Abstract
We investigated the roles of nascent and recycling MHC class II molecules (MHC II) in the presentation of two well-defined I-Ed-restricted epitopes that are within distinct regions of the influenza virus hemagglutinin (HA) protein. The site 3 epitope (S3; residues 302–313) lies in the stalk region that unfolds in response to mild acidification, while the site 1 epitope (S1; residues 107–119) is situated in the stable globular domain. In a murine B lymphoma cell line and an I-Ed-transfected fibroblast cell line, presentation from inactivated virus of S3 is inhibited by primaquine, a compound that prevents recycling of cell surface proteins, including MHC II, while S1 presentation is unaffected. In contrast, brefeldin A, an agent that inhibits exit of proteins from the endoplasmic reticulum, selectively inhibited S1 presentation without affecting S3 presentation, suggesting that S1 presentation requires nascent MHC II. The use of agents that perturb endosomal function revealed a requirement for acidification of internalized viral particles for presentation of both epitopes. Notably, all compounds tested had similar effects on presentation of the two epitopes derived from endogenously synthesized HA. Thus, recycling I-Ed molecules appear to be crucial for capturing and presenting an epitope that is revealed in mild acidic conditions following the uptake of virions or the synthesis of Ag, while nascent I-Ed molecules are required for presentation of a second epitope located in a structurally constrained region of the same polypeptide. Viral glycoproteins, such as HA, may have been a major impetus for the evolutionary establishment of this recycling pathway.
CD4+ T cells that help establish the effector functions of both CD8+ T and B cells are triggered by at least two cohorts of MHC class II (MHC II)3/peptide complexes. In the classical pathway, MHC II composed of α- and β-chains combine in the endoplasmic reticulum (ER) with invariant chain (Ii). The MHC II-Ii complex is then targeted to the endosomal compartment due to a sorting signal present in the cytoplasmic tail of Ii (1). In the endosomes, Ii is proteolytically processed (2), leaving the class II-associated Ii peptide (CLIP) in the peptide binding groove of MHC II (3, 4, 5). Removal of CLIP and loading of antigenic peptides are facilitated by HLA-DM (in humans; H2-M in mice; referred to henceforth as DM) (6). DM acts as both a peptide editor (7) and a chaperone by stabilizing MHC II (8, 9). Ags taken up by APCs become available for class II binding in a sequential manner (10). Many epitopes require the excessive proteolytic processing of the highly acidic late endocytic vesicles where the bulk of nascent class II molecules exchange CLIP for antigenic peptide. The presentation of such epitopes is sensitive to depletion of nascent MHC II through the use of protein synthesis inhibitors and strategies that ablate Ii and DM functions.
In the alternative pathway, epitopes that are made available in less acidic and proteolytic environments can be bound by MHC II that have recycled from the cell surface to early endocytic vesicles (11, 12, 13, 14, 15, 16). This pathway does not require newly synthesized MHC II and is independent of Ii and DM functions. While the significance of this alternative pathway is not fully understood, it is clearly distinct from the conventional pathway and could have evolved to present a distinct set of peptides that would be presented poorly or not at all via the classical pathway. A common property of such epitopes appears to be their location in relatively unconstrained domains of the parent Ags (17). The introduction of structural constraints in the Ag through chemical cross-linking can cause epitopes normally presented by recycling MHC II to require nascent MHC II- and DM-independent loading of epitopes to become DM dependent (16, 18).
For several years we have exploited the influenza virus hemagglutinin (HA) molecule to address fundamental issues in Ag processing and presentation due to the presence of well-defined epitopes, its ability to undergo specific conformational changes that are induced by acidification following virus adsorption, and structural information on both pre- and postacidification states. There are two well-defined I-Ed-restricted epitopes, termed site 1 (S1; aa 107–119) and site 3 (S3; aa 302–313) in A/Puerto Rico/8/34 influenza virus (referred to as PR8 hereafter) HA protein (19). Although these epitopes are presented by the same MHC II restriction element, they are strikingly different in several respects (18, 20, 21). Most notable here are the DM dependence of S1 presentation compared with the DM independence of S3 presentation, and the generation of S3, as detected by mAb staining, in an early endosome immediately following virion uptake vs the much later appearance of S1 in a late endosome. This latter observation is probably due to the location of S3 in the stalk region and of S1 in the globular domain of HA. Following virion uptake, HA undergoes a radical change in conformation in response to early endosomal acidification that includes unfolding of the stalk region (22, 23). This leads to fusion of viral and endosomal membranes and delivery of the viral genome to the cytosol. The compact globular domain, in contrast, is unaffected by acidification and appears to require proteolysis to be disassembled. While S1 presentation is sensitive to treatment with the protease inhibitor leupeptin, S3 presentation is actually enhanced following treatment with this inhibitor (20). Given these various points, we have speculated that the S1/I-Ed complex is generated in late endocytic vesicles and presented by nascent MHC II, while S3 is loaded in an early endosomal compartment and presented by recycling MHC II. The experiments reported here address this hypothesis.
Another dichotomy in class II presentation concerns the source of Ag. It is generally understood that MHC II-presented peptides are derived from exogenous (extracellular) Ags (24). However, some endogenous Ags, those synthesized within the APC, can gain access to the MHC II presentation pathway (25). Indeed, it appears that some epitopes can be generated only through the processing of endogenous Ag (26, 27). Recent studies have implicated both cytosolic proteases, including the proteasome, and endosome resident proteases in the generation of epitopes from endogenous proteins, including transmembrane proteins (28, 29, 30). Like recycling MHC II, the significance of the endogenous pathway(s) has not been fully explored. The issue is of special interest to us because the presentation of S3, relative to that of S1, is much higher when the APC synthesizes HA than when inactivated virions are provided. One possible explanation is that the pathways for the generation of S3 from exogenous and endogenous sources of HA are distinct, with different requirements for recycling class II molecules. Thus, the presentation of S1 and S3 from exogenous and endogenous sources of HA under various conditions was investigated in this work.
Materials and Methods
Cell lines
The B cell lymphoma cell line A20.2J was originally described by Kim et al. (31) and was provided by Dr. V. Engelhard (University of Virginia, Charlottesville, VA). This cell line was maintained in RPMI 1640 (Fisher Scientific, Pittsburgh, PA) supplemented with 5% FBS (HyClone, Logan, UT) and 0.05 mM 2-ME (Sigma-Aldrich, St. Louis, MO). L-I-Ed cells (L929 cells stably transfected with I-Ed) (32) were provided by Dr. R. Germain (National Institutes of Health, Bethesda, MD) and were maintained in IMDM (Fisher Scientific) supplemented with 5% FBS and hypoxanthine/aminopterin/thymine (Sigma-Aldrich).
T cell hybridomas
The generation of S1- and S3-specific T cell hybridomas that express β-galactosidase upon Ag-driven stimulation has been described previously (33). The T cell hybridomas were maintained in RPMI 1640 supplemented with 10% FBS, 0.05 mM 2-ME, and 1 mM sodium pyruvate.
Viruses
The influenza viruses PR8 (H1N1), A/England/333/80 (Eng; H1N1), antigenically distinct PR8 mutant virus Rv6 (Rv6; H1N1) (34), B/Lee/40 (B/Lee), and Japanese/57 (Jap; H2N2) were grown in the allantoic cavity of 10-day-old embryonated eggs and harvested on day 12. The virus titer was determined by hemagglutination of chicken erythrocytes. Virus purification was conducted according to standard methods by differential sedimentation using sucrose density gradients.
Ag presentation assays and inhibitors
In Ag presentation assays, APCs were pulsed with 500 hemagglutinating units (HAU) of the indicated virus that was inactivated by short wave UV light (254 nm, 1800 mJ; Stratalinker 1800; Stratagene, La Jolla, CA) or infected with 50 HAU of live virus/1 million cells for 1 h at 37°C in a tube rotator. Cells were washed once with complete medium, resuspended at 1 million cells/ml, and cultured in 96-well, flat-bottom tissue culture plates before T cell hybridomas were added. In assays involving drug-treated APCs, APCs were fixed with 0.5% paraformaldehyde for 2 min on ice and washed with complete medium before culturing with T cell hybridomas. APCs were pretreated with the indicated drugs 30–60 min before infection/virus pulse, pulsed/infected in the presence of drugs, and maintained in the presence of drugs before being fixed, unless otherwise indicated. APCs were treated with Con A or bafilomycin A1 (both drugs used at final concentrations of 1, 10, or 100 nM) or brefeldin A (at 5 μg/ml final concentration; all drugs were purchased from Sigma-Aldrich) or primaquine (at the indicated final concentrations; Aldrich, Milwaukee, WI). APCs and T cell hybridomas were cocultured for 16–18 h, and T cell responses were measured using the fluorogenic substrate methyl-umbelliferyl-β-d-galactoside (MUG) according to the method described by Sanderson and Shastri (35). All assays were performed at least twice, and the results of a representative assay are presented in this paper. Error bars represent the SEM of experimental replicates.
Synthetic peptides
Synthetic peptides corresponding to the S1 epitope of HA (aa 107–119, SVSSFERFEIFPK) and S3 epitope (aa 302–313, CPKYVRSAKLRM) were purchased from ResGen, Invitrogen (San Diego, CA). Peptides were reconstituted in DMSO.
Flow cytometry
Following infection of untreated control cells or cells treated with various inhibitors, cells were fixed 7 h postinfection with 2% paraformaldehyde. Cells were washed with PBS, and surface Fc receptors were blocked with normal rabbit serum. Cells were then incubated with an HA-specific mAb (H28-E23) (34) on ice for 1 h. Subsequently, cells were washed and treated with FITC-conjugated anti-mouse IgG for 1 h on ice. Cells were then washed with PBS and analyzed by flow cytometry.
Immunoprecipitation and SDS-PAGE
Untreated or inhibitor-treated A20 cells were infected with infectious PR8 or pulsed with UV-inactivated PR8 as described above. Cells were incubated in complete medium for 3 h in the presence or the absence of the inhibitor, starved for 30 min in cysteine- and methionine-free MEM (Sigma-Aldrich), and labeled for 4 h with [35S]methionine (ICN Pharmaceuticals, Costa Mesa, CA; 100 μCi/5 × 106 cells/1 ml medium). Cells were then washed and lysed in 1% Triton X-100 (Fisher Scientific) containing lysis buffer. Cell lysates were incubated with a mixture of two mAbs (H16-L10-4R5 and H19-S24-4) (36) to nucleoprotein (NP) bound to protein A-Sepharose for 3 h at 4°C and washed several times with wash buffer containing 300 mM NaCl, 1% Nonidet P-40, and 0.1% SDS in 25 mM Tris-Cl pH 7.4 (all from Sigma-Aldrich). Protein A-Sepharose beads (Repligen, Cambridge, MA) containing Ab-NP complexes were then resuspended in SDS-PAGE loading buffer, boiled, and loaded onto a 10% SDS-polyacrylamide gel. The gel was dried, exposed to x-ray film overnight, and developed according to standard procedures.
Internalization and re-emergence studies
Internalization and re-emergence of I-Ed molecules in A20 cells were studied as described previously (12). Briefly, cell surface Fc receptors were blocked with normal mouse serum (Pierce, Rockford, IL) on ice, and cells were treated with biotinylated mAbs against I-Ed (14.4.4s; BD PharMingen, San Diego, CA) for 2 h on ice. After washing three times with ice-cold PBS to remove unbound Abs, cells were shifted to 37°C for various lengths of time. Aliquots of cells were transferred to ice to prevent further internalization. Subsequently, cells were treated with streptavidin-conjugated 125I (sp. act., 20–40 mCi/mg; purchased from Amersham Pharmacia Biotech, Little Chalfont, U.K.) for 30 min on ice. Cells were washed, and bound radioactivity was measured in a gamma counter. For re-emergence studies, cells were treated as described above with 14.4.4s coupled to a reversible biotinylation agent NHS-SS-Biotin (Pierce) coupled according to the recommendations of the manufacturer. After incubation at 37°C for 1 h to allow internalization, biotin (linked to 14.4.4s) that still remained bound at the cell surface I-Ed molecules was cleaved using a stripping buffer. Cells were washed and shifted back to 37°C for different lengths of time to induce re-emergence of internalized I-Ed back to the cell surface, and aliquots of cells were transferred to 4°C to quantitate re-emerged I-Ed molecules as described above.
Results
Specificity of T cell hybridomas
To demonstrate the specificity of the T cell hybridomas that are specific for S1 or S3 epitopes, A20 cells were pulsed with a panel of UV-inactivated influenza viruses (see below) that possessed or lacked S1 and S3 epitopes, and hybridoma activation was measured. S1-specific T cell hybridoma recognizes A20 cells pulsed with PR8 and Eng in which S3 epitope is mutated (Fig. 1⇓a), while S3-specific T cell hybridoma recognizes A20 cells infected with PR8 and Rv6 in which S1 is mutated (Fig. 1⇓b). None of these hybrids recognizes cells infected with control viruses B/Lee and Jap. Further, S1-specific T cell hybridoma recognizes A20 cells pulsed with S1 peptide, but not S3 peptide (Fig. 1⇓c), while S3-specific T cell hybridoma recognizes cells pulsed with S3 peptide, but not S1 peptide (Fig. 1⇓d). Additionally, dilution of virus-pulsed APCs (Fig. 1⇓, a and b) and pulsing of APCs with decreasing doses of virus (data not shown) or synthetic peptides (Fig. 1⇓, c and d) showed that the T cell hybridomas can distinguish between varying doses of the antigenic peptide displayed on the surface of APCs and respond in a graded fashion, making them suitable for the questions posed in the studies described here.
Specificity of T cell hybridomas. A20 cells were pulsed with 500 HAU of UV-inactivated PR8 (possesses both S1 and S3), B/Lee (lacks both S1 and S3), Jap (lacks both S1 and S3), Eng (possesses S1 but mutated S3), or Rv6 (lacks S1 but possesses S3) influenza virus for 1 h in HBSS-BSA and cocultured with 1 × 105 S1 (a) or S3 (b)-specific T cell hybridoma for 18 h before T hybridoma activation was measured using MUG substrate as described by Sanderson and Shastri (35 ). A20 cells (1 × 105) were also pulsed with varying concentrations of synthetic peptides corresponding to S1 (c) or S3 (d) and cocultured with 1 × 105 specific T cell hybridomas as described above. This experiment is performed routinely to confirm specificity, and results shown here are a typical outcome. Error bars represent the SEM of experimental replicates.
Primaquine treatment selectively inhibits S3 presentation
To begin testing the hypothesis that the S3 epitope is presented by recycling class II molecules, we made use of primaquine, a compound that has been demonstrated to selectively inhibit recycling of cell surface molecules through a direct interaction with endosomes without neutralization of endosomal pH (37). When primaquine-treated A20 cells are pulsed with UV-inactivated PR8, S1 presentation is unaffected (Fig. 2⇓a). However, primaquine treatment severely impairs presentation to S3-specific T cell hybrids (Fig. 2⇓b), suggesting that recycling MHC II are involved in the presentation of S3. S3 presentation is also inhibitable by primaquine treatment when A20 cells are pulsed with purified PR8, while S1 presentation is unaffected (Fig. 2⇓, c and d). These two sources of Ag are not infectious and hence provide only exogenous sources of HA. Interestingly, similar results were obtained when APCs presented endogenous HA following their infection with PR8 (Fig. 2⇓, e and f). Treatment with primaquine does not affect the ability of A20 cells to present synthetic peptides (Fig. 2⇓, g and h). In line with previous observations (26), S3 presentation is more potent in cells infected with the virus than in cells pulsed with the inactivated virus (compare S3 presentation from UV-inactivated and infectious viruses; Fig. 2⇓, b and f). One explanation for the effect of primaquine on the presentation of S3 from endogenous HA is that it inhibits viral infection, thereby eliminating a significant supply of Ag and limiting S3 presentation. This would seem unlikely as the pH environment of endosomes, critical for inhibiting influenza virus replication, is unperturbed by treatment with primaquine. Nevertheless, we immunostained for HA expression at the cell surface following virus infection of mock-treated as well as primaquine-treated cells and analyzed them by flow cytometry. HA expression is not significantly affected by primaquine treatment of A20 cells (Fig. 3⇓a). We also collected supernatants from mock-treated and primaquine-treated cells and assayed for hemagglutination of chicken erythrocytes, a measure of viral replication and budding. We found that equal amounts of virion progeny are present in the supernatants from mock-treated and primaquine-treated cells (Fig. 3⇓b). Further, we immunoprecipitated newly synthesized PR8-NP following viral infection from mock- and primaquine-treated cells. Although the band corresponding to NP appears to be less intense in primaquine-treated cells compared with untreated control cells, when NP bands were normalized by densitometry to the nonspecific band that is present in all lanes, NP levels were comparable and, in fact, somewhat enhanced in primaquine-treated cells (Fig. 3⇓c). These results suggest that inhibition of S3 presentation from infectious PR8 is not due to deleterious effects of primaquine treatment on viral protein synthesis.
S3 presentation is primaquine sensitive. A20 cells were pretreated for 30 min with 100 μM primaquine (Pq), pulsed with 500 HAU of UV-inactivated PR8 (uvPR8; a and b) or 100 HAU of sucrose density gradient purified PR8 (pPR8; c and d), or infected with 50 HAU of infectious PR8 (iPR8; e and f) or in the presence of the inhibitor and cultured in complete medium supplemented with the drug for 7 h. Cells were then fixed in 0.5% paraformaldehyde, washed, and cocultured with S1-specific (a, c, and e) or S3-specific (b, d, and f) T cell hybridomas and assayed as described in Fig. 1⇑. A20 cells treated with the inhibitor as described above were pulsed with 10−8 M S1 (g) or 10−10 M S3 (h) peptide and cocultured with specific T cell hybridomas before activation was measured using MUG substrate. This experiment was performed several times, and the results of a representative experiment are shown here. Error bars represent the SEM of experimental replicates.
Effect of primaquine on viral protein synthesis. a, Primaquine (Pq)-treated or mock-treated cells were infected with PR8 as described in Fig. 2⇑ and fixed with 0.5% paraformaldehyde 7 h postinfection. Cells were then stained for HA using mAbs against HA (H28-E23) (34 ) and FITC-conjugated anti-mouse IgG and were analyzed by flow cytometry. Results with live unfixed cells were identical. b, Supernatants from uninfected (A20), UV-PR8-pulsed (UV-PR8), PR8-infected (PR8), or primaquine-treated, PR8-infected (Pq) A20 cells were collected, and the HA titer was determined using chicken erythrocytes. Double-diluted supernatants starting at 1/5 dilution were used, and the highest dilution that resulted in visible HA is presented as the HA titer. c, Uninfected (U); UV-inactivated, PR8-pulsed (UV); or PR8-infected A20 cells that were mock treated (PR8) or treated with primaquine (Pq) as described above were labeled with [35S]methionine and lysed, and nascent NP was immunoprecipitated using mAbs against NP (H16-L10-4R5 and H19-S24-4) (36 ). Immunoprecipitates were resolved on a 10% SDS-PAGE, and autoradiography was performed according to standard methods. ∗, A nonspecific band that is present in all lanes. The ratio between the NP band and the nonspecific band by densitometry is 1.48 for untreated control cells and 1.57 for primaquine-treated cells.
To confirm that primaquine treatment affects recycling of MHC II in A20 cells, we analyzed internalization of cell surface MHC II and recycling back to the cell surface in primaquine-treated and untreated cells. Cell surface I-Ed molecules were marked with biotinylated anti-I-Ed mAb (14.4.4s) during incubation of A20 cells on ice. Cells were then shifted to 37°C for various periods of time to induce internalization of cell surface I-Ed. Internalization was measured over varying periods of time by assessing the loss of cell surface biotin-conjugated Ab using 125I-labeled streptavidin. Re-emergence of internalized I-Ed was assessed following stripping of biotinylated cell surface Ab and warming the cells to 37°C for various periods of time to allow for the return of internalized I-Ed to the cell surface and detection as described above. As expected, primaquine does not inhibit internalization of cell surface I-Ed (Fig. 4⇓a), while it drastically affects the re-emergence of internalized molecules at the cell surface (Fig. 4⇓b). In primaquine-treated cells, the rate of internalization appears to be more rapid than in untreated control cells. This can be attributed to the inability of internalized I-Ed molecules to return to the cell surface in primaquine-treated cells.
Primaquine treatment inhibits re-emergence of internalized I-Ed in A20 cells. A20 cells were mock treated or treated with 100 μM primaquine (Pq), and internalization/re-emergence of internalized I-Ed were studied employing the method developed by Pinet et al. (12 ). For assessing the internalization of I-Ed (a), cell surface I-Ed molecules were labeled with 14.4.4s coupled to the reversible biotinylation agent NHS-SS-Biotin, and the loss of cell surface label following incubation of cells at 37°C for various lengths of time was measured using streptavidin-conjugated 125I as described in Materials and Methods. For re-emergence studies (b), biotin molecules attached to I-Ed remaining at the cell surface following 1-h incubation at 37°C were stripped, and the cells were shifted back to 37°C to allow re-emergence of internalized I-Ed. Re-emerging I-Ed molecules were captured using streptavidin-conjugated 125I at different time points. Treatment of A20 cells with biotinylated 14.4.4s and streptavidin-conjugated 125I produced 45,688 ± 107 cpm, that of primaquine-treated cells produced 34,367 ± 453 cpm, and that with a biotinylated irrelevant Ab produced 2,542 ± 74 cpm. This experiment was performed twice with similar outcomes. Error bars represent the SEM of experimental replicates.
We next asked whether primaquine-mediated inhibition of S3 presentation is cell type dependent by performing similar assays in a fibroblast line stably expressing I-Ed (L-I-Ed). S3 presentation is inhibited when L-I-Ed cells are treated with increasing doses of primaquine, while S1 presentation is unaffected when cells were pulsed with UV-inactivated PR8 (Fig. 5⇓, a and b). Further, primaquine treatment inhibits S3 presentation from infectious PR8 without affecting S1 presentation (Fig. 5⇓, c and d). Again, primaquine treatment does not significantly affect the presentation of synthetic peptides (Fig. 5⇓e). The results of the experiments outlined above suggest that S3 presentation is dependent on recycling MHC II from both UV-inactivated and infectious viruses in two distinct types of APCs.
The effect of primaquine treatment on S3 presentation is not cell type dependent. L929 fibroblast cells stably transfected with I-Ed (L-I-Ed) were mock treated or treated with varying concentrations of primaquine (Pq), pulsed with UV-inactivated PR8 (a and b), or infected with infectious PR8 (c and d) and assayed for S1 (a and c) and S3 (b and d) presentations as described in Fig. 2⇑. A20 cells treated with varying concentrations of primaquine were pulsed with 10−8 M S1 or 10−10 M S3 (e) peptide, and T cell hybridoma activation was measured. This experiment was performed three times with similar outcomes. Error bars represent the SEM of experimental replicates.
S1 presentation requires nascent MHC class II molecules
A prediction based upon the results presented above is that nascent MHC class II molecules are necessary for S1, but not S3, presentation. To test this, we used brefeldin A, a fungal metabolite that inhibits transport of proteins, including nascent MHC class II molecules, from the ER without affecting recycling MHC II. We treated A20 cells with this inhibitor for different periods of time before pulsing them with UV-inactivated PR8. Cells were pulsed with the virus in the presence of the inhibitor and cultured in medium supplemented with the inhibitor for 7 h following virus pulse. Subsequently, cells were fixed, and S1 and S3 presentation was assessed. As expected, while S3 presentation was unaffected by this inhibitor, S1 presentation gradually declined and was reduced to ∼10% in cells that were pretreated for 3 h before virus pulse (Fig. 6⇓a). These data demonstrate that S1 presentation is dependent on nascent MHC II, whereas S3 presentation is unaffected when nascent MHC II are blocked in the ER by brefeldin A. To demonstrate that brefeldin A treatment does not inhibit recycling of I-Ed, we treated A20 cells with brefeldin A and assessed internalization/re-emergence of I-Ed, as described above. The results indicated that brefeldin A treatment significantly affects neither internalization (Fig. 6⇓b) nor re-emergence (Fig. 6⇓c) of internalized I-Ed. We did not study the effect of brefeldin A treatment on the presentation of S1 and S3 from biosynthesized HA following PR8 infection, as brefeldin A will block the transport of newly synthesized HA (38), complicating the interpretation of results.
Effect of brefeldin A treatment on S1 and S3 presentations. a, A20 cells were pretreated with 5 μg/ml brefeldin A for different lengths of time before pulsing with UV-inactivated PR8 for 1 h and then were cultured for 7 h in the presence of brefeldin A. Cells were then fixed, T cell hybridomas were added, and Ag presentation assay was performed as described in the text. The percentage of the T hybridoma response compared with mock-treated cells and maximal values is presented at each time point. Internalization (b) and re-emergence (c) of surface I-Ed in mock-treated or brefeldin A-treated A20 cells were determined as described in Fig. 4⇑. Treatment of A20 cells with biotinylated 14.4.4s and streptavidin-conjugated 125I produced 11,281 ± 364 cpm, for brefeldin A-treated cells the result was 9,904 ± 407 cpm, and for cells treated with a biotinylated irrelevant Ab the value was 1,645 ± 115 cpm. Two independent experiments were performed with similar results. Error bars represent the SEM of experimental replicates.
Treatment of APCs with inhibitors that perturb endosomal functions affect both S1 and S3 presentations, but by different mechanisms
Influenza viral infection following endocytosis depends on the fusion-initiating conformational change undergone by HA (22). Based on our earlier findings, we anticipated that such changes are a prerequisite for S3 exposure and its subsequent capture by I-Ed, whereas delivery of HA to late endocytic vesicles may be essential for the generation and loading of S1. Both steps depend upon acidification. Hence, raising endosomal pH may have significant effects on the presentation of both epitopes, though perhaps for different reasons. To test this prediction, we used the vacuolar proton-ATPase inhibitors, Con A and bafilomycin A1. In these experiments A20 cells were pretreated for 1 h with the indicated concentration of the inhibitor, incubated with the Ag source in the presence of the inhibitor for 1 h, and cultured for 7 h in medium supplemented with the inhibitor before the cells were fixed and T cell hybridomas added. Con A treatment of A20 cells inhibits both S1 and S3 effectively at 10 nM, while at 1 nM the inhibitory effect is more pronounced for S3 (Fig. 7⇓a). A 10-fold higher concentration of bafilomycin (100 nM) is required to inhibit both S1 and S3 presentation from UV-inactivated PR8 (Fig. 7⇓c). At the tested concentrations of bafilomycin A1, the effects are similar on both S1 and S3 presentations, unlike Con A, which selectively inhibits S3 presentation at lower concentrations without significantly affecting S1 presentation. Similar effects of Con A and bafilomycin A1 are observed when presentation of S1 and S3 from infectious PR8 is assessed (Fig. 7⇓, b and d, respectively). Treatment does not significantly affect presentation of synthetic peptides (Fig. 7⇓e). Furthermore, treatment with these agents, even at the highest concentration tested, does not have a significant effect on the viability of the APCs, as determined by trypan blue exclusion studies (viability >95% compared with the untreated control cells; data not shown).
Effect of drugs that perturb endosomal functions on S1 and S3 presentations. A20 cells were pretreated for 1 h with varying concentrations of Con A, pulsed with UV-inactivated PR8 (a) or infected with PR8 (b) in the presence of the indicated concentrations of Con A for 1 h, and cultured in the presence of the inhibitor for 7 h before the cells were fixed and cocultured with specific T cell hybridomas. c and d, T hybridoma responses against cells that were treated with varying concentrations of bafilomycin A1 and tested exactly as described for Con A-treated cells. A20 cells, treated with the compounds described above, were pulsed with 10−8 M S1 or 10−10 M S3 (e) peptide and cocultured with specific T cell hybridomas before activation was measured using MUG substrate. Results are presented as the percent T hybridoma responses against treated APCs compared with untreated cells that were either pulsed with UV-inactivated PR8 or infected with PR8. This experiment was performed three times with similar outcomes in each case. Error bars represent the SEM of experimental replicates.
For exogenous presentation our hypothesis predicts a requirement for the transport of internalized viral proteins from early to late endosomes for S1, but not S3, presentation. However, if this class of compounds also inhibits acidification of early endosome, as shown in some cell types (39, 40), it may prevent conformational changes in HA that we hypothesize to be a prerequisite for exposing S3 epitope. To test the idea that different aspects of Con A and bafilomycin A1 action underlie the inhibition of S1 and S3 presentation, we varied the time when cells were treated with the inhibitor relative to when virus was added. If presentation of S3 depends upon the acid-induced conformational change, an early and transient step, then its inhibition will require treatment with these compounds at a relatively early time. In contrast, if S1 depends upon delivery to the late endosome, a location where Ag may accumulate before being loaded onto nascent class II molecules, a constant supply of the inhibitor at a higher concentration may be required to block S1 presentation, and the effect may be expected to be lost if treatment is withheld. We focused upon Con A for this test due to its differential effect on S1 and S3 presentation at particular concentrations (see Fig. 7⇑, a and b). Thus, cells were incubated transiently in 10 nM Con A 1 h before, at the beginning of, and immediately following a 1-h pulse with inactivated or infectious virus or continuously with the inhibitor starting 1 h before virus pulse (Fig. 8⇓i). As shown in Fig. 8⇓ii, presence of Con A starting either 1 h before or at the point of virus infection/pulse is sufficient to prevent S3 presentation. Addition of Con A 1 h after infection/pulse only moderately inhibits S3 presentation (Fig. 8⇓iia). In contrast, S1 presentation is not significantly inhibited under any of these conditions used (Fig. 8⇓iib). As expected, treatment of cells with 10 nM Con A throughout the course of the experiment inhibits both S1 and S3 presentation. As mentioned above, the acid-induced conformational change that we predict to be necessary for S3 presentation is also necessary for viral penetration and subsequent viral replication. Therefore, to determine whether the sensitivity of S3 presentation to early treatment with Con A affects inhibition of the conformational change, we monitored the effect of such treatments on infectivity, as assessed by flow cytometry. Treatment of cells with Con A starting 1 h after viral infection only slightly affects cell surface expression of HA, but treatment before and during viral infection has a substantial effect (Fig. 8⇓iiia). We also estimated the HAU in the supernatants from PR8-infected cells that were mock treated or treated with Con A as described above. Treatment with Con A before and during infection of cells drastically reduced the levels of quantifiable HA in the supernatants, while treatment of cells following virus infection only partially reduced hemagglutinating activity in the supernatant (Fig. 8⇓iiib). The results presented here are from infectious virus as the Ag source. However, similar results were obtained when UV-inactivated virus was used as the Ag (data not shown). These results support the idea that neutralization of the early endosome, a rapid effect of Con A treatment, negates the presentation of S3, but not S1, while endosomal maturation, requiring prolonged high concentrations of Con A, is a key step for the presentation of S1.
Effects of varying lengths of Con A treatment on S1 and S3 presentations. i, Schematic representation of Con A treatment of A20 cells. ii, A20 cells were treated with 10 nM Con A as indicated in i and fixed with 0.5% paraformaldehyde after 7 h, and T hybridoma responses to infectious PR8 (a, S1; b, S3) were assessed as described earlier. iii, A20 cells treated as described in i were fixed 7 h postinfection with 0.5% paraformaldehyde, and cell surface HA was analyzed by flow cytometry using mAbs against HA- and FITC-conjugated anti-mouse IgG (a). Similar results were obtained with live unfixed cells. Supernatants collected from A20 cultures treated as described above were subjected to HA assay using chicken erythrocytes. The highest dilution of the supernatant that resulted in visible agglutination of erythrocytes is presented as the HA titer (b). Similar results were obtained in two independent experiments. Error bars represent the SEM of experimental replicates.
Discussion
Although widely considered minor, the pathway for presentation of epitopes by recycling MHC II may play a key role in protection against certain pathogens. Unlike MHC I that require peptides of defined length for binding and presentation, MHC II can bind large peptides due to their open ended structure (41, 42). Thus, epitopes that are revealed following mild acidification without any aggressive proteolytic processing can bind to MHC II even when they are a part of a large peptide or a whole protein Ag. Further, some fragile epitopes may be destroyed rather than generated in highly acidic and proteolytic late endocytic vesicles, a description that fits S3 given its much higher presentation in the presence of the protease inhibitor leupeptin (citation). Thus, presentation of epitopes with such characteristics would be greatly enhanced by the presence of recycling MHC II in early endocytic vesicles.
To the growing list of epitopes that are presented by recycling MHC II (11, 12, 13, 14, 15, 17, 43), we now add the S3 of influenza HA molecule. This epitope is notable within the group in that its presentation is consistent with a great deal of structural information available on the HA molecule and the extensive unfolding that it naturally undergoes in response to acidification (23). Superimposition of PR8 HA onto the crystal structure of A/HK/68 HA (44) reveals that S1 is located in the globular domain of HA, which is not substantially altered following acidification. In contrast, the S3 epitope is positioned in the stalk region in proximity to the fusion domain of the HA2 peptide, which is known to be radically modified (22), leading to partial unfolding (45, 46, 47). Such unfolding is probably an essential step in S3 processing. Indeed, acidification of HA appears to be the single most important step for S3 loading onto MHC II. Firstly, S3, but not S1, can be presented from acid-treated virions by chemically fixed APC (21). Secondly, immediately after internalization of PR8, S3 appears to be made available in early endocytic vesicles, based on the use of a conformation-dependent mAb that recognizes HA in early endosomes (18). Given these points, it will be interesting to determine the form of S3 that is complexed with recycling MHC II. It is theoretically possible that a rather large portion of HA is attached to MHC II via S3 if unfolding is, indeed, the only required step. Experiments are currently underway to address this question. Additionally, while S1 requires DM for its loading, S3 presentation is DM independent. Indeed, S3 presentation is enhanced in DM-deficient A20 cells compared with DM-expressing A20 cells (18). Although this may be due at least in part to elevated I-Ed expression in DM-deficient A20 cells, another possibility is that S3 has a lower affinity for I-Ed compared with S1 and, in the presence of DM, is prevented from loading onto I-Ed in late endocytic vesicles where DM is present in high levels. This would render S3 presentation almost exclusively dependent on recycling I-Ed. Interestingly, the S3 epitope appears to have a weak consensus sequence for I-Ed binding compared with the S1 epitope, based on sequence comparison (http://syfpeithi.bmi-heidelberg.com/). Our ongoing studies to measure relative affinities of S1 and S3 to I-Ed may shed light on this possibility.
Epitopes presented by nascent and recycling MHC II have been shown to require distinct endosomal functions. While the majority of epitopes reported to be presented by recycling MHC II are not sensitive to compounds that perturb endosomal functions (16, 17, 43, 48), some have been reported to be inhibited (14, 15). In the experiments described in this paper both S1 and S3 presentations are inhibited by Con A and bafilomycin A1 depending on the concentration used and the time points of APC treatment. The results with neutralizers are striking in having a similar effect on S1 and S3 presentation given the many ways that presentation is differently affected by strategies that modulate Ag processing. However, when one further dissects the action of Con A (Fig. 8⇑), it can be appreciated that the two epitopes are inhibited by different mechanisms. A low concentration of Con A is sufficient to inhibit S3 and is effective when the APCs are treated during the initial hours of addition of virus (Fig. 8⇑), while a higher concentration and longer periods of treatment of APCs are required to abolish S1 presentation (Fig. 7⇑). These results suggest that different steps, namely, acid-induced conformational changes in HA for S3 presentation and delivery of internalized virus particles from early to late endosomes for S1 presentation, are inhibited by Con A. This observation is in contrast to the finding made by Pinet and Long (16) that the presentation of H3 epitope of influenza HA by recycling HLA DR molecules is insensitive to neutralizers. It may be that a requirement for endosomal acidification depends on the structural context of the epitope and, in turn, accessibility of the epitopes to recycling MHC II.
While S1 presentation is comparable whether processed from UV-inactivated PR8 or infectious PR8, S3 presentation is more pronounced from infectious PR8, suggesting that endogenously synthesized HA is a particularly favorable substrate for S3 production. Despite the possibility that exogenous and endogenous HA are processed via very different pathways, presentation of S3 from both UV-inactivated and infectious PR8 is primaquine sensitive, suggesting similar, if not identical, sites of loading onto MHC II. One of the several possibilities by which endogenous HA can reach a common loading compartment is through internalization of HA that has trafficked from the ER to the plasma membrane, thereby gaining the same location as exogenously acquired Ag. However, HA is internalized from the plasma membrane very inefficiently (49), making this possibility remote. Another possibility to consider is that the forms of S3 presented from UV-inactivated and infectious viruses may not be identical. This may be due to loading in different endocytic compartments, with S3 generated from UV-inactivated virus forming a less stable complex with MHC II than that generated from endogenous HA, perhaps due to the pH environment where the epitope is loaded onto MHC II (50). Finally, primaquine treatment does not significantly affect the biosynthesis of viral Ags (HA and NP) and trafficking of biosynthesized HA to the cell surface (Fig. 3⇑). However, whether this inhibitor impairs any other step that is required to deliver biosynthesized HA for processing and presentation via another pathway that does not involve recycling MHC II is a possibility. These issues remain to be addressed.
Based on the results provided here and a number of other reports, recycling MHC II appear to be crucial for presenting epitopes that are vulnerable to destruction by proteases in the highly acidic late endocytic vesicles and are primarily revealed in the early endosome through unfolding, triggered in the case of HA by acidification. This is probably the case with fusogenic proteins of a number of viruses, including members of orthomyxo, toga, flavi, rhabdo, bunya, and arena families. Such a mode of presentation is probably vital, as the availability of such epitopes for capture by MHC II may be transient. Further, such epitopes will be presented quite rapidly following Ag uptake, thereby providing one of the earliest triggers to the adaptive immune response. Rapid presentation of epitopes that might otherwise be unavailable due to the instability that attends unfolding could contribute significantly to the defense against certain infections.
Acknowledgments
We thank Dr. Nia Tatsis, Mona Tewari, and Matthew B. Zook for the critical reading of this manuscript.
Footnotes
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↵1 This work was supported by National Institutes of Health Grant AI36331 (to L.C.E.).
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↵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: l_eisenlohr{at}hendrix.jci.tju.edu
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↵3 Abbreviations used in this paper: MHC II, MHC class II; B/Lee, B/Lee/40; CLIP, class II-associated invariant chain peptide; Eng, A/England/333/80; ER, endoplasmic reticulum; HA, hemagglutinin; HAU, hemagglutinating units; Ii, invariant chain; Jap, Japanese/57; MUG, methyl-umbelliferyl-β-d-galactosidase; NP, nucleoprotein; PR8, A/Puerto Rico/8/34 influenza virus; Rv6, PR8 mutant virus Rv6; S1, site 1 epitope; S3, site 3 epitope.
- Received October 1, 2002.
- Accepted January 22, 2003.
- Copyright © 2003 by The American Association of Immunologists