Lysosomal V-ATPases in maturing dendritic cells

After stimulation, the immune system`s most potent antigen-presenting cells, dendritic cells (DCs), transform from an immature phenotype specialized for antigen capture into a mature phenotype specialized for the presentation of peptide-MHC complexes (pMHC) and consequently for T cell stimulation. A key mechanism responsible for this alteration is the generalized activation of lysosomal function causing enhanced acidification in mature DCs compared to immature DCs. The more acidic pH in lysosomes of mature DCs augments antigen proteolysis and thus the efficient formation of pMHC. Generally, the interior of lysosomes is acidified by an ATP-dependent proton pump that pumps protons into the lumen of lysosomes thereby decreasing lysosomal pH. Previous data from Dr. Mellmans group indicate that the enhanced lysosomal acidification of mature DCs reflects the assembly of V1 and V0 subunits to functional V-ATPases. Thus, lysosomal function in DCs appears to be specialized for the developmentally regulated processing of internalized antigens. Since the enhanced lysosomal acidification of mature DCs has not yet been investigated in detail, this project is aimed at characterizing the mechanisms regulating lysosomal function during DC maturation in vitro and in vivo. The aims are: 1/ Examination of the kinetics wherby the DCs gain acidification capacity as a function of time after stimulation in vitro. Immature DCs will be prepared from bone marrow precursor cells. DC lysosomes will be labeled with pH- sensitve markers. After stimulation with lipopolysaccharide (LPS), the lysosomal pH of intact cells or isolated lysosomes will be determined as a function of time by FACS or single-organelle flow analysis. 2/ Identification of the molecular features of the V-ATPase and characterization of the ion transport mechanism during DC maturation in vitro. We will use RT-PCR to investigate which V-ATPase subunit isoforms are expressed in DCs and whether this is affected by DC maturation. Immunofluorescence microscopy will be used to identify the subcellular localization of selected subunits. The initial rate of acidification and the ion permeabilities of the lysosomal membrane as a function of DC maturation will be determined using in vitro acidification assays. 3/ Do DCs in peripheral tissues and in lymph nodes have active V-ATPases? DCs will be labeled with fluorochrome coupled antigen and stimulated in vivo by LPS prior to isolation from peripheral tissues and lymph nodes. FACS will be used to measure lysosomal pH in DC populations expressing various levels of costimulatory molecules to determine the maturation state of the DCs.