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The Journal of Immunology, 2004, 172: 6607-6614.
Copyright © 2004 by The American Association of Immunologists

Differential Requirements for Endosomal Reduction in the Presentation of Two H2-Ed-Restricted Epitopes from Influenza Hemagglutinin1

Gomathinayagam Sinnathamby*, Maja Maric2,{dagger}, Peter Cresswell{dagger} and Laurence C. Eisenlohr3,*

* Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107; and {dagger} Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the role of reduction in the presentation of two H2-Ed-restricted epitopes (site 1 epitope (S1) and site 3 epitope (S3)) occupying distinct domains of the influenza hemagglutinin major subunit that contains four intrachain disulfide bonds and is connected to the virion by one interchain bond. S3 is situated within the stalk region that unfolds in response to mild acidification, and loads onto recycling H2-Ed in the early endosome, while S1, located in the structurally constrained globular domain, loads onto nascent H2-Ed in the late endosome. Predicting dependence upon reduction for either epitope seemed plausible but the results from several approaches were clear: presentation of S1 but not S3 is reduction dependent. Surprisingly, IFN-{gamma}-inducible lysosomal thiol reductase (GILT), the only reductase thus far known to be involved in MHC class II-restricted processing, is not necessary for the generation of S1. However, GILT is necessary for presentation of either epitope when the virus is pretreated with a reducible cross-linker. The results suggest that unfolding of the Ag, perhaps a prerequisite for proteolytic processing in many cases, proceeds either spontaneously in the early endosome or via reduction in a later endosome. They further imply mechanisms for GILT-independent reduction in the late endosome, with GILT perhaps being reserved for more intractable Ags.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Major histocompatibility complex class II (MHC II)4 molecules present peptides to CD4+ T cells through distinct intracellular pathways. Nascent MHC II are delivered to endocytic vesicles via the associated invariant chain (Ii). Ii is then proteolytically processed (1), leaving the class II-associated Ii peptide in the binding groove of MHC II (2, 3, 4). Removal of the class II-associated Ii peptide from MHC II and loading of antigenic peptides is mediated by a nonclassical MHC II known as H2-M (in mice, and HLA-DM in humans) (5) that acts both as a peptide editor (6) and a chaperone (7, 8). Mature MHC II molecules that recycle from cell surface can bind antigenic peptides in early endosomes and present them independent of Ii (9, 10, 11, 12). Unlike MHC class I molecules, MHC II molecules have open-ended peptide-binding pockets that enable them to acquire large peptides (13) and denatured proteins (14). In fact, acidification and disulfide bond reduction in the absence of proteolysis provides sufficient processing for a variety of intact Ags (15).

Glutathione and cysteine constitute the major physiological thiols necessary for creating an intracellular reducing environment, with glutathione being the most prevalent cellular thiol accounting for >90% of the total nonprotein sulfur (16). The importance of intracellular glutathione in processing Ags with disulfide bonds has been well documented (17, 18, 19, 20). Though intracellular reducing activity has been found to be mainly associated with late endosomes (21, 22), the acidic conditions of late endosomes and lysosomes have been reported to be unfavorable for disulfide bond reduction while favoring proteolysis (23). Hence, reduction in late endosomes and lysosomes would appear to require catalysis. Indeed, such a catalyst has recently been identified. GILT (IFN-{gamma}-inducible lysosomal thiol reductase) is expressed constitutively in APCs and is optimally active at low pH (24). The importance of GILT in the processing of Ags with disulfide bonds has been documented both in vitro (25, 26, 27) and in vivo (28). Although GILT plays a key role in the processing of many Ags, there may be other enzymes involved in endosomal redox reactions.

Although many Ags are actively unfolded via reduction as an early or, perhaps, only step in their processing, the fusogenic proteins of many viruses spontaneously unfold upon entering the endosomal compartment. Given this "self processing," the role of disulfide bond reduction in the presentation of such proteins is of particular interest. The prototypical influenza virus hemagglutinin (HA), is an especially attractive subject due to a wealth of structural and functional information (29). HA is composed of two subunits, HA1 and HA2. HA1 mediates binding to host cells and, in the version from influenza virus A/Puerto Rico/8/34 (PR8), harbors two well-defined H2-Ed-restricted epitopes (site 1 epitope (S1), aa 107–119; site 3 epitope (S3), aa 302–313) (30) with strikingly dissimilar characteristics (31, 32, 33, 34). S1 is located in the compact globular head of HA and is bound to nascent MHC II in late endosomes through the action of H2-M. Because this globular head contains all of the intrachain disulfide bonds within HA1 (see Fig. 1A), reduction might be necessary for S1 presentation. Indeed, reduction and alkylation of purified HA allows for the presentation of S1 by fixed APCs (35). At the same time, as the epitope is generated in the catabolically active late endosome, proteolysis alone may effectively liberate S1. This notion is supported by the observation that leupeptin, an inhibitor of endosomal thiol proteases, significantly reduces S1 presentation (31). In contrast, S3 resides in the stalk region of the HA1 subunit that undergoes rapid acid-induced unfolding in the early endosome (36). This epitope is presented by recycling H2-Ed without the involvement of H2-M (33, 34). As there is limited proteolytic activity in the early endosome (37) and S3 is available for binding following the conformational change, we have long speculated that S3 is presented as part of a much larger polypeptide, perhaps the entire HA1 subunit, although direct evidence of this is still lacking. In any event, it is almost certainly necessary for the S3-containing ligand to be separated from the virion to be loaded onto recycling H2-Ed. Ostensibly, this could be accomplished by reduction of the single disulfide bond between HA1 and HA2, although the capacity for reduction in the early endosome is unclear. Of note, while mature GILT has been located exclusively in late endosomes and lysosomes (28), at least in human cells, the enzymatically active precursor form can be found in early endosomes (24). The other means of separation would be proteolysis, although proteolytic activities within the early endosome are limited (37). Thus, for the generation of either epitope, there are compelling arguments for and against reduction as a mandatory step. The experiments presented in this study were designed to settle these questions and show rather clearly that for S1, but not S3 presentation, disulfide bond reduction is mandatory.



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  		FIGURE 1. A, Secondary structure of PR8 HA. The two subunits of HA (HA1 and HA2) with the intramolecular disulfide bonds are illustrated with the amino acid positions of cysteine residues involved in disulfide bonds formation. Approximate locations of S1 and S3 are represented. This schematic representation is adapted from Ref. 43 . B, Effect of BSO on Ag presentation. Mock-treated or BSO-treated L-I-Ed cells were pulsed with 25 hemagglutinating units (HAU) of purified PR8 per 1 x 106 cells, fixed with 0.5% paraformaldehyde, washed, double diluted (starting at 1 x 105 cells/well), and cultured with S1- or S3-specific T cell hybrids for 16 h and T hybrid activation was measured using MUG substrate. As a control, cells were also pulsed with S1 synthetic peptide and used as APC.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and viruses

L929 cells stably transfected with H2-Ed (L-I-Ed) were provided by Dr. R. Germain (National Institutes of Health, Bethesda, MD) (38). The hamster ovary fibroblast WAB4 and the somatic cell hybrid WALC (18) were a kind gift of Dr. K. L. McCoy (Virginia Commonwealth University, Richmond, VA). S1- and S3-specific T cell hybridomas that express {beta}-galactosidase upon Ag-driven stimulation have been described (34, 39). T cell hybridomas were maintained in RPMI 1640 supplemented with 10% FBS (HyClone Laboratories, Logan, UT), 0.05 mM 2-ME and 1 mM sodium pyruvate (Invitrogen, Carlsbad, CA). Primary fibroblasts from wild-type and GILT–/– mice were generated from skin biopsies according to standard procedures.

The PR8 was propagated in the allantoic cavity of embryonated eggs and titrated by hemagglutination of chicken erythrocytes. Virus particles were purified using sucrose density gradients. Purified virus was cross-linked with a cleavable agent dimethyl 3,3'-dithiobispropionimidate · 2HCl (DTBP; Pierce, Rockford, IL) as described earlier (33).

Ag presentation assays

Ag presentation assays were performed as described earlier (34). T hybridoma activation was measured using the fluorogenic substrate methyl-umbelliferyl-{beta}-D-galactoside (MUG) according to the method of Sanderson and Shastri (40). In assays involving the chemical inhibitor L-buthionine-(S,R)-sulfoximine (BSO; Sigma-Aldrich), APCs were treated with this inhibitor for 24 h at the indicated concentration, pulsed with PR8 in the presence of the inhibitor, and cultured for an additional 7 h in complete medium supplemented with the inhibitor. Cells were then fixed with 0.5% paraformaldehyde for 1 min on ice, and washed with complete medium before culturing with T cell hybrids. All assays were performed at least twice and the results of representative assays are presented in this paper. Error bars represent the SEM of experimental replicates.

Plasmid constructs, synthetic peptides, and Abs

H2-Ed {alpha} and {beta} cDNAs in pBlueScript SK+ plasmid, provided by Dr. J. Miller (University of Chicago, Chicago, IL), were PCR-amplified and cloned into the eukaryotic expression vector pRc-CMV. pcDNA3 carrying human class II transcriptional activator (hCIITA) was a kind gift of Dr. M. Marks (University of Pennsylvania, Philadelphia, PA). Murine GILT (mGILT) cDNA (24) was PCR-amplified from a BSKSII+ construct and cloned into pIRES-Bleo (Clontech Laboratories, Palo Alto, CA). Synthetic peptides corresponding to S1 (aa 107–119, SVSSFERFEIFPK) and S3 (aa 302–313, CPKYVRSAKLRM) were purchased from Invitrogen. A rabbit polyclonal antiserum against mGILT has been described earlier (24).

Transfections

Cells (2.5 x 105) were plated in 6-well tissue culture plates overnight. At 70–80% confluency, cells were transfected with 1 µg of the indicated plasmid construct using Lipofectamine 2000 reagent (Invitrogen) in OPTI-MEM (Invitrogen). Twelve hours later, transfection mix was removed and complete medium was added. After 36 h, cells were trypsinized and used in Ag presentation assays or lysed in 1% Triton X-100 (Fisher Scientific, Pittsburgh, PA) containing PBS for use in Western blot analysis.

Generation of recombinant retroviruses and transduction of cells

cDNA for H2-Ed {alpha} and {beta}, hCIITA, and mGILT were cloned into the MSCV-based retrovirus vector CMV IRES GFP {alpha} (kind gift of Dr. J. Zhang, Thomas Jefferson University, Philadelphia, PA) and transfected along with pCL-Eco helper plasmid (Imgenex, San Diego, CA) into 293T cells using calcium phosphate reagent (CellPhect transfection kit; Amersham Biosciences, Piscataway, NJ). After overnight incubation, cells were washed once and DMEM supplemented with 5% FBS was added. Supernatant containing recombinant retroviruses was harvested after 24 h and stored in –70°C.

Primary skin fibroblasts were transduced with the indicated recombinant retroviruses in the presence of polybrene (10 µg/ml; Sigma) in a 6-well tissue culture plate overnight. Cells were then cultured in DMEM supplemented with 10% FBS (HyClone Laboratories) for 48 h before they were used in Ag presentation assays.

iRNA-mediated gene silencing

Three iRNA duplexes (control iRNA: GCGCGCUUUGUAGGAUUCGdTdT, GILT1 iRNA: UGUCAGCGGUACGUGGGAGdTdT, and GILT2 iRNA: GCUGGAAAAGGAGGCAGCGdTdT) were synthesized by Dharmacon (Lafayette, CO). L-I-Ed cells were plated in 24-well plates overnight and transfected at 70–80% confluency with 0.5 and 1 µg of iRNA duplexes in Oligofectamine reagent (Invitrogen) overnight. Transfection mix was then removed and complete medium was added. Cells were trypsinized at the indicated time points and used in Ag presentation assays or lysed in 1% Triton X-100 containing PBS for use in Western blot analysis.

Western blot analysis

The indicated amounts of cell lysates were resolved on a 10% SDS-polyacrylamide gel and the proteins were transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA) using a wet transfer apparatus (Bio-Rad). mGILT and transferrin receptor were probed with rabbit anti-mGILT serum and mouse anti-human transferrin receptor Ab (Zymed Laboratories, San Francisco, CA), followed by HRP-conjugated secondary reagents. Blot was developed using ECL detection system (Kirkegaard and Perry Laboratories, Gaithersburg, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
S1 but not S3 presentation is sensitive to depletion of intracellular glutathione

As a first step in determining the role of reduction in the generation of S1 and S3, we treated cells with BSO that reduces intracellular glutathione levels by inhibiting {gamma}-glutamyl-cysteine synthetase, an enzyme involved in glutathione synthesis. Treatment of L-I-Ed cells with BSO inhibits S1 presentation but not S3 presentation (Fig. 1B) from purified PR8. BSO treatment of cells affects neither cell viability, as determined by trypan blue exclusion (data not shown), nor the ability of APCs to present synthetic peptides. These results suggest to us that inhibition of intracellular reduction by depleting glutathione levels adversely affects S1 presentation without impairing S3 presentation.

S1 presentation is impaired in a processing-defective hamster cell line

We next investigated S1 and S3 presentation in a hamster cell line variant (WAB4) previously shown to be defective in processing Ags with disulfide bonds, owing to reduced levels of intracellular glutathione. For a positive control we used the somatic hybrid WALC, resulting from a fusion between WAB4 and a murine fibroblast cell line, a strategy that corrected the Ag-processing defect (18, 19). We transfected these cell lines with eukaryotic expression constructs carrying H2-Ed {alpha} and {beta} cDNA and hCIITA. CIITA globally regulates the expression of genes involved in Ag presentation such as MHC II, Ii, and HLA-DM (41). When WAB4 cells are pulsed with purified PR8, S3 presentation is readily discernable while S1 presentation is not (Fig. 2). In contrast, both the epitopes were presented by the somatic hybrid WALC. All transfectants activated T cell hybrids when pulsed with S1 or S3 synthetic peptides. These results suggest that S1 but not S3 presentation requires disulfide bond reduction.



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  		FIGURE 2. Ag presentation in disulfide bond-processing defective WAB4 and competent WALC cells. WAB4 and WALC (18 ) were transfected with 1 µg each of expression plasmids carrying H2-Ed {alpha} and {beta}, and hCIITA genes for 48 h. Cells were then pulsed with 25 HAU of purified PR8 per 1 x 106 cells or synthetic peptides, double diluted (starting at 1 x 105 cells/well), and cultured with S1- or S3-specific T hybrids for 16 h. T hybrid activation was measured using MUG substrate.

 
S3 presentation can be forced to depend upon endosomal reduction by treating virus particles with a reducible cross-linker

Earlier and ongoing experiments have indicated that S3 is made available in the early endosomal compartment for presentation by recycling H2-Ed in a H2-M-independent fashion (33, 34). The basis for this presentation phenotype appears to be the location of S3 within HA. Thus, we speculated that if S3 were placed in a more constrained context, it might require endosomal reduction for presentation. To test this notion, we used the cleavable cross-linker DTBP that contains a disulfide bridge. Interestingly, when WAB4 cells are pulsed with the cross-linked virus, neither epitope is presented. S3, but not S1, is presented from mock cross-linked virus (Fig. 3). In contrast, WALC is able to present both epitopes from cross-linked virus. We consistently observed a slight but significant reduction in the level of T hybrid activation when cross-linked PR8 is the source of the Ag. We attribute this reduction to modification of HA protein that compromises generation of both S1 and S3. WAB4 and WALC transfectants activated T hybrids equally well when pulsed with the synthetic peptides. Thus, cross-linking of virus particles with a reducible agent renders endosomal reduction necessary for both S1 and S3 presentation.



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  		FIGURE 3. Ag presentation in WAB4 and WALC cells from chemically cross-linked PR8. WAB4 and WALC were transfected with 1 µg each of eukaryotic expression plasmids carrying H2-Ed {alpha} and {beta}, and hCIITA genes for 48 h. Transfected WAB4 and WALC cells were then pulsed with 100 HAU of PR8 that was chemically cross-linked with DTBP (xPR8) or treated with the cross-linking HEPES buffer alone (cPR8) per 1 x 106 cells or synthetic peptides, double diluted (starting at 1 x 105 cells/well), and cultured with S1- or S3-specific T hybrids for 16 h. T hybrid activation was measured using MUG substrate.

 
Expression of GILT does not rescue S1 presentation in WAB4 cells

The results shown thus far demonstrate a role for endosomal reduction in the presentation of S1. The only enzyme that has been directly implicated in the endosomal reduction process is GILT (28), suggesting its possible role in the reduction of HA. The inability of WAB4 to process Ags with disulfide bonds efficiently has been attributed to low levels of intracellular glutathione (19). We considered the possibility that over-expression of GILT might be able to compensate for this defect and rescue S1 presentation. Hence, we cotransfected WAB4 with plasmids expressing H2-Ed {alpha} and {beta}, and CIITA along with murine GILT, and analyzed S1/S3 presentation. As can be seen from Fig. 4A, S1 presentation remains undetectable from vector-transfected as well as GILT-transfected cells, while S3 presentation is unaltered. WAB4 transfectants pulsed with synthetic peptides activate both S1 and S3 T hybrids. Immunoblot analysis reveals that an abundant level of GILT is expressed in transfected cells (Fig. 4B). These results demonstrate that the defect associated with WAB4 cannot be reversed by overexpressing GILT, perhaps due to the reduced levels of intracellular glutathione, a thiol donor for reduction process.



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  		FIGURE 4. Effect of GILT over-expression on Ag presentation in WAB4 cells. A, WAB4 cells were transfected with 1 µg each of eukaryotic expression plasmids carrying H2-Ed {alpha} and {beta}, and hCIITA genes with pIRES-bleo expression plasmid or 1 and 2 µg of pIRES-bleo carrying mGILT cDNA for 48 h. Cells were then pulsed with 25 HAU of purified PR8 (top panels) per 1 x 106 cells or synthetic peptides (bottom panels), double diluted (starting at 1 x 105 cells/well) and used as APC in Ag presentation assay. B, Twenty micrograms of total cell extracts from WAB4 cells transfected with eukaryotic plasmids coding for hCIITA (1 µg), mGILT (1 and 2 µg), or untransfected cells were subjected to SDS-PAGE followed by immunoblot analysis using rabbit anti-mouse GILT, HRP-conjugated anti-rabbit IgG, and ECL system.

 
Diminishing the levels of intracellular GILT has no impact on Ag presentation

GILT expression is constitutive in professional APCs and inducible via IFN-{gamma} in other cell types (24). However, as the murine fibroblast cell line L-I-Ed presents S1 and S3 efficiently, we asked whether GILT is expressed in L-I-Ed. As shown in Fig. 5Aa, abundant levels of GILT can be detected by immunoblot analysis. To determine the role of GILT in processing of HA and presentation of S1 and S3, we used iRNA-mediated gene-silencing technology. We designed a control iRNA duplex and two duplexes based on the GILT open reading frame. When L-I-Ed cells are transfected with the designed duplexes and lysates analyzed after 36 and 60 h posttransfection, GILT2 but not GILT1 or control duplex transfected cells show reduced GILT expression (Fig. 5A, b and c). However, transfection with both GILT1 and GILT2 duplexes does not result in greater suppression of GILT expression than that achieved by GILT2 alone. We subjected lysates to immunoblot analysis with anti-transferrin receptor Ab as an internal control (Fig. 5Ad). We could not achieve complete silencing of GILT expression with higher iRNA concentrations or even following sequential transfections at 24 h intervals (data not shown). However, we asked the question whether the reduced level of GILT expression has any impact on Ag presentation.



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  		FIGURE 5. iRNA-mediated silencing of mGILT expression in L-I-Ed cells and its effect on Ag presentation. Aa, Total extracts (10, 25, and 50 µg) of L-I-Ed cells were resolved on a 10% SDS-PAGE and immunoblotted with rabbit anti-mGILT antiserum. L-I-Ed cells were plated in 24-well plates overnight, transfected with 0.5 or 1 µg of control or GILT-specific (GILT1 and GILT2) iRNA duplexes for the indicated periods of time. Twenty micrograms of cell extracts were then subjected to SDS-PAGE followed by immunoblot analysis using anti-mGILT (Ab and Ac) or anti-transferrin receptor (Ad) Abs. B, L-I-Ed cells were transfected with 1 µg of GILT-specific (GILT2 or GILT1 + 2) or control iRNA duplexes for 48 h. Cells were then pulsed with a high dose (50 HAU; top panels) or a low dose (10 HAU; bottom panels) of purified PR8 and used as APC at the indicated densities in Ag presentation assays. S1- and S3-specific T hybrid responses were then measured using MUG substrate. C, Twenty micrograms of cell extracts were then subjected to SDS-PAGE followed by immunoblot analysis using anti-mGILT or anti-transferrin receptor Abs.

 
L-I-Ed transfectants were pulsed with purified PR8 48 h posttransfection and used as APCs in Ag presentation assays. As seen from Fig. 5B, L-I-Ed cells transfected with control as well as GILT iRNA duplexes present S1 and S3 identically. Immunoblot analysis of GILT and transferrin receptor expression is shown in Fig. 5C. These results may be due to the following reasons: 1) iRNA-mediated gene silencing does not lead to complete elimination of GILT, leaving sufficient levels to bring about reduction and/or (2) GILT is not involved in mediating reduction of HA, suggesting the possibility that a yet unidentified reductase is responsible for endosomal reduction of HA.

Presentation of S1 and S3 from cross-linked PR8 is impaired in GILT-depleted cells

We observed earlier that when PR8 is cross-linked with the reducible cross-linker DTBP, both S1 and S3 presentation becomes dependent on endosomal reduction (Fig. 3). Hence, we asked whether iRNA-mediated diminution of GILT has any impact on the presentation of cross-linked virus. Interestingly, both S1 and S3 (Fig. 6) are presented poorly by cells transfected with GILT-specific but not control iRNA duplexes. The various transfectants presented synthetic peptides identically. These results indicate that when PR8 is constrained structurally with DTBP, GILT plays a prominent role in Ag presentation. Further, they show that the reduction of GILT expression by iRNA treatment, though incomplete, is physiologically significant.



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  		FIGURE 6. Effect of diminishing mGILT expression on Ag presentation from chemically cross-linked PR8. L-I-Ed cells were transfected with control or GILT-specific iRNA duplexes for 48 h, pulsed with 100 HAU of DTBP cross-linked (xPR8) or mock cross-linked (cPR8) PR8 per 1 x 106 cells, double diluted (starting at 1 x 105 cells/well), and cultured with S1- (top panels) or S3-specific (middle panels) T hybrids. Transfected cells were also pulsed with S3 synthetic peptide (bottom panel) and T hybrid activation was measured using MUG substrate.

 
Presentation of S1 and S3 in primary GILT–/– fibroblasts

Because processing of untreated virus may require lower levels of GILT activity than that required for processing of cross-linked virus, we cannot conclusively rule out a role for GILT in the generation of the epitopes under standard conditions. Hence, we established primary fibroblasts from GILT-free mice and examined their ability to present S1 and S3 from untreated and cross-linked virus. Because the cells are derived from C57BL/6 mice, we transduced them with recombinant retroviruses expressing H2-Ed {alpha} and {beta}, along with retroviruses expressing hCIITA and GILT. As can be seen from Fig. 7A, both S1 and S3 are presented efficiently from GILT–/– cells and coexpression of GILT does not impact presentation levels. When pulsed with synthetic peptides, both the transducers induce similar levels of T hybridoma activation suggesting that H2-Ed expression is nearly identical. As anticipated, GILT–/– cells present both S1 and S3 poorly from cross-linked PR8 and reconstitution with retrovirally expressed GILT significantly enhances their presentation (Fig. 7B). Thus, while reduction is a necessary step in the generation of S1, GILT does not appear to be involved unless HA is further constrained by cross-linking.



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  		FIGURE 7. Presentation of S1 and S3 by GILT–/– primary fibroblasts. Primary fibroblasts from a GILT–/– C57BL/6 mouse were transduced with retroviruses carrying cDNA for H2-Ed {alpha} and {beta}, and hCIITA with or without mGILT for 48 h. 1 x 106 cells were then pulsed with 10 HAU of purified PR8 (A, top panels) or synthetic peptides (A, bottom panels), double diluted (starting at 1 x 105 cells/well), and S1 and S3 presentation was assessed using specific T hybridomas. B, Cells were also pulsed with 100 HAU of PR8 that was chemically cross-linked using DTBP (xPR8) or treated with the cross-linking HEPES buffer alone (cPR8) per 1 x 106 cells, double diluted (starting at 1 x 105 cells/well), and S1 hybridoma responses were measured using MUG substrate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Influenza virus undergoes profound changes, mediated by HA, within the endosomal compartment. Fusion of viral and endosomal membranes is triggered by a radical, acid-induced, conformational change that involves unfolding of the stalk region within the HA1 subunit and exposure of the fusion domains within the HA2 subunit. These orchestrated interactions with the endosomal compartment are of great utility for understanding MHC II-restricted presentation (31, 32, 33, 34, 35, 42). In this study, we have investigated the role of reduction in the generation of two epitopes within HA that, despite being restricted to the same class II element (H2-Ed), have strikingly different presentation phenotypes. The evidence strongly suggests that the differences can be ascribed to positioning within HA with respect to the acid-induced conformational change rather than intrinsic properties such as affinity for H2-Ed. Concerning the question of reduction, the context of the two epitopes is also strikingly different. S1 is situated in the globular domain of the HA1 subunit that harbors four disulfide bonds (36, 43), while S3 is within the stalk region that unfolds in response to endosomal acidification and contains a single disulfide bond that connects HA1 to HA2.

Several different lines of evidence suggest that S3 is made available for binding to recycling MHC II as a result of the acid-induced conformational change in an early endosome (31, 32, 33, 34, 35, 42). Given the open-ended nature of the class II peptide-binding groove (13), the well-established ability of class II to bind large proteins provided that the epitope resides in a disordered region (14, 44, 45), and the low proteolytic activity within early endosomes, we envision S3 loading onto recycling MHC II molecules as part of a large molecule, perhaps the entire HA1 subunit. Separation of HA1 from the virion would seem to be necessary for MHC II loading. This could be achieved by proteolysis or by reduction of the disulfide bond connecting HA1 and HA2. Neither was a compelling choice at the outset of this investigation, given the low proteolytic activity within early endosomes (37) and the fact that endosomal-reducing activity has been mapped mainly to late endocytic/lysosomal compartments (21, 22, 24, 28). The two independent approaches taken in this study show clearly that S3 presentation does not require endosomal reducing activity. Thus, we now favor the notion that there is sufficient proteolytic activity in the early endosome to effect the separation of S3 from the virion.

Given the much greater proteolytic activity of the late endosome, it seemed plausible that presentation of S1 might not require reduction. In contrast, disulfide bonds can be inhibitory for protease action (46). Results were equally clear in the case of S1, whose dependence upon reduction is profound. Certainly proteolysis, necessary or not, is involved, because S1 is presented as part of a relatively short peptide (47).

As GILT is the only endosomal reductase known to participate in Ag processing, is localized to late endosomes, and is most active at low pH (24, 28), we expected that it would be instrumental in the generation of S1. Neither overexpression of GILT in the disulfide-processing defective WAB4 cell line nor depletion of intracellular GILT using iRNA approach in a mouse L cell line affected S1 or S3 presentation, suggesting that GILT does not provide the reducing activity necessary for S1 presentation. Because iRNA treatment did not completely eliminate GILT, it was possible that the residual GILT is sufficient for processing HA. Alternatively, the data might reflect the presence of other reductase(s) in the endocytic pathway. To distinguish between these two possibilities unambiguously, we performed presentation assays with primary fibroblasts obtained from GILT–/– mice. Cells from GILT-free mice presented S1 and S3, and introduction of GILT had no impact on presentation (Fig. 7). Thus, we conclude that S1 requires reduction for its generation but GILT is not involved. It will be of interest to determine the source of reductive activity that is necessary for the generation of S1 and almost certainly other epitopes as well. Enzymes such as protein disulfide isomerase, that has been detected in endosomes (48), and thioredoxin oxidoreductase, that has been shown to enhance in vitro proteolysis of an Ag by cysteine-proteases (49), are some candidates, but their involvement in endosomal reduction in intact cells has not been demonstrated and they do not appear to be active at low pH.

Our experiments with cross-linked virus shed some light on the relative roles of GILT and the S1-generating reducing activity. Previously, we observed that treatment of PR8 with a reducible cross-linker, which prevents HA from undergoing the acid-induced conformational change necessary for fusion, caused a substantial portion of S3 presentation to become H-2M-dependent (33). In the current set of experiments, we observed that cross-linking additionally caused presentation of both epitopes to become strongly GILT dependent, with levels of GILT remaining after iRNA treatment being insufficient for presentation of either (Fig. 6). Perhaps GILT is critical for the processing of Ags that are more constrained by disulfide bonds than untreated PR8. Alternatively, GILT may preferentially act on particular disulfide bonds, including that within the cross-linker.

Our results provide additional support for the division of labor that we have proposed for recycling and nascent MHC II. Many pathogen-associated proteins, including HA-like fusogens and bacterial toxins, are programmed to unfold in the early endosome (29, 50, 51), with unfolding being potentially the only necessary processing step for presentation by open-ended MHC II. We have speculated in the past (33, 34) that such proteins strongly influenced the evolution of a MHC II-recycling pathway for the capture of epitopes that would otherwise be destroyed on their way to the late endosome for loading via the classical pathway. In contrast, many other proteins, exemplified in this study by the globular domain of HA1, do not spontaneously unfold and require the more aggressive environment of the late endosome to be made accessible for MHC II binding. In this case, active reduction by GILT and/or at least one other reductase is needed. Thus, we propose that either an Ag, or the domain of an Ag, is self-processing and loads onto a recycling MHC II in the early endosome, or it requires active unfolding, a process that is restricted to the late endosome where subsequent loading onto nascent MHC II takes place.


   Acknowledgments
 
We thank Dr. Kathleen L. McCoy for the hamster cell lines used in this study, Dr. Olga Shestova for valuable suggestions, and Mona Tewari for a critical reading of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI 36331 (to L.C.E), and Howard Hughes Medical Institute and National Institutes of Health Grant AI 23081 (to P.C). L.C.E. is a Leukemia and Lymphoma Society Scholar (1121-00). Back

2 Current address: Department of Microbiology and Immunology, Georgetown University, Med/Dent Building, C302, 3900 Reservoir Road NW, Washington, DC 20057. Back

3 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}mail.jci.tju.edu Back

4 Abbreviations used in this paper: MHC II, MHC class II; BSO, L-buthionine-(S,R)-sulfoximine; CIITA, class II transcriptional activator; DTBP, dimethyl 3,3'-dithiobispropionimidate · 2HCl; GILT, IFN-{gamma}-inducible lysosomal thiol reductase; h, human; HA, hemagglutinin; HAU, hemagglutinating units; i, inhibitory; Ii, invariant chain; L-I-Ed, L929 cells stably transfected with H2-Ed; m, murine; MUG, methyl-umbelliferyl-{beta}-D-galactoside; PR8, influenza virus A/Puerto Rico/8/34; S1, site 1 epitope; S3, site 3 epitope. Back

Received for publication January 29, 2004. Accepted for publication March 26, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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