DC activation abrogates expression of March-I (11, 12), thereby preventing pMHC-II ubiquitination (13, 14) and turnover of pMHC-II complexes that were generated at the time of DC activation (15). Activation of DCs that acquire antigen in their immature state results in a cell that is an extremely potent stimulator of na?ve CD4 T cells and, as a consequence of diminished soluble antigen endocytosis and MHC-II biosynthesis it has been proposed that fully-activated DCs are poor antigen sampling, processing, and presenting cells (2, 16, 17). which immature DCs preferentially present antigens acquired in Rab11a? DM+ ITX3 late endosomes whereas mature DCs use recycling MHC-II to present antigenic peptides acquired in both Rab11a+ early endosomes and Rab11a? endosomes for CD4 T cell activation. Introduction Dendritic cells (DCs) are potent antigen (Ag)-presenting cells that initiate adaptive immune responses (1). ITX3 Resting (immature DCs) constitutively sample their microenvironment and present a diverse repertoire of (mostly self) pMHC-II complexes on their surface (2). After encounter with pathogens or antigens, and in response to inflammatory stimuli, immature DCs become activated and acquire a mature DC phenotype that consists of increased expression of T cell costimulatory molecules and abundant pMHC-II enriched with the peptides that are generated at the time of DC activation (3-5). Immature DCs continuously synthesize MHC-II molecules that are directed to late endosomal antigen processing compartments by their binding to the MHC-II chaperone Invariant chain (Ii) (6). These compartments contain the proteolytic enzymes required for proteolysis of both Ii and internalized antigens and the peptide editor DM that assists in the generation of immunodominant T cell epitopes ITX3 from internalized antigens (7). After their arrival on the immature DC surface, pMHC-II complexes internalize (8, 9), are ubiquitinated by the E3 ubiquitin ligase March-I (10), and are targeted for degradation in lysosomes (9, 11). This process ensures continuous turnover ITX3 of irrelevant pMHC-II in DCs until the cell receives an activation signal. DC activation abrogates expression of March-I (11, 12), thereby preventing pMHC-II ubiquitination (13, 14) and turnover of pMHC-II complexes that were generated at the time of DC activation (15). Activation of DCs that acquire antigen in their immature state results in a cell that is an extremely potent stimulator of na?ve CD4 T cells and, as a consequence of diminished soluble antigen endocytosis and MHC-II biosynthesis it has been proposed that fully-activated DCs are poor antigen sampling, processing, and presenting cells (2, 16, 17). However, receptor-mediated endocytosis and receptor-dependent phagocytosis persist after DC activation (18-21) and there have been reports showing that for 2 h at 4C. The lentivirus pellet was resuspended in 100 L PBS, aliquoted, and frozen at ?80C. For DC transduction, concentrated lentivirus Rab11a shRNA and protamine sulfate (10?g/ml) were added to a culture of bone marrow cells with GM-CSF on day 2 of culture. After culture for an additional Rabbit Polyclonal to EPHA2/3/4 3 days, the medium containing lentivirus was removed and fresh complete DC media containing GM-CSF was added to the cells for an additional 2 days. When indicated, transduced DCs were activated with LPS overnight. Venus-Rab11 lentiviral supernatant was prepared from HEK293T cells and concentrated Rab11 lentivirus was added to bone marrow cells as described above to generate transduced DCs. Puromycin (5 g/ml) was added to the medium in ITX3 the final 24 hr of culture to enrich for transduced cells. Transduced DCs were activated with LPS overnight, stained, and analyzed by immunofluorescence microscopy. Immunofluorescence microscopy DCs were stained with anti-pMHC-II mAb Y3P on ice for 30 min, washed, and then incubated either on ice (to allow visualization of surface pMHC-II) or at 37C for 15 min (to allow visualization of internalized pMHC-II). To reveal only internalized pMHC-II, surface pMHC-II mAb was blocked by incubation of cells with goat anti-mouse IgG on ice for 1 h. Cells were then fixed using 4% paraformaldehyde in PBS, permeabilized using 0.1% saponin in PBS, washed, and incubated with Alexa-fluor-conjugated secondary antibodies to detect pMHC-II mAb. Fixed DCs were plated on poly-L-lysine-coated glass coverslips for 30 min at room temperature for analysis by confocal microscopy. Cells were imaged using a Zeiss LSM 880 confocal microscope using a 63X oil immersion objective lens. Fluorescence intensity of pMHC-II staining was done by masking individual cells and quantitating pixel intensity within the masked area (correcting for variations in cell size) using Zeiss software ZEN 2 (blue release). At least 20 cells in each condition were analyzed and statistical variations between samples were determined using a College students t-test. Colocalization of internalized pMHC-II with endosome markers was determined by.