Monocytes immunoselected via the novel monocyte specific molecule, CD300e, differentiate into active migratory dendritic cells.

Monocytes, immunoselected using MMRI-1, a monoclonal antibody specific for CD300e, were used to generate dendritic cells (DC). These CD300e immunoselected monocyte-derived DC (MoDC) were compared phenotypically and functionally to CD14 immunoselected MoDC. CD300e and CD14 immunoselected mature MoDC expressed similar levels of the DC marker, CD83 and costimulatory molecules, CD80, CD86, and CD40. Both preparations took up soluble antigen with similar efficiency by pinocytosis and receptor mediated uptake. The CD300e and CD14 immunoselected MoDC also induced comparable CD4+ T lymphocyte allogeneic responses and recall responses to tetanus toxoid. Similar magnitude CD8 T lymphocyte responses to the naive antigen, MART-1 and the recall antigen, FMP, were induced by both MoDC preparations. Cytokine secretion by each type of MoDC preparation was similar; each secreted interleukin-12, tumor necrosis factor-alpha, and low levels of interferon-gamma but in most cases no interleukin-10. Migration studies confirmed that both types of MoDC migrated towards the chemokine, CCL21 although CD300e immunoselected showed greater migration. Overall, the CD14 immunoselected MoDC had higher spontaneous background migration, compared with the CD300e immunoselected MoDC. Differential signaling from the antibodies used to immunoselect the monocytes may account for the slight differences in migratory capacity. These data identify the CD300e antigen as another monocyte-specific marker that can be used to purify monocytes for differentiation into functionally active MoDC.

GAIP participates in budding of membrane carriers at the trans-Golgi network.

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans-Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.