned a cell surface receptor with a single Ig (V) domain, a transmembrane lysine residue, and a short cytoplasmic domain. By homology screening of BALB / c macrophage libraries, we identified a second cDNA for a highly homologous receptor. These receptors appear to be the mouse orthologues of a recently identified human cDNA, TREM‐2, so we have designated the receptors as mouse TREM‐2a and TREM‐2b. By Northern blotting, transcripts for TREM‐2 were found in each of three macrophage cell lines but not in a variety of other hematopoietic cell lines. We further demonstrate that TREM‐2a is associated with endogenous DAP12 in macrophage cells, and cross‐linking of TREM‐2a on the surface of macrophages leads to the release of nitric oxide. Our studies define TREM‐2 as a receptor family in mouse macrophages and demonstrate the capacity of these receptors to activate macrophage function through DAP12.

TIM-2 is expressed on B cells and in liver and kidney and is a receptor for H-ferritin endocytosis

T cell immunoglobulin-domain and mucin-domain (TIM) proteins constitute a receptor family that was identified first on kidney and liver cells; recently it was also shown to be expressed on T cells. TIM-1 and -3 receptors denote different subsets of T cells and have distinct regulatory effects on T cell function. Ferritin is a spherical protein complex that is formed by 24 subunits of H- and L-ferritin. Ferritin stores iron atoms intracellularly, but it also circulates. H-ferritin, but not L-ferritin, shows saturable binding to subsets of human T and B cells, and its expression is increased in response to inflammation. We demonstrate that mouse TIM-2 is expressed on all splenic B cells, with increased levels on germinal center B cells. TIM-2 also is expressed in the liver, especially in bile duct epithelial cells, and in renal tubule cells. We further demonstrate that TIM-2 is a receptor for H-ferritin, but not for L-ferritin, and expression of TIM-2 permits the cellular uptake of H-ferritin into endosomes. This is the first identification of a receptor for ferritin and reveals a new role for TIM-2.

Phage versus Phagemid Libraries for Generation of Human Monoclonal Antibodies

Non-immune (naïve) phage antibody libraries have become an important source of antibodies for reagent, diagnostic, and therapeutic use. To date, reported naïve libraries have been constructed in phagemid vectors as fusions to pIII, yielding primarily single copy (monovalent) display of antibody fragments. For this work, we subcloned the single chain Fv (scFv) gene repertoire from a naïve phagemid antibody library into a true phage vector to create a multivalently displayed scFv phage library. Compared to monovalently displayed scFv, multivalent phage display resulted in improved efficiency of display as well as antibody selection. A greater number of antibodies were obtained and at earlier rounds of selection. Such increased efficiency allows the screening for binding antibodies after a single round of selection, greatly facilitating automation. Expression levels of antigen-binding scFv were also higher than from the phagemid library. In contrast, the affinities of scFv from the phage library were lower than from the phagemid library. This could be overcome by utilizing the scFv in a multivalent format, by affinity maturation, or by converting the library to monovalent display after the first round of selection.

TREM2, a DAP12-associated receptor, regulates osteoclast differentiation and function

Deficiency of the signaling adapter protein DAP12 or its associated receptor TREM2 is associated with abnormal OC development in humans. Here we examine the role of TREM2 in mouse OC development and function, including migration and resorption in vitro. These results provide new evidence that TREM2 regulates OC function independent of its effects on multinucleated OC differentiation.

The signaling adapter protein DAP12 regulates multinucleation during osteoclast development

Deficiency of the signaling adapter protein DAP12 is associated with bony abnormalities in both mice and humans. We identify specific DAP12‐associated receptors expressed by osteoclasts and examine function of DAP12 in murine osteoclasts in vivo and in vitro. These data show a new role for DAP12 signaling in regulating formation of multinucleated osteoclasts.

CMRF-35-like molecule-1, a novel mouse myeloid receptor, can inhibit osteoclast formation

By homology to triggering receptor expressed by myeloid cells-2, we screened the mouse expressed sequence tag database and isolated a new single Ig domain receptor, which we have expressed and characterized. The receptor is most similar in sequence to the human CMRF-35 receptor, and thus we have named it CMRF-35-like molecule (CLM)-1. By screening the mouse genome, we determined that CLM-1 was part of a multigene family located on a small segment of mouse chromosome 11. Each contains a single Ig domain, and they are expressed mainly in cells of the myeloid lineage. CLM-1 contains multiple cytoplasmic tyrosine residues, including two that lie in consensus immunoreceptor tyrosine-based inhibitory motifs, and we demonstrate that CLM-1 can associate with Src-homology 2 containing phosphatase-1. Expression of CLM-1 mRNA is down-regulated by treatment with receptor activator of NF-κB ligand (RANKL), a cytokine that drives osteoclast formation. Furthermore, expression of CLM-1 in the osteoclastogenic cell line RAW (RAW.CLM-1) prevents osteoclastogenesis induced by RANKL and TGF-β. RAW.CLM-1 cells fail to multinucleate and do not up-regulate calcitonin receptor, but they express tartrate-resistant acid phosphatase, cathepsin K, and β3 integrin, suggesting that osteoclastogenesis is blocked at a late-intermediate stage. Thus, we define a new family of myeloid receptors, and demonstrate that the first member of this family, CLM-1, is an inhibitory receptor, able to block osteoclastogenesis.

Characterization of TREM-3, an activating receptor on mouse macrophages: definition of a family of single Ig domain receptors on mouse chromosome 17

We recently reported the cloning of two triggering receptors expressed by myeloid cells (TREM), TREM‐2a and TREM‐2b, which are highly homologous to each other. These receptors associate with DAP12, and ligation of TREM‐2 on the surface of macrophages leads to the release of nitric oxide. Using the immunoglobulin (Ig) domain of TREM‐2 to screen a mouse EST database we have isolated a novel receptor, derived from a WEHI‐3 macrophage library, which shows homology to TREM‐2 (20%). The DNA sequence of this receptor has been submitted to Genbank with the name TREM‐3. The predicted amino acid sequence contains a single Ig domain and a transmembrane lysine residue. We found transcripts for TREM‐3 in two macrophage cell lines (RAW264.7 and MT2) but not in P388D1 macrophage cells. TREM‐3 transcripts could also be detected at low levels in T cell lines, but were not detectable in NK, B cell, or mast cell lines. Furthermore, in macrophage cells, transcripts for TREM‐3 were up‐regulated by LPS, but were down‐regulated by IFN‐γ. Like TREM‐1 and TREM‐2, TREM‐3 signals through DAP12, and when TREM‐3 is transfected into an NK cell line it mediates redirected lysis. Thus, TREM‐3 functions as an activating receptor. Analysis of the mouse genome reveals that the gene for TREM‐3 lies adjacent to the gene for TREM‐1 and in close proximity to a number of other single Ig domain receptors, including TREM‐2. Thus, TREM‐3 is a novel member of a family of immunoglobulin receptors that form an innate immune gene complex on chromosome 17.

Mincle, the receptor for mycobacterial cord factor, forms a functional receptor complex with MCL and FcεRI-γ.

Upon receptor activation, the myeloid C‐type lectin receptor Mincle signals via the Syk‐CARD9‐Bcl10‐MALT1 pathway. It does so by recruiting the ITAM‐bearing FcεRI‐γ. The related receptor macrophage C‐type Lectin (MCL) has also been shown to be associated with Syk and to be dependent upon this signaling axis. We have previously shown that MCL co‐precipitates with FcεRI‐γ, but were unable to show a direct association, suggesting that MCL associates with FcεRI‐γ via another molecule. Here, we have used rat primary cells and cell lines to investigate this missing link. A combination of flow cytometric and biochemical analysis showed that Mincle and MCL form heteromers on the cell surface. Furthermore, association with MCL and FcεRI‐γ increased Mincle expression and enhanced phagocytosis of Ab‐coated beads. The results presented in this paper suggest that the Mincle/MCL/FcεRI‐γ complex is the functionally optimal form for these C‐type lectin receptors on the surface of myeloid cells.

Physiologic functions of activating natural killer (NK) complex-encoded receptors on NK cells.

Summary: Natural killer (NK) cells express a superfamily of surface proteins that share common structural features: dimeric type II integral membrane proteins with extracellular domains resembling C‐type lectins. These receptors are encoded by a single genetic region called the NK complex (NKC). The NKC encompasses several families of genes including NKR‐P1, Ly‐49, CD94/NKG2, and NKG2D. Different NKC‐encoded receptors have been shown to activate or to inhibit NK‐cell function, and different receptors within the same family can have opposing functions. Within an individual NK cell, inhibitory receptors typically predominate over stimulatory receptors, calling into question the teleologic requirement or physiologic significance of lectin‐like activating receptors in NK cells. Despite the widespread expression of inhibitory receptors, however, subtle features of activating receptor biology enable them to stimulate effector functions in vivo and in vitro. Activating receptors and inhibitory receptors differ in their subset expression, in their structural constraints for binding to common ligands, in their ligand repertoires, and in that divergent families of activating receptors utilize different signaling pathways. These subset, binding, repertoire, and signaling diversities may allow activating receptors to manifest their effects in spite of inhibitory receptor functions during pathologic conditions in vivo. In this review, we will present a detailed analysis of the data supporting this hypothesis with particular relevance toward physiologic NK‐cell functions.