Purification of circulating plasmacytoid dendritic cells using counterflow centrifugal elutriation and immunomagnetic beads

Background aimsPlasmacytoid dendritic cells (pDC) are a dendritic cell (DC) subset specialized in the production of high amounts of interferon (IFN) type I (IFN-α, -β) in response to viruses. They can be purified from peripheral blood mononuclear cells (PBMC), usually using magnetic bead sorting.MethodsIn this study, we set up a counterflow centrifugal elutriation (CCE) procedure to enrich pDC from PBMC. We first analyzed each CCE fraction for the presence of pDC using CD123 and BDCA-2 as markers. We then purified pDC using CCE and magnetic beads and verified that their functions were not affected by this procedure.ResultspDC were sorted by CCE into intermediate fractions between those containing lymphocytes and monocytes. The pDC frequency in these intermediate fractions was 3-fold that in PBMC. Using negative-magnetic bead sorting, starting with the same number of cells and beads, we obtained more than twice as many pDC from intermediate fractions as from PBMC. The phenotypes and IFN-α production capacities of sorted pDC from PBMC and from intermediate fractions were similar, both immediately after sorting and after stimulation with CpG-A oligodeoxynucleotides. In addition, we showed that intermediate fractions could be cryopreserved and that magnetic bead sorting could be performed with the same efficiency after thawing.ConclusionsAltogether, our results show that CCE can be used to enrich lymphocytes, monocytes and pDC from the same donor, without magnetic beads on their surface. Our method should be useful for the purification of these cells for experimental research and may also be adaptable for clinical use in immunotherapy.     

An interaction between U2AF 65 and CF I(m) links the splicing and 3' end processing machineries

The protein factor U2 snRNP Auxiliary Factor (U2AF) 65 is an essential component required for splicing and involved in the coupling of splicing and 3' end processing of vertebrate pre-mRNAs. Here we have addressed the mechanisms by which U2AF 65 stimulates pre-mRNA 3' end processing. We identify an arginine/serine-rich region of U2AF 65 that mediates an interaction with an RS-like alternating charge domain of the 59 kDa subunit of the human cleavage factor I (CF I(m)), an essential 3' processing factor that functions at an early step in the recognition of the 3' end processing signal. Tethered functional analysis shows that the U2AF 65/CF I(m) 59 interaction stimulates in vitro 3' end cleavage and polyadenylation. These results therefore uncover a direct role of the U2AF 65/CF I(m) 59 interaction in the functional coordination of splicing and 3' end processing.     

Interactions of the Cytoplasmic Domains of Human and Simian Retroviral Transmembrane Proteins with Components of the Clathrin Adaptor Complexes Modulate Intracellular and Cell Surface Expression of Envelope Glycoproteins

The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXØ, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXØ motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXØ motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXØ motifs interact with the μ1 and μ2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the β2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXØ-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved.