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First published online August 17, 2004
doi: 10.1242/10.1242/jcs.01288

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The pathway for MHCII-mediated presentation of endogenous proteins involves peptide transport to the endo-lysosomal compartment

¹ National Centre for Biological Sciences, UAS-GKVK Campus, Bellary Road, Bangalore 560065, India
² National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India

View larger version (113K): [in a new window]   Fig. 4. Peptide-MHC class II complexes derived from transmembrane anchored I-E localize to late lysosomal compartments of APCs. Macrophages from B10A. (5R) mice that express I-Ab and IE were fixed, permeabilized and stained with Y-Ae and antibodies recognizing LAMP-1, MPR (MPR-300) and cathepsin-D. Y-Ae complexes colocalize with LAMP-1 (A) and cathepsin-D (C), but not with MPR (B). Images are single representative confocal sections. All images on the very right are respective overlays of the stained images of A, B and C. Scale bar, 10 µm.

View larger version (15K): [in a new window]   Fig. 7. Lack of Ii diminishes the efficiency of cytosolic antigen presentation on MHC class II. (A-C) Macrophage APCs from wild-type or Ii-/- H-2b mice were loaded with the antigen using the loading pathway indicated. These were tested in titrating concentrations for their ability to stimulate the T cell line identified in each panel. B3 recognizes an OVA peptide in the context of the class I molecule, H-2Kb, whereas 13.8 recognizes an OVA peptide in the context of the class II molecule, I-Ab.

View larger version (32K): [in a new window]   Fig. 1. Peptide-MHCII complexes are formed and presented to T cells from a cytosolically expressed as well as from a cytosolically delivered protein. (A) BMC-2 cells in 6-well dishes were transfected with 10 µg of either E52-68-eGFP or eGFP (control) DNA and stained 24 hours later with the Y-Ae antibody. Fluorescence of GFP and Y-Ae was analysed by flow cytometry. Data are shown as twocolour plots and as single-parameter graphs for Y-Ae fluorescence of appropriately gated eGFP+ and eGFP- cells as indicated. Shaded area under curve, untransfected cells; thin lined curves, eGFP transfected cells; thick lined curves, E52-68-eGFP transfected cells. (B) BMC-2 cells pulsed cytosolically (via osmotic pinosome lysis method) or exogenously via the fluid phase with 5 mg/ml GST E52-68-myc fusion protein were stained with Y-Ae after 1 hour and analysed by flow cytometry. Shaded curve, unstained cells; thin line, unloaded cells; thick line, exogenously loaded cells; grey line, cytosolically loaded cells. (C) Responses of the 1H3.1 T cell line to titrating numbers of BMC-2 cells (APCs/well), transfected with E52-68-eGFP DNA (squares) or eGFP DNA (triangles), or exogenously pulsed with GST-E52-68-myc fusion protein (circles) for 24 hours are shown as measured by IL-2 ELISA.

View larger version (22K): [in a new window]   Fig. 2. MHCII-mediated presentation of cytosolically introduced protein is not the result of peptide regurgitation and subsequent bystander or cross presentation. IL2 secretion was monitored to determine specific TCR activation of the OVA-specific MHCII (H-2b)-restricted T cell line 13.8, to C57BL/6 (H-2b) and/or C3H/HeJ (H-2k) macrophages cytosolically loaded with 10 mg/ml OVA (+Ag). Bystander presentation was assessed by incubating T cell line 13.8 with C3H/HeJ (H-2k) macrophages cytosolically loaded with antigens (+Ag) along with unloaded (-Ag) C57BL/6 (H-2b) macrophages. IL2-secretion was monitored by a proliferation assay as described in Materials and Methods.

View larger version (109K): [in a new window]   Fig. 3. Peptide-MHCII complexes derived from cytosolic proteins are formed in lysosomal compartments in different APCs. (A,B) BMC-2 cells transfected as in Fig. 2A with E52-68-eGFP (A) or eGFP (B) were fixed 12 hours after transfection, permeabilized and stained with Y-Ae and anti-LAMP-1 antibodies. (C,D) C57Bl/6 macrophages were pulsed cytosolically (C) or exogenously via the fluid phase (D) with GST E52-68-myc protein (5 mg/ml), chased for 15 minutes, fixed, permeabilized and stained with YAe and anti-LAMP-1 antibodies. Images are single representative confocal sections. All images on the very right are respective overlays of the stained images of A, B, C and D. Scale bar, 10 µm.

View larger version (100K): [in a new window]   Fig. 5. Peptide-MHC class II complexes derived from endogenous transmembrane or cytosolic protein are localized to lysosomal compartments in bone marrow-derived dendritic cells. GM-CSF cultured BM-DCs from B10A.(5R) (A) or C57B1/6 (B) mice were either directly fixed (A) or first pulsed cytosolically with GST-E52-68-myc protein, chased, fixed and then stained for Y-Ae, anti-LAMP-1 antibodies. Images are single representative confocal sections. All images on the very right are respective overlays of the stained images of A and B. Scale bar, 10 µm.

View larger version (79K): [in a new window]   Fig. 6. Rapid formation of peptide-MHC class II complexes derived from cytosolically delivered protein in endosomal compartments and their subsequent appearance on the cell surface of APCs. (A,B) Macrophages from C57Bl/6 mice were pulsed either exogenously in isotonic medium (A) or cytosolically in hypertonic medium (B) for 10 minutes with 5 mg/ml GST-E protein. Pulsed protein was washed off and cells were chased for different times indicated, fixed-permeabilized and stained with the Y-Ae antibody to detect formation of peptide-MHC class II complexes. (C,D) BMC-2 cells incubated with 5 mg/ml GST-E52-68-myc protein coupled to the ChariotTM reagent for 15 minutes at 0°C, were washed, chased for 15 minutes at 37°C, fixed-permeabilized and co-stained for LAMP-1 and Y-Ae antibodies to detect the intracellular Y-Ae+ complexes formed at this time. Right image is an overlay of the stained images in C. (D) A time course of detection by flow cytometry of intracellular Y-Ae+ complexes formed in BMC-2 cells after pulsing by the indicated methods and chased for different lengths of time. Scale bars, 10 µm.

View larger version (29K): [in a new window]   Fig. 8. H-2M is required for cytosolic antigen presentation on MHC class II. (A,B) Macrophage APCs from wild-type or H-2M-/- H-2b mice were loaded with OVA, by the fluid phase loading pathway (A, OVA Exogenous) or via osmotic lysis of pinosomes (OVA, Cytosolic) and tested in titrating concentrations for their ability to stimulate the MHCII restricted T cell line 13.8 by monitoring IL2 secretion in an ELISA reaction, absorbance at 570 nm, A 570. (C,D) Total cell associated Y-Ae and MHCII I-Ab-specific antibody, Y-3P, fluorescence were quantified from APCs derived from H-2M-/- (grey circles) and control C57/Bl6 (black circles) mice, either loaded via the fluid phase (C) or by the osmotic lysis of pinosomes (D) show that Y-Ae-positive peptide-MHCII complexes are absent in the H-2M-/- mice. (E,F) Human B cell lines (T1-IAb) or its HLADM-deficient mutant counterpart (T2-IAb) stably transfected with IAb were transiently transfected with eGFP (thin line) or with E52-68-eGFP (thick line) as indicated and stained 20 hours later with Y-Ae and analysed by flow cytometry after gating for eGFP+ cells. The shaded area under the curve represents isotype control signals for the Y-Ae antibody staining.

View larger version (61K): [in a new window]   Fig. 9. TAP transporter is not required for cytosolic antigen presentation on MHC class II. (A-C) Responses of the (A) OVA-specific MHC class I-restricted T cell line, B3Z, and (B,C) the E-specific MHC class II-restricted T cell line, 1H3.1. APCs used were either wild-type or TAP-1-/- H-2b macrophages as indicated. Antigens indicated were loaded either cytosolically or exogenously and used to stimulate the relevant T cell lines in titrating numbers. IL2-secretion was monitored by ELISA as indicated in the text. (D) Macrophage APCs from TAP-1-/- mice were loaded cytosolically with GST-E52-68-myc and stained with the Y-Ae and anti-LAMP-1 antibodies. Image on the right shows overlay; green, Y-Ae; red, anti-LAMP-1. Scale bar, 10 µm. (E) Bone marrow-derived macrophages from TAP-1-/- and wild-type mice were transiently transfected with E52-68-eGFP or eGFP and stained for E-specific MHCII complexes with Y-Ae antibodies and detected by flow cytometry. Thin lined curve shows the signal from TAP-1-/-, thick lined curve from wild-type cells and shaded area represent isotype control signals for Y-Ae antibody staining.

View larger version (21K): [in a new window]   Fig. 10. Cytoplasmic loading onto MHCII is insensitive to reduction of PI 3-kinase activity and 3-methyladenine treatment. BMC-2 cells were transfected and treated with autophagy inhibitors as indicated to the right of each plot (3MA, 10 mM; LY294002, 50 µM). The generation of Y-Ae+ peptide MHCII complexes was assayed by flow cytometry at 20 hours post-transfection. The numbers in the top right quadrant indicate that the percentage of eGFP-expressing cells that are also positive for Y-Ae+ complexes is unaffected by the different treatments tested. Numbers in the top left quadrant indicate percentage of eGFP-expressing cells; numbers in the bottom right quadrant indicate percentage of cells gated for positive Y-Ae signals; numbers in the bottom left quadrant indicate percentage of unstained cells.

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