ABSTRACT
On encountering a danger signal, dendritic cells (DCs) undergo a complex maturation process and become specialized in antigen presentation. We previously reported that engagement of major histocompatibility complex (MHC) class II molecules located on immature DCs in membrane rafts by lymphocyte activation gene-3 (LAG-3; CD223) leads to DC maturation. In contrast, exposure of DCs to class II-specific monoclonal antibodies (mAbs) did not lead to maturation. Here, we have investigated the signal transduction pathways involved in the LAG-3-induced maturation of human monocyte-derived DCs. We first show that areas of raft aggregation (both cholesterol rich and CDw78 microdomains) could be visualized using a soluble LAG-3 protein and confocal microscopy. Engagement of class II molecules by both its natural ligand LAG-3 and class II mAb induces rapid protein phosphorylation of phospholipase Cgamma2 (PLCgamma2) and p72syk as well as activation of phosphatidyl inositol 3-kinase/Akt, p42/44 extracellular signal-regulated protein kinase, and p38 mitogen-activated protein kinase pathways. Studies using inhibitors demonstrate that these 3 pathways are all important in inducing the maturation process of LAG-3-stimulated DCs. When class II molecules were ligated with LAG-3 versus specific antibody, differences in the phosphorylation pattern of c-Akt were observed. Thus, MHC class II signaling in DCs involves several pathways that have to be finely regulated to lead to cell activation and maturation.
Subject(s)
Antigens, CD , Dendritic Cells/enzymology , Histocompatibility Antigens Class II/metabolism , Membrane Proteins/metabolism , Protein Serine-Threonine Kinases , Signal Transduction/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , Cross-Linking Reagents/metabolism , Dendritic Cells/immunology , Down-Regulation/drug effects , Down-Regulation/immunology , Enzyme Activation/immunology , Enzyme Inhibitors/pharmacology , Enzyme Precursors/antagonists & inhibitors , Enzyme Precursors/metabolism , Humans , Immunophenotyping , Intracellular Signaling Peptides and Proteins , Ligands , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Monocytes/immunology , Monocytes/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phospholipase C gamma , Phosphorylation , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Stilbenes/pharmacology , Syk Kinase , Type C Phospholipases/metabolism , Tyrosine/metabolism , p38 Mitogen-Activated Protein Kinases , Lymphocyte Activation Gene 3 ProteinABSTRACT
Lymphocyte activation gene-3 (LAG-3) is an MHC class II ligand expressed on activated T and NK cells. A LAG-3Ig fusion protein has been used in mice as an adjuvant protein to induce antitumor responses and specific CD8 and CD4 Th1 responses to nominal Ags. In this work we report on the effect of LAG-3Ig on the maturation and activation of human monocyte-derived dendritic cells (DC). LAG-3Ig binds MHC class II molecules expressed in plasma membrane lipid rafts on immature human DC and induces rapid morphological changes, including the formation of dendritic projections. LAG-3Ig markedly up-regulates the expression of costimulatory molecules and the production of IL-12 and TNF-alpha. Consistent with this effect on DC maturation, LAG-3Ig disables DC in their capacity to capture soluble Ags. These events are associated with the acquisition of professional APC function, because LAG-3Ig increases the capacity of DC to stimulate the proliferation and IFN-gamma response by allogeneic T cells. These effects were not observed when using ligation of MHC class II by specific mAb. Class II-mediated signals induced by a natural ligand, LAG-3, lead to complete maturation of DC, which acquire the capacity to trigger naive T cells and drive polarized Th1 responses.
