MHC class II molecules are found on the basolateral plasma membrane domain of polarized epithelial cells, where they can present Ag to intraepithelial lymphocytes in the vascular space. We have analyzed the sorting information required for efficient intracellular localization and polarized distribution of MHC class II molecules in stably transfected Madin-Darby canine kidney cells. These cells were able to present influenza virus particles to HLA-DR1-restricted T cell clones. Wild-type MHC class II molecules were located on the basolateral plasma membrane domain, in basolateral early endosomes, and in late multivesicular endosomes, the latter also containing the MHC class II-associated invariant chain and an HLA-DM fusion protein. A phenylalanine-leucine residue within the cytoplasmic tail of the β-chain was required for basolateral distribution, efficient internalization, and localization of the MHC class II molecules to basolateral early endosomes. However, distribution to apically located, late multivesicular endosomes did not depend on signals in the class II cytoplasmic tails as both wild-type class II molecules and mutant molecules lacking the phenylalanine-leucine motif were found in these compartments. Our results demonstrate that sorting information in the tails of class II dimers is an absolute requirement for their basolateral surface distribution and intracellular localization.

Epithelial cells from various organs are involved in MHC class II-restricted Ag presentation (for reviews see Ref. 1 and 2). MHC class II molecules are transported to the endocytic pathway, where they bind peptides derived from proteins entering the endosomal-phagosomal pathway, which are then at the plasma membrane presented to CD4+ T cells. Sorting of newly synthesized class II molecules to endosomes depends on specific regions within the cytoplasmic tail of the associated protein invariant chain (Ii)5 (3, 4, 5, 6). In endosomes, Ii becomes proteolytically processed, leaving the class II molecules free to bind internalized processed Ag, a process which is catalyzed by HLA-DM (for reviews see Refs. 7 and 8). Ag presentation can also occur independently of Ii expression and newly synthesized class II mol- ecules by recycling of mature class II molecules from the plasma membrane into acidic compartments (9, 10, 11, 12). Signals in the cytoplasmic tails of the class II α- and β-chains are important for presentation by mature recycling class II molecules (11, 13). The various requirements for presentation of different Ags indicate that class II peptide loading can occur in several distinct endocytic compartments.

In epithelial cells, MHC class II molecules, like MHC class I molecules (14, 15) and CD4 (16), are located at the basolateral surface (17, 18, 19, 20, 21). The basolateral membrane faces the vascular space, where the class II molecules may present Ag to emigrating T cells. Class II molecules have also been detected in intracellular vesicles in tissue epithelial cells of human and rodents in vivo (17, 18, 19, 20, 21), but little is known about the actual endocytic compartment to which class II molecules traffic and the signals involved in this transport in epithelial cells. Polarized cells may have a more complex sorting system than nonpolarized cells, as they have separate apical and basolateral plasma membrane domains, in addition to separate apical and basolateral early endosomal populations (22). However, cognate apical and basolateral pathways are also found in nonpolarized cells (23, 24).

In Madin-Darby canine kidney (MDCK) cells, apical and basolateral proteins are generally sorted in the trans-Golgi network for direct delivery to the respective plasma membrane domains (25), and signals for sorting to either the apical or the basolateral plasma membrane have been identified. Basolateral sorting signals are located in the cytoplasmic tail of several proteins and can generally be divided into two classes: 1) basolateral signals that are colinear with signals for internalization through clathrin-coated pits, either tyrosine- or leucine-based motifs; and 2) basolateral signals that are different from signals for coated pit localization, either tyrosine-dependent or -independent motifs (for reviews see Refs. 26, 27, 28). Some basolateral sorting signals can also mediate sorting in the endocytic/transcytotic pathways (26, 29).

We have previously shown that human class II molecules (HLA-DR1) and Ii are located at the basolateral surface and in endosomes when transfected stably in polarized MDCK cells (30). Here, we have extended these studies and show that an FL (single letter amino acid code) motif is required for localization of class II molecules at the basolateral plasma membrane and in early endosomes.

The mouse mAb L243 (American Type Culture Collection, Manassas, VA; Ref. 31) recognizes the luminal domains of HLA-DRβ molecules. The IgG1 mAb BU45 (The Binding Site, Birmingham, U.K.; Ref. 32) and Vic Y1 (a gift from Dr. W. Knapp, Vienna, Austria) recognize the C-terminal and the N-terminal domain of human Ii, respectively. The mAb VP3 (a gift from Sreenivasan Ponnambalam, Dundee, Scotland) recognizes the luminal domain of CD8. The cells were labeled for immunofluorescence with FITC-conjugated goat anti-mouse (GαM) IgG (Dianova, Hamburg, Germany) Abs. The cells were labeled for immuno-electron microscopy (EM) with 15-nm and/or 20-nm gold particles conjugated to GαM-IgG (GαM15/GαM20) Abs (British BioCell International Cardiff, U.K.).

MDCK cells (strain II) were grown in DMEM (BioWhittaker, Walkersville, MD) supplemented with 10% FCS (Life Technologies, Rockville, MD). Cells (3.3 × 105/cm2) were cultured on Transwell polycarbonate filter units with a pore size of 0.4 μm (Costar, Cambridge, MA) for 4–5 days to form tight monolayers before the experiments. Fresh medium was added every day after plating onto filters. Transepithelial resistance was measured using a Millipore ERS apparatus (Millipore, Bedford, MA). All cell lines exhibited resistance of more than 200 Ω cm2. The relative surface area of the apical and the basolateral domains in MDCK II cells grown under the present conditions has been measured to be 1.1 (apical/basolateral; Ref. 33).

The generation and maintenance of the HLA-DR1-restricted T cell clones C1.6, specific for the influenza virus A hemaglutinin H3 (peptide 307–318), and the clone C3.5, specific for the influenza virus A matrix protein M1 (peptide 18–31), have been described (34). Influenza virus A, strain X-31 (a gift from J. Yewdell, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) was purified as described (10) and inactivated by exposure to 1200 μW/cm2 of UV light for 15 min on ice.

MDCK cells stably expressing human MHC class II molecules, HLA-DR1 α- and β-chain, were made as described (30). cDNA-encoding truncated HLA-DR molecules, Δα and Δβ, lacking the last 10 or 13 aa of the cytoplasmic tail, respectively, in the vector CDM8 (11) were used. Point mutations in the cytoplasmic tail of the β-chain were introduced by site-directed mutagenesis in a single-stranded M13 mp19 vector by the method of Kunkel (35). The cytoplasmic tail region used for mutagenesis was controlled by DNA sequencing. The mutant cDNA (DR βFL-AA) was then subcloned into the CDM8 vector. The CDM8-cDNA was cotransfected with the RSV.5(neo) vector (36) for cell selection. Cells expressing full-length or truncated HLA-DR molecules were cotransfected with cDNA encoding the p33 form of human Ii (p33Ii) in the expression vector pMEP4 (Hygro; Invitrogen, San Diego, CA).

MDCK cells were stably transfected by the DNA-calcium phosphate procedure as described (37). Clones expressing the neomycin or hygromycin resistance markers were selected in the presence of G418 (geneticin; 0.5 mg/ml active weight; Sigma, St. Louis, MO) or hygromycin B (0.3 mg/ml; Sigma) in the culture medium, respectively. Stably transfected cells were cloned by isolating resistant colonies using cloning cylinders. Colonies expressing the proteins of interest were identified by immunofluorescence, as described. To screen for expression of Ii, stable hygromycin-resistant colonies were induced with 10 μM CdCl2 for 16 h before the assays. The cell line used for transfection showed correct polarity, as confirmed by measuring methionine uptake (38) and secretion of the endogenous glycoprotein complex gp80 (Ref. 39 and data not shown).

The mouse mAbs BU45 and L243 were purified from mouse ascites fluid by precipitation with sodium sulfate (0.18 g/ml Na2SO4) and purification with a protein A-Sepharose (Pharmacia, Piscataway, NJ) column at pH 8.0. The Abs were >90% pure IgG as determined by SDS-PAGE. Purified Ab molecules were labeled with Na125I using chloramine-T-catalyzed iodination and were purified by gel-filtration chromatography on a Sephadex column (Pharmacia). The spec. act. of the Abs and the amount of acid-soluble and -precipitable material was determined by TCA precipitation and counting in a Cobra Auto-Gamma counter (Downers Grove, IL). The amount of soluble radioactivity was generally <2%.

Binding assays were all performed on ice at 4°C. Cells were cooled on ice, and 125I-Ab, diluted in DMEM containing 1% FCS and 10 mM HEPES, was added to either the apical or the basolateral side of the monolayer for 2 h. Integrity of the monolayers was assessed by measuring the radioactivity in the medium of both chambers, and generally <0.1% of the added radioactivity diffused across the monolayer. Unbound Ab was removed by extensive washing (five times) with PBS supplemented with 1 mM CaCl2, 1 mM MgCl2, and 2% FCS. The filters were then excised, and bound radioactivity was measured in a gamma counter. Nonspecific binding was determined by quenching binding of the iodinated Ab with a 100-fold excess of nonlabeled Ab. These values, which generally represented <10% of the total binding, were subtracted from the total bound radioactivity to give the specific binding to expressing cells. Total binding in cpm × 103 ranged from 100 to 200 for HLA-DR molecules and from 5 to 20 for Ii. All given values represent the mean values ± SEM derived from at least three identical experimental setups, each performed in duplicates.

Stably transfected MDCK cells were grown on polycarbonate filters and processed for immunofluorescence by a variation of the method described by Berod (40). Briefly, plasma membrane staining was visualized by adding specific mAbs, diluted in DMEM/1% FCS/10 mM HEPES, to the apical or the basolateral side of the monolayer for 1 h at 4°C. The cells were then washed extensively with PBS supplemented with 1 mM CaCl2, 1 mM MgCl2, and 2% FCS and fixed by the pH shift paraformaldehyde fixation procedure. The filters were then excised, and bound IgG was visualized by incubation with an FITC-conjugated GαM-IgG. To study internalization of the transfected proteins, cell monolayers were incubated with Abs on the apical or the basolateral side for 30 min at 37°C, before fixation, permeabilization by Triton X-100, and staining with FITC-conjugated GαM-IgG. To visualize total protein, cell monolayers were fixed and permeabilized before labeling with primary and secondary Abs for 1 h each at 37°C in a humidified chamber. Fluorescence was detected, and images were acquired by a Leica TCS-NT digital scanning confocal microscope equipped with a 60/1.2 water immersion objective (Leica, Heidelberg, Germany). Images were averaged four times during acquisition to reduce background noise and processed for presentation with Adobe Photoshop (Adobe Systems, Mountain View, CA).

MDCK cells (2 × 105) were labeled for 15 h with sodium [51Cr] chromate (100 μCi in a six-well plate) in 1 ml DMEM supplemented with 2 mM glutamine, 10 mM HEPES, pH 7.4, 12 μg/ml gentamicin, and 10% heat-inactivated FCS. The adherent cells were then incubated for 4 h with either 10 μM synthetic peptide (H3 307–318 or M1 18–31) or with different dilutions of UV-inactivated influenza virus in 1 ml of DMEM containing 5% heat-inactivated FCS. The cells were then trypsinized, washed, counted, and plated at 5 × 103 cells in V-bottom 96-well plates containing the effector cells C1.6 (H3 specific) or C3.5 (M1 specific). After 4 h, supernatants were harvested and counted. The maximum lysis was measured by lysis of target cells with 2% Triton X-100. All cytotoxic assays included a titration of E:T cells, and all experiments were performed at least twice. The data are presented as percentage of maximal lysis after subtraction of the spontaneous lysis.

To identify endosomal compartments, cell monolayers were incubated with 5-nm and 10-nm colloidal gold particles (Zymed, San Francisco, CA) coated with BSA (41) in the apical or the basolateral medium, respectively, for 1 h at 37°C (procedure A). Apical early endosomes (AP-EE) and basolateral early endosomes (BL-EE) were defined as compartments containing only 5-nm or 10-nm gold particles, respectively, whereas late endosomes (LE-1h) were defined as compartments containing both endocytosed markers. As the late endosomal fraction was morphologically heterogeneous, a different assay was used to separate different late endosomal populations. The 10-nm gold particles were internalized from the basolateral side for 3 h, followed by an 18-h chase and a subsequent 1-h apical uptake of 5-nm colloidal gold particles (procedure B). After washing in PBS, the cells were fixed by immersing the filters in Sörensens phosphate buffer, pH 7.4, containing 4% paraformaldehyde together with 0.1% glutaraldehyde for 1 h at room temperature. After fixation, the filters were incubated in 2.3 M sucrose for 1 h at room temperature, and then cut into triangular segments and mounted on silver pins perpendicular to the plane of sectioning. Mounted filters were frozen and stored in liquid N2. The specimens were sectioned on a Reichert Ultracut S ultramicrotome (Vienna, Austria) with a Reichert FCS cryo attachment using Drukker International Diamond knifes (Cuijk, The Netherlands). Single and double immunocytochemical labeling of thawed cryosections was performed mainly as described (42) using mouse mAbs followed by 15-nm and/or 20-nm gold particles coated with GαM-IgG (GαM15/GαM20) Abs (British BioCell International). The sections were examined on a JEOL 100CX and JEOL 1200EX transmission electron microscope (Tokyo, Japan).

To estimate the distribution of a transfected protein in the endocytic pathway, the number of positively labeled compartments with a defined character was related to the total number of positively labeled compartments. The fraction of a defined endosomal population containing the transfected protein was estimated by relating the number of positively labeled compartments to the total number of endosomes having the defined character. Quantitation was performed on randomly acquired micrographs.

We have found that human MHC class II molecules (HLA-DR) are located at the basolateral plasma membrane and in vesicles both when expressed alone and together with human Ii in stably transfected MDCK cells (30). The class II α and β cytoplasmic tails have been shown to jointly contribute a signal for internalization (11), and to study whether the cytoplasmic tails were equally important for basolateral targeting, stably transfected MDCK cells expressing class II molecules having either one or both cytoplasmic tails truncated (outlined in Fig. 1) were eventually stably supertransfected with Ii. Cells expressing truncated class II molecules alone or together with Ii were analyzed to determine the apical vs the basolateral cell surface distribution of αβ and Ii. 125I-labeled anti-Ii (BU45) or anti-HLA-DR (L243) mAbs were added to the apical or the basolateral medium of the cell monolayers at 4°C. After removal of unbound Ab, filters were excised and bound radioactivity was determined in a gamma counter. The percentage specific binding to either the apical or the basolateral surface is shown in Table I. Full-length class II molecules were predominantly located basolaterally (80%), whereas class II molecules having either tail truncated were distributed in a nonpolarized fashion (∼40% apical and 60% basolateral), indicating that both α and β cytoplasmic tails were required for efficient class II basolateral targeting. Ii coexpression had little or no effect on the steady-state polarized surface distribution of wild-type or truncated class II molecules, although >90% of surface-expressed Ii was detected basolaterally in these cells (Table I). This was expected because class II molecules in complex with Ii only account for a few percent of total class II at the plasma membrane at steady state (43). Together, these data show that basolateral distribution of class II molecules depended on the cytoplasmic tails of the α- and β-chains but were independent of Ii coexpression.

FIGURE 1.

Amino acid sequence in the single letter code of the HLA-DR α- and β-chain cytoplasmic tails. Δα and Δβ lack the C-terminal 10 or 13 aa of the cytoplasmic tail, respectively. In β FL-AA, the FL residues of the β tail were point mutated to AA, as indicated with bold letters.

FIGURE 1.

Amino acid sequence in the single letter code of the HLA-DR α- and β-chain cytoplasmic tails. Δα and Δβ lack the C-terminal 10 or 13 aa of the cytoplasmic tail, respectively. In β FL-AA, the FL residues of the β tail were point mutated to AA, as indicated with bold letters.

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Table I.

Polarized cell-surface distribution of class II molecules (HLA-DR) and Ii in stably transfected MDCK cellsa

125I-L243 Binding (apical/basolateral ± SE)125I-BU45 Binding (apical/basolateral ± SE)
− Ii+ Ii
αβ 19/81 ± 3.1 10/90 ± 2.7 Ii (αβ) 6/94 ± 2.1 
Δαβ 38/62 ± 5.1 27/73 ± 0.3 Ii (Δαβ) 2/98 ± 0.5 
αΔβ 38/62 ± 2.2 35/65 ± 0.9 Ii (αΔβ) 3/97 ± 3.0 
ΔαΔβ 42/58 ± 2.7 41/59 ± 0.5 Ii (ΔαΔβ) 7/93 ± 4.0 
αβFL-AA 53/47 ± 4.1    
125I-L243 Binding (apical/basolateral ± SE)125I-BU45 Binding (apical/basolateral ± SE)
− Ii+ Ii
αβ 19/81 ± 3.1 10/90 ± 2.7 Ii (αβ) 6/94 ± 2.1 
Δαβ 38/62 ± 5.1 27/73 ± 0.3 Ii (Δαβ) 2/98 ± 0.5 
αΔβ 38/62 ± 2.2 35/65 ± 0.9 Ii (αΔβ) 3/97 ± 3.0 
ΔαΔβ 42/58 ± 2.7 41/59 ± 0.5 Ii (ΔαΔβ) 7/93 ± 4.0 
αβFL-AA 53/47 ± 4.1    
a

Full-length or cytoplasmic tail-truncated α- and β-chains of HLA-DR were expressed either alone or together with Ii, and the cells were grown on Transwell units to form tight monolayers. 125I-L243 (anti-HLA-DR) or 125I-BU45 (anti-Ii) were added either to the apical or the basolateral compartment for 2 h on ice. Unbound Ab was removed by extensive washing, filters were excised, and bound radioactivity was determined. Values are given as the percent specific binding to the apical or the basolateral surface and represent mean values (±SE) of at least three independent experiments performed in duplicates. Values for nonspecific binding, determined in the presence of a 100-fold excess of nonlabeled Ab, were subtracted and generally represented <10% of the total binding. Total binding in cpm × 103 was between 100 and 200 for 125I-L243 and ∼5 for 125I-BU45.

Efficient basolateral sorting of several transmembrane proteins depends on sorting signals in their cytoplasmic domains. These signals can be colinear with signals for internalization through clathrin-coated pits and often depend on a critical tyrosine residue or on two hydrophobic amino acids, one being a leucine (for reviews see Refs. 26, 27, 28). The cytoplasmic tail of the DR1 β-chain contains a putative leucine motif (FL), which is conserved as LL or FL in several species (13). To analyze whether the FL residues were involved in internalization and basolateral targeting of class II molecules, these residues were mutated to alanines (Fig. 1) and the mutant β-chain (αβFL-AA) was cotransfected with wild-type α-chain. The polarized plasma membrane distribution of αβFL-AA was analyzed by binding of 125I-L243 to the apical or the basolateral side of the cell monolayer at 4°C. αβFL-AA molecules were located at both apical and basolateral plasma membrane domains in a nonpolarized fashion (50% apical, 50% basolateral) (Table I). Thus, the FL residues in the β-chain cytoplasmic tail were essential for efficient basolateral distribution of the class II molecules.

The polarized surface distribution of wild-type and mutant class II molecules found by binding of iodinated Abs on ice was confirmed by confocal immunofluorescence microscopy (Fig. 2,A) and immuno-EM (Fig. 3). Both wild-type and mutant class II molecules were also detected in vesicles (micrographs not shown). To determine whether vesicular class II localization was due to internalization from the plasma membrane, cells expressing αβ or αβFL-AA molecules were incubated with L243 at the apical or the basolateral side of the cell monolayer for 30 min at 37°C. Vesicular staining was seen when Abs were added at the basolateral, but not at the apical, surface of cells expressing αβ (Fig. 2,B, upper panel). No vesicular localization was observed upon the addition of Ab to cells expressing αβFL-AA molecules (Fig. 2 B, lower panel) or truncated class II molecules (micrographs not shown). Thus, the FL motif in the β-chain tail is required for internalization of class II molecules at the basolateral plasma membrane domain.

FIGURE 2.

Polarized distribution of wild-type and mutant class II molecules. Immunofluorescence micrographs show the polarized localization of class II molecules in cells expressing αβ (upper panels) and αβFL-AA (lower panels) after incubation with L243 at the apical (right panels) or the basolateral (left panels) side of the monolayer for 1 h at 4°C (A) or 37°C (B). After fixation, bound L243 were visualized by incubation with an FITC-conjugated GαM-IgG.

FIGURE 2.

Polarized distribution of wild-type and mutant class II molecules. Immunofluorescence micrographs show the polarized localization of class II molecules in cells expressing αβ (upper panels) and αβFL-AA (lower panels) after incubation with L243 at the apical (right panels) or the basolateral (left panels) side of the monolayer for 1 h at 4°C (A) or 37°C (B). After fixation, bound L243 were visualized by incubation with an FITC-conjugated GαM-IgG.

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FIGURE 3.

Immuno-EM micrographs show the polarized distribution of class II molecules in cells expressing αβ. While almost no class II was found at the apical plasma membrane (A), the basolateral plasma membrane (B) showed strong labeling. Class II molecules were detected by labeling with mouse anti-DRβ Abs followed by GαM-coated 20-nm colloidal gold particles (arrowhead). The cells were fixed following incubation with BSA-gold particles (procedure B), and the small gold particles seen at the apical plasma membrane represent 5-nm BSA-gold particles present in the apical medium. Bars, 500 nm.

FIGURE 3.

Immuno-EM micrographs show the polarized distribution of class II molecules in cells expressing αβ. While almost no class II was found at the apical plasma membrane (A), the basolateral plasma membrane (B) showed strong labeling. Class II molecules were detected by labeling with mouse anti-DRβ Abs followed by GαM-coated 20-nm colloidal gold particles (arrowhead). The cells were fixed following incubation with BSA-gold particles (procedure B), and the small gold particles seen at the apical plasma membrane represent 5-nm BSA-gold particles present in the apical medium. Bars, 500 nm.

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To further identify the intracellular localization of wild-type and mutant class II molecules, cells were grown on filter supports and processed for immuno-EM analysis. MDCK cells have been found to possess separate apical and basolateral early endosomes, but common late endosomes (22). To identify the different endosomal compartments, cell monolayers were incubated with 5-nm and 10-nm BSA-coated colloidal gold particles in the apical or the basolateral medium, respectively, for 1 h at 37°C (procedure A). Apical early endosomes (AP-EE) and basolateral early endosomes (BL-EE) were defined as compartments containing only 5-nm or 10-nm gold particles, respectively, and late endosomes-1h (LE-1h) as compartments containing both endocytosed tracers. The compartments defined as LE-1h were morphologically heterogenous and may also include a common recycling compartment and transcytotic compartments. Sections were labeled with different Abs against class II molecules (anti-DRβ or L243) and/or Ii (VicY1 or BU45). Both Abs used against class II and Ii gave similar results. The endosomal distribution of class II and Ii was then estimated by relating the number of positively labeled compartments with a defined character to the total number of positively labeled compartments, as shown in Table II.

Table II.

Intracellular distribution of class II and Ii-positive vesiclesa

CompartmentCell lineProcedure
αβαβIiαβIiΔαΔβΔαΔβIiΔαΔβIiαβFL-AA
AP-EE 
BL-EE 24 ± 8 20 ± 4 15 ± 8 3 ± 3 
LE-1h 76 ± 8 80 ± 4 85 ± 8 100 100 97 ± 3 100 
 n = 66 n = 54 n = 36 n = 55 n = 23 n = 36 n = 60  
         
Mvb-1h 69 ± 6 88 ± 2 82 ± 3 63 ± 3 94 ± 4 91 ± 5 70 ± 4 
LE-ON 30 ± 5 9 ± 2 12 ± 4 25 ± 3 4 ± 3 8 ± 4 25 ± 2 
Lysosomes 1 ± 1 3 ± 3 6 ± 1 12 ± 4 2 ± 2 1 ± 1 5 ± 2 
 n = 60 n = 60 n = 108 n = 42 n = 61 n = 53 n = 124  
CompartmentCell lineProcedure
αβαβIiαβIiΔαΔβΔαΔβIiΔαΔβIiαβFL-AA
AP-EE 
BL-EE 24 ± 8 20 ± 4 15 ± 8 3 ± 3 
LE-1h 76 ± 8 80 ± 4 85 ± 8 100 100 97 ± 3 100 
 n = 66 n = 54 n = 36 n = 55 n = 23 n = 36 n = 60  
         
Mvb-1h 69 ± 6 88 ± 2 82 ± 3 63 ± 3 94 ± 4 91 ± 5 70 ± 4 
LE-ON 30 ± 5 9 ± 2 12 ± 4 25 ± 3 4 ± 3 8 ± 4 25 ± 2 
Lysosomes 1 ± 1 3 ± 3 6 ± 1 12 ± 4 2 ± 2 1 ± 1 5 ± 2 
 n = 60 n = 60 n = 108 n = 42 n = 61 n = 53 n = 124  
a

The intracellular distribution of class II and Ii-positive vesicles in the endocytic pathway of MDCK cells stably expressing αβ, αβIi, ΔαΔβ, ΔαΔβIi, or αβFL-AA was characterized by two separate endocytic uptake procedures and by morphology. Procedure A, Cell monolayers were incubated with 5-nm and 10-nm colloidal gold particles in the apical or the basolateral medium, respectively, for 1 h at 37°C. AP-EE and BL-EE were defined as compartments containing only 5-nm or 10-nm gold particles, respectively, and LE-1h as compartments containing both endocytosed markers. Procedure B, Cell monolayers were incubated with 10-nm gold particles at the basolateral side for 3 h at 37°C, followed by an 18-h chase and a subsequent 1-h apical uptake of 5-nm colloidal gold particles. Vesicles containing 5-nm gold particles only generally were multivesicular bodies (mvb-1h), whereas vesicles containing both 5- and 10-nm gold particles (LE-ON) were multilamellar. Vesicles containing only 10-nm gold particles were typically electron-dense lysosomes.

The endosomal distribution of class II and Ii-positive vesicles was estimated by EM of thawed cryosections labeled for class II with anti-DRβ and/or Ii with BU45. Bound Abs were visualized using secondary Abs conjugated with 15-nm or 20-nm gold particles. Values are given as percentage of the total number of positively labelled intracellular compartments and represent mean values (±SEM) of three independent experiments (n, total number of positive vesicles counted).

Both in cells expressing either class II alone (αβ) or together with Ii, 20–25% of the class II-positive compartments were BL-EE and correspondingly 75–80% LE-1h (Table II). A similar distribution of Ii-positive vesicles (15% BL-EE and 85% LE-1h) was detected in the αβIi cells. No class II or Ii was detected in AP-EE. The class II expression levels in these cells were similar as judged by immunoprecipitation analysis (data not shown). Moreover, the fraction of class II-positive BL-EE and LE-1h compared with the total number of BL-EE and LE-1h was similar in αβ and αβIi cells (data not shown). Thus, the class II molecules were mainly located in compartments accessible to both apical and basolateral tracers, both when expressed alone and together with Ii. Class II and Ii molecules were also located in BL-EE, probably due to internalization from the basolateral plasma membrane.

In contrast, point-mutated (αβFL-AA) or truncated class II molecules (ΔαΔβ) were only detected in LE-1h and neither in AP-EE or BL-EE, both when expressed alone and together with Ii (Table II). Moreover, in ΔαΔβIi cells 97% of the Ii-positive compartments were LE-1h and only 3% BL-EE (Table II), suggesting that signals in the class II cytoplasmic tails influenced the intracellular sorting of Ii. The fraction of class II-positive LE-1h compared with the total number of LE-1h was similar in cells expressing wild-type or mutant class II molecules (data not shown). These data indicate that transport of class II molecules to BL-EE, but not to late endosomes, requires the FL motif in the β-chain tail.

The late endosomal population (LE-1h) defined above was morphologically heterogeneous. To further elucidate the precise intracellular class II localization, we used a different assay to separate distinct late endocytic populations. Cell monolayers were incubated with 10-nm BSA-gold particles at the basolateral side for 3 h at 37°C followed by an 18-h chase and a subsequent 1-h apical uptake of 5-nm BSA-gold particles (procedure B). As found by procedure A, 5-nm BSA-gold particles apically internalized for 1 h localized to AP-EE as well as more complex compartments corresponding to LE-1h. Using procedure B, the late endosomes could be separated into at least two different subpopulations: multivesicular bodies containing 5-nm BSA-gold particles only (mvb-1h) located apically to the nucleus, and compartments containing both 5-nm and 10-nm BSA-gold particles (LE-ON). The morphology of the LE-ON compartments varied from multivesicular to more multilamellar, and the LE-ON contained more endocytosed BSA-gold particles (both 5 and 10 nm) compared with mvb-1h. Compartments containing 10-nm BSA-gold particles only resembled typically electron-dense lysosomes. Sections were labeled with Abs against class II or Ii, and the endosomal distribution of class II and Ii was estimated as described above. In cells expressing wild-type (αβ) (Fig. 4,B), point-mutated (αβFL-AA), or truncated (ΔαΔβ) class II molecules alone, ∼70% of the class II-positive compartments were mvb-1h and ∼30% were LE-ON, with little or no class II detected in lysosomes (Table II). In cells coexpressing Ii and wild-type (Fig. 4,C), point-mutated, or truncated (Fig. 4,D) class II molecules, ∼90% of the class II-positive vesicles were mvb-1h and <10% LE-ON (Table II). In these cells, 80–90% of the Ii-positive compartments were defined as mvb-1h. A corresponding shift in class II localization from LE-ON to mvb-1h upon Ii coexpression was also observed when the number of class II-positive LE-ON/mvb-1h was compared with the total number of LE-ON/mvb-1h in these cells (data not shown). Thus, the major fraction of both wild-type and mutant class II molecules was located in multivesicular compartments, which were reached by an apical endocytic marker within 1 h. The efficiency of class II localization to these compartments was increased upon Ii coexpression, which could be caused by Ii-induced retention of the gold particles in early endocytic compartments (44), although we cannot exclude that Ii may regulate endosomal distribution of class II molecules in a more specific way.

FIGURE 4.

Immuno-EM micrographs show the intracellular localization of class II molecules in cells expressing αβ (A and B), αβIi (C), ΔαΔβIi (D), and αβIiCD8DM (E). Before fixation, polarized monolayers of the transfected cells were incubated either with 5-nm (small arrowheads) and 10-nm (large arrowheads) BSA-gold particles in the apical and basolateral medium, respectively, for 1 h (A); or with 10-nm BSA-gold particles (large arrowheads) in the basolateral medium for 3 h, followed by an 18-h chase and a subsequent 1-h incubation with 5-nm BSA-gold particles (small arrowheads) in the apical medium (B–E). Sections were then either labeled for class II molecules, using mouse anti-DRβ Abs followed by GαM-coated 20-nm colloidal gold particles (A–D), or double-labeled for class II and Ii using Abs to Ii (VicY1) and GαM-coated 15-nm colloidal gold particles after the class II labeling (E). Class II molecules (thin arrows) were found in LE-1h (A), mvb-1h (B–D), and LE-ON (C). Double labeling for class II and Ii revealed colocalization of class II (thin arrows) and Ii (bold arrows) in mvb-1h (E). Bars, 100 nm.

FIGURE 4.

Immuno-EM micrographs show the intracellular localization of class II molecules in cells expressing αβ (A and B), αβIi (C), ΔαΔβIi (D), and αβIiCD8DM (E). Before fixation, polarized monolayers of the transfected cells were incubated either with 5-nm (small arrowheads) and 10-nm (large arrowheads) BSA-gold particles in the apical and basolateral medium, respectively, for 1 h (A); or with 10-nm BSA-gold particles (large arrowheads) in the basolateral medium for 3 h, followed by an 18-h chase and a subsequent 1-h incubation with 5-nm BSA-gold particles (small arrowheads) in the apical medium (B–E). Sections were then either labeled for class II molecules, using mouse anti-DRβ Abs followed by GαM-coated 20-nm colloidal gold particles (A–D), or double-labeled for class II and Ii using Abs to Ii (VicY1) and GαM-coated 15-nm colloidal gold particles after the class II labeling (E). Class II molecules (thin arrows) were found in LE-1h (A), mvb-1h (B–D), and LE-ON (C). Double labeling for class II and Ii revealed colocalization of class II (thin arrows) and Ii (bold arrows) in mvb-1h (E). Bars, 100 nm.

Close modal

To further characterize the class II-positive compartments, MDCK cells coexpressing wild-type class II molecules and Ii were transfected stably with the fusion protein CD8-DMβ, containing the cytoplasmic tail of the HLA-DMβ-chain fused to the transmembrane and luminal domains of the plasma membrane resident protein CD8. CD8-DMβ has been shown to colocalize with HLA-DM molecules in HeLa cells (45). Polarized cells expressing class II, CD8-DMβ, and Ii were processed for immuno-EM analyses by uptake of endocytic markers as described in procedure B. The extent of colocalization of class II, Ii, and CD8-DMβ was determined by indirect sequential double labeling.

We found extensive colocalization of class II and Ii (Fig. 4 E), of class II and CD8-DMβ, and of Ii and CD8-DMβ molecules (micrographs not shown) in mvb-1h. Some colocalization was also detected in LE-ON, but not in AP-EE or BL-EE. A different intracellular distribution of intact HLA-DM cannot be excluded due to the protease sensitivity of CD8-DMβ as opposed to HLA-DM. A tyrosine-based signal in the DMβ cytoplasmic tail has been identified as essential for endosomal targeting of HLA-DM molecules (45, 46, 47). CD8-DMβY-A molecules, having the tyrosine mutated to alanine, were not detected in mvb-1h, although some labeling of LE-ON has been observed,6 indicating that this is a signal for sorting to the mvb-1h compartments. The specific localization of class II, Ii, and HLA-DM molecules, all components required for class II peptide loading, in the mvb-1h suggest that these structures may be a class II peptide loading compartment.

As the majority of class II molecules were found in late multivesicular endocytic compartments both when expressed alone and together with Ii, we were interested in whether MDCK cells expressing HLA-DR1 molecules alone were able to process and present the hemaglutinin H3 and the matrix protein M1 epitope of influenza virus to HLA-DR1-restricted T cells. Previous studies have shown that the H3 epitope can bind to recycling class II molecules in early endocytic compartments, whereas binding of the M1 epitope occurs in later endocytic compartments and requires newly synthesized class II molecules and Ii (11, 12).

Nonpolarized MDCK cells expressing αβ were labeled with 51Cr, pulsed with H3 or M1 synthetic peptides or UV-inactivated influenza virus (UV-flu) particles, and tested for lysis by the H3- or M1-specific cytotoxic T cell clones, C1.6 and C3.5, respectively. As shown in Fig. 5,a, the synthetic H3 peptide and the H3 epitope of processed influenza virus were presented efficiently to C1.6. The cells were also able to present synthetic M1 peptide and the M1 epitope of processed UV-flu particles to C3.5 T cells (Fig. 5 b). Together, these data show that MDCK cells expressing DR1-molecules were able to process and present influenza virus Ags and that both the H3 and the M1 epitope are presented independently of Ii coexpression. These results are consistent with our EM data, showing that class II molecules are present in typical peptide loading compartments independent of Ii or intact cytoplasmic tails.

FIGURE 5.

Presentation of inactivated influenza virus by MDCK αβ cells to HLA-DR1-restricted T cell clones. Cells expressing αβ were either untreated, pulsed with synthetic peptide (H3 307–318 or M1 18–31), or pulsed with increasing concentrations of UV-flu at different E:T ratios and then analyzed for lysis by the H3-spesific clone C1.6 (a) or the M1-specific clone C3.5 (b). The data are presented as percentage of maximum lysis after subtraction of the spontaneous lysis.

FIGURE 5.

Presentation of inactivated influenza virus by MDCK αβ cells to HLA-DR1-restricted T cell clones. Cells expressing αβ were either untreated, pulsed with synthetic peptide (H3 307–318 or M1 18–31), or pulsed with increasing concentrations of UV-flu at different E:T ratios and then analyzed for lysis by the H3-spesific clone C1.6 (a) or the M1-specific clone C3.5 (b). The data are presented as percentage of maximum lysis after subtraction of the spontaneous lysis.

Close modal

MHC class II molecules may be involved in Ag presentation in epithelial cells of various organs (1, 2). The class II molecules are located at the basolateral surface of transfected polarized MDCK cells (30). Here, MDCK cells stably transfected with wild-type or mutant HLA-DR1 molecules, alone or together with Ii, were used to further characterize the intracellular localization of class II molecules and the signals involved in their polarized transport.

We found that class II molecules were located on the basolateral plasma membrane, in BL-EE, and in multivesicular compartments that were accessible to both apical and basolateral endocytic markers, but not at the apical plasma membrane or in AP-EE. Hershberg et al. (48) found that class II-restricted peptides were only presented when fed basolaterally. Our data are in agreement with this observation if HLA-DR were loaded at the plasma membrane or in early (recycling?) basolateral endosomes. The FL residues in the cytoplasmic tail of the HLA-DR1 β-chain were required for efficient basolateral sorting and internalization at the basolateral plasma membrane and thus for localization in BL-EE. A dihydrophobic motif (LL or FL) is found in the β-chain cytoplasmic tail of several species (13), indicating an important role of this evolutionary conserved motif in the function of class II molecules. The α-chain cytoplasmic tail was also required for efficient basolateral transport of DR1 molecules, as truncation of either tail lead to nonpolarized sorting of the class II molecules. The α-chain tail may contribute to correct folding of the FL motif to be recognized by a sorting machinery or the α-chain may contain sorting information. These results are consistent with a previous study (11) where we found that both the class II α and β cytoplasmic tails were required for internalization of HLA-DR1 from the plasma membrane. Moreover, Zhong et al. (13) identified a dileucine motif in the cytoplasmic tail of the mouse Ak class II β-chain, corresponding to the FL residues in DRβ, as essential for Ag presentation by recycling class II molecules. Several studies have shown that tailless and full-length class II molecules differed in their Ag presenting capacity (10, 11, 49, 50, 51), and truncation of the β-chain were found to reduce the level of class II-associated invariant chain peptide complexes (52), possibly caused by different intracellular routing of wild-type and truncated class II molecules.

The cytoplasmic tail of Ii contains two leucine-based basolateral sorting signals, which also function as internalization signals (30). Ii coexpression only marginally increased the basolateral distribution of wild-type and truncated class II molecules, which is not surprising, considering that only a few percent of the surface class II molecules are in complex with Ii and that this fraction is efficiently internalized (43). Thus, the main role of Ii may be to target newly synthesized class II molecules to endosomes. Basolateral sorting signals within the class II cytoplasmic tails may then be important for transport of these molecules to the basolateral surface after dissociation from Ii in endosomes. Together, our data suggest that intracellular transport of class II-Ii is directed by the function of several different leucine-based signals. The amino acid context requirements for motifs involved in both basolateral sorting and internalization may be identical or differ (27, 28, 53), and it is tempting to speculate that the motifs have different affinities for components of the sorting machinery and may even work differently at different sorting stations, resulting in a fine tuning of the final routing and destination of the class II-Ii complex within the cell.

Class II-Ii complexes can be transported to the endocytic pathway directly from the trans-Golgi network (54, 55) or via the plasma membrane (6, 43). Class II molecules can also enter endosomes in the absence of Ii (for reviews see Refs. 8, 56, 57). In polarized MDCK cells, class II molecules were detected both in BL-EE and in late endosomes, containing internalized apical and basolateral markers, both in the presence and the absence of Ii. Localization to BL-EE, but not to late endosomes, required both the α- and the β-chain cytoplasmic tails, including the FL motif in the β tail. Interestingly, less Ii-positive BL-EE were observed in cells expressing truncated αβ, suggesting that information in the class II cytoplasmic tails maybe influenced the initial sorting of Ii before degradation. Ii-independent localization of class II molecules to late endosomes correlates with the data obtained in the cytotoxic T cell assay showing that both the H3 and the M1 epitope of inactivated influenza virus can be presented by cells expressing DR1 molecules. Previous studies in human fibroblasts demonstrated that presentation of the M1 epitope requires Ii-dependent sorting of newly synthesized class II molecules to late endocytic compartments, whereas the H3 epitope could be presented by class II molecules recycling from the plasma membrane into earlier endosomal loading compartments (11, 12). We have used the same HLA-DR1, Ag, and T cells as in Pinet et al. (11, 12), so the discrepancy is most likely due to cell type-specific differences in protein sorting and/or Ag processing.

The class II-positive late endosome population was morphologically heterogeneous, and we distinguished between two different class II-positive LE-1h populations: multivesicular bodies located apically of the nucleus which contained 1-h apically endocytosed marker (mvb-1h), and more complex multivesicular and multilamellar vesicles containing both 1-h apical marker and basolateral marker internalized for 3 h with a subsequent 18-h chase (LE-ON). The mvb-1h contained less endocytosed marker and was probably earlier in the endocytic pathway than the LE-ON. The majority of class II-positive vesicles in cells expressing wild-type class Ii molecules were mvb-1h. Interestingly, also in cells expressing mutant class II molecules, most class II-positive late endosomes were defined as mvb-1h. As mutant class II molecules were not detected in early endosomes, this indicates that newly synthesized class II molecules may be sorted directly to the mvb-1h compartments independently of information within their tails. This may be due to the luminal 80–82 aa segment of the class II β-chain, reported to control late events in the intracellular sorting of class II molecules (58, 59). A different explanation may be that a slow delivery of mutant class II molecules to mvb-1h through the endocytic pathway by normal plasma membrane turnover leads to accumulation in these compartments as the class II molecules are relatively resistant to degradation. We could detect typical mvb-1h also in cells lacking class II molecules, indicating that these structures were not induced by the class II expression. The mvb-1h may be apical recycling compartments, previously described to contain other basolateral proteins as the transferrin receptor and transcytosed IgA (60). These data are in line with other studies showing that APC harbor a major pool of their intracellular class II molecules in special endocytic compartments, termed MHC class II compartments (MIICs) (61) or class II containing vesicles (62). Both multivesicular and multilamellar MIICs have been described (61), where the multivesicular MIICs, thought to represent the main entry site of newly synthesized class II molecules in the endocytic pathway, represented an earlier endocytic compartment than the multilamellar MIICs (63).

Several studies have shown that MIICs contain high levels of HLA-DM molecules (64, 65, 66), suggesting that these compartments are peptide loading compartments. In our study, most Ii-positive vesicles were mvb-1h, and the fraction of class II-positive mvb-1h were increased upon Ii coexpression, indicating that Ii facilitated transport to or retention in mvb-1h. Moreover, we found that the fusion protein CD8-DMβ, containing the cytoplasmic tail of HLA-DMβ fused to CD8, was sorted to the same mvb-1h as class II and Ii, whereas a fusion protein having the tyrosine-sorting motif of the DMβ tail mutated to alanine was excluded from the mvb-1h. This is in line with previous data from several groups (45, 46, 47) who found this tyrosine signal to be required for sorting of HLA-DM molecules to MIIC. Localization of class II, Ii, and HLA-DM molecules in compartments that are accessible to both apical and basolateral endocytic markers is consistent with a specific function of such compartments in epithelial Ag presentation. However, the steady-state distribution of HLA-DM may be different from the fusion protein, as CD8-DMβ is protease sensitive whereas intact HLA-DM is long lived. The localization of class II molecules expressed in the absence of Ii and HLA-DM to similar peptide loading compartments is in line with the studies of Hershberg et al. (48), who showed that an HLA-DR-positive intestinal epithelial cell line may present apically internalized Ag in the absence of detectable Ii and HLA-DM. Thus, the specific localization of class II molecules in compartments that are accessible for endocytic markers internalized both from the apical and the basolateral side of the epithelial cell monolayer makes APC able to survey both external environments for foreign intruders.

We thank Hege Hardersen and Målfrid Røe for technical assistance and Thomas Halder for critical reading of the manuscript.

1

A.S. was supported by grants from the Research Counsil of Norway, and T.W.N was supported by a grant from the Norwegian Cancer Society.

5

Abbreviations used in this paper: Ii, invariant chain; GαM, goat anti-mouse; MDCK, Madin-Darby canine kidney; EM, electron microscopy; AP-EE, apical early endosomes; BL-EE, basolateral early endosomes; LE-1h, late endosomes containing both 1 h apical and basolateral endocytosed marker; mvb, multivesicular bodies; mvb-1h, multivesicular bodies with 1 h uptake of endocytosed marker; LE-ON, late endosomes with endocytosed markers after both 1 h and overnight incubation; MIIC, MHC class II compartments; UV-flu, UV-inactivated influenza virus particles.

6

Røe, M., K. Egdalen, A. Simonsen, A. Kelly, and O. Bakke. Polarized targeting of HLA-DM by a tyrosine motif in the β-chain. Submitted for publication.

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