Figure 1.
Constitutive MHC-I internalization in DCs is differentially controlled by cytoplasmic tyrosine- and exon 7-dependent mechanisms.
(A) Amino acid sequences of the cytoplasmic domains of wild-type H-2Kb and the two cytoplasmic tail mutants Δ7 and ΔY. The asterisk denotes a known conserved serine phosphorylation site. The Δ7 mutant contains a deletion of the 13 amino acids comprising exon 7, indicated as dashed lines. Highlighted amino acids indicate conserved tyrosine and serine residues. TM, transmembrane domain. (B) Splenic dendritic cells isolated from KbWT, and Δ7 and ΔY transgenic mice were labeled with FITC-conjugated H-2Kb-specific mAb, washed, and incubated at 37°C for the indicated time points. DCs were then imaged using confocal fluorescence microscopy to visualize internalized MHC-I-containing vesicles. Data are representative of at least 3 images captured from 2 independent experiments.
Figure 2.
Quantification of MHC-I internalization in DCs.
The dynamics of MHC Class I internalization is assessed by the reduced mean fluorescence units of FITC-labeled H-2Kb surface expression. Following labeling with (A and C) FITC-conjugated H-2Kb- or (B and D) PE-conjugated H-2Kk-specific antibodies and internalization at 37°C for the indicated time points, flow cytometric analysis was conducted to assess internalization of Kb and Kk molecules, as measured by the reduction in FITC and PE mean fluorescence intensities over time, respectively. Data are representative of 3 different experiments performed in triplicate.
Figure 3.
MHC-I cell surface-to-endosome trafficking in DCs is differentially abrogated by mutations in cytoplasmic tyrosine or exon 7-encoded determinants.
(A to C) Splenic DCs isolated from KbWT, and Δ7 and ΔY transgenic mice were mounted on coverslips, labeled with FITC-conjugated H-2Kb-specific mAb, washed, then incubated at 37°C for the indicated times. DCs were then fixed, permeabilized and counterstained for EEA-1. Images were acquired using a multiphoton fluorescence confocal microscope. Yellow color indicates co-localization of surface-derived H-2Kb (green) with EEA-1 (red). Data are representative of at least 3 images captured from 2 independent experiments.
Figure 4.
MHC-I cell surface-to-lysosome trafficking in DCs is impaired by mutation in the cytoplasmic tyrosine residue.
(A to D) Splenic DCs isolated from KbWT, and Δ7 and ΔY transgenic mice were mounted on coverslips, labeled with FITC-conjugated H-2Kb-specific mAb, washed, and incubated at 37°C for the indicated times. DCs were then fixed, permeabilized, and counterstained for LAMP-1. Yellow color indicates co-localization of surface-derived H-2Kb (green) with LAMP-1 (red). Data are representative of at least 3 images captured from 2 independent experiments.
Figure 5.
Cytoplasmic tail mutations significantly reduce the contribution of surface MHC-I molecules to endosomal and lysosomal peptide-loading compartments.
(A to C) Splenic DCs isolated from KbWT, Δ7, and ΔY transgenic mice were labeled with FITC-conjugated H-2Kb-specific mAb, washed and incubated for 6 hr at 37°C in 5 mg/mL ovalbumin protein. DCs were then labeled with mAbs specific for (A) early endosomal antigen, EEA-1, (B) lysosomal marker LAMP-1 and (C) Golgi marker Giantin. All DCs were simultaneously co-stained with purified 25.D1.16 (anti-H-2Kb/OVA257–264) antibody. Cellular markers EEA-1, LAMP-1 and Giantin were visualized by staining with secondary antibodies coupled to Alexa-568 (red) whereas H-2Kb/OVA257–264 complexes were visualized by staining with secondary antibody conjugated to Alexa-647 (blue). Three-color fluorescence was detected by laser scanning confocal microscopy of 488-nm (green), 568-nm (red), and 633-nm (blue) wavelengths. Photographs depict three-color image overlays to assess colocalization of the three markers. White color indicates a triple overlap of all three markers (green+red+blue), whereas yellow (green+red), pink (red+blue), and light blue (green+blue) indicate overlap of two of the three markers. D to F shows a quantitative assessment of internalized MHC-I and MHC-I/peptide complexes within intracellular compartments of DCs. Three-color confocal overlay images of DCs, as shown in Figure 5, were analyzed for relative fluorescent color (pixel) intensity in order to obtain a quantitative measure of fluorophore colocalization. For each data set, 30 to 50 individual DCs derived from each of the indicated mouse strains were analyzed. The green color indicates surface-labeled H-2Kb, the blue color indicates Kb-OVA257–264 peptide complexes, and red color indicates either (A) early endosomal antigen (EEA-1), (B) LAMP-1, or (C) Giantin. White pixels indicate triple overlap of all three markers (green+red+blue), whereas yellow (green+red), pink (red+blue), and light blue (green+blue) indicate overlap of two of the three markers. Graph depicts individual color pixel mean percentages and standard deviations, as calculated by dividing the number of pixels of a given color by the total number of colored pixels counted. Data are representative of at least 3 images captured from 4 independent experiments.
Figure 6.
T cell activation is severely compromised in protein-pulsed DCs of transgenic mice containing the cytoplasmic tail mutation.
bmDCs from KbWT, Δ7 and ΔY transgenic mice were isolated, incubated for 6 hours with 10 mg/mL OVA and co-cultured for 24 and 48 hours with B3Z-T hybridoma cells previously labeled with 1 µM of CFSE. Mixed cultures were then stained with anti-CD3-PE antibody and flow cytometry was conducted to examine the CFSE/CD3+ T cell population and proliferation. Histograms depict proliferating T cells following incubation with OVA-pulsed DCs (including one representative of OVA257–264) of transgenic mice for 24 (A) and 48 (B) hours. Data are representative of 1 experiment performed in triplicate.
Figure 7.
Model of dendritic cell MHC-I trafficking and cross-presentation.
Schematic representation of trafficking routes for KbWT, Δ7, and ΔY molecules in dendritic cells, depicting proposed intracellular sites of antigen acquisition. In the direct presentation pathway (bottom), endogenously-synthesized proteins (green) are degraded by cytosolic proteosome complexes into antigenic peptides, which are transported by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER) for binding to nascent class Iα chain/β2-microglobulin dimers. KbWT, Δ7, and ΔY molecules initially loaded in the ER with endogenously-derived peptides (green) are then transported through the cis- and trans-Golgi (TGN) and to the cell surface via the secretory pathway. Alternatively, a subset of KbWT and Δ7, but not ΔY, molecules may be re-routed from the secretory pathway directly into endolysosomal compartments, although this pathway remains largely uncharacterized. In cross-presentation, exogenous protein antigens (orange, top) are internalized into endocytic vesicles, where they can be transported into the cytosol via ER-associated retrotranslocation (asterisk) and subsequently enter the direct presentation pathway. Exogenous antigens may also be transported into endolysosomes, where they are degraded by resident proteases and Cathepsin S into antigenic peptides (orange). Surface MHC-I molecules are constitutively internalized and transported through the endocytic pathway by a mechanism that requires the MHC-I cytoplasmic tyrosine (Y) residue. MHC-I molecules lacking Exon 7 (Ex7), despite abundant colocalization within endosomes and lysosomes, are significantly delayed in their endocytic transport. MHC-I transport through early endosomes and late endosomes/lysosomes seems to be required for acquisition and cross-presentation of exogenously-derived peptides, suggesting that such peptides are bound to recycling MHC-I molecules directly within endocytic loading compartments (ELCs).