Mobilization of MHC class I molecules from late endosomes to the cell surface following activation of CD34-derived human Langerhans cells - PubMed (original) (raw)
Mobilization of MHC class I molecules from late endosomes to the cell surface following activation of CD34-derived human Langerhans cells
P A MacAry et al. Proc Natl Acad Sci U S A. 2001.
Abstract
Langerhans cells are a subset of dendritic cells (DCs) found in the human epidermis with unique morphological and molecular properties that enable their function as "sentinels" of the immune system. DCs are pivotal in the initiation and regulation of primary MHC class I restricted T lymphocyte immune responses and are able to present both endogenous and exogenous antigen onto class I molecules. Here, we study the MHC class I presentation pathway following activation of immature, CD34-derived human Langerhans cells by lipopolysaccharide (LPS). LPS induces an increase in all components of the MHC class I pathway including the transporter for antigen presentation (TAP), tapasin and ERp57, and the immunoproteasome subunits LMP2 and LMP7. Moreover, in CD34-derived Langerhans cells, the rapid increase in expression of MHC class I molecules seen at the cell surface following LPS activation is because of mobilization of MHC class I molecules from HLA-DM positive endosomal compartments, a pathway not seen in monocyte-derived DCs. Mobilization of class I from this compartment is primaquine sensitive and brefeldin A insensitive. These data demonstrate the regulation of the class I pathway in concert with the maturation of the CD34-derived Langerhans cells and suggest potential sites for antigen loading of class I proteins.
Figures
Figure 1
Surface phenotype of CD34-derived LCs (A) compared with CD14 monocyte-derived DCs (B). Cells generated under distinct culture conditions were analyzed by flow cytometry for the expression of the cell markers indicated. Thick lines indicate the cells stained in the absence of LPS, whereas the dotted lines show the profiles obtained following activation with LPS (0.5 μg).
Figure 2
LPS induced changes in the MHC class I antigen presentation pathway in DCs. Immature CD34-derived LCs (A) and monocyte-derived DCs (B) were activated by addition of 0.5 μg of LPS. At time 0, 6, and 48 h post-LPS, cells were harvested and fixed, and the expression of DC activation marker CD83, MHC class II, and MHC class I was monitored by flow cytometry. (C) LPS mediated a coordinate induction of MHC class I components. CD34-derived LCs were induced with LPS for 0–48 h, after which the cells were extracted in 1% Triton X-100, and membranes from separated lysates were probed with antibodies specific for the indicated proteins. MCP20 is a constitutive, noninducible component of the human proteasome and serves as a loading control for each time point. (D) LPS increases TAP-mediated peptide transport in CD34-derived LCs. Streptolysin O-permeabilized immature (□) and LPS-matured (▵) CD34-derived LCs were incubated with the iodinated RRYQNSTEL peptide at 37°C for the indicated time periods. Translocation was assessed by binding of the hot, glycosylated reporter peptide to Con A-Sepharose beads.
Figure 3
Cellular localization of MHC class I molecules in DCs. (A) MHC class I colocalizes in MIIC. (i–iii) Day 7 immature CD34-derived LCs and (iv–vi) monocyte-derived DCs were fixed, stained with R.DRAB (anti-class II αβ) and 4E (anti-class I), and examined by confocal immunofluorescence microscopy. The class I and class II molecules show colocalization in CD34-derived LCs (iii) but not in the monocyte-derived DCs (vi). Day 7 immature CD34-derived LCs (vii–ix) were stained with 4E (anti-MHC class I) and R.DMB-C (anti-HLA-DM). Merged images show colocalization of class I with HLA-DM. (x–xii) Time course of activation of immature CD34-derived LCs with LPS. 6 h postactivation, intracellular class I staining (rabbit anti-class I heavy chain antisera) is less apparent, and by 12 h class I is seen predominantly at the cell surface. (xiii–xv) Immature CD34-derived LCs stained with antibody recognizing free class I heavy chains (HC10) and HLA-DM (R.DMB-C) show colocalization. (B,i–iii) Immature CD34-derived LCs were stained for immunological markers for late endosomes/lysosomes LAMP1 (H4A3) (i–iii), for early endosomes, CD1a (10D12) (iv–vi), and for Birbeck organelles LAG (anti-LAG) (vii–ix). Merged images show colocalization between MHC class I and LAMP1, but no colocalization between HLA-DM and the early endosome marker, CD1a, or the Birbeck granule marker, Langerin. Size markers represent 10 μm.
Figure 4
Class I colocalizes in MIICs. (A) Ultrathin cryosections of immature CD34-derived LCs were double immunolabeled for MHC class II (rabbit anti-human HLA-DR, small gold) and MHC class I (rabbit anti-heavy chain serum, large gold). PM, plasma membrane. (B–D) Endosomes, double immunolabeled with class I (small gold), together with HLA-DR (large gold) (B), class I (large gold) together with CD63 (small gold) (C), and mannose-6-phosphate receptor (small gold) (D). Large gold particles are 15 nm and small gold particles (arrows) 5 nm; size bars represent 200 nm.
Figure 5
Primaquine inhibits the rapid LPS-induced increase in cell-surface MHC class I molecules. Day 7 cluster purified immature DC were incubated with LPS alone (I), LPS and brefeldin A (II), and LPS and primaquine (III). Cells were harvested at 0, 5, and 24 h after addition of the LPS and fixed; expression of MHC class I molecules was examined by flow cytometry.
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