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The Journal of Immunology, 1998, 161: 5187-5192.
Copyright © 1998 by The American Association of Immunologists

HSV-1 Glycoprotein I-Reactive TCR{gamma}{delta} Cells Directly Recognize the Peptide Backbone in a Conformationally Dependent Manner1

Roger Sciammas2 and Jeffrey A. Bluestone3

Committee on Immunology and Ben May Institute for Cancer Research, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Despite the description of numerous antigenic ligands recognized by TCR{gamma}{delta} cells, detailed information concerning the structural nature of these antigenic epitopes is lacking. In addition, the recent descriptions of human TCR{gamma}{delta} cells recognizing mycobacterium-derived low m.w. lipid molecules confirms that the spectrum and nature of biologic structures that are capable of being recognized by TCR{gamma}{delta} cells are unclear. We have previously described a murine TCR{gamma}{delta} cell clone, TgI4.4, that is reactive to herpes simplex virus (HSV)-1 glycoprotein I (gI). Unlike TCR{alpha}ß-mediated, MHC-restricted Ag recognition but similar to Ig Ag recognition, TgI4.4 recognizes purified gI directly, in the absence of Ag processing or presentation. Since gI is a complex glycoprotein, the nature of the antigenic epitope was investigated. First, gI recognition by TgI4.4 is conformationally dependent, as revealed by denaturation and proteolytic experiments. Secondly, the epitope recognized by TgI4.4 was mapped to the amino terminus by using insertion mutants of gI. Lastly, TgI4.4 recognizes the gI protein directly since completely deglycosylated forms of gI are efficiently recognized. Therefore, TCR{gamma}{delta} cells are capable of recognizing a variety of molecular structures, including proteins. The ability of TgI4.4 to recognize a nonglycosylated form of gI suggests that HSV-1 recognition by TCR{gamma}{delta} cells in vivo is not limited by cell-specific glycosylation patterns or glycosylation-dependent conformational influences.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tcell receptor {gamma}{delta} cells prove to be a critical immunoregulatory population in both bacterial and viral pathogenesis (1, 2), including herpes simplex virus (HSV)4-1 infections (3). However, in most cases the Ags recognized by the activated TCR{gamma}{delta} cells isolated from draining lymph nodes or pathogenic lesions remain unknown (2, 4, 5). Despite the paucity of Ag-specific clones of TCR{gamma}{delta} cells, the TCR{gamma}{delta} CDR3 regions sequenced from lymphoid tissues and gut are very diverse, suggesting a potential for broad Ag reactivity (6, 7). An abundance of recent evidence suggests that TCR{gamma}{delta} cells recognize unprocessed Ags directly (5, 8). For instance, as documented for HSV-1 glycoprotein I-reactive cells (3, 9) as well as two MHC alloreactive TCR{gamma}{delta} cell clones (10), none of the known factors involved in MHC class I or II Ag processing affect TCR{gamma}{delta} Ag recognition. Furthermore, purified "whole" Ags are able to stimulate TCR{gamma}{delta} cells directly (9, 11). This mode of Ag recognition suggests Ig type recognition properties. Two structural lines of evidence support these conclusions. First, Ag receptor CDR3 structures, analyzed from sequence databases, show that TCR{gamma}{delta} chains are structurally more similar to Ig heavy and light chains than to TCR{alpha}ß-chains (12). Second, recent crystallographic data acquired from a human TCRV{delta}-chain reveal distinct configurations within both the framework and CDR regions that implicate structures similar to both the TCR and Ig molecules (13).

Structurally diverse types of glycoproteins ranging from heat shock proteins (14, 15, 16, 17, 18); MHC class II (19), MHC class Ia (4), MHC class Ib (20, 21); CD1c (22); bacterial superantigens (23), CD48 (24, 25); and HSV-1 glycoprotein I can be recognized by TCR{gamma}{delta} cells (3, 26). In fact, a subset of human TCR{gamma}{delta} cells have been described that are reactive to mycobacterium-derived, phosphorylated low m.w. protease resistant (LMP) lipid compounds (27, 28, 29). Interestingly, the presence of the phosphate moiety is essential for recognition of the lipid compound (30). Therefore, given the array of biologic structures capable of being recognized by TCR{gamma}{delta} cells-lipids and glycoproteins, it is essential to examine the nature of these molecular interactions. We have described the recognition of HSV-1 glycoprotein I by murine TCR{gamma}{delta} cells (3, 9). gI recognition by a representative clone, TgI4.4, is direct and is independent of Ag processing or presentation. In this study we further explore the nature of this direct interaction and show that recognition of gI is highly dependent on conformation, maps to the amino terminus, and is independent of glycosylation. This structural analysis of TCR{gamma}{delta} cell Ag recognition will have important implications toward the interpretation of molecular structures and models of TCR{gamma}{delta}-mediated Ag recognition derived from x-ray crystallographic data.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions

CHO gIIg-expressing cells were grown and used to produce large quantities of soluble gIIg, as previously described (9). LgI-1 cells are LMtk- cells obtained from American Type Culture Collection (ATCC, Manassas, VA) and stably transfected with the gI DNA, as previously described (26). The TCR{gamma}{delta} clone TgI4.4 (105 cells) was passaged once a week with irradiated (20 Gy) splenic feeder cells (5 x 106 cells), mitomycin C-treated (40 µg/ml for 30 min) LgI-1 cells (6 x 105 cells), and 30 U/ml rhIL-2, as previously described (26). Ldld CHO mutant cells were obtained from ATCC with permission from Dr. Monty Krieger (Massachusetts Institute of Technology, Boston, MA) (31). All cells were grown in DMEM medium supplemented with 10% FCS, 2 mM [l]-glutamine, 2 mM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 x 10-5 M 2-ME. The Ldld cells were cultured in Ham’s F12 medium instead of the DMEM and contained the same supplements. Tunicamycin was dissolved in DMSO and incubated with the CHO gIIg cells at the indicated concentrations following addition of fresh medium. The serum-free medium supplement, ITS, used for the Ldld glycosylation experiments, consisted of insulin 0.625 mg/ml, transferrin 0.625 mg/ml, and selenium 0.625 mg/ml and was obtained from Collaborative Research (Bedford, MA). Depending on the glycosylation conditions, FCS (3%), UDP-galactose (20 µM), and UDP-galactosamine (200 µM) were used to supplement the F12-ITS media.

TgI4.4 bioassay

Resting TgI4.4 cells were stimulated with gIIg immobilized on plastic, gIIg adsorbed to plastic immobilized goat anti-hIgG1 Fc-specific Abs, or gI-expressing cells for 48 h. The supernatant was harvested and analyzed for secreted IFN-{gamma} by ELISA. This consisted of a sandwich protocol of sequential steps of anti-IFN-{gamma}- (H22 clone) coated wells, incubation of cell supernatants, incubation of a secondary goat anti-IFN-{gamma} antisera, and, finally, detection with alkaline phosphatase-coupled donkey anti-goat Abs.

Abs and reagents

Goat anti-hIgG1 Fc Ab, affinity purified, (Cappel, Malvern, PA) was used to coat tissue culture wells for adsorption of soluble gIIg. Alkaline phosphatase-coupled goat anti-hIgG1 Fc Ab, affinity purified (Cappel) was used for gIIg detection in Western blots. Abs for the IFN-{gamma} ELISA have been previously described and were obtained from Dr. Schrieber (Washington University, St. Louis, MO). The tunicamycin, UDP-galactose, UDP-galactosamine, and trypsin-coupled agarose beads were obtained from Sigma (St. Louis, MO). The protease endo-lys-c and the N-glycanase were obtained from Boehringer Mannheim (Indianapolis, IN). The G418 sulfate used for negative selection of cells following DNA transfections was obtained from Life Technologies (Rockville, MD).

Plasmids

The gIIg expression plasmid used for transfection of Ldld cells has been previously described (9). Linker scanning mutants of HSV-1 gI were constructed by S. Basu, et al., and consisted of the insertion of 10- to 12-mer linkers into various positions of the gI DNA, based on restriction enzyme sites (32). The mutants were sequenced to confirm the restoration of the correct reading frame.

Western analysis

gIIg was enriched from 1 ml of cell supernatant using protein A-Sepharose beads (Pharmacia, Uppsala, Sweden). Beads were washed with PBS, and the bound gIIg was eluted using Laemmli sample buffer and boiling. Samples were resolved using 10% SDS-PAGE in a minigel apparatus and then transferred to nitrocellulose membrane (Schleicher & Schuell, Keene, NH) using a minigel transfer apparatus (Bio-Rad, Hercules, CA). The membrane was blocked with 3% BSA dissolved in TBST buffer (10 mM TRIS, 0.15 M NaCl, and 0.05% Tween 20) and then probed with alkaline phosphatase-coupled goat anti-human IgG Fc-specific Ab. Excess Ab was washed off, and the membrane was developed using BCIP and NBT substrates (Promega, Madison, WI).

gIIg treatments

gIIg in PBS was treated with trypsin-coupled Sepharose beads (Sigma) or with purified endo-lys-c (Boehringer Mannheim) at 37°C overnight. Trypsin beads were pelleted, and the supernatant, containing the proteolytic fragments, was used to coat plastic tissue culture wells for a TgI4.4 bioassay. The endo-lys-c sample was directly coated to plastic tissue culture wells, up to 100 µg/ml, for a TgI4.4 bioassay. Digestion was confirmed by SDS-PAGE and Coomassie blue staining. Reduction of gIIg was accomplished using various concentrations of 2-ME for 1 h at 25°C. Samples were dialyzed against PBS and then immobilized on plastic tissue culture wells for a TgI4.4 bioassay.

DNA transfections

Ldld CHO mutant cells were transfected using a kit from Specialty Media (Lavallette, NJ) that is based on a calcium-phosphate precipitation method. Cells were cotransfected with 10 µg gIIg expression plasmid and 1 µg of a neomycin resistance plasmid, pSV2neo. G418 sulfate was added at 24 h posttransfection at a concentration of 1 mg/ml. Colonies were isolated using cloning cylinders (Specialty Media) and screened for expression by the ability to stimulate TgI4.4. LMtk- cells were transiently transfected using a DEAE-dextran method. Cells in log-phase growth in a 100-mm plate were washed extensively and incubated with DMEM medium containing HEPES (10 mM), chloroquine (50 µM), DEAE-dextran (0.25 mg/ml), and 10 µg DNA in a final volume of 5 ml. After 3–4 h, the medium was aspirated and the cells were pulsed with DMEM medium containing 10% (v/v) DMSO for 80 s. Following the DMSO pulse, the cells were incubated in complete media and used in a TgI4.4 bioassay 40 h post transfection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
gI recognition by TgI4.4 is highly dependent on conformation

A soluble fusion protein, gIIg, constructed with the extracellular region of gI and the Fc portion of human IgG1 was secreted from CHO cells and purified over an immunoaffinity column of protein A-Sepharose. Purified gIIg was able to stimulate the TCR{gamma}{delta} clone TgI4.4 to proliferate and to secrete IFN-{gamma} when immobilized directly on plastic tissue culture wells or indirectly by adsorption to immobilized anti-human IgG Fc-specific Abs, as previously shown (9). The tertiary structure of gI is critical for TgI4.4 recognition since thermal denaturation of gIIg abrogated recognition (data not shown). Furthermore, mild reduction of intermolecular disulfide bonds, accorded by treatment of gIIg with the reducing agent ß-mercaptoethanol also abrogated recognition by TgI4.4, (Fig. 1GoA). These observations suggest that perturbations in the conformation of gIIg results in the disordering of the antigenic epitope. Second, protease digestion of gIIg was pursued to identify fragments capable of stimulating TgI4.4. Fig. 1GoB shows that trypsin-digested gIIg failed to stimulate TgI4.4 when immobilized on plastic tissue culture wells. Since there are 17 putative trypsin cleavage sites in gI, it is possible that none of the fragments maintain the correct conformation. However, even digestion of gIIg with endo-lys-C, a protease that generates only four fragments of gI, failed to stimulate TgI4.4 when these peptides were immobilized (Fig. 1GoB). Thus, the conformation of the tertiary structure of gI plays a crucial role in forming the epitope recognized by TgI4.4.



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  		FIGURE 1. TgI4.4-mediated recognition of gI is dependent on tertiary structure. A, gIIg was treated with various doses of 2-ME for 1 h at room temperature, dialyzed against PBS, and used to stimulate TgI4.4 by immobilization to tissue culture wells. B, gIIg was incubated with the proteases trypsin or endo-lys-c at 37°C overnight and used to stimulate TgI4.4 by immobilization to tissue culture wells. Shown in all panels is IFN-{gamma} secretion by TgI4.4.

 
The epitope recognized by TgI4.4 maps to the amino terminus of gI

The identification of the antigenic epitope of gI recognized by TgI4.4 was pursued to gain a greater understanding of the molecular interactions between the TCR and Ag. gI is expressed by HSV-1 and localizes both on the cell surface of infected cells and on the viral envelope in heterodimeric form with another glycoprotein, gE (33, 34). TgI4.4 is capable of recognizing virus-infected cells as well as purified gIIg, suggesting that the recognized epitope is independent of, and not affected by, heterodimer formation. Interestingly, heterodimeric association between gI and gE confers high avidity-monomeric IgG Fc binding activity, whereas gE alone exhibits low avidity-aggregated IgG Fc binding activity (33, 34). Since gE, but not gI, contains sequence homology to mammalian Fc receptors (35), the role of gI in converting the low avidity IgG Fc binding activity of gE to high avidity binding is unknown. To elucidate the role of gI in Fc binding, Basu. et al. generated linker scanning mutants of gI, characterized these mutants, and identified regions of gI required to confer high avidity Fc binding activity by the heterodimer (32). These mutants were used to dissect the epitope specificity of TgI4.4. As seen in Table IGo, wt gI, transiently expressed in L cells, was efficiently recognized by TgI4.4 as determined by the induction of IFN-{gamma} secretion. Mutation of the core region of gI prevented TgI4.4 recognition; however, these mutations appear to render the gI molecule sensitive to misfolding since both Ab recognition and Fc binding activity were abolished (32). Two closely spaced mutants, at positions 43 and 63, also eliminated TgI4.4 recognition despite these mutants’ ability to bind monomeric IgG Fc. Therefore, it is likely that the amino terminus of gI, surrounding amino acids 40–70, contains the epitope recognized by TgI4.4.


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  		Table I. The epitope of gI recognized by TgI4.4 maps to the amino terminus1

 
Carbohydrate modifications of gI are not required for TgI4.4 recognition

gI is a complex glycoprotein containing three potential N-linked carbohydrate sites and a putative domain of O-linked carbohydrates. The role of these carbohydrates in TgI4.4-mediated gI recognition was investigated. To assess the role of N-linked carbohydrates, the glycosylation inhibitor tunicamycin, which prevents the transfer of carbohydrates from the dolichol core in the endoplasmic reticulum to the asparagine acceptor in the protein, was used (36). CHO gIIg cells treated with tunicamycin inhibited all N-linked glycosylations of gIIg as analyzed by increased mobility on SDS-PAGE and subsequent Western blotting (Fig. 2GoB). The absence of N-linked glycosylation was confirmed by comparing the mobility of both tunicamycin and N-glycanase-treated gIIg (Fig. 2GoB). As shown in Fig. 2GoA, despite efficient deglycosylation, TgI4.4 was still able to recognize non-N-linked carbohydrate-modified gIIg when adsorbed to anti-human IgG1-coated tissue culture wells.



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  		FIGURE 2. N-linked glycosylations of gI are not required for efficient recognition by TgI4.4. A, CHO gIIg cells were incubated with the glycosylation inhibitor tunicamycin for 2 days, and the supernatant containing modified gIIg was used to stimulate TgI4.4 by adsorption to anti-human IgG1-coated tissue culture wells. Shown is IFN-{gamma} secretion by TgI4.4. B, The gIIg from the tunicamycin-treated CHO gIIg cells was analyzed for increased mobility on SDS-PAGE and compared with untreated and N-glycanase-treated gIIg. Shown is detection of the various forms of gIIg by Western blot.

 
The removal of O-linked glycosylation required the use of a novel CHO mutant cell, Ldld (31). This cell line is a glycosylation mutant that exhibits reversible defects in the synthesis of protein and lipid-linked oligosaccharide. Ldld is deficient in UDP-galactose/UDP-galactosamine 4 epimerase activity, which is required for isomerizing pools of UDP-glucose/UDP-glucosamine into UDP-galactose/UDP-galactosamine. UDP-galactose (GAL) and UDP-galactosamine (GALNAC) precursors are necessary for subsequent protein modifications that result in the processing of either N-linked or O-linked modifications, respectively. When cultured in serum-free medium, the Ldld cells do not induce any carbohydrate modifications. However, when the serum-free media is reconstituted with serum, synthesized UDP-galactose, or UDP-galactosamine, proteins are modified. Therefore, gIIg-transfected Ldld cells were cultured in the ITS serum-free medium alone or supplemented with FCS, GAL, GALNAC, or GAL/GALNAC. As shown in Fig. 3GoA, TgI4.4 was able to recognize all of the modified forms of gIIg, including the completely deglycosylated form, when the resultant supernatants were adsorbed to anti-human IgG1-coated tissue culture wells. The slope of the dose response curves, obtained by titrating the various supernatants, suggests that all forms were recognized in a similar manner, independent of differences in the types of glycosylations present. Interestingly, the supernatant of the normally glycosylated form (in the presence of serum) titrates further out. Thus, either the non-serum-supplemented forms are produced less well or there is a slight change in conformation of the normally glycosylated form. Furthermore, the differentially glycosylated forms of gIIg could be discriminated by differential mobility on SDS-PAGE, and the completely deglycosylated form migrated close to its expected molecular mass of 57 kDa as determined by the primary structure of gIIg (Fig. 3GoB). The differences in the maxima of the ability of the various glycosylated forms of gIIg to stimulate TgI4.4 suggest either qualitative or quantitative differences. Since the fully deglycosylated form stimulates TgI4.4, any qualitative differences reflect subtle conformational modifications of the recognized epitope rather than carbohydrate moieties comprising a portion of the epitope. This is not surprising since TgI4.4 recognizes gIIg in a conformationally dependent manner. The differences in stimulation may reflect a quantitative difference. This interpretation is consistent with the Western blot analysis demonstrating that the variable amounts of gIIg in each of the samples correlated with the stimulatory activity. Therefore, recognition of gI by TgI4.4 is directed to a proteinaceous epitope and can occur independently of glycosylations.



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  		FIGURE 3. The epitope of gI recognized by TgI4.4 is independent of glycosylation and is proteinaceous. A, Ldld gIIg cells were cultured in serum-free medium, ITS. ITS + GAL, ITS supplemented with UDP-galactose; ITS + GALNAC, ITS supplemented with UDP-galactosamine; ITS + GAL + GALNAC, ITS supplemented with UDP-galactose and UDP-galactosamine; ITS + FCS, ITS supplemented with 3% FCS. Treatment of Ldld cells with serum-free ITS medium prevents carbohydrate modifications whereas the various ITS supplements of either GAL, GALNAC, GAL/GALNAC, or FCS result in differentially glycosylated proteins. The gIIg-containing supernatant from the treated Ldld gIIg cells was used to stimulate TgI4.4 following serial twofold titrations and subsequent adsorption to anti-human IgG1-coated tissue culture wells. Shown is IFN-{gamma} secretion (U/ml) by TgI4.4 in response to 0.5, 0.25, and 0.125 (v/v) of supernatant. B, The altered mobility of the differentially modified gIIgs was analyzed by SDS-PAGE and Western blotting. The completely deglycosylated form, obtained by culturing the Ldld gIIg cells in serum-free media without carbohydrate supplements, designated ITS, migrates close to 57 kDa, the predicted molecular mass of the gIIg fusion protein based on its primary structure.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report extends the current concept of TCR{gamma}{delta} cell-mediated Ag recognition by defining the molecular requirements of recognition by an Ag-specific TCR{gamma}{delta} cell clone. The HSV-1 glycoprotein I (gI)-specific TCR{gamma}{delta} cell clone TgI4.4 recognizes gI in a direct manner, independent of classical Ag processing or presentation (9). To gain a greater understanding of the molecular interactions involved in gI recognition by TgI4.4, conditions that alter the tertiary structure of gI were used, and it was determined that the conformational status of gI controls recognition. Using insertion mutants of gI, the epitope recognized by TgI4.4 was located in the amino terminus, surrounding amino acids 40–70. The overall conformation and properties of gI were not altered by these mutations since high avidity-monomeric IgG Fc binding by the gIgE heterodimer was still intact. In fact, the closely related gI glycoprotein derived from HSV-2 is not recognized by TgI4.4 (26), consistent with the high degree of divergence between these two proteins in the N terminus (37). While it is clear that the amino terminus contains the epitope, it is not clear whether the insertions disrupted the primary structure of the epitope or whether the insertions disrupted the local conformational status of the amino terminus and thereby prevented recognition. Without an additional measure of the conformation or function of this region of the amino terminus, this cannot be conclusively determined. Reduction of gIIg with ß-mercaptoethanol abrogated recognition by TgI4.4, suggesting that the epitope is dependent on tertiary structure. Although the epitope lacks cysteine residues, it is adjacent to a cluster of cysteines that are conserved among HSV-1 glycoproteins (37), suggesting that the disulfide bonded cysteines are somehow positioning the amino terminus in an appropriate conformation/orientation. In addition, the protease endo-lys-c used in this study did not cleave within the identified epitope but instead separates the epitope from the rest of the protein just before the cluster of cysteine residues. These data suggest that the epitope of gI recognized by TgI4.4 is highly conformationally dependent, independent of the ability of gI to heterodimerize with gE, is solvent exposed, and, perhaps, is distal to and protrudes away from cell membrane.

Given the recent descriptions of lipid reactive TCR{gamma}{delta} cells, it has become important to define the nature of biologic structures recognized by Ag-specific TCR{gamma}{delta} cells. The glycoprotein gI afforded a system in which to determine whether or not TgI4.4 Ag specificity was directed at the peptide, carbohydrate, or both. Interestingly, it was found that gI recognition by TgI4.4 is directed at the peptide backbone and can occur in the absence of any glycosylations. Since TgI4.4 appears to recognize the fully deglycosylated gIIg less well, these results do not rule out the possibility of subtle, qualitative differences in the conformation of the epitope induced by carbohydrate modifications. However, due to the variability in the amounts of gIIg produced in response to the various treatments, it is just as possible that the differences in stimulation of TgI4.4 by the various forms reflect quantitative differences. In fact, the amino terminus, the location of TgI4.4’s epitope, is distal from predicted N-linked and O-linked glycosylation sites (37). In parallel with the lack of direct carbohydrate reactivity, it is interesting to point out that the majority of CDR3 amino acids in the TCR{delta} chain are hydrophobic in nature (9), suggesting 1) a direct interaction between these two regions and 2) epitope-driven selection for this TCR structure.

Finally, it was shown that TCR{gamma}{delta} cells play a critical role in HSV-1 immunity and that gI-reactive TCR{gamma}{delta} cells may constitute a large portion of the observed protective response (3). The findings that gI recognition is both direct and independent of carbohydrates suggests that TCR{gamma}{delta} cells may operate in a niche of recognition not only of infected cells incapable of efficient Ag presentation but also of many distinct cell types, independently of cell-specific glycosylation patterns. Secondly, it is not clear whether gI constitutes a major protective target of TCR{gamma}{delta} cell-mediated protection in vivo. The use of an engineered gI-deficient HSV-1 to address this question is not possible since this virus does not propagate well in vivo and its virulence is attenuated (38, 39, 40). It might now be possible to address this question by creating a recombinant HSV-1 that contains mutations in the amino terminus that disrupt TCR{gamma}{delta} cell gI recognition without altering the essential functions of gI in HSV-1 virulence.


   Acknowledgments
 
We thank Drs. Thandavarayan Nagashunmugam and Harvey M. Friedman for intellectual discussions and for providing us with the gI mutant expression plasmids. We are also indebted to Dr. Monty Krieger for providing the glycosylation-defective CHO cell line Ldld. We also thank J. Auger and Drs. P. Fields, E. Klotz, and U. Korthauer for numerous discussions and insightful suggestions.


   Footnotes
 
1 R.S. is a recipient of a National Institutes of Health Interdisciplinary Training Grant in Immunology (5T32 AI 07090). This research has been supported by National Institutes of Health Grants RO1 AI 26847 and P30 CA 14599. Back

2 Current address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305. Back

3 Address correspondence and reprint requests to Dr. Jeffrey A. Bluestone, Committee on Immunology and Ben May Institute for Cancer Research, MC1089, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. Back

4 Abbreviations used in this paper: HSV, herpes simplex virus; wt, wild type; GAL, galactose; GALNAC, galactosamine; gI, glycoprotein I; CHO, Chinese hamster ovary. Back

Received for publication March 19, 1998. Accepted for publication July 7, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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