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HSV-1 Glycoprotein I-Reactive TCRγδ Cells
Directly Recognize the Peptide Backbone in a
Conformationally Dependent Manner
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J Immunol 1998; 161:5187-5192; ;
http://www.jimmunol.org/content/161/10/5187
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Copyright © 1998 by The American Association of
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References
Roger Sciammas and Jeffrey A. Bluestone
HSV-1 Glycoprotein I-Reactive TCRgd Cells Directly
Recognize the Peptide Backbone in a Conformationally
Dependent Manner1
Roger Sciammas2 and Jeffrey A. Bluestone3
cell receptor gd 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
TCRgd cells isolated from draining lymph nodes or pathogenic
lesions remain unknown (2, 4, 5). Despite the paucity of Ag-specific clones of TCRgd cells, the TCRgd 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 TCRgd 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 TCRgd cell
clones (10), none of the known factors involved in MHC class I or
II Ag processing affect TCRgd Ag recognition. Furthermore, purified “whole” Ags are able to stimulate TCRgd 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 TCRgd chains are structurally more similar to
T
Committee on Immunology and Ben May Institute for Cancer Research, University of
Chicago, Chicago, IL 60637
Received for publication March 19, 1998. Accepted for publication July 7, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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.
2
Current address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305.
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.
4
Abbreviations used in this paper: HSV, herpes simplex virus; wt, wild type; GAL,
galactose; GALNAC, galactosamine; gI, glycoprotein I; CHO, Chinese hamster
ovary.
Copyright © 1998 by The American Association of Immunologists
Ig heavy and light chains than to TCRab-chains (12). Second,
recent crystallographic data acquired from a human TCRVd-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 –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
TCRgd cells (3, 26). In fact, a subset of human TCRgd cells have
been described that are reactive to mycobacterium-derived, phosphorylated low m.w. protease resistant (LMP) lipid compounds
(27–29). 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 TCRgd 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 TCRgd 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 TCRgd cell Ag recognition will have important
implications toward the interpretation of molecular structures and
models of TCRgd-mediated Ag recognition derived from x-ray
crystallographic data.
Materials and Methods
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 LMtk2
cells obtained from American Type Culture Collection (ATCC, Manassas,
VA) and stably transfected with the gI DNA, as previously described (26).
0022-1767/98/$02.00
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Despite the description of numerous antigenic ligands recognized by TCRgd cells, detailed information concerning the structural
nature of these antigenic epitopes is lacking. In addition, the recent descriptions of human TCRgd 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 TCRgd cells are unclear. We have previously described a murine TCRgd cell clone, TgI4.4, that is reactive to herpes
simplex virus (HSV)-1 glycoprotein I (gI). Unlike TCRab-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, TCRgd 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 TCRgd cells in vivo is not limited by
cell-specific glycosylation patterns or glycosylation-dependent conformational influences. The Journal of Immunology, 1998, 161:
5187–5192.
5188
STRUCTURAL REQUIREMENTS OF RECOGNITION BY TCRgd CELLS
The TCRgd clone TgI4.4 (105 cells) was passaged once a week with irradiated (20 Gy) splenic feeder cells (5 3 106 cells), mitomycin C-treated
(40 mg/ml for 30 min) LgI-1 cells (6 3 105 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 mg/ml streptomycin, and 5 3 1025
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 mM), and
UDP-galactosamine (200 mM) were used to supplement the F12-ITS
media.
TgI4.4 bioassay
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-g 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 mg/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.
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 mg gIIg expression plasmid and 1 mg 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. LMtk2 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 mM), DEAE-dextran (0.25 mg/ml), and
10 mg 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
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 TCRgd clone
TgI4.4 to proliferate and to secrete IFN-g 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
b-mercaptoethanol also abrogated recognition by TgI4.4, (Fig.
1A). 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. 1B shows that trypsindigested 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 endolys-C, a protease that generates only four fragments of gI, failed to
stimulate TgI4.4 when these peptides were immobilized (Fig. 1B).
Thus, the conformation of the tertiary structure of gI plays a crucial
role in forming the epitope recognized 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 I, wt
gI, transiently expressed in L cells, was efficiently recognized by
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Resting TgI4.4 cells were stimulated with gIIg immobilized on plastic, gIIg
adsorbed to plastic immobilized goat anti-hIgG1 Fc-specific Abs, or gIexpressing cells for 48 h. The supernatant was harvested and analyzed for
secreted IFN-g by ELISA. This consisted of a sandwich protocol of sequential steps of anti-IFN-g- (H22 clone) coated wells, incubation of cell
supernatants, incubation of a secondary goat anti-IFN-g antisera, and, finally, detection with alkaline phosphatase-coupled donkey anti-goat Abs.
DNA transfections
The Journal of Immunology
5189
Table I. The epitope of gI recognized by TgI4.4 maps to the amino
terminus a
L Cells Transiently
Transfected
1
1
1
1
1
1
1
1
1
nothing
wt gI
gI 43
gI 63
gI 104
gI 128
gI 145
gI 165
gI 200
TgI4.4 Recognition
(IFN-g Secretion:
U/ml)
Conformationally
Dependent mAb
Recognitionb
High AvidityMonomeric IgG Fc
Binding Activityc
ND
120
ND
ND
101
ND
ND
87
108
2
1
2
2
1
2
2
2
1
2
1
1
1
1
2
2
1
1
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-g
secretion by TgI4.4.
TgI4.4 as determined by the induction of IFN-g 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.
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. 2B). The
absence of N-linked glycosylation was confirmed by comparing
the mobility of both tunicamycin and N-glycanase-treated gIIg
(Fig. 2B). As shown in Fig. 2A, 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.
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. 3A, 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 IgG1coated 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. 3B). The
differences in the maxima of the ability of the various glycosylated
forms of gIIg to stimulate TgI4.4 suggest either qualitative or
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a
wt and linker scanning mutants of gI were expressed in L cells by transient
transfection methods and used to stimulate TgI4.4. At 40 h posttransfection, cells
were harvested and plated with TgI4.4 cells, both at 105 cells/well for 48 h prior to
harvesting supernatants and measuring IFN-g secretion. Shown are the results of the
IFN-g ELISA in U/ml. ND (not detected), indicates that the IFN-g was below the
detection level of the assay. The various mutants are designated by the amino acid
position where the linker was inserted.
b,c
Also shown are the results, obtained by S. Basu et al. (32) of the ability of the
gI mutants to exhibit high avidity-monomeric IgG Fc binding activity and to be
recognized by a conformationally dependent mAb.
5190
STRUCTURAL REQUIREMENTS OF RECOGNITION BY TCRgd CELLS
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.
Discussion
This report extends the current concept of TCRgd cell-mediated
Ag recognition by defining the molecular requirements of recognition by an Ag-specific TCRgd cell clone. The HSV-1 glycoprotein I (gI)-specific TCRgd 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
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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-g 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 Nglycanase-treated gIIg. Shown is detection of the various forms of gIIg by
Western blot.
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 b-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 TCRgd cells, it
has become important to define the nature of biologic structures
recognized by Ag-specific TCRgd 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
TCRd 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 TCRgd cells play a critical role in
HSV-1 immunity and that gI-reactive TCRgd 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 TCRgd 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 TCRgd cellmediated 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 – 40).
It might now be possible to address this question by creating a
The Journal of Immunology
5191
recombinant HSV-1 that contains mutations in the amino terminus
that disrupt TCRgd 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.
References
1. Bluestone, J. A., R. Khattri, R. Sciammas, and A. I. Sperling. 1995. TCR g d
cells: a specialized T-cell subset in the immune system. Annu. Rev. Cell Dev.
Biol. 11:307.
2. Haas, W., P. Pereira, and S. Tonegawa. 1993. g/d cells. Annu. Rev. Immunol.
11:637.
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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 1 GAL,
ITS supplemented with UDP-galactose; ITS 1 GALNAC, ITS supplemented with UDP-galactosamine;
ITS 1 GAL 1 GALNAC, ITS supplemented with UDP-galactose and
UDP-galactosamine; ITS 1 FCS,
ITS supplemented with 3% FCS.
Treatment of Ldld cells with serumfree 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-g 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 SDSPAGE 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.
3. Sciammas, R., P. Kodukula, Q. Tang, R. L. Hendricks, and J. A. Bluestone. 1997.
T cell receptor-g/d cells protect mice from herpes simplex virus type 1-induced
lethal encephalitis. J. Exp. Med. 185:1969.
4. Bluestone, J. A., R. Q. Cron, M. Cotterman, B. A. Houlden, and L. A. Matis.
1988. Structure and specificity of T cell receptor g/d on major histocompatibility
complex antigen-specific CD31, CD42, CD82 T lymphocytes. J. Exp. Med.
168:1899.
5. Chien, Y. H., R. Jores, and M. P. Crowley. 1996. Recognition by g/d T cells.
Annu. Rev. Immunol. 14:511.
6. Elliott, J. F., E. P. Rock, P. A. Patten, M. M. Davis, and Y. H. Chien. 1988. The
adult T-cell receptor d-chain is diverse and distinct from that of fetal thymocytes.
Nature 331:627.
7. Davis, M. M., and P. J. Bjorkman. 1988. T-cell antigen receptor genes and T-cell
recognition. Nature 334:395.
8. Sciammas, R., Y. Tatsumi, A. I. Sperling, K. Arunan, and J. A. Bluestone. 1994.
TCR g d cells: mysterious cells of the immune system. Immunol. Res. 13:268.
9. Sciammas, R., R. M. Johnson, A. I. Sperling, W. Brady, P. S. Linsley,
P. G. Spear, F. W. Fitch, and J. A. Bluestone. 1994. Unique antigen recognition
by a herpesvirus-specific TCR-gd cell. J. Immunol. 152:5392.
10. Schild, H., N. Mavaddat, C. Litzenberger, E. W. Ehrich, M. M. Davis,
J. A. Bluestone, L. Matis, R. K. Draper, and Y. H. Chien. 1994. The nature of
major histocompatibility complex recognition by g d T cells. Cell 76:29.
5192
STRUCTURAL REQUIREMENTS OF RECOGNITION BY TCRgd CELLS
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
phocyte clone specific for herpes simplex virus glycoprotein I. J. Immunol. 148:
983.
Pfeffer, K., B. Schoel, H. Gulle, S. H. Kaufmann, and H. Wagner. 1990. Primary
responses of human T cells to mycobacteria: a frequent set of g/d T cells are
stimulated by protease-resistant ligands. Eur. J. Immunol. 20:1175.
Tanaka, Y., S. Sano, E. Nieves, G. De Libero, D. Rosa, R. L. Modlin,
M. B. Brenner, B. R. Bloom, and C. T. Morita. 1994. Nonpeptide ligands for
human g d T cells. Proc. Natl. Acad. Sci. USA 91:8175.
Constant, P., F. Davodeau, M. A. Peyrat, Y. Poquet, G. Puzo, M. Bonneville, and
J. J. Fournie. 1994. Stimulation of human g d T cells by nonpeptidic mycobacterial ligands. Science 264:267.
Schoel, B., S. Sprenger, and S. H. Kaufmann. 1994. Phosphate is essential for
stimulation of V g 9V d 2 T lymphocytes by mycobacterial low molecular weight
ligand. Eur. J. Immunol. 24:1886.
Krieger, M., P. Reddy, K. Kozarsky, D. Kingsley, L. Hobbie, and M. Penman.
1989. Analysis of the synthesis, intracellular sorting, and function of glycoproteins using a mammalian cell mutant with reversible glycosylation defects. Methods Cell Biol. 32:57.
Basu, S., G. Dubin, T. Nagashunmugam, M. Basu, L. T. Goldstein, L. Wang,
B. Weeks, and H. M. Friedman. 1997. Mapping regions of herpes simplex virus
type 1 glycoprotein I required for formation of the viral Fc receptor for monomeric IgG. J. Immunol. 158:209.
Johnson, D. C., and V. Feenstra. 1987. Identification of a novel herpes simplex
virus type 1-induced glycoprotein which complexes with gE and binds immunoglobulin. J. Virol. 61:2208.
Johnson, D. C., M. C. Frame, M. W. Ligas, A. M. Cross, and N. D. Stow. 1988.
Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and gI. J. Virol. 62:1347.
Dubin, G., S. Basu, D. L. Mallory, M. Basu, R. Tal-Singer, and H. M. Friedman.
1994. Characterization of domains of herpes simplex virus type 1 glycoprotein E
involved in Fc binding activity for immunoglobulin G aggregates. J. Virol. 68:
2478.
Tkacz, J. S., and O. Lampen. 1975. Tunicamycin inhibition of polyisoprenyl
N-acetylglucosaminyl pyrophosphate formation in calf-liver microsomes. Biochem. Biophys. Res. Commun. 65:248.
McGeoch, D. J. 1990. Evolutionary relationships of virion glycoprotein genes in
the S regions of alphaherpesvirus genomes. J. Gen. Virol. 71:2361.
Longnecker, R., S. Chatterjee, R. J. Whitley, and B. Roizman. 1987. Identification of a herpes simplex virus 1 glycoprotein gene within a gene cluster dispensable for growth in cell culture. Proc. Natl. Acad. Sci. USA 84:4303.
Dingwell, K. S., L. C. Doering, and D. C. Johnson. 1995. Glycoproteins E and I
facilitate neuron-to-neuron spread of herpes simplex virus. J. Virol. 69:7087.
Dingwell, K. S., C. R. Brunetti, R. L. Hendricks, Q. Tang, M. Tang,
A. J. Rainbow, and D. C. Johnson. 1994. Herpes simplex virus glycoproteins E
and I facilitate cell-to-cell spread in vivo and across junctions of cultured cells.
J. Virol. 68:834.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
11. Crowley, M. P., Z. Reich, N. Mavaddat, J. D. Altman, and Y. Chien. 1997. The
recognition of the nonclassical major histocompatibility complex (MHC) class I
molecule, T10, by the g d T cell, G8. J. Exp. Med. 185:1223.
12. Rock, E. P., P. R. Sibbald, M. M. Davis, and Y. H. Chien. 1994. CDR3 length in
antigen-specific immune receptors. J. Exp. Med. 179:323.
13. Li, H., M. I. Lebedeva, A. S. Llera, B. A. Fields, M. B. Brenner, and
R. A. Mariuzza. 1998. Structure of the Vd domain of a human g d T-cell antigen
receptor. Nature 391:502.
14. O’Brien, R. L., M. P. Happ, A. Dallas, E. Palmer, R. Kubo, and W. K. Born.
1989. Stimulation of a major subset of lymphocytes expressing T cell receptor g
d by an antigen derived from Mycobacterium tuberculosis. Cell 57:667.
15. Holoshitz, J., F. Koning, J. E. Coligan, J. De Bruyn, and S. Strober. 1989. Isolation of CD42 CD82 mycobacteria-reactive T lymphocyte clones from rheumatoid arthritis synovial fluid. Nature 339:226.
16. Janis, E. M., S. H. Kaufmann, R. H. Schwartz, and D. M. Pardoll. 1989. Activation of g d T cells in the primary immune response to Mycobacterium tuberculosis. Science 244:713.
17. Haregewoin, A., G. Soman, R. C. Hom, and R. W. Finberg. 1989. Human g d2
T cells respond to mycobacterial heat-shock protein. Nature 340:309.
18. Fisch, P., M. Malkovsky, S. Kovats, E. Sturm, E. Braakman, B. S. Klein,
S. D. Voss, L. W. Morrissey, R. DeMars, W. J. Welch, et al. 1990. Recognition
by human V g 9/V d 2 T cells of a GroEL homolog on Daudi Burkitt’s lymphoma
cells. Science 250:1269.
19. Matis, L. A., A. M. Fry, R. Q. Cron, M. M. Cotterman, R. F. Dick, and
J. A. Bluestone. 1989. Structure and specificity of a class II MHC alloreactive g
d T cell receptor heterodimer. Science 245:746.
20. Matis, L. A., R. Cron, and J. A. Bluestone. 1987. Major histocompatibility complex-linked specificity of g d receptor-bearing T lymphocytes. Nature 330:262.
21. Bonneville, M., K. Ito, E. G. Krecko, S. Itohara, D. Kappes, I. Ishida,
O. Kanagawa, C. A. Janeway, D. B. Murphy, and S. Tonegawa. 1989. Recognition of a self major histocompatibility complex TL region product by g d T-cell
receptors. Proc. Natl. Acad. Sci. USA 86:5928.
22. Porcelli, S., M. B. Brenner, J. L. Greenstein, S. P. Balk, C. Terhorst, and
P. A. Bleicher. 1989. Recognition of cluster of differentiation 1 antigens by human CD4-CD8-cytolytic T lymphocytes. Nature 341:447.
23. Rust, C. J., F. Verreck, H. Vietor, and F. Koning. 1990. Specific recognition of
staphylococcal enterotoxin A by human T cells bearing receptors with the V g 9
region. Nature 346:572.
24. Mami-Chouaib, F., C. Miossec, P. Del Porto, C. Flament, F. Triebel, and
T. Hercend. 1990. T cell target 1 (TCT.1): a novel target molecule for human
non-major histocompatibility complex-restricted T lymphocytes. J. Exp. Med.
172:1071.
25. Del Porto, P., F. Mami-Chouaib, J. M. Bruneau, S. Jitsukawa, J. Dumas,
M. Harnois, and T. Hercend. 1991. TCT.1, a target molecule for g/d T cells, is
encoded by an immunoglobulin superfamily gene (Blast-1) located in the CD1
region of human chromosome 1. J. Exp. Med. 173:1339.
26. Johnson, R. M., D. W. Lancki, A. I. Sperling, R. F. Dick, P. G. Spear, F. W. Fitch,
and J. A. Bluestone. 1992. A murine CD42, CD82 T cell receptor-g d T lym-