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The Journal of Immunology, 1998, 161: 1599-1607.
Copyright © 1998 by The American Association of Immunologists

Antigen Processing of Two H2-IEd-Restricted Epitopes Is Differentially Influenced by the Structural Changes in a Viral Glycoprotein1

Kimberly A. Chianese-Bullock*, Helena I. Russell{dagger}, Christopher Moller{ddagger}, Walter Gerhard{ddagger}, John J. Monaco{dagger} and Laurence C. Eisenlohr2,*

* Department of Microbiology and Immunology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA 19107; {dagger} Department of Molecular Genetics, Howard Hughes Medical Institute, University of Cincinnati, Cincinnati, OH 45267; and {ddagger} Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The factors that influence the intracellular location(s) of MHC class II-restricted epitope loading remain poorly understood. We present evidence that two I-Ed-restricted epitopes of the influenza hemagglutinin (HA) molecule, termed site 1 (S1; encompassing amino acid residues 107–119) and site 3 (S3; encompassing amino acid residues 302–313), are generated in distinct endocytic compartments. By means of an epitope-specific mAb, we show that S1 becomes detectable in late endocytic/lysosomal vesicles; using a mutant cell line, we also show that the presentation of S1 is dependent upon H2-DM expression. In contrast, S3; presentation is H2-DM-independent and appears in early endosomes as a result of acid-induced structural changes in HA. Presentation of both epitopes can be made H2-DM-independent by denaturing HA and made H2-DM-dependent by preventing the acid-induced conformational changes from occurring. These findings indicate that the structural context of a given epitope can determine where it is processed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Major histocompatibility complex class II molecules are expressed by B cells, macrophages, dendritic cells, or IFN-{gamma}-activated cells and function to present peptides that have been derived from exogenous Ags to CD4+ Th cells (1, 2, 3, 4). The {alpha}- and ß-chains of class II molecules coassemble in the endoplasmic reticulum and directly associate with an additional protein that has been termed invariant chain (Ii)3 (5). One Ii trimer assembles with three class II {alpha}ß heterodimers to form a nonameric complex (6) that travels from the endoplasmic reticulum to the Golgi apparatus. A sorting signal found within the amino-terminal cytoplasmic domain of Ii (7, 8) targets nonameric complexes to vesicular compartments within the endocytic pathway (7, 9, 10, 11), with a fraction traveling to these compartments indirectly via the plasma membrane (12). Ii is proteolytically digested within the endocytic pathway (13, 14), leaving the class II-associated Ii peptide (CLIP) associated with the groove of MHC class II molecules (15, 16, 17). Although CLIP has a sequence at its N terminus that facilitates its removal from class II at endosomal pH (18), the efficient removal of CLIP from many class II alleles requires the participation of HLA-DM (termed H2-DM in the mouse). This heterodimer accumulates in late endosomal compartments (19, 20), in which it is believed to mediate the catalytic exchange of CLIP for antigenic peptides (21, 22, 23). Recent evidence has assigned two additional roles to HLA-DM. The first role is as an editor, facilitating the exchange of peptides with a low affinity and low stability for a given class II allele with high affinity and high stability peptides (22, 24). The second role is as a chaperone molecule for class II molecules. DM coprecipitates with Ii-associated and empty class II molecules, preserving the peptide-binding capacity of the latter in lysosomal compartments (25, 26). Peptide loading correlates with the dissociation of class II from DM and with transit to the plasma membrane, where class II/peptide complexes trigger CD4+ T lymphocytes with the appropriate receptor specificity.

The extent to which individual organelles contribute to the processing and class II binding of epitopes derived from host or foreign proteins remains controversial. Two different approaches, immunoelectron microscopy and subcellular fractionation, have been used to address this issue. Early immunoelectron microscopy studies by Guagliardi et al. (11) demonstrated the colocalization of MHC class II molecules, Ii, and exogenous Ag in early endosomes, suggesting that these compartments are the sites for class II processing and presentation. The involvement of early endocytic vesicles in class II peptide binding was supported by subcellular fractionation studies in which class II-containing vesicles termed CIIV(27), and lysozyme-loading compartments (28) were identified and characterized as early processing compartments that were distinct from early endosomes. In contrast to these results, similar studies identified newly synthesized class II molecules in a prelysosomal compartment, termed MIIC, in which the absence of Ii is compatible with MIIC as a site of peptide loading (29). Subcellular fractionation studies have also described the existence of a prelysosomal compartment that is involved in peptide loading (30, 31, 32, 33). Differences in the descriptions of the class II peptide-loading compartment(s) may be attributed to the use of different cell lines or to differences in the resolution of the various protocols employed. Alternatively, these data suggest that class II peptide binding is supported in multiple endocytic compartments. Accumulating evidence indicates that this possibility is likely the case (34, 35, 36, 37). As has been suggested previously (34, 35, 37), such an arrangement would be advantageous, since each compartment within the endocytic pathway would provide a unique environment to accommodate the processing of a wide variety of class II-restricted epitopes. Some epitopes may be revealed in early endosomes and destroyed before reaching later compartments, whereas others may require the more active proteolytic environment of late endosomes and lysosomes for release. Furthermore, the properties of the epitope itself, such as the sequence of acidic residues, may determine the site of optimal binding (38).

At least some of the heterogeneity in peptide loading may be due to the involvement of two distinct populations of class II molecules (35, 37); these populations are definitively established by the mutation of internalization signals within the cytosolic tails of the class II {alpha}- and ß-chains (35, 37). Cells transfected with these mutant class II molecules are unable to present a peptide sequence derived from hemagglutinin (HA) while still maintaining the presentation of a peptide sequence derived from the matrix protein of an influenza virus (37). These results suggest that antigenic fragments that are made accessible in early endosomal compartments can be loaded at these sites onto mature, recycling, class II molecules following the removal of CLIP or previously bound peptides (37). A requirement for class II internalization has been inversely correlated with a requirement for Ii expression, which is consistent with the notion that class II molecules do not require Ii for cycling to early endocytic compartments. In contrast, newly synthesized class II molecules require the expression of Ii for delivery to late endosomal compartments where Ag is aggressively processed (35, 37). This dichotomy has recently been extended to the expression of DM. Epitopes that load onto recycling class II molecules can do so in the absence of DM, whereas epitopes that load onto nascent class II molecules require functional DM (36, 39, 40). Whether a DM-like molecule facilitates the loading of recycling class II molecules is not known.

The processing requirements and class II populations involved in the presentation of individual epitopes may be dictated to a large degree by the structural context of these epitopes. Of particular interest and utility are class II epitopes derived from viral proteins that undergo programmed conformational changes following delivery to the early endosome. One such protein is the HA molecule of the influenza virus. HA is a trimeric glycoprotein that is responsible for the attachment of the virus to host cell surfaces via association with terminal sialic acid residues (41). Following receptor-mediated endocytosis, HA undergoes specific structural changes in response to acidification; these changes initiate fusion between viral and host cell membranes, resulting in the delivery of the viral genome to the host cell cytosol (41). Studies on protease sensitivity and reactivity with peptide-specific Abs suggest that the structural changes are profound, with considerable unfolding of some regions of the molecule (42, 43, 44). These observations have been substantiated by recent crystallographic data showing major rearrangements of an HA fragment following acidification (45). Thus, acid-induced structural changes in HA have the potential to expose antigenic epitopes in early endocytic vesicles in which class II molecules may be capable of capturing and presenting such epitopes to CD4+ T lymphocytes.

The studies reported here focus upon two well-defined, I-Ed-restricted, class II epitopes within the HA molecule of the A/Puerto Rico/8/34 influenza virus (PR8). Reactivity against site 1 (S1) has been mapped to the region of amino acids (aa) 107–119, and reactivity against site 3 (S3) maps to the region of aa 302–313 (46). Previously reported experiments have suggested different processing requirements for the presentation of these two epitopes. First, a kinetic analysis demonstrated that the presentation of S3 precedes that of S1 (47). Second, treating APCs with the protease inhibitor leupeptin enhances the presentation of S3 while significantly decreasing the presentation of S1 (47). Finally, S3 but not S1 is presented on prefixed cells pulsed with an acid-modified form of HA (48). Altogether, these results suggest that the S3 epitope is efficiently processed for class II binding in an early endocytic compartment, relying on the programmed structural changes that HA undergoes following endocytosis. In contrast, the availability of S1 is not impacted by this early conformational change, but instead requires the proteolytic environment of late endocytic compartments for processing. We present here experiments that directly test and substantiate this model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female BALB/c mice (H-2d, National Cancer Institute, Frederick, MD) of 6 wk of age or older were maintained in the Laboratory Animal Facility at Thomas Jefferson University (Philadelphia, PA).

Peptides

A synthetic peptide for HA S1, encompassing aa 107–119 (SVSSFERFEIFPK), was purchased from Research Genetics (Huntsville, AL). The synthetic peptide for HA S3, encompassing aa 302–313 (CPKYVRSAKLRM), was generated by the Kimmel Cancer Center Peptide Facility (Philadelphia, PA).

Abs and staining reagents

HB65 (American Type Culture Collection, Manassas, VA) is a mouse mAb that is specific for the nucleoprotein of influenza A viruses. Y8–10C2 (Sa11) and H28-E23 (Sb9) are mouse mAbs that are specific for either the monomeric (or acid-modified) or both the monomeric and trimeric forms of HA, respectively (49). CM1–1.2 is a mouse mAb that is specific for HA S1. 1D4B (Developmental Studies Hybridoma Bank, Johns Hopkins University School of Medicine, Baltimore, MD; Department of Biology, University of Iowa, Iowa City, IA) is a rat mAb that is specific for the lysosomal resident protein, lysosomal-associated membrane protein-1 (LAMP-1). MAS-753b (Accurate Chemical, Westbury, NY) is a rat mAb that is specific for the murine transferrin receptor, CD71. Secondary fluorescein-labeled horse anti-mouse Ig (IgG), biotinylated rabbit anti-rat Ig (IgG), and Texas Red-labeled avidin D were purchased from Vector Laboratories (Burlingame, CA).

Cell lines

The parent H-2d-positive B cell lymphoma cell line A20.2J (A20) was originally described by Asofsky and coworkers (50). The use of this cell line, which was kindly provided by Dr. V. Engelhard (University of Virginia, Charlottesville, VA), in CD4+-mediated cytolysis was established by Williams and Engelhard (51). The A20 class II processing and presentation mutant (A20 3A5) has been described previously (52). The A20 3A5 cell line was determined to be defective in the H2-DM {alpha}-chain and was reconstituted by stable transfection with the wild-type murine DM {alpha}-chain cDNA (A20 3A5 R) in pcDNA1/neo (Invitrogen, Carlsbad, CA) as described elsewhere.4 All three B cell lymphoma cell lines were maintained in vitro in RPMI 1640 (Fisher Scientific, Pittsburgh, PA) supplemented with 5% FBS (Sigma, St. Louis, MO), 0.05 mM 2-ME (Sigma), and Ser X-tend (5% FBS equivalent, Irvine Scientific, Santa Ana, CA).

The CD4+ cytotoxic T cell clone that was specific for HA S1 (vir 1.2) was generated as described previously (53). The HA S3 clone (5.1.1R20) (Th20) was generated in a manner similar to the other S3 clones described previously (54). At the beginning of each passage, the T cell clones (1–2 x 105/ml) were maintained by a biweekly stimulation with irradiated (2200 rads), PR8-pulsed (~100–200 HA units (HAU) per 106 cells), BALB/c spleen cells (2 x 106/ml). The irradiated spleen cells were infected with PR8 for 1 h at room temperature in Iscove’s modified Dulbecco’s medium (IMDM) (Fisher Scientific) supplemented with 0.05% BSA fraction V (Sigma) and gentamycin (0.01 mg/ml, Sigma). The cells were washed, adjusted to the desired concentration, and incubated at 37°C/9% CO2 in a total of 5 ml of culture medium consisting of IMDM supplemented with 5% FBS, 0.05 mM 2-ME, and gentamycin (0.01 mg/ml). On day 5 following restimulation, 1 x 105/ml T cell clones were maintained in culture medium supplemented with 25 U/ml human rIL-2 (National Cancer Institute). The clones were passaged every 2 to 3 days in IL-2-supplemented culture medium and were used on day 3 after each passage.

L-Kd cells, which are L929 cells transfected with H2-Kd (55), were maintained in DMEM (Fisher Scientific) supplemented with 5% FBS.

Viruses

The influenza viruses PR8 (H1N1) and A/England/333/80 (ENGL) (H1N1) and the antigenically distinct PR8 mutant virus (Rv6) (H1N1) (49, 56) were grown in the allantoic cavity of 10-day-old embryonated hen eggs. Viruses were concentrated by differential sedimentation (60,000 x g) or were purified in some cases by banding in sucrose density gradients. The virus concentrations were determined by the hemagglutination of human E. Titers are expressed as HAU, with 8.75 ng of protein being equivalent to 1 HAU of virus.

Virus with uncleaved HA was generated by inoculating chicken embryo fibroblasts (CEFs) with 5000 HAU of PR8 in PBS containing 0.1% BSA (balanced salt solution (BSS)/BSA, Sigma) for 48 h at 37°C. The virus was purified by differential sedimentation and its titer determined as described above. The presence of uncleaved virus was confirmed by running the samples on SDS-polyacrylamide gels under reducing conditions followed by Western blot analyses using the HA-specific mAb H28-E23.

Cross-linked PR8 was generated by incubating purified virus for 15 h at room temperature in either 10 mg/ml of dimethyl adipimidate (DMA) (Pierce, Rockford, IL) in 0.1 M HEPES (Sigma) cross-linking buffer or in cross-linking buffer alone as a control. The cross-linking reaction was stopped by the addition of a fivefold volume of IMDM supplemented with 0.05% BSA fraction V and gentamycin (0.01 mg/ml).

Inactivation of influenza viruses

Virus was inactivated by exposure to short wave (254 nm) UV light (1800 µJ; Stratalinker 1800, Stratagene, La Jolla, CA). L cell fibroblasts pulsed with UV-inactivated influenza virus showed no evidence of de novo viral protein synthesis when tested for the production of nucleoprotein using the HB65 mAb. UV inactivation did not alter the integrity of the HA protein as determined by a hemagglutination assay.

Viral Ags

HA was isolated by treating purified PR8 with bromelain as described by Brand and Skehel (57). HA purified in this manner is termed bromelain-cleaved HA (BHA) and consists of the soluble ectodomain of HA.

Reduced and alkylated HA was generated by incubating 150 µg of BHA in a final volume of 2 ml of 0.2 M Tris-HCl (pH 8.5) containing 6 M guanidine HCl (Sigma) and 10 mM DTT (Boehringer Mannheim, Indianapolis, IN) at 37°C for 2 h. The mixture was cooled on ice for 10 min, and 200 µl of 0.2 M 2-iodoacetamide (Eastman Kodak, Rochester, NY) in 0.2 M Tris-HCl (pH 8.5) containing 6 M guanidine HCl was added and incubated on ice for 30 min. Protein concentrations were determined with the Bio-Rad dye reagent (Hercules, CA).

Staining

A total of 4 x 104 L-Kd cells were seeded onto coverslips in 24-well plates and incubated overnight at 37°C/9% CO2. The coverslips were washed twice with ice-cold PBS and incubated with 200 µl (~200–400 HAU in BSS/BSA) of ice-cold UV-treated PR8 for 20 min in the presence of 1 mM 2,3-dehydro-2-deoxy-N-acetyl neuraminic acid (DDAN) (Sigma). The plates were incubated at 37°C/9% CO2 and were washed twice at each time point with ice-cold PBS before fixation with 3% paraformaldehyde (Tousimis, Rockville, MD) for 15 min at room temperature. Between each of the remaining steps, the coverslips were washed three times with PBS/azide. Autofluorescence was quenched with 50 mM NH4Cl (Sigma) for 15 min before permeabilization with 0.1% Triton X-100 (Fisher Scientific) for 2 min at room temperature. CM1–1.2, Y8–10C2, 1D4B, or a combination of these reagents were used as primary Abs and were incubated with the cells at 4°C for at least 2 h. A fluorescein-labeled horse anti-mouse Ig (IgG) mAb and a biotinylated rabbit anti-rat Ig (IgG) mAb were used as secondary Abs followed by the addition of Texas Red avidin D. Both sets of reagents were incubated with the cells for at least 2 h at 4°C. Background staining was determined following incubation with the secondary reagents. All images were viewed at the Kimmel Cancer Institute Confocal Facility.

Cytotoxicity assay

Target cells (2 x 107/ml) were coincubated with dilutions of the influenza virus in BSS/BSA for 1 h at 37°C. RPMI 1640 (5 ml) supplemented with 5% FBS, 0.05 mM 2-ME, and 0.01 mg/ml gentamycin (assay medium) was added to each target, and the cells were incubated for an additional 4 h. Each target was labeled using 100 µCi/106 cells of Na51CrO4 (Amersham, Arlington Heights, IL) in assay medium for 1 to 2 h at 37°C. After two washes in PBS, 104 radiolabeled targets in assay medium were added to each well of a 96-well round-bottom plate. Effector T cells were added to obtain the E:T ratios indicated for each experiment in a total volume of 200 µl. Peptide, BHA, or reduced and alkylated HA were added during the overnight incubation with the CD4+ T cells. After 12 to 14 h of incubation at 37°C/6%CO2, 100 µl of supernatant was harvested and counted using a gamma detector (Pharmacia, Uppsala, Sweden). The percentage of specific lysis was calculated as 100 x ([experimental cpm - spontaneous cpm]/[total cpm - spontaneous cpm]).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Presentation of S1 and S3 in murine A20 B cells that are deficient in H2-DM

We speculated that if S3 is processed in an early endocytic compartment, its presentation might be independent of H2-DM expression, as has been shown for other class II alleles (36, 39, 40). In contrast, the presentation of S1 was predicted to depend upon H2-DM expression if processing of this epitope occurs late in the endocytic pathway. To test this possibility, we compared the presentation of HA S1 and S3 epitopes in both A20 B cells and A20 B cells that had been rendered deficient in the murine HLA-DM equivalent, H2-DM (52).4

Wild-type or H2-DM deficient A20 B cells were pulsed with UV-inactivated influenza virus and tested as targets for cytolytic CD4+ T cells specific for S1 or S3 in a 51Cr release assay. As with most class II epitopes derived from exogenous Ags, the presentation of S1 was dependent upon H2-DM expression. In A20 cells, the presentation of S1 is quite high, whereas only background levels of killing were seen with the A20 3A5 mutant cell line (Fig. 1Go). In contrast to S1, the presentation of S3 was not dependent upon H2-DM expression; CD4+ T cell recognition of this epitope was maintained and, in fact, enhanced in the mutant cell line (Fig. 1Go). To confirm the specificity of the CD4+ cytotoxic T cell responses, we tested two additional UV-inactivated influenza viruses expressing point mutations in either S1 (Rv6) or S3 (ENGL) (Fig. 1Go). Rv6 was recognized by the S3-specific but not the S1-specific CD4+ cytotoxic T cell clone, while the reverse was true for the ENGL strain of influenza virus. We speculated that the enhanced killing of the A20 3A5 mutant cell line by the S3-specific clone was due to the availability of a greater number of peptide-receptive class II molecules in the early endosomal compartments, which is consistent with the low affinity of I-Ed for CLIP and the role of DM in CLIP removal and peptide editing (24, 58). To test the relative availability of peptide-receptive class II molecules in the A20 and A20 3A5 cell lines, we generated dose-response curves using the synthetic peptide versions of S1 and S3. The curves showed an increase in presentation efficiency by the mutant cell line of at least 10-fold as compared with the A20 cell line (Fig. 2Go) despite surface class II levels being essentially equivalent (Ref. 52 and data not shown).



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  		FIGURE 1. Role of H2-DM expression in the presentation of S1 and S3. Murine A20 B cells (A20), A20 B cells deficient in H2-DM (A20 3A5), or H2-DM-deficient cells reconstituted with functional H2-DM (A20 3A5 R) were pulsed with equivalent HAUs of the indicated UV-treated influenza virus or with UV-treated allantoic fluid (AF) as a negative control. Lysis of the target cells by the S1-specific CD4+ T cell clone, vir 1.2, and the S3-specific CD4+ T cell clone, Th20, at the indicated E:T ratios was determined. The specificity of the T cell responses was determined using influenza viruses that have a point mutation in either S1 (Rv6) or S3 (ENGL). S1 presentation depends upon H2-DM expression, whereas S3 presentation is maintained in the absence of H2-DM. Similar results were obtained using purified viruses.

 


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  		FIGURE 2. S1 and S3 peptide dose-response curves indicate that the mutant cell line has a greater number of receptive class II molecules. The A20 and A20 3A5 cell lines were incubated with the indicated concentrations of a S1 synthetic peptide (aa 107–119) or a S3 synthetic peptide (aa 302–313). Lysis of the target cells by vir 1.2 (a S1-specific clone) and Th20 (a S3-specific clone) was determined at E:T ratios of 1 and 0.5, respectively.

 
To confirm that the loss of S1 presentation in the mutant A20 3A5 cell line was due to the lack of H2-DM expression and was not the result of an additional mutation generated by the chemical mutagenesis protocol, we used A20 3A5 cells that had been reconstituted by stable transfection with the murine DM {alpha}-chain cDNA. As seen in Figure 1Go, the presentation of S1 was restored following transfection with a functional H2-DM {alpha}-chain (A20 3A5 R). The presentation of S3 was also maintained in the reconstituted cell line (Fig. 1Go). Greater levels of lysis for S1 and S3 in the reconstituted A20 3A5 R are likely due, at least in part, to the higher expression levels of I-Ed class II molecules as determined by FACS analysis (data not shown).

Availability of S1 occurs late in the endocytic pathway

The H2-DM dependence of S1 presentation along with the earlier evidence described above suggests that S1 becomes available for binding to I-Ed only late in the endocytic pathway. To test this directly, we used confocal immunomicroscopy to trace the appearance of S1 following the endocytosis of UV-inactivated PR8. We employed a mAb (CM1–1.2) that had been developed against a synthetic version of the epitope with the key feature, as demonstrated below, that it does not react with HA in its native context.

We employed L cell fibroblasts for this analysis, because the A20 cell line proved to be a poor candidate for these studies due to its small size and relatively high nucleus to cytoplasm ratio. PR8 was adsorbed to the cell surface at 0°C in the presence of DDAN, a neuraminidase inhibitor that enhances viral uptake (59). The temperature was then shifted to 37°C for various periods of time before fixation. Using confocal immunomicroscopy, we determined that the S1 epitope becomes available for binding by CM1–1.2 between 30 and 60 min following viral uptake (Fig. 3Go). The CM1–1.2 signal at 60 min was punctate, with the majority representing a perinuclear staining pattern. The intensity of the staining began to decrease by 120 min and was significantly reduced by 240 min.



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  		FIGURE 3. Time course for S1 availability in the endocytic pathway. L-Kd cells were incubated on ice for 20 min with UV-inactivated PR8 in the presence of 1 mM DDAN. Following incubation at 37°C for the indicated times, the cells were fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were either stained with a mouse mAb reactive with S1 (CM1–1.2) or double-stained with CM1–1.2 and a rat mAb reactive with LAMP-1 (1D4B). Reactivity with these Abs was determined by fluorescein-labeled horse anti-mouse Ig (IgG) and biotinylated rabbit anti-rat Ig (IgG) followed by the addition of Texas Red-labeled avidin. Arrows indicate points of overlap.

 
The staining by CM1–1.2 overlaps to a great extent with that of 1D4B, a mAb that is specific for the lysosomal resident protein, LAMP-1 (Fig. 3Go), indicating the appearance of S1 within a late endosomal/lysosomal compartment. The areas of CM1–1.2 staining not indicated by 1D4B could represent LAMP-1-negative late endosomes or sorting compartments within the endocytic pathway. Together, these results demonstrate that S1 availability occurs after HA is trafficked into the late endocytic/lysosomal compartments in which the presence of H2-DM is required for the exchange of S1 for CLIP (Fig. 1Go).

S3 is available in early endocytic compartments, and its appearance precedes that of S1

Although a mAb that is specific for S3 is not available, it has been previously reported that acid-treatment alone is sufficient to render S3, but not S1, presentable by prefixed APCs (48). The Y8–10C2 mAb recognizes a determinant on the globular domain of HA that becomes exposed following acidification (60). Therefore, the appearance of the Y8–10C2 epitope correlates with S3 availability for class II binding. Similar confocal studies revealed that staining with Y8–10C2 is observed in peripheral vesicles at 5 to 10 min after viral uptake (Fig. 4Go). At these early time points, the Y8–10C2 signal does not colocalize to any appreciable degree with LAMP-1 (Fig. 4Go and data not shown). These observations suggest that S3 is available for class II binding in an early endocytic vesicle, well in advance of S1. The acid-modified version of HA is capable of penetrating into late endocytic compartments, as the perinuclear staining pattern that develops at 30 min persists until the 60-min time point (Fig. 4Go). The overlap between Y8–10C2 and LAMP-1 staining at the later time points demonstrates the presence of acid-modified HA in the late endosomal/lysosomal compartments but in no way implies the presence of intact S3, since Y8–10C2 reactivity does not map to the hinge region where S3 resides. As with S1, the signal for Y8–10C2 declined significantly by 120 min (Fig. 5Go).



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  		FIGURE 4. Time course for S3 availability in the endocytic pathway. L-Kd cells were incubated with UV-inactivated PR8 and treated as described in the legend to Figure 3Go. Following an incubation at 37°C for the indicated time, the cells were stained with a mouse mAb that recognizes the acid-modified form of HA (Y8–10C2) or double-stained with Y8–10C2 and 1D4B. Reactivity with these Abs was determined by a fluorescein-labeled horse anti-mouse Ig (IgG) or a biotinylated rabbit anti-rat Ig (IgG) followed by the addition of Texas Red-labeled avidin D. Arrows indicate points of overlap.

 


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  		FIGURE 5. S1 presentation becomes H2-DM-independent following preprocessing of HA. A20 and A20 3A5 cells were incubated with decreasing concentrations of BHA, a reduced and alkylated form of BHA (R&A BHA), or PBS/azide as a negative control. Lysis of the target cells by vir 1.2 or Th20 was determined. S1 presentation is H2-DM-independent when HA is preprocessed by reduction and alkylation. One mole of BHA corresponds to 3 moles of reduced and alkylated BHA. The E:T ratio for this assay was 1.

 
Defect in S1 presentation by H2-DM-deficient cells can be overcome if HA is preprocessed

The results presented thus far suggest that S3 is presented by H2-DM-deficient cells due to its availability for class II binding shortly after the acidification of HA in early endocytic vesicles. Conversely, the location of S1 within a portion of HA that is not processed until the late endocytic/lysosomal compartments may underlie its lack of presentation by the H2-DM mutants. To address this and also eliminate the possibility that S1 presentation is intrinsically H2-DM-dependent, we compared the presentation of S1 and S3 that had been derived from either a native or denatured form of HA.

Native HA molecules were isolated from the core proteins of influenza viral particles following the incubation of purified virus with bromelain (57). Although lacking the transmembrane region of HA, BHA maintains its trimeric structure and undergoes conformational changes in response to acidification similar to those of full-length HA (43). The presentation of S1 and S3 derived from BHA mirrored that seen with whole UV-inactivated virus (Fig. 5Go). Both epitopes were presented by the parental A20 cell line, whereas only S3 was presented by the H2-DM mutant cell line.

BHA was then converted to denatured monomeric subunits by reduction and alkylation as described in Materials and Methods. In the A20 cell line, both S1 and S3 are presented from the reduced and alkylated form of this Ag (Fig. 5Go). Note that the stimulation of the S3 clone is quite high when the epitope is provided as part of denatured HA. The same is true for synthetic peptide (Fig. 2Go), suggesting that the processing pathway for the presentation of S3 from native protein is relatively inefficient. Notably, when HA is "preprocessed" by denaturation, S1 becomes presentable by the A20 3A5 mutant cell line (Fig. 5Go). Whether S1 that is derived from this preprocessed form of HA combines primarily with internal or surface-bound class II is not known. However, earlier experiments demonstrated that this epitope is presented on prefixed cells following the denaturation of HA (61), suggesting that at least some S1 combines with class II molecules at the cell surface.

Sufficient constraint of HA structural changes significantly reduces S3 presentation in H2-DM mutant cell line

We predicted that if S3 presentation relied on the structural changes in HA following acid-modification, its presentation could be forced into H2-DM dependence by constraining the HA molecule. The full maturation and function of HA requires cleavage by cell-associated, trypsin-like activity of the nascent HA0 polypeptide chain to a ~50-kDa HA1 peptide and a ~20-kDa HA2 peptide containing the fusion domain (62, 63). This event occurs shortly after biosynthesis and before viral assembly. Of note, uncleaved HA has receptor-binding activity and can undergo limited structural changes upon acidification, although the fusion peptide is not free for insertion into host cell membranes (19). Uncleaved HA was generated in CEFs, which are deficient in trypsin-like activity. Western blot analysis indicated that the vast majority (>90%) of HA in the CEF-grown virus is in the uncleaved state, with essentially complete conversion to HA1 and HA2 following trypsin treatment (Fig. 6GoA, inset). Dilutions of these UV-inactivated virus preparations were tested. The presentation of these two forms by the A20 or A20 3A5 mutant cell line to the S1-specific clone was essentially identical (Fig. 6GoA). Similarly, S3 presentation was unchanged in both the normal and H2-DM-deficient cell lines (Fig. 6GoA). These data suggest that the acid-modified changes that accompany the uncleaved form of PR8 are sufficient to permit S3 presentation in the A20 3A5 mutant cell line.



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  		FIGURE 6. S3 presentation becomes H2-DM-dependent when HA is sufficiently constrained. A20 and A20 3A5 cells were incubated with 30 HAU of an uncleaved or cleaved source of PR8 (A) or with 75 HAU of a cross-linked (DMA PR8) or mock-treated source of PR8 (control DMA PR8) (B). PBS/azide was used as a negative control. Lysis of the target cells by vir 1.2 (S1) and Th20 (S3) was determined at the indicated E:T ratios. The inset within A shows a Western blot analysis of egg-grown virus (P), PR8 grown in CEFs and mock-treated following purification (U), and PR8 grown in CEFs and treated with trypsin following purification (C).

 
To constrain HA even further, we treated purified virus with the chemical cross-linker DMA. This treatment prevents HA from undergoing detectable structural changes in response to acidification (60). The presentation of S1 in both the A20 and A20 3A5 mutant cell lines was unchanged following the cross-linking of PR8 (Fig. 6GoB), demonstrating that this treatment does not substantially alter the ability of HA to be processed. Similar to that of S1, the presentation pattern of S3 was unchanged for the cross-linked PR8 virus as compared with control virus in the A20 cell line. However, S3 presentation by the H2-DM mutant cell line was significantly reduced when the epitope was derived from cross-linked PR8 (Fig. 6GoB). Thus, with sufficient constraint of HA, the more aggressive processing activity of the late endosome/lysosome is required for S3 presentation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have demonstrated that the S1 and S3 epitopes derived from HA have strikingly different processing phenotypes despite being restricted to the same class II molecule. The disparate presentation patterns of S1 and S3 in the H2-DM mutant cell line are consistent with a model in which the S3 epitope is exposed and combines with class II molecules in an early endocytic compartment, a step that is contingent upon the acid-induced structural changes in HA. In contrast, S1 presentation is uninfluenced by these changes and requires the harsh proteolytic environment of later endocytic/lysosomal compartments for excision. This model is in line with earlier data showing a more rapid appearance of S3 vs S1 at the cell surface and the effects of the protease inhibitor leupeptin, which enhances S3 presentation while significantly inhibiting S1 presentation (47). The model is at odds, however, with earlier reports on work with A20s suggesting that epitopes combine with class II in an early endocytic compartment (27). At present, we cannot formally rule out the possibility that the S1 epitopes made available in a late compartment undergo retrograde transport. We note a minor population of vesicles that stained for S1 but not LAMP-1 (Fig. 4Go) that has not been further characterized. The direct identification of intracellular S1/I-Ed and S3/I-Ed complexes will be an important future study. However, much of the evidence shown previously and described here, including the stark differences in DM dependence exhibited by S1 and S3, strongly suggest the existence of at least two separate loading compartments.

The positioning of S1 and S3 with respect to the structure of HA before and after acidification is consistent with their distinct processing requirements. Superimposition of the A/PR/8/34 sequence onto the crystallographic structure of HA derived from A/HK/68 (64) shows that S1 is part of the globular domain of the molecule, proximal to the receptor binding pocket (Fig. 7Go). The structure of this domain is not substantially altered following acidification (65), providing a basis for our observation that the presentation of S1 is equivalent when derived from either an untreated or a cross-linked source of Ag (Fig. 6Go). The cross-linking results and the H2-DM-independent presentation of S1 following the reduction and alkylation of HA indicate that the location of S1 processing is dictated by its structural context rather than by its own sequence or the sequences immediately flanking it.



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  		FIGURE 7. The positioning of S1 and S3 within HA. The globular head of HA is made up of the HA1 polypeptide and contains the S1 epitope. The S3 epitope is located within the hinge region of HA1 in close proximity to the fusion peptide domain. The fusion peptide resides within the HA2 peptide and lies at the HA interface following formation of the trimer. In response to acidification, radical changes in the secondary structure of HA place the fusion peptide in close proximity with host cell membrane. This figure was adapted from Wilson et al. (64).

 
Before acidification in early endosomes, the S3 epitope is positioned at the HA interface in proximity to the fusion domain of the HA2 peptide (64). Upon acidification, the secondary structure of the HA2 peptide is radically modified, causing a large displacement of the fusion peptide in a way that allows contact with host cell membranes (45). As a result, the interface regions of HA become partially unfolded (42, 43, 60). We propose that these structural changes are sufficient to accommodate the binding of S3 to class II molecules in early endosomes as indicated by the availability of S3 using the conformational-dependent Ab, Y8–10C2. This proposition is supported by an earlier study, which demonstrated that S3 is presentable on prefixed cells following the acidification of PR8 (48), and is also consistent with our finding that constraint of HA forces its presentation to become H2-DM-dependent (Fig. 6Go).

We have yet to determine whether S3 is presented by mature recycling class II molecules or by nascent class II molecules that have trafficked to early endocytic vesicles. The former option is favored by the limited proteolysis of Ii in early endocytic vesicles and the involvement of recycling class II molecules in the presentation of other class II-restricted epitopes (35, 37). The latter possibility is consistent with several reports demonstrating the trafficking of nascent class II to the plasma membrane and to early endosomes (12, 20, 27, 34). Experiments in our laboratory using brefeldin A and emetine to assess the role of recycling class II molecules in the presentation of S1 and S3 have been inconclusive. The deletion of internalization signals in the cytosolic domains of the I-Ed molecule may better address this issue (35, 37).

It remains to be seen how frequently the early endosomal pathway is used for the processing and presentation of class II epitopes. We suspect that, at least for the presentation of S3, this pathway is relatively inefficient. Stimulation of the S3-specific clone is quite high when the epitope is provided as part of denatured HA (Fig. 5Go) or by synthetic peptide (Fig 2Go). However, when HA is provided in native form, presentation is relatively modest compared with that of S1 (Fig. 1Go). The inability of most high-affinity peptides to be removed from class II molecules in the compartment in which S3 is made available may contribute to its inefficient presentation by H2-DM-expressing cells. Alternatively, if S3 binds to class II in the context of full-length HA, as has been recently demonstrated for a partially denatured fragment of hen egg lysozyme (40), then these complexes may be unstable at the cell surface. Preliminary results in our laboratory support this possibility, in that full-length HA can be coimmunoprecipitated with I-Ed class II molecules at 10 min following viral uptake. The majority of these complexes are not detectable by 60 min, suggesting that the association between full-length HA and I-Ed occurs in early endocytic vesicles, and that HA is proteolyzed or dissociates from I-Ed (K.A.C-B., B. Dudenhoeffer, and L.C.E., unpublished observations). We speculate that S3 mediates this interaction. The possibility of other I-Ed binding regions being exposed following acidification has not yet been tested, although all other I-Ed-restricted epitopes identified thus far have been localized to the globular domain (66). As with the H2-DM-independent presentation of S1 following the denaturation of HA, the cross-linking results indicate that the structural context of S3 influences its processing. A recent report has suggested that the presence of acidic residues within a given epitope biases toward loading in a late (low pH) compartment (38). Of note, S1 contains two acidic residues (both glutamic acid), while S3 contains none. Acid content may prove to be an important determinant of peptide loading but is likely to be superseded by structural context.

It is possible that some presentation of S3 as part of untreated virus is H2-DM-dependent. The fact that leupeptin treatment greatly enhances S3 presentation suggests that the epitope is labile and might not reach nascent class II molecules for binding (47). However, the H2-DM-dependent presentation of S3 derived from cross-linked virus indicates that S3 is capable of surviving in a later compartment. The mAb used to determine the appearance of S3, Y8–10C2, maps to the globular domain (60) and therefore confirms the integrity in LAMP-1-positive vesicles of the globular domain, in which S1 resides, but not the hinge region, in which S3 resides (Fig. 4Go). We are currently developing the tools needed to trace S3 directly.

As the autocatalytic removal of CLIP from I-Ed is likely given the presentation of S3 in the absence of H2-DM, why is S1 not presented by the H2-DM mutants? DM has been shown to play a role in peptide editing by exchanging low-affinity and low-stability peptides with high-affinity and high-stability peptides (22, 24). It is unlikely that this function explains the H2-DM dependence of S1, as we have no reason to believe that S1 would compete poorly with other epitopes in the late compartment, particularly since the supply of epitopes in these compartments appears to be limited (26, 67). In support of this notion, S1 competes with other epitopes and is efficiently presented by a denatured form of HA in the H2-DM mutants (Fig. 5Go). The role of DM as a chaperone provides a more compelling explanation. In the absence of DM, empty class II molecules aggregate in an acidic environment and lose their peptide-binding capacity (25, 26). The relative abundance of DM in late endosomal and lysosomal compartments suggests that the absence of this molecule would have the greatest effect on epitopes generated late in the endocytic pathway (26, 68). Thus, one possibility is that S1 is made available in H2-DM-negative cells only after most receptive class II molecules have been incapacitated. Unlike S1, S3 presentation is maintained in H2-DM mutants, as aggregation is unlikely to occur in the pH environment of early endosomes.

Recently, Ma and Blum reported that DM-dependent presentation could be overcome by changing the mode of entry to receptor-mediated endocytosis via transferrin receptor or surface Ig (69). Their observation is in contrast to our findings, since S1 presentation is DM-dependent despite the fact that receptor-mediated endocytosis is the normal mode of entry for the influenza virus (70). This discrepancy could be due to differences in the cell types used. A more intriguing possibility is that the intracellular routes taken by the Ags following internalization are different and, consequently, so are the sites of processing and loading.

In addition to influenza, a number of other viruses in the orthomyxo, toga, flavi, rhabdo, bunya, and arena families have proteins that undergo acid-induced structural changes following endocytosis. As has been suggested previously (4), this group of proteins may provide one of the more compelling reasons for the existence of early endosomal loading of antigenic fragments. The open-ended nature of the class II binding groove suggests that unfolding is an important component of Ag processing (71). This may be particularly true in the early endosome, in which processing appears to involve unfolding more than proteolysis.


   Acknowledgments
 
We thank J. Keen, F. Santini, and P. Hingorani for their assistance with the confocal imaging and W. S. Kwan for excellent technical assistance. We also thank M. M. Marks and J. R. Drake for critical reading of this manuscript, I. A. York and K. L. Rock for reagents and information in advance of publication, and J. W. Yewdell for suggesting the use of cross-linked virus for such studies.


   Footnotes
 
1 This study was supported by National Institutes of Health Grant AI36331 to L.C.E. K.A.C.-B. was supported by a Foerderer Fellowship and a George M. Speck Fellowship (both from Jefferson Medical College) and also by a National Institutes of Health Training Grant (T32CA09683). Back

2 Address correspondence and reprint requests to Dr. Laurence C. Eisenlohr, Thomas Jefferson University, BLSB Room 726, 233 South 10th Street, Philadelphia, PA 19107-5541. E-mail address: Back

3 Abbreviations used in this paper: Ii, invariant chain; CLIP, class II-associated invariant chain peptide; HA, hemagglutinin; S1, site 1; S3, site 3; LAMP-1, lysosomal-associated membrane protein-1; HAU, hemagglutinating units; IMDM, Iscove’s modified Dulbecco’s medium; CEF, chicken embryo fibroblast; BSS, balanced salt solution; DMA, dimethyl adipimidate; BHA, bromelain-cleaved HA; DDAN, dehydro-2-deoxy-N-acetyl neuraminic acid. Back

4 H. I. Russell, I. A. York, K. L. Rock, and J. J. Monaco. Altered expression of H2-DM alpha in two class II antigen processing-defective H2d mouse cell lines. Submitted for publication. Back

Received for publication February 18, 1998. Accepted for publication March 20, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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