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Colocalization of FcRI-Targeted Antigen With Class I MHC: Implications for Antigen Processing¹

Cheryl A. Guyre^*, Marc E. Barreda, Sharon L. Swink and Michael W. Fanger^2,

Departments of ^* Physiology and Microbiology, Dartmouth Medical School, Lebanon, NH 03756

	Abstract

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The high-affinity receptor for IgG (CD64 or FcRI) is constitutively expressed exclusively on professional APCs (monocytes, macrophages, and dendritic cells). When Ag is targeted specifically to FcRI, Ag presentation is markedly enhanced, although the mechanism of this enhancement is unknown. In an effort to elucidate the pathways involved in FcRI targeting, we developed a model targeted Ag using enhanced green fluorescent protein (eGFP). This molecule, wH22xeGFP, consists of the entire humanized anti-FcRI mAb H22 with eGFP genetically fused to the C-terminal end of each CH3 domain. wH22xeGFP binds within the ligand-binding region by its Fc end, as well as outside the ligand-binding region by its Fab ends, thereby cross-linking FcRI. Confocal microscopy studies revealed that wH22xeGFP was rapidly internalized by the high-FcRI-expressing cell line U937 10.6, but did not associate with intracellular proteins Rab4, Rab5a, or Lamp-1, suggesting that the targeted fusion protein was not localized in early endosomes, recycling vesicles, or lysosomes. Interestingly, wH22xeGFP was found colocalized with intracellular MHC class I, suggesting that FcRI-targeted Ags may converge upon a class I processing pathway. These data are in agreement with studies in the mouse showing that FcRI targeting can lead to Ag-specific activation of cytotoxic T cells. Data obtained from these studies should lead to a better understanding of how Ags targeted to FcRI are processed and under what conditions they lead to presentation of antigenic peptides in MHC class I, as a foundation for the use of FcRI-targeted Ags as vaccines.

	Introduction

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The targeting of Ag to certain cell surface molecules, including FcR, has been shown in many systems to lead to more efficient presentation (reviewed in Refs. 1 and 2). It has been shown that FcR-mediated uptake of Ag-Ab complexes can enhance Ag presentation by monocytes (3) and B cells (4) and that directing Ag to FcR in mice increases the effectiveness of immunization (5). Studies by Liu et al. demonstrated that Ag presentation could be markedly enhanced by targeting tetanus toxoid (TT)³ to FcRII on human B cells using a chemical conjugate of TT and a mAb specific for FcRII (4). In a related study, enhanced Ag presentation was demonstrated using an immunogenic Th epitope of TT targeted to FcRI on monocytes using a fusion protein of peptide and anti-FcRI mAb (6). Similar results were observed when dendritic cells (DCs) were used as APC (7). Although this enhancement has been well documented in systems using T cell proliferation assays and CD4⁺ T cell lines, few groups have demonstrated the effects of Ag targeting on class I-restricted Ag presentation. Recently, however, Regnault et al. showed that targeting Ag to FcR on murine DCs using immune complexes led to enhanced MHC class I-restricted Ag presentation (8). Likewise, Machy et al. demonstrated a striking enhancement (up to 100,000-fold) in both class I and class II presentation of OVA when it was targeted to FcR on murine DCs using liposomes (9). Although both of these groups demonstrated that enhanced Ag presentation was mediated through Fc receptors, and were able to rule out the participation of FcRII, they were unable to distinguish between FcRI and FcRIII as being the critical receptor. Studies by Wallace et al. using the human myeloid cell line THP-1 as the APC showed that targeting Ag to human FcRI led to enhanced killing of those cells by Ag-specific CTL (10).

It is unclear how certain exogenous Ags, which were once thought to be processed only through the class II processing pathway, end up being presented in the context of class I. A few years ago, Rock proposed two models of processing, one that involved the delivery of the Ag into the cytosol for proteosomal degradation and entry into the classical class I pathway, and a second that involved the proteolysis of Ag in phagolysosomes and subsequent fusion of these vesicles with those containing newly synthesized or recycled class I molecules (11). Recent studies focusing on processing pathways using confocal microscopy have provided evidence for the existence of both pathways (12, 13).

In an effort to elucidate the processing pathways involved following uptake of FcRI-targeted Ags, we have developed an FcRI-targeted enhanced green fluorescent protein (eGFP) fusion protein, wH22xeGFP, that binds to and cross-links FcRI. The eGFP molecule serves as a model Ag and can be tracked using confocal microscopy. wH22xeGFP has been shown to internalize rapidly (C. Guyre, manuscript in preparation), and the intracellular location of the fluorochrome can be determined after various treatment times by fixing the cells and then performing intracellular staining with Abs known to localize to a particular subcellular location. Therefore, this fusion protein can be used as a tool to evaluate the internalization and processing of Ags targeted to FcRI via an anti-FcRI mAb, a strategy currently being pursued for the development of vaccines (2). This paper outlines studies that were performed to determine the intracellular trafficking and colocalization of wH22xeGFP with intracellular molecules known to be involved in the endosomal/lysosomal pathway. Importantly, these studies provide the first direct evidence of colocalization of an FcRI-targeted fusion protein with intracellular MHC class I.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and reagents

A high-FcRI-expressing subclone of the parent U937 human myeloid cell line, 10.6, was a kind gift of Dr. Paul M. Guyre (Dartmouth Medical School, Lebanon, NH). These cells were originally derived by FACS sorting for the 0.1% brightest U937 cells stained for FcRI and cloned by limiting dilution (14). Cells were cultured in RPMI 1640 supplemented with 10% FBS, L-glutamine, 25 mM HEPES, and 50 µg/ml gentamicin (all from BioWhitaker, Walkersville, MD) (complete medium) in the presence or absence of 25 ng/ml IFN- (Genentech, South San Francisco, CA) at 37°C/5% CO₂. wH22xeGFP is a fusion protein in which the eGFP molecule is genetically fused to the CH3 domain of the H chain of H22, a humanized mAb that binds FcRI outside of the ligand-binding domain by its Fab ends, and within the ligand-binding domain by its Fc end (C. Guyre, manuscript in preparation).

Antibodies

Unlabeled Abs used included W6/32, a murine IgG2a Ab specific for human class I (American Type Culture Collection (ATCC), Manassas, VA) (15); 32.2, a murine IgG1 Ab specific for human FcRI (Medarex, Annandale, NJ); anti-Rab4, a rabbit polyclonal Ab specific for the endosomal marker Rab4 (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-Rab5a, a rabbit polyclonal Ab specific for the endosomal marker Rab5a (Santa Cruz Biotechnology). Both anti-Rab4 and anti-Rab5a are specifically reactive to endosomal proteins of human, mouse, and rat species. Control Abs included RPC5.4, a murine IgG2a isotype control (ATCC) (16); P3, a murine IgG1 isotype control (ATCC); and normal rabbit IgG (Santa Cruz Biotechnology). Secondary Abs included Cy3-conjugated F(ab')₂ goat anti-mouse IgG⁺IgM and Cy5-conjugated goat anti-rabbit IgG (both from Jackson ImmunoResearch, West Grove, PA). W6/32 and a murine IgG2a isotype control (R&D Systems, Minneapolis, MN) were directly labeled with Cy3 using FluoroLink Cy3 monofunctional dye (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions. Other directly labeled Abs included CyChrome-conjugated anti-CD107a, a murine IgG1 specific for human Lamp-1; CyChrome-conjugated anti-HLA-A,B,C, a murine IgG1 specific for human class I; and CyChrome-conjugated murine IgG1 isotype control (all from PharMingen, San Diego, CA). In some studies, human globulin (Sigma, St. Louis, MO) was used at a final concentration of 3 mg/ml in complete medium containing 0.5 mg/ml BSA.

Internalization assay and confocal staining

Cells were treated on ice or at 37°C with wH22xeGFP from 30 min to 16 h. Following treatment, cells were washed twice with ice-cold PBS containing 1% BSA (Sigma) and 0.05% sodium azide (PBA; Fisher Scientific, Fair Lawn, NJ) and fixed in ice-cold 1% methanol-free formaldehyde (MFF; Polysciences, Warrington, PA) in PBS for a minimum of 1 h. In some studies, a second fixation was performed using 1% MFF in PBA containing 0.5% saponin (PBAS; Sigma). Cells were then transferred to a 96-well round-bottom polypropylene microtiter plate (Costar, Corning, NY) and washed twice with ice-cold PBAS. In some experiments, cells were incubated for 1 h on ice with 3 mg/ml human globulin (Sigma) in PBAS to block nonspecific binding. Abs specific for intracellular markers diluted in PBAS were then added at a final concentration of 10–30 µg/ml for an additional 1 h on ice. For CyChrome-labeled Abs, 10 µl was used per 10⁶ cells, according to the manufacturer’s recommendation. After staining, cells were washed twice with ice-cold PBAS. For samples stained with directly conjugated Abs, cells were fixed in 1% MFF in PBS and stored at 4°C. For samples stained with unconjugated Abs, cells were resuspended in the appropriate secondary Ab (1:40 dilution in PBAS) and incubated on ice for 1 h. Cells were then washed twice with ice-cold PBAS, fixed in 1% MFF in PBS, and stored at 4°C.

Confocal microscopy

Fixed cells were pelleted and 3.5 µl of pelleted cells was placed on a glass slide (Gold Seal Products, Portsmouth, NH). Approximately 3.5 µl of Prolong Antifade reagent (Molecular Probes, Eugene, OR) was added, followed by an 18-mm coverslip (Corning), which was then sealed with nail varnish. Imaging was performed on a Bio-Rad MRC-1024 confocal scanning laser microscope system (Bio-Rad, Richmond, CA) using a krypton/argon laser and LaserSharp version 3.2 software (Bio-Rad). 605DF32, 522DF32, and 680DF32 band pass filters were used for photomultiplier tubes (PMTs) 1, 2, and 3, respectively. Laser power was set to 3% on all lines (488, 568, and 647 nm wavelength). All cells were imaged using a x63/1.4NA PlanApo objective (Zeiss, Oberkochen, Germany) with oil and zooms of either 1.0, 3.0, or 5.0, with corresponding pixels sizes of 0.298, 0.099, and 0.060 µm, respectively. Iris size was 3.0 for all PMTs used in a given experiment. PMT 2 was set to photon-counting mode with low signal on to image eGFP fluorescence. PMT gains and black levels, as well as mixer settings, were determined for each individual experiment using unstained isotype control and single-color-stained samples to minimize autofluorescence and fluorochrome crossover. PMTs corresponding to fluorochromes not used in a given experiment were turned to the lowest iris, gain, and black level settings. Under optimal conditions, this microscope system is capable of an axial (z-axis) resolution approaching 0.5 µm and a lateral resolution of 0.2 µm. Using the settings described above, the section thickness is estimated to be slightly higher (1 µm) than the optimal axial resolution. The optimal lateral resolution of 0.2 µm for this microscope is attained with a zoom 3.0. Typical image acquisition settings included one to three accumulations of slow scans, which were determined by the minimum number of scans required to reach fluorochrome saturation for any part of the image field. Images were captured as a single section from the center plane of cells and are representative of two to seven fields captured per slide. Color levels were adjusted using Adobe Photoshop 4.0.1 software (Adobe Systems, Mountain View, CA) to provide the necessary contrast for color reproduction on slides and prints.

Flow cytometric analysis

Cells were treated at 37°C with 10 µg/ml of wH22xeGFP in complete medium. Following treatment, cells were washed twice with ice-cold PBA and stained on ice in a 96-well round-bottom polypropylene microtiter plate (Costar). Surface expression of FcRI and class I was determined using mAbs 32.2 (Medarex) and W6/32 (ATCC), respectively. Briefly, cells were resuspended in 12 mg/ml human IgG for 15 min to block nonspecific binding. mAb was then added at a final concentration of 20 µg/ml, and cells were incubated on ice for 1 h. Cells were then washed three times with ice-cold PBA, resuspended in a 1:40 dilution of R-PE-conjugated goat anti-mouse IgG, and incubated on ice for 45 min. Cells were washed twice with ice-cold PBA and resuspended in 1% MFF in PBS. Fixed cells were stored at 4°C for at least 16 h before flow cytometric analysis was performed. Fixed cells were analyzed by flow cytometry using a FACScan (BD Biosciences, San Jose, CA). For each experiment, compensation was set using unstained ("autofluorescent") or isotype control, green-only, and red-only samples to eliminate fluorochrome crossover between FL-1 and FL-2. Mean fluorescence intensity (MFI) values from the FL-2 channel were used to determine the percentage of cell surface expression following treatment with wH22xeGFP using the following equation: [(MFI treated with wHxeGFP - MFI isotype control)/(MFI untreated - MFI isotype control)] x 100%, where the isotype control is mAb P3 or RPC5.4, which reflects the background fluorescence due to nonspecific binding of Abs and cell autofluorescence.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intracellular colocalization of wH22xeGFP with MHC class I

To assess whether FcRI-targeted fusion proteins can converge upon a class I processing pathway, IFN--treated 10.6 cells were treated with wH22xeGFP for 5 h, then washed and fixed. Cells were next permeabilized with a saponin-containing buffer and stained for intracellular class I using mAb W6/32, followed by Cy3-conjugated goat anti-mouse IgG. Following fixation and mounting on glass slides, cells were imaged on a confocal microscope. Representative images were taken from the center plane of the cells to assess the internal staining of wH22xeGFP and class I molecules. Fig. 1 illustrates that regardless of whether cells were kept on ice or incubated at 37°C, intracellular class I appeared throughout the cytoplasm and in many cells existed largely in an intracellular pool. After 5 h at 37°C, most of the internalized wH22xeGFP fusion protein colocalized with class I MHC in a perinuclear staining pattern, as evidenced by the yellow (green + red) staining (Fig. 1). Importantly, the GFP moiety did not contribute to the binding of wH22xeGFP to cells, as shown by the ability of unlabeled H22 to completely block the binding of wH22xeGFP (Table I).

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Intracellular colocalization of wH22xeGFP with MHC class I. Cells were treated either on ice or at 37°C with 10 µg/ml wH22xeGFP for 5 h, washed with PBA, and fixed. Cells were permeabilized using PBAS, treated with 3 mg/ml human IgG, and then stained using W6/32, followed by Cy3-conjugated goat anti-mouse IgG. Green stain represents eGFP fluorescence, red stain represents class I staining, and yellow staining is the result of green plus red staining. Zoom = 1.0. Images are representative of at least five similar experiments.

In an effort to further characterize the nature of wH22xeGFP/intracellular class I interaction, a time course study was performed in which cells were stained on ice with wH22xeGFP, shifted to 37°C for various times, then washed and fixed. Samples were subsequently stained for intracellular class I using mAb W6/32, followed by Cy3-conjugated goat anti-mouse IgG. As seen in Fig. 2A, internalized fusion protein was found to be colocalized with class I as early as 0.5 h (yellow staining), although some wH22xeGFP was not colocalized with class I (green staining). By 2 h, most internalized fusion protein appeared to be colocalized with class I (yellow staining).

View larger version (41K): [in this window] [in a new window]   FIGURE 2. Kinetics of wH22xeGFP/MHC class I colocalization. Cells were treated at 37°C with 10 µg/ml wH22xeGFP for 0.5–4 h, washed with PBA, and fixed. Intracellular staining for class I was performed as described in Fig. 1. Green stain represents eGFP fluorescence, red stain represents class I staining, and yellow staining is the result of green plus red staining. A, Time course, zoom = 1.0; B, 4 h, zoom = 5.0. Images are representative of at least five similar experiments.

Surface anti-class I mAb internalizes at 37°C

To better understand the mechanisms involved, we examined whether colocalization of wH22xeGFP with MHC class I resulted from the intracellular association of fusion protein with class I or occurred as a result of the simultaneous entry of wH22xeGFP with surface class I. In particular, cells were surface-labeled on ice with wH22xeGFP, followed by human IgG (to block any potentially unoccupied Fc receptor), and an anti-class I mAb, Cy3-W6/32, was then added. After warming to 37°C, aliquots were removed, washed, and fixed at various time points. Cells were then analyzed by confocal microscopy. Results showed the internalization and colocalization (yellow) of wH22xeGFP (green) and of the anti-class I Ab (red), particularly evident at 2 and 4 h (Fig. 3A). A similar experiment using a different mAb, CyChrome-anti-HLA-A,B,C, confirmed colocalization of wH22xeGFP with class I (Fig. 4). One possible interpretation of this result is that the fusion protein caused the internalization of class I. We believe this to be unlikely, in light of data demonstrating that, unlike surface FcRI, which was down-modulated following wH22xeGFP treatment, surface class I expression did not change significantly over time (Fig. 5). Analysis of control samples treated with anti-class I mAb alone indicated that the red label appeared inside cells, particularly at 2 and 4 h (Fig. 3B), suggesting that the entry of class I (or the detection Ab) was independent of the fusion protein treatment. Taken together with data showing that no significant change of surface class I occurred following wH22xeGFP treatment, these images are consistent with a class I recycling pathway that is independent of fusion protein treatment.

View larger version (33K): [in this window] [in a new window]   FIGURE 3. Anti-class I Abs and wH22xeGFP both internalize from the surface of the cell. Cells were treated on ice with 10 µg/ml wH22xeGFP (A) or medium alone (B) for 30 min, followed by 8 mg/ml human IgG for 15 min (to block the binding of W6/32 by its Fc end in the unlikely event that any Fc receptors were unoccupied). Cy3-W6/32 was then added at a final concentration of 30 µg/ml for 15 min. An aliquot was then removed for a t = 0 sample, and the remaining cells were shifted to 37°C. Aliquots of cells were removed at various times, washed,and fixed. Green stain represents eGFP fluorescence, red stain represents class I staining, and yellow staining is the result of green plus red staining. Zoom = 1.0. Images are representative of at least three similar experiments.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Determination of colocalization of wH22xeGFP with MHC class I using a different anti-class I Ab. Cells were incubated with 10 µg/ml wH22xeGFP and CyChrome-conjugated anti-HLA-A, B, C (1:6 dilution) at 37°C for 1 h. Cells were then washed with ice-cold PBA and fixed. Green stain represents eGFP fluorescence, blue stain represents class I staining, and aqua staining is the result of green plus blue staining. Zoom = 3.0. Images are representative of at least three similar experiments.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Surface expression of FcRI, but not MHC class I, is decreased following treatment with wH22xGFP. Cells were incubated at 37°C with 10 µg/ml wH22xeGFP for various time periods. Cells were then washed and stained for surface FcRI () or class I (), as described in Materials and Methods. A, Values (±SD) are plotted as percentage of control cells (based on MFI values of the FL-2 channel as described in Materials and Methods) treated in the absence of wH22xeGFP. *, p < 0.05; **, p < 0.01; ***, p < 0.001; from Tukey-Kramer Multiple Comparisons Test following ANOVA, indicating significant differences in FcRI expression, but not class I expression, between cells treated with wH22xeGFP and cells incubated in medium alone. B, Class I expression, plotted as MFI (anti-class I - isotype control) ± SD, of cells treated with wH22xeGFP () or medium alone (). Class I expression was not significantly different in the presence or absence of prebound wH22xeGFP. Data are representative of at least two similar experiments.

Internalization of wH22xeGFP does not lead to colocalization with early or late endosomal markers

In an attempt to identify the pathways in which FcRI-targeted Ags are internalized and processed, IFN--treated 10.6 cells were incubated with wH22xeGFP at 37°C for 5 h, then washed and fixed. Cells were subsequently stained for intracellular markers associated with early endosomes (EEs) and late endosomes (LEs)/lysosomes. Representative images were taken from the center plane of the cells to assess the internal staining of wH22xeGFP molecules and markers that cocompartmentalized with the fusion protein. Fig. 6 illustrates the internal distribution of EE markers Rab4 (Fig. 6, A and D) and Rab5a (Fig. 6, B and E) and LE/lysosome marker Lamp-1 (Fig. 6, C and F), stained in blue. As expected, at 4°C wH22xeGFP (green) remained on the cell surface (Fig. 6, A–C), but internalization was evident after incubation at 37°C (Fig. 6, D–F). These photomicrographs suggest that little, if any, of the internalized wH22xeGFP was colocalized with any of these three endosome/lysosome markers after 5 h, as evidenced by the green (rather than aqua) color of the internalized fusion protein.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. Lack of intracellular colocalization of wH22xeGFP with Rab4, Rab5a, or Lamp-1 after 5 h. Cells were treated with 10 µg/ml wH22xeGFP for 5 h either on ice (A–C) or at 37°C (D–F), washed with PBA, and fixed. Cells were permeabilized using PBAS, and intracellular staining was performed using anti-Rab4 or anti-Rab5a, followed by Cy5-conjugated goat anti-rabbit IgG, or using CyChrome-conjugated anti-Lamp-1. Green stain represents eGFP fluorescence, blue stain represents endosome/lysosome marker staining, and aqua staining is the result of green plus blue staining. Arrows indicate examples of internalized fusion protein. Zoom = 1.0. A and D, Rab4. B and E, Rab5a. C and F, Lamp-1. Images are representative of three to five similar experiments.

View larger version (38K): [in this window] [in a new window]   FIGURE 7. Lack of intracellular colocalization of wH22xeGFP with Rab4, Rab5a, or Lamp-1 at 30 or 60 min. Cells were pretreated on ice for 30 min with 10 µg/ml wH22xeGFP and then incubated at 37°C. Aliquots of cells were removed at 0.5 or 1 h, washed, and fixed. Cells were then permeabilized using PBAS, and intracellular staining was performed as described in Fig. 6. Green stain represents eGFP fluorescence, blue stain represents endosome/lysosome marker staining, and aqua staining is the result of green plus blue staining. Zoom = 5.0. A, Rab4; B, Rab5a; C, Lamp-1. Images are representative of three to five similar experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

IFN- treatment was used throughout the study to up-regulate surface FcRI. Because IFN- is known to affect surface expression of both FcRI and class I (17), as well as to alter the proteosome make-up and protein processing (18), the intracellular pathways described in these studies may be more characteristic of an IFN--stimulated cell, such as an "activated monocyte."

Of interest, studies in which cells were first prebound on ice with wH22xeGFP and with Abs labeling surface class I demonstrated intracellular colocalization of the labels when the cells were subsequently warmed, suggesting a mechanism by which the fusion protein and class I could localize in the same subcellular compartment. However, the apparent internalization of class I was not dependent on treatment with fusion protein, as anti-class I Ab staining was observed inside cells even in control samples containing no fusion protein. We first considered that manipulation of the cells (i.e., cooling them for staining and then warming them for the internalization assay) may have led to the internalization of class I. However, repeated studies performed without cooling the cells gave similar results. Recycling pathways of class I have been described by others (12, 13), and it is possible that we captured recycling events in our analysis. Studies by Chiu et al. showed that surface class I could internalize through endosomes and that intracellular pools of class I similar to what was observed in our studies appeared to arise, at least in part, by endocytosed surface class I (13). Although wH22xeGFP and surface class I could enter cells simultaneously, perhaps resulting in colocalization, it is also possible that colocalization of the fusion protein with class I could be due to the merging of wH22xeGFP with an intracellular pool of class I. Indeed, in some instances we have observed the appearance of a portion of intracellular wH22xeGFP that is not colocalized with class I (e.g., Fig. 2A, green staining), suggesting that at least some fusion protein enters the cell independent of surface class I. Together, these findings suggest that FcRI-targeted Ags colocalize with recycling, as well as with intracellular, pools of class I made up of recycled or newly synthesized class I molecules.

To understand how FcRI-targeted Ags traffic through cells, experiments were performed to dissect out parts of the endosomal/lysosomal pathway. Rab4 and Rab5, small GTPases involved in endosome fusion events, have been shown to be involved in endocytosis and phagocytosis and are frequently used as markers for EEs (reviewed in Refs. 19 and 20). Recycling vesicles (RVs) also contain Rab5, but are Rab4 negative. LEs/lysosomes contain neither Rab4 nor Rab5, but are associated with a lysosome-associated membrane glycoprotein, Lamp-1 (reviewed in Refs. 20, 21, 22). Abs to each of these Rab and Lamp-1 proteins are used to define the intracellular compartment with which they are associated. Colocalization with the LE/lysosome marker Lamp-1 was absent at 0.5 and 1 h. At 5 h it appeared that the majority of wH22xeGFP was not colocalized with Lamp-1. Although we believe these data to be inconclusive, we cannot rule out the possibility that some portion of wH22xeGFP may traffic to lysosomes, especially at later time points (e.g., 5 h), when wH22xeGFP may be processed by other pathways potentially induced by FcRI activation. Interestingly, we did not observe colocalization of intracellular fusion protein with markers that define EEs (Rab4 and Rab5a) or RVs (Rab5a).

We first considered that the entry of wH22xeGFP into endosomes or lysosomes may be difficult to detect using eGFP, because this fluorochrome is sensitive to low pH. However, Kneen et al. found that only up to 50% of eGFP fluorescence can be quenched at pH 6.0 or lower (23). Because endosomes have a pH between 6.3 and 6.8 (reviewed in Ref. 20), more than half of the eGFP fluorescence should be retained when in endosomes, allowing for detection of wH22xeGFP using confocal microscopy. Indeed, we observed eGFP fluorescence inside cells, suggesting that at least a portion of wH22xeGFP can be tracked after it has been internalized. Furthermore, we found that in vitro exposure of wH22xeGFP to a pH of 5.0, followed by neutralization, reduced neither the binding nor the fluorescence of the molecule (data not shown), suggesting that the fluorescence of wH22xeGFP is not irreversibly quenched when exposed to this pH. By contrast, in vitro exposure of wH22xeGFP to a pH of 3.0 led to a drastic (90%) reduction of eGFP fluorescence, indicating that detection of that molecule in the acidic environment of lysosomes is unlikely.

The lack of colocalization of wH22xeGFP with Rab4 and Rab5a suggests that wH22xeGFP was not located in endosomes. One explanation of these findings is that transit of wH22xeGFP through endosomes occurs, but is so rapid that colocalization events with endosomal markers were missed by the first time point of the assay. Mellman has proposed that trafficking through EEs can be very rapid, transiting in 2–3 min (20), suggesting that our earliest time point, 30 min, may have been too late to capture wH22xeGFP in EEs. However, because wH22xeGFP appears to internalize over time (at least 4 h, Fig. 5), fusion protein should be continuously entering the cell during the assay period, allowing for the observation of any colocalization events. Therefore, we believe that FcRI-targeted Ags may traffic through a novel pathway, rather than through the endosomal pathway defined by Rab4 and Rab5a.

Interestingly, if wH22xeGFP were continuously recycled through RVs, colocalization with Rab5a should have been observed. However, our studies indicate that the internalization of wH22xeGFP correlates with the down-modulation of FcRI and that surface FcRI levels did not rebound over 16 h (C. Guyre, manuscript in preparation). Therefore, the lack of colocalization with Rab5a is consistent with our hypothesis that FcRI-bound wH22xeGFP does not recycle.

To determine whether FcRI-targeted fusion proteins traffic to lysosomes, we stained for Lamp-1 at various times after internalization with wH22xeGFP. The kinetics of Ag transport to Lamp-1-containing LEs/lysosomes has largely been studied using whole bacteria as Ag. Read et al. have described the arrival of significant amounts of bacteria to phagolysosomes of human macrophages in 30–60 min (24). Other studies have shown maximal colocalization of internalized bacteria with Lamp-1 at 80 min (25). In studies using the plasma protein ₂-macroglobulin as Ag, transit time to endosomes was 15 min (26). In our studies, wH22xeGFP was clearly not colocalized with Lamp-1 at 0.5 h or at 1 h (Fig. 7C). Although this finding may indicate that wH22xeGFP did not traffic to lysosomes, it is more likely that the eGFP fluorochrome would have been quenched in the low-pH environment (pH 3.0) of lysosomes. However, the observation of intracellular eGFPfluorescence that is clearly not colocalized with Lamp-1 indicates that at least some portion of wH22xeGFP did not traffic to lysosomes, nor did it transit through a Lamp-1 compartment, because our studies indicate that the eGFP component would not have survived the environment and could not have been detected. One hypothesis is that the fusion protein is diverted into the cytosol, a mechanism that has been proposed for cross-priming effects (class I-restricted CTL responses to exogenous Ag) observed in dendritic cells (27). Such a mechanism would be consistent with the cytosolic model of Ag processing described by Rock, in which exogenous Ag escapes the endocytic pathway and enters the cytosol for proteosomal degradation (11).

Studies by Gromme et al. showed that a fraction of class I colocalized in a class II-rich compartment known as the MIIC. They further demonstrated that acidic pH (equivalent to that of a late endosome or lysosome) caused the release of peptide from class I molecules, resulting in "empty" class I molecules that could conceivably be loaded with peptides from exogenous Ag (12). It has been shown that the cytoplasmic domain of FcRI contains a class II localization sequence (28), which could potentially deliver an FcRI-targeted Ag to an intracellular compartment where both class II and class I are localized. Based on these data, we propose that in APCs the FcRI-targeted fusion protein traffics to a compartment of the cell that contains both class I and class II MHC. There, the fusion protein is degraded as the pH drops, antigenic peptides are loaded onto class I and class II molecules, and the peptides are presented to CD8⁺ and CD4⁺ T cells, respectively. The model presented here can be expanded using such APC as DC. Such studies would allow for correlations between functional responses and colocalization of wH22xeGFP with proteins known to play a role in Ag presentation, such as class I and class II.

In summary, we have shown the colocalization of an FcRI-targeted fusion protein, wH22xeGFP, with intracellular class I molecules through a potentially novel pathway. At least two potential mechanisms exist to explain this finding: 1) wH22xeGFP is delivered to a subcellular compartment containing intracellular class I molecules, or 2) wH22xeGFP and recycling surface class I molecules enter cells simultaneously and colocalize. These two models are not mutually exclusive and are both consistent with the noncytosolic pathway proposed by Rock, in which peptides are generated from exogenous Ag in an acidic phagolysosomal compartment that then fuses with a vesicle containing class I (11).

	Acknowledgments

 
We thank Drs. Paul M. Guyre, Tibor Keler, Robert Graziano, William Wade, Mark Lang, and Geza Fejes-Toth for helpful discussions, and Kenneth Orndorff and Dr. Alice L. Givan for technical assistance with image analysis.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants DK07508 and AI19053 and by the Albert J. Ryan Foundation. Confocal scanning laser microscopy was performed in the Herbert C. Englert Cell Analysis Laboratory, which was established with equipment grants from the Fannie E. Rippel Foundation and the National Institutes of Health Shared Instrument Program and is supported in part by the core grant of the Norris Cotton Cancer Center (CA-23108).

² Address correspondence and reprint requests to Dr. Michael W. Fanger, Department of Microbiology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756.

³ Abbreviations used in this paper: TT, tetanus toxoid; DC, dendritic cell; EE, early endosome; eGFP, enhanced green fluorescent protein; LE, late endosomes; MFF, methanol-free formaldehyde; PBA, PBS-BSA-azide; PBAS, PBA-saponin; PMT, photomultiplier tube; RV, recycling vesicles; MFI, mean fluorescence intensity.

Received for publication August 2, 2000. Accepted for publication November 28, 2000.

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