targeting of the adaptor molecule DAP12 in mice caused abnormal distribution and impaired antigen presentation capacity of dendritic cells (DCs). by yet another signaling pathway which involves the adaptor molecule DAP12 (also called KARAP). DAP12 is associated with several NK and myeloid cells activating receptors 434445464748495051525354. Like FcRγ DAP12 contains a cytoplasmic ITAM recruits the PTKs ZAP70 and p72/syk and promotes activation of ERK 44455556. Knock-in mice bearing a nonfunctional mutation within the ITAM of DAP12 showed a dramatic accumulation of DCs in mucocutaneous epithelia and were resistant to hapten-specific contact sensitivity 57. In addition DAP12-deficient mice were resistant to experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin oligodendrocyte glycoprotein PF-03814735 peptide 58. These phenotypes suggested a role of DAP12 in regulating migration and antigen presentation capacity of DCs. Three DAP12-associated receptors have been identified in myeloid cells. One of these myeloid DAP12-associating lectin-1 (MDL-1) is a member of the C-type lectin superfamily 50. The others signal-regulatory protein β (SIRP-β) and triggering receptor expressed on myeloid cells-1 (TREM-1) belong to the Ig superfamily 5359. TREM-1 is preferentially expressed on neutrophils and a subset of blood monocytes 53. SIRP-β and MDL-1 are mainly expressed on blood monocytes and macrophages 5060. When monocytes are differentiated toward DCs by culturing them in vitro PF-03814735 in the presence TRKA of GM-CSF and IL-4 expression of MDL-1 SIRP-β and TREM-1 PF-03814735 is completely downregulated 505360. Recently we have cloned a cell surface receptor distantly related to TREM-1 called TREM-2. TREM-2 is a member PF-03814735 of the Ig-superfamily characterized by a single V-type PF-03814735 extracellular domain a transmembrane region with a charged residue of lysine and a short cytoplasmic tail with no signaling motifs 53. Here we found that TREM-2 is associated with DAP12 and in contrast to TREM-1 SIRP-β and MDL-1 is not expressed on monocytes but it is strongly upregulated on human DCs derived in vitro from monocytes. This observation provided the opportunity to investigate the role of TREM2/DAP12-mediated signaling pathways in DC migration and maturation. Materials and Methods Production of TREM-2 Human IgM Fusion Protein. Soluble TREM-2 was produced as a chimeric protein consisting of TREM-2 extracellular domain and human IgM constant regions (TREM-2 human IgM [TREM-2-HuIgM]) as previously described 61. TREM-2 extracellular domain was amplified from the cloned full length cDNA by polymerase chain reaction using the following oligonucleotides: 5′-ACTCTGCTTCTGCCCTTGGCTGGGG 3 Purification of TREM-2-HuIgM from culture supernatants was performed by affinity chromatography on Sepharose-coupled mouse anti-human IgM mAb (Sigma-Aldrich) according to manufacturer’s protocols. Transfections. 293 cells were transiently transfected with a cDNA encoding human TREM-2 as a FLAG peptide NH2-terminal fusion protein (Eastman Kodak Co.) using cytofectene (Bio-Rad Laboratories). Production and Modifications of Anti-TREM-2 and Control mAbs. 6 BALB/c mice (Iffa-Credo) were immunized with purified TREM-2-HuIgM. Spleen cells were fused with the SP2/0 myeloma cells and hybridoma supernatants were screened by ELISA using TREM-2-HuIgM as capturing protein and human-adsorbed horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (BD PharMingen) as detecting Ab. ELISA-positive hybridoma supernatants were then tested by flow cytometry for staining 293 cells expressing FLAG-tagged TREM-2. mAb 29E3 (anti-TREM-2 IgG1..