Presentation by Recycling MHC Class II Molecules of an Influenza Hemagglutinin-Derived Epitope That Is Revealed in the Early Endosome by Acidification

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-E^d) (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-E^d 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-E^d (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-E^d 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-E^d back to the cell surface, and aliquots of cells were transferred to 4°C to quantitate re-emerged I-Ed molecules as described above.

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.

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FIGURE 1.

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.

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FIGURE 2.

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.

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FIGURE 3.

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-E^d molecules were marked with biotinylated anti-I-E^d 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-E^d. 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-E^d 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-E^d to the cell surface and detection as described above. As expected, primaquine does not inhibit internalization of cell surface I-E^d (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-E^d molecules to return to the cell surface in primaquine-treated cells.

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FIGURE 4.

Primaquine treatment inhibits re-emergence of internalized I-E^d in A20 cells. A20 cells were mock treated or treated with 100 μM primaquine (Pq), and internalization/re-emergence of internalized I-E^d were studied employing the method developed by Pinet et al. (12 ). For assessing the internalization of I-E^d (a), cell surface I-E^d 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-E^d 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-E^d. Re-emerging I-E^d 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-E^d (L-I-E^d). S3 presentation is inhibited when L-I-E^d 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.

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FIGURE 5.

The effect of primaquine treatment on S3 presentation is not cell type dependent. L929 fibroblast cells stably transfected with I-E^d (L-I-E^d) 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-E^d, we treated A20 cells with brefeldin A and assessed internalization/re-emergence of I-E^d, 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-E^d. 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.

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FIGURE 6.

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-E^d 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-E^d, 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).

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FIGURE 7.

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.

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FIGURE 8.

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.