Major histocompatibility complex class II-peptide complexes internalize using a clathrin- and dynamin-independent endocytosis pathway

Abstract

Major histocompatibility complex (MHC) class II molecules (MHC-II) function by binding antigenic peptides and displaying these peptides on the surface of antigen presenting cells (APCs) for recognition by peptide-MHC-II (pMHC-II)-specific CD4 T cells. It is known that cell surface MHC-II can internalize, exchange antigenic peptides in endosomes, and rapidly recycle back to the plasma membrane; however, the molecular machinery and trafficking pathways utilized by internalizing/recycling MHC-II have not been identified. We now demonstrate that unlike newly synthesized invariant chain-associated MHC-II, mature cell surface pMHC-II complexes internalize following clathrin-, AP-2-, and dynamin-independent endocytosis pathways. Immunofluorescence microscopy of MHC-II expressing HeLa-CIITA cells, human B cells, and human DCs revealed that pMHC enters Arf6(+)Rab35(+)EHD1(+) tubular endosomes following endocytosis. These data contrast the internalization pathways followed by newly synthesized and peptide-loaded MHC-II molecules and demonstrates that cell surface pMHC-II internalize and rapidly recycle from early endocytic compartments in tubular endosomes.

FIGURE 1.

pMHC-II and MHC-II-Ii internalize with similar kinetics in both HeLa-CIITA and human B cells. A, HeLa-CIITA cells were lysed in Triton X-100 and aliquots of the lysate were subjected to immunoprecipitation using an isotype control IgG, anti-pMHC-II mAb L243, or the anti-MHC-II α-chain mAb DA6.147. The immunoprecipitates were then analyzed by immunoblot analysis for the total content of MHC-II α-chain (top panel) or Ii (bottom panel). B, HeLa-CIITA cells and monocyte-derived human immature DCs were fixed, permeabilized, and stained with mAb recognizing pMHC-II (green) and LAMP-2 (red). C, HeLa-CIITA cells (circles), JY B cells (squares), and KG-1 DCs (triangles) were incubated on ice with mAb recognizing pMHC-II (L243) or MHC-II-Ii (LL1). The cells were extensively washed and re-cultured at 37 °C. At different times the cells were washed on ice and the amount of tagged pMHC-II or MHC-II-Ii remaining on the cell surface was determined by incubating the cells with fluorescently labeled secondary antibodies on ice. The cells were analyzed by FACS analysis and representative histograms of pMHC-II and MHC-II-Ii expression on HeLa-CIITA cells are shown. The median fluorescence intensity of each time point was expressed as a fraction of the percentage of amount of tagged pMHC-II or MHC-II-Ii present on the cell surface at time 0. The data shown are the mean ± S.D. obtained from more than three independent experiments. HeLa-CIITA cells (circles) or KG-1 DCs (triangles) were incubated with anti-pMHC-II mAb L243 at 37 °C, washed, and the relative amount of internal pMHC-II recycling back to the cell surface was determined as described under “Experimental Procedures.” The data shown are the mean ± S.D. obtained from more than three independent experiments. D, upper panels, HeLa-CIITA cells were incubated with mAb recognizing pMHC-II for 30 min on ice. The cells were then fixed with paraformaldehye, pMHC-II molecules remaining on the surface were either “blocked” as described under “Experimental Procedures” or sham-blocked. Immunolabeled cell surface MHC-II was visualized using Alexa 546-conjugated secondary antibodies and confocal immunofluorescence microscopy. Lower panels, HeLa-CIITA cells were incubated with mAb recognizing pMHC-II (L243) or MHC-II-Ii (LL1) for 30 min on ice, extensively washed, and re-cultured at 37 °C for 15 min. The cells were then fixed with paraformaldehye, pMHC-II molecules remaining on the surface were blocked, and internalized pMHC-II or MHC-II-Ii visualized with Alexa 546-conjugated secondary antibodies after permeabilization.

FIGURE 2.

Kinetics of internalization of pMHC-II in HeLa-CIITA cells. HeLa-CIITA cells were biotinylated on ice using sulfo-NHS-SS-biotin as described under “Experimental Procedures.” The cells were kept on ice or incubated at 37 °C for various times and incubated with ice-cold reduced glutathione (or not) as indicated. The cells were lysed and pMHC-II isolated by immunoprecipitation using mAb L243. The immunoprecipitates were analyzed by SDS-PAGE and biotinylated pMHC-II detected using streptavidin-horseradish peroxidase and total pMHC-II present in the sample detected using the β-chain-specific mAb XD5.A11. The amount of biotin-labeled pMHC-II present in each sample was expressed as a fraction of the total amount of biotin-labeled pMHC-II present on cells before glutathione treatment.

FIGURE 3.

Depletion of clathrin or AP-2 inhibits MHC-II-Ii but not pMHC-II endocytosis. A, HeLa-CIITA cells were treated with control siRNA or siRNA targeted against clathrin or the μ2 subunit of AP-2. After 3 days the expression of clathrin or the μ2 subunit of AP-2 in the cell population was determined by immunoblot analysis. B, the expression of MHC-II-Ii (LL1) or pMHC-II (L243) on the plasma membrane of siRNA-treated cells was determined by FACS analysis. Representative histograms showing staining of isotype control antibody (filled gray), control siRNA (solid line), or either clathrin- or AP-2 siRNA (dashed line) are shown. The expression of pMHC-II or MHC-II-Ii on the cell surface under each condition is shown as a -fold increase in plasma membrane expression by normalizing the median fluorescence intensity of expression of the clathrin- or AP-2-siRNA-treated cells as compared with the expression in control siRNA-treated cells. C, HeLa-CIITA cells treated with control siRNA or AP-2 siRNA were incubated on ice with mAb recognizing MHC-II-Ii or pMHC-II. The cells were extensively washed and either kept on ice or re-cultured for either 5 or 15 min at 37 °C. The cells were washed on ice and the amount of tagged pMHC-II or MHC-II-Ii remaining on the cell surface was determined by incubating the cells with fluorescently labeled secondary antibodies on ice. The percentage of tagged MHC-II-Ii or pMHC-II remaining on the cell surface of control siRNA- or AP-2 siRNA-treated cells after 5 or 15 min of culture at 37 °C was expressed as a percentage of the total amount present on the surface on cells maintained on ice.

FIGURE 4.

AP-2 depletion does not alter surface pMHC-II endocytosis. HeLa-CIITA cells treated with control siRNA or siRNA targeting the μ2 subunit of AP-2. After 3 days the cells were biotinylated on ice using sulfo-NHS-SS-biotin, washed, and either incubated on ice or cultured at 37 °C for 10 min. The cells were then incubated with reduced glutathione on ice to remove surface biotin, washed, and pMHC-II isolated by immunoprecipitation analysis. The immunoprecipitates were analyzed by SDS-PAGE and biotinylated pMHC-II detected using streptavidin-horseradish peroxidase. A representative gel indicating the amount of biotinylated pMHC-II on control or AP-2 siRNA-treated cells is shown. The percentage of biotinylated pMHC-II remaining on the plasma membrane after incubation of the cells at 37 °C was expressed as a fraction of the total amount of biotin-labeled pMHC-II present on cells before glutathione treatment. The results shown are representative of two independent experiments.

FIGURE 5.

Stable surface expression of pMHC-II is dynamin- and AP180-independent in HeLa-CIITA cells. HeLa-CIITA cells were transfected with plasmids encoding HA-dynamin mutant or GFP-AP180. After 2 days the cells were stained on ice with Alexa 548-conjugated mAb recognizing MHC-II-Ii (LL1) or pMHC-II (L243) and fixed with paraformaldehyde. Dynamin-transfected cells were then permeabilized and stained with Alexa 488-labeled anti-HA epitope antibody. The cell populations were analyzed by two-color FACS analysis and cells expressing HA-tagged dynamin mutant or GFP-AP180 were analyzed for MHC-II-Ii or pMHC-II expression and were compared with cells from the same transfections that did not express HA-tagged dynamin mutant or GFP-AP180. Representative histograms showing staining of isotype control antibody (filled gray), MHC-II-Ii or pMHC-II expression in dynamin- or GFP-AP180-negative cells (solid line), and MHC-II-Ii or pMHC-II expression in dynamin- or GFP-AP180-positive cells (dashed line) are shown. The expression of MHC-II-Ii or pMHC-II on the cell surface under each condition is shown as a -fold increase in plasma membrane expression by normalizing the median fluorescence intensity of expression of the dynamin mutant- or AP180-expressing cells as compared with the expression in non-expressing cells.

FIGURE 6.

Stable surface expression of pMHC-II is dynamin- and AP180-independent in professional APCs. JY B cells and KG-1 DCs were transfected with a plasmid encoding GFP-AP180. After 2 days the cells were stained on ice with Alexa 548-conjugated mAb recognizing MHC-II-Ii (LL1) or pMHC-II (L243) and fixed with paraformaldehyde. The cell populations were analyzed by two-color FACS analysis and cells expressing GFP-AP180 were analyzed for MHC-II-Ii or pMHC-II expression and were compared with cells from the same transfections that did not express GFP-AP180. Representative histograms showing staining of isotype control antibody (filled gray), MHC-II-Ii or pMHC-II expression in GFP-AP180-negative cells (solid line), MHC-II-Ii or pMHC-II expression in GFP-AP180-positive cells (dashed line) are shown. The results shown are representative of those obtained in two independent experiments.

FIGURE 7.

Internalized pMHC-II is present on tubular endosomes. Untransfected HeLa cells (panel A), HeLa-CIITA cells (panel A), and monocyte-derived human DCs (panel B) were incubated with mAb recognizing pMHC-II (L243) or MHC-II-Ii (LL1) for 30 min at 37 °C. The cells were then fixed with paraformaldehye, pMHC-II molecules remaining on the surface were blocked, and internalized MHC-II visualized using Alexa 546-conjugated secondary antibodies after permeabilization. Confocal microscopy reveals the presence of intracellular pMHC-II in tubular structures and MHC-II-Ii in perinuclear vesicles only in HeLa-CIITA cells and human DCs. Insets show higher magnification images of the indicated regions of the cells.

FIGURE 8.

Internalized pMHC-II is present in Arf6⁺ Rab35⁺ endosomal tubules. HeLa-CIITA cells were transfected with plasmids encoding wild-type HA-Arf6, GFP-Rab35, GFP-EHD1, or GFP-CD63 (panel A) or the HA-Arf6 T27N mutant or GFP-Rab35 S22N mutant (panel B). The distribution of each endosomal protein (green) and internalized pMHC-II (red) was analyzed by confocal microscopy as described above. Insets show higher magnification images of the indicated regions of the cells. The overlays reveal considerable colocalization of internalized pMHC-II with Arf6-Rab35, and EHD1-containing tubules in cells expressing the wild-type proteins and colocalization with the disrupted, vesicular structures in cells expressing the mutant proteins. C, HeLa-CIITA cells were incubated with trace amounts of the Alexa 488-conjugated pMHC-II mAb L243 at 37 °C and immediately analyzed by confocal microscopy. Individual images were acquired every 2 min and videos were generated by overlaying successive images. Shown is a representative series of images (of a single focal plane) showing the movement of a bolus of internalizing pMHC-II along tubular internal structures back to the plasma membrane.