JOURNAL OF VIROLOGY, May 1994, p. 2803-2810
Copyright © 1994, American Society for Microbiology
Vol. 68, No. 5
Antigenic Specificities of Human CD4+ T-Cell Clones
Recovered from Recurrent Genital Herpes Simplex
Virus Type 2 Lesions
DAVID M. KOELLE,l* LAWRENCE COREY,1 RAE LYN BURKE,2 ROSELYN J. EISENBERG,3
GARY H. COHEN,3 RATH PICHYANGKURA,4 AND STEVEN J. TRIEZENBERG4
Departments ofMedicine and Laboratory Medicine, University of Washington, Seattle, Washington 98195';
Chiron Corporation, Emeryville, Califomia 946082; University ofPennsylvania, Philadelphia,
Pennsylvania 191043; and Michigan State University, East Lansing, Michigan 488244
Received 22 November 1993/Accepted 24 January 1994
Lesions resulting from recurrent genital herpes simplex virus (HSV) infection are characterized by
infiltration ofCD4+ lymphocytes. We have investigated the antigenic specificity of47 HSV-specific CD4+ T-cell
clones recovered from the HSV-2 buttock and thigh lesions of five patients. Clones with proliferative responses
to recombinant truncated glycoprotein B (gB) or gD of HSV-2 or purified natural gC of HSV-2 comprised a
minority of the total number of HSV-specific clones isolated from lesions. The gC2- and gD2-specific CD4+
clones had cytotoxic activity. The approximate locations of the HSV-2 genes encoding HSV-2 type-specific
CD4+ antigens have been determined by using HSV-1 X HSV-2 intertypic recombinant virus and include the
approximate map regions 0.30 to 0.46, 0.59 to 0.67, 0.67 to 0.73, and 0.82 to 1.0 map units. The antigenic
specificity of an HLA DQ2-restricted, HSV-2 type-specific T-cell clone was mapped to amino acids 425 to 444
ofVP16 ofHSV-2 by sequential use of an intertypic recombinant virus containing VP16 ofHSV-2 in an HSV-1
background, recombinant VP16 fusion proteins, and synthetic peptides. Each of the remaining four patients
also yielded at least one type-specific T-cell clone reactive with an HSV-2 epitope mapping to approximately
0.67 to 0.73 map units. The antigenic specificities of lesion-derived CD4+ T-cell clones are quite diverse and
include at least 10 epitopes. Human T-cell clones reactive with gC and VP16 are reported here for the first time.
Recurrent herpes simplex virus (HSV) infection in humans
is a localized cutaneous disease in individuals with intact
cellular immunity. The roles of infiltrating cells in host defense
are incompletely understood. The cellular infiltrate is enriched
for CD4+ cells during early stages of lesion formation (13). In
murine models of HSV infection, adoptively transferred bulk
(28) or cloned (24) CD4+ T cells can protect against viral
challenge. Keratinocytes, the predominant infected cell type in
HSV skin lesions, are capable of presenting HSV antigen to
CD4+ T cells after gamma interferon treatment (12). Func-
tional roles of lesion-infiltrating CD4+ T cells in recurrent
HSV lesions may include lymphokine release, inhibition of
viral replication, cytotoxicity, and B-cell help (48-50).
The antigens recognized by human HSV-specific CD4+ T
cells have not been fully characterized. The membrane glyco-
proteins B, C, and D (gB, gC, and gD) stimulate proliferation
of T lymphocytes from peripheral blood mononuclear cells
(PBMC) of HSV-seropositive individuals (43, 52). Secondary
in vitro stimulation of PBMC with HSV antigen has been
required to expand and enrich HSV-specific T cells prior to
derivation of HSV-specific T-cell clones (51). A large propor-
tion of the T-cell clones isolated and maintained by periodic
restimulation with whole HSV antigen were reactive with gB or
gD (52), and a substantial proportion of these clones also had
cytotoxic T-lymphocyte (CTL) activity (48). It is not known
whether secondary in vitro stimulation with crude viral antigen
influences the relative proportions of resultant clones reactive
*Corresponding author. Mailing address: Pacific Medical Center,
Room 9301, 1200 12th Ave. S., Seattle, WA 98144. Phone: (206) 326-
4162 (laboratory); (206) 326-4177 (office). Fax: (206) 326-4178. Elec-
tronic mail address: email@example.com.
with specific HSV proteins or displaying CTL or other effector
CD4+ T cells recognize primarily exogenously synthesized
antigen after processing by appropriate antigen-processing
cells (7). Abundant virion proteins are thus candidates for
CD4+ T-cell antigens. Strong serum antibody responses to
viral glycoproteins and the abundant tegument protein VP16
are present in human HSV-1 and HSV-2 infections (1),
consistent with the presence of a CD4+ helper T-cell response.
However, the presence of a human CD4+ response to VP16
has not been previously demonstrated.
We have recently developed techniques for isolating HSV-
specific CD4+ T-cell clones from recurrent human HSV
lesions (23). We report here the identification of the antigens
recognized by a subset of these T-cell clones and the mapping
of additional HSV-2 type-specific CD4+ T-cell antigens with
the use of HSV-1 x HSV-2 intertypic recombinant viruses
MATERIALS AND METHODS
Viruses. HSV-2 strain 333 (22) and HSV-1 strain El 15 (40)
were used throughout unless otherwise specified. HSV-1 x
HSV-2 IRV RH1G7, RSlG25, RS1G31, and R7015 (31, 32)
were the kind
MP801-1, expressing the gene for gC of HSV-2, and HSV-2
strain GpSl, containing a deletion of gC, were the kind giftof
Patricia Spear (53). An IRV (designated RP-2) bearing the
VP16 gene from HSV-2 (strain HG52) in the HSV-1 (KOS)
genome was constructed by complementation of the VP16-
deleted virus 8MA (45). The HSV-2 fragment includes the
entire VP16 open reading frame with 30 bp of 5' flanking
sequence and 43 bp of 3' flanking sequence.
gift of Beruard Roizman. HSV-1
KOELLE ET AL.
Virus stocks were prepared by infecting HDF cells at a
multiplicity of infection of 0.001 to 0.01 or 1.0 (strain RP-2
only) and allowing the infection to progress until cytopathic
effect involved 80 to 100% of the cells. Scraped cells were
sonicated and then centrifuged at 400 x g for 10 min after
addition of 2 ml of minimal essential medium-10% fetal calf
serum per original 150-cm2 flask of HDF cells. Supernatant
was frozen in aliquots, and virus titers were determined by a
plaque assay on Vero cells (37). Titers ranged from 107 to 3 x
109 PFU/ml. Mock virus preparations were made from the
same batch of HDF. Recombinant vaccinia viruses containing
the genes for gB and gD of HSV-2 (42) and wild-type vaccinia
virus New York were grown on HDF and prepared in the same
fashion as HSV strains, and titers were determined by a plaque
assay on BHK-21 cells (37).
Cell lines. PBMC were prepared by Ficoll-Hypaque density
gradient centrifugation and cryopreserved for use as antigen-
presenting cells (APC). Epstein-Barr virus-transformed lym-
phoblastoid cell lines (LCL) were prepared from PBMC as
described previously (42) and maintained in RPMI-FC (RPMI
1640, 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid [HEPES], 2 mM L-glutamine, 50 ,ug of streptomycin per
ml, 50 U ofpenicillin per ml, 2 x 10-5 MP-mercaptoethanol,
1 mM pyruvate, 10% fetal calf serum). HDF cells (37) were
maintained in minimal essential medium-10% fetal calf serum.
Cells used for preparation of viral stocks and functional assays
were negative for mycoplasma (Mycotect; GIBCO, Grand
T-lymphocyte culture. Lymphocytes recovered from swabs
or biopsies of culture-positive HSV-2 lesions were cloned by
using a high efficiency procedure with phytohemagglutinin
(PHA) and interleukin 2 (IL-2) (27) as previously described
(23). Briefly, vesicle fluid and scraped cells from vesicle bases,
or cells recovered from 3-mm skin biopsies minced with
scissors and expressed through a 190-pLm-pore-size sieve (Cel-
lector; Bellco Glass, Inc., Vineland, N.J.), were underlaid with
anequal volume of Ficoll-Hypaque. Cells were recovered from
the interface after centrifugation at 400 x g for 30 min at room
temperature. These cells were 90 to 95% polymorphonuclear
leukocytes, with occasional eosinophils and monocytes, 1% or
less small round mononuclear cells, and 5 to 10% epithelial
cells with or without multiple nuclei or inclusions. The cells
wereplated at 10 to 200 cells per well in T-cell medium (RPMI
1640 containing 25 mM HEPES, 2 mM L-glutamine, 50 ,ug of
streptomycin per ml, 50 U ofpenicillin per ml, and 10% human
blood type AB' serum) and stimulated with PHA, allogeneic
irradiated PBMC as feeder cells, and 20 to 50 U of IL-2
(Pharmacia, Piscataway, N.J.) per ml. Acyclovir (50 ,uM) was
included for the first 2 weeks to inhibit HSV replication.
Between 7.9 and 48.7% of wells were positive for growth by
microscopic examination. Clones were periodically restimu-
lated with PHA, allogeneic feeder cells, and IL-2 (42). Sub-
clones were derived at one cell per well (27).
In addition, cells (4.2 x 105) recovered from swabs from
patient 1, lesion A, were stimulated in bulk with 106 allogeneic
irradiated (3,300 rads of gamma irradiation) PBMC and 0.4 ,ug
of PHA-P (Murex Diagnostics Ltd., Dartford, England) per ml
in 2 ml of T-cell medium plus 50 ,uM acyclovir in a 1.88-cm2
well. After 48 h, 1 ml of medium was exchanged for medium
containing 50 U of IL-2 per ml. Cells were split as necessary
and fed with T-cell medium containing 50 U of IL-2 per ml.
Clones were derived after 19 days of growth by plating the cells
at one cell per well with allogeneic irradiated PBMC, PHA,
and IL-2 (27).
Proliferation assays. Screening assays for lesion-derived
T-cell clones were performed in duplicate as described previ-
ously (23). Cloned T cells or bulk lesion-derived cells (104 per
well) and antigen were added to a final volume of 200 pl of
T-cell medium in 96-well U-bottomplates.Autologous gam-
ma-irradiated (3,300 rads) PBMC (10 per well) were included
as APC. After 3 days, [3H]thymidine (1 jiCi per well) was
added for 18 h; cells were harvested with a semiautomated
harvester and counted by liquid scintillation. All subsequent
assays were performed in triplicate. Clones were judged to be
specific for HSV strains or purified antigens if the stimulation
index (cpm of [3H]thymidine incorporation for HSV strain or
antigen/cpm of [3H]thymidine incorporation for mock HDF
antigen) was greater than 4.0. Stimulation indices are reported
with two significant figures to reflect the precision of the
experimental measurements. For experiments with allogeneic
APC, proliferation in response to both mock and HSV anti-
gens was measured with allogeneic irradiated PBMC, and
stimulation indices were calculated from these two measure-
Antibody blocking of proliferation. Monoclonal antibodies
(MAbs) L243 (38), B7/21 (35), and SPV-L3 (39) recognize
HLA DR, DP, and DQ framework determinants, respectively.
Supernatant of L243 cells, obtained from the American Type
Culture Collection, was used at 1:4 (final dilution). Purified
B7/21, obtained from Tom Cotner, was used at 1:400 (final
dilution). Purified SPV-L3, obtained from Hans Yssel, was
used at 10 jig/ml (final concentration). These concentrations
were determined in preliminary titration experiments to inhibit
proliferation of CD4+ T-cell clones restricted by the relevant
HLA class II loci by greater than 80% and to inhibit prolifer-
ation of T-cell clones restricted at irrelevant HLA class II loci
by less than 15%.
Antigens. Antigens were prepared from viral stocks by
exposure to UV light for 30 min at 10 cm from a new GT038
bulb, eliminating infectious virus. Antigens were routinely used
in lymphoproliferation assays at 1:100 (final dilution), corre-
sponding to 105 to 107 PFU/ml prior to UV treatment.
Recombinant gB and gD of HSV-2 were purified from trans-
fected Chinese hamster ovary cells (8). gB2 is truncated at
amino acid 696, and gD2 is truncated at amino acid 302.
Recombinant glycoproteins were used at 2 jig/ml (final con-
centration) (52). gC of HSV-2 was obtained by affinity chro-
matography of infected cells (43) and used at 2 jig/ml (final
concentration). Fusion proteins containing the DNA-binding
domain of the yeast protein GAL4 (amino acids 1 to 147) and
the activation domains of VP16 (amino acids 413 to 490 or 402
to 490 for HSV-1 ) and HSV-2, respectively, were purified
from Escherichia coli to greater than 95% homogeneity by
(37a). Peptides corresponding to amino acids 400 to 490 of
VP16 of HSV-2, 15 amino acids long and overlapping by 10
amino acids (Chiron Mimotopes, Clayton, Australia), were
dissolved in 20% acetonitrile-0.1 M acetic acid at 2.0 mg/ml
and used at 10 jig/ml (final concentration).
Cytotoxicity assays. Target LCL were infected for 18 h with
HSV or vaccinia viruses at a multiplicity of infection of 5,
loaded with 5'Cr, and washed as previously described (42).
Cloned lymphocytes were plated in 100[lIof RPMI-FC in
96-well U-bottom plates, and targets (5 x 103 per well) added
in 100 ,ul of RPMI-FC. After 4 h at 37°C, the plates were
centrifuged at 50 x g for 2 min, and 100 ,u of supernatant was
counted with a gamma counter. Allogeneic target cells were
mismatched at HLA class I and II loci. Results are reported as
percent specific release, determined as [(mean experimental
cpm - mean spontaneous cpm)/(mean maximal cpm - mean
spontaneous cpm)] x 100. Spontaneous release was always
less than 20% of total release.
HUMAN LESIONAL HSV-SPECIFIC CD4+ T CELLS
TABLE 1. HSV-specific CD4+ T-cell clones derived directly from
HSV-2 lesions and available for antigenic specificity analysis
available for detailed study
aStimulation index for HSV-2 antigen greater than 4.0.
bStimulation indices for HSV-1 and HSV-2 antigens greater than 4.0.
cIncludes five HSV-2 type-specific CD4+ T-cell clones derived from bulk
culture of lesion-derived T lymphocytes (see text).
Flow cytometry. T-cell clones were characterized for cell
surface expression of CD3, CD4, and CD8
Serology. Type-specific serologies for HSV-1 and HSV-2
were performed by immunoblotting (2).
Cloning of lesion CD4+ HSV-specific T cells. In this report,
we describe the antigenic specificities of 47 lesion-derived
HSV-specific CD4+ T-cell clones from five patients (Table 1),
representing all of the HSV-specific clones available in suffi-
cient number for detailed study. A total of 42 HSV-specific
CD4+ T-cell clones obtained directly from swabs or biopsies of
recurrent HSV-2 buttock or thigh lesions were available in
sufficient number for detailed studies. Details of the cloning
procedure and reactivity with HSV-1 and HSV-2 antigens have
been previously reported for patient 1, lesion A, and patients 2,
3, and 4 (23). In addition, five HSV-2 type-specific CD4+ T-cell
clones, designated 1A.Bx, were obtained by cloning bulk
lesion-derived cells from patient 1, lesion A, after 19 days of
bulk culture. All clones were CD3+, CD4+, and CD8- by flow
cytometry. Overall, 26 of 47 (55%) of the lesion-derived
HSV-specific T-cell clones recognized HSV-2 type-specific
gB- and gD-specific CD4+ T-cell clones. The HSV-specific
CD4+ T-cell clones were each tested for reactivity with recom-
binant truncated gB and gD of HSV-2. Three clones from
three different patients had stimulation indices of greater than
5.0 with gB2 (3B.268, 4.4D3, and 5.5), and one clone had a
stimulation index of greater than 5.0 with gD2 (3B.134) (Table
2, experiment 1). Proliferation of clone 3B.134 was inhibited by
Effector to Target Ratio
FIG. 1. Cytotoxic activity of lesion CD4+ T-cell clone 3B.134
against HSV-2- and vaccinia virus-infected and uninfected autologous
(Auto) and allogeneic (Allo) LCL. Allogeneic LCL were mismatched
at HLA class I and II loci.
greater than 90% by anti-HLA DP MAb but not by anti-HLA
DR MAb (data not shown). The gD2-specific clone 3B.134
also lysed vaccinia virus-gD2- and HSV-2-infected autologous
target cells, but not uninfected or wild-type vaccinia virus-
infected autologous cells or HSV-2-infected allogeneic cells, in
a cytotoxicity assay (Fig. 1). The gB2-specific clones did not
have cytolytic activity against HSV-2- or vaccinia virus-gB2-
infected target cells (data not shown). All four clones reactive
with gB2 or gD2 recognized type-common determinants (Ta-
ble 2, experiment 2). Subclones of each of the gB- and
gD-reactive clones obtained at one cell per well retained the
same pattern of reactivity with purified recombinant glycopro-
teins, HSV-1 antigens, and HSV-2 antigens (data not shown).
The remaining T-cell clones all had stimulation indices with
gB2 and gD2 of less than 3.0 (data not shown).
Epitope mapping using IRV. Fourteen of the 26 (54%)
HSV-2 type-specific clones displayed stimulation indices of
greater than 4.0 to at least one viral antigen prepared from a
panel of IRV (Table 3). Twelve HSV-2 type-specific T-cell
clones did not proliferate in response to antigen prepared from
any of the IRV. Clone 4.3C12 is shown as an example (Table
3). All IRV elicited specific proliferative responses from bulk
PBMC of HSV-seropositive individuals, demonstrating their
antigenicity. As expected, T-cell clones reactive with type-
common epitopes reacted with all IRV. Clone 1A.2 is shown as
an example (Table 3). Data for the 20 additional type-common
and 11 additional unmappable HSV-2 type-specific T-cell
clones are not included in Table 3. The T-cell clones all
displayed identical patterns of reactivity in at least two assays.
Type-specific T-cell clones recognized diverse HSV-2 anti-
gens. Three HSV-2 type-specific clones (1A.1, 1B.9F8, and 5.4)
TABLE 2. Lesion-derived CD4+ clones specific for gB2 and gD2
Mean [3H] incorporation (SD)'
aDetermined from triplicate samples. Glycoprotein-specific reactivityis indicated in boldface.
bUsed at 2 ,ug/ml (final concentration).
VOL. 68, 1994
KOELLE ET AL.
TABLE 3. Proliferative responses of lesion-derived, HSV-2 type-specific CD4+ T-cell clones to antigens prepared from HSV-1, HSV-2, and
HSV-1 x HSV-2 IRV
HSV-2 type-specific clones
aData defining specificity for an HSV-2 map region are indicated here and in Tables 4 and 5 in boldface. ND, not done.
bApproximate HSV-2 map region.
c Bulk nonirradiated PBMC (105) from an HSV-1- and HSV-2-seropositive donor were cultured with viral antigen at 1:100 (final dilution) and labeled with
[3Hlthymidine from days 5 to 6.
dRepresentative clone recognizing a type-common epitope. Data for remaining type-common clones are not shown but are similar.
eRepresentative clone recognizing an HSV-2 type-specific epitope not included ip the panel of IRV. Data for additional clones unreactive with the panel of IRV
are not shown but are similar.
reacted with IRV RH1G7 (10, 32), corresponding to approx-
imately 0.30 to 0.46 map units within the HSV-2 genome.
Clones 1A.1 and 1B.9F8 were recovered from lesions from the
One HSV-2type-specificclone(5.19)reactive with IRV R7015
(32), corresponding to approximately 0.82 to 1.0 map units
within the HSV-2genome,was isolated frompatient5. A total
of 10 HSV-2 type-specific clones reacted with IRV RS1G25
(31,32), correspondingtoapproximately0.59 to 0.73mapunits
within the HISV-2genome.Of these 10clones,8(1A.B.25, 2.3,
3B.294, 4.1A11, 4.2E1, 4.2F10, 4.3C3, and 5.1) also reacted
with IRV RS1G31 (31), indicating specificity for an epitope
encodedbyHSV-2 betweenapproximately0.67 and 0.73 map
units. Alleightclones reactive with RS1G31 were also reactive
with RS1G25. Two clones (2.1 and 3B.230) reacted with
antigen prepared from RS1G25 but not RS1G31, indicating
specificityfor anantigenencodedby HSV-2 between approx-
imately0.59 and 0.67map units.
gC-specific CD4+ T-cell clone. As the gene for gC2 is
located atapproximately 0.64 map units (41), the two clones
reactive with RS1G25 but not RS1G31 (2.1 and 3B.230) were
evaluated for proliferative responses to affinity-purified gC2
and special HSV strains (Table 4). Clone 3B.230 had a specific
proliferative response to gC2. This clone also proliferated in
response to the recombinant HSV-1 strain MP801-1, express-
ing the HSV-2 gC gene, and failed to proliferate in response to
the HSV-2 strainGpS1,which does not express gC2. Subclones
of 3B.230 obtained at one cell per well displayed an identical
pattern of reactivity (data not shown). Clone 3B.230 was also
reactive in cytotoxicity assays with RS1G25 and MP801-1 but
not RS1G31 orGpS1 (Fig. 2), the same pattern of reactivity
obtained in proliferation assays. In contrast, clone 2.1 did not
proliferate in response to MP801-1 or purified gC2 and did
react with the GpS1, indicating reactivity with a HSV-2 type-
specific epitope not contained within gC2.
CD4+ T-cell clones with specificity for VP16 and other
antigens mapping to RS1G31. Eight lesion-derived CD4+
T-cell clones, including at least one from each patient, reacted
with an HSV-2 epitope contained within IRV RSiG31 and
thus mapping to approximately 0.67 to 0.73 map units (Table
3). One of these, clone 1A.B.25.1, proliferated in response to
the IRV RP-2 (Table 5), which contains only the HSV-2 gene
TABLE 4. Proliferativeresponseof lesion-derivedCD4+clones 2.1 and 3B.230 topurified gC2 and antigens prepared from various
aHSV-1 straincontainingthegeneforgCof HSV-2.
"HSV-2 strain which does notexpress gC.
cUsed at 2
HUMAN LESIONAL HSV-SPECIFIC CD4+ T CELLS
IoSpecific release at E:T ratio 10:1
FIG. 2. Cytotoxic activity of gC2-specific CD4+ T-cell clone 3B.230
against infected and uninfected autologous (Auto) and allogeneic
(Allo) LCL. HSV-1 x HSV-2 IRV RS1G25 contains HSV-2 DNA
from approximately 0.59 to 0.73 map units within an HSV-1 back-
ground. MP801-1 is an HSV-1 strain containing the gene for gC of
HSV-2. GpSI is an HSV-2 strain which does not express gC. Alloge-
neic LCL were mismatched at HLA class I and II loci. Data are
percent specific release at an effector-to-target (E:T) ratio of 10:1.
for VP16 within an HSV-1 background. Clone 1A.B.25.1 is a
subclone of 1A.B.25 (Table 3). Clones 2.3, 3B.294, 4.1A11,
4.2E1, and 4.2F10 did not react with RP-2 (Table 5). Clones
4.3C3 and 5.5 were not available for evaluation with RP-2
Allogeneic PBMC from patient 5, sharing HLA DR3 and
DQ2 with patient
1A.B.25.1 (Table 6). Alloreactivity against the patient 5 PBMC
was not present, as incorporation of [3H]thymidine in response
to these allogeneic PBMC and mock antigen was less than 500
cpm (data not shown). Proliferation of clone 1A.B.25.1 to
autologous PBMC and HSV-2 antigen was inhibited more than
90% by anti-HLA DQ MAb SPV-L3 but not inhibited by
anti-HLA DR or anti-HLA DP MAbs (Table 6), identifying
the restriction element as DQ2. A fusion protein bearing the
activation domain of VP16 of HSV-1 or HSV-2 linked to the
DNA-binding domain of the yeast GAL4 protein was purified
after expression in E. coli. Dose-dependent proliferative re-
sponses were observed at HSV-2 fusion protein concentrations
of between 0.5 and 8 jig/ml (Fig. 3). No proliferation was
elicited by the HSV-1 fusion protein. Peptides containing
amino acids 425 to 439 (LRLDGEEVDMTPADA) and 430 to
444 (EEVDMTPADALDDFD) of VP16 of HSV-2 (11) both
induced specific proliferative responses (Fig. 3).
1, presented HSV-2 antigen to clone
TABLE 5. Proliferative responses of type-specific lesion-derived
CD4+ clones recognizing an HSV-2 antigen mapping to RS1G31
(approximately 0.67 to 0.73 map units) to RP-2 antigen
aAntigens used at 1:100 (final dilution) except for clones 4.1A11 and 4.2E1
(1:30 [final dilution]).
TABLE 6. Lesion-derived CD4+ T-cell clone 1A.B.25 is restricted
by HLA DQ2
aAutologous PBMC are HLA DR3,11, DQ2, 7; allogeneic PBMC are HLA
Limited information concerning the antigenic specificity of
human HSV-specific T lymphocytes is available. Secondary in
vitro restimulation of PBMC with HSV antigen prior to
cloning has previously been necessary to obtain CD4+ HSV-
specific T-cell clones (52). It is not known to what extent the
antigenic specificities and effector functions of clones obtained
with this method reflect the properties of T lymphocytes that
have migrated to, and been activated within, recurrent HSV
lesions. CD4+ T-cell clones obtained in this manner have been
primarily specific for gB and gD (52). We have used the in situ
enrichment of HSV-specific T cells in recurrent HSV-2 lesions
(23) to obtain T cells for cloning without the requirement for
in vitro restimulation with HSV antigen.
The T-cell cultures used in this study were believed to be
clonal. It was necessary to initially plate the lesion cells at
greater than one cell per well because of the low purity of
lymphocytes (<1%) in the lesion cell preparations. Since the
maximum percentage of wells positive for growth was 48.7%
(23), the average number of lymphocytes plated per well is
estimated to be less than one (20, 23). Since approximately
10% of all lesion-derived T-cell cultures were HSV specific
(23), it is very unlikely that more than one HSV-specific T-cell
clone would be represented within an individual culture of
lesion lymphocytes. All of the cultures tested were >97%
CD4+ and <3% CD8+. Multiple subclones were evaluated for
HSV-2 no APC
HSV-2 allo APC
VP16(l) 413-490 8 ug/ml
VP16(2) 402-490 8 ug/ml
FIG. 3. Proliferative responses of lesion CD4+ clone 1A.B.25.1 to
HSV antigens, purified recombinant GAL4-VP16 fusion proteins
containing amino acids 1 to 147 ofyeast GAL4 linked to amino acids
413 to 490 of VP16 (HSV-1) or amino acids 402 to 490 of VP16
(HSV-2), and peptides from VP16 of HSV-2. Allogeneic (allo)PBMC
were mismatched at HLA class I and II loci.
VOL. 68, 1994
KOELLE ET AL.
clones 1A.B.25, 2.3, 3B.134, 3B.230, 3B.268, 4.2E1, and 4.2F10,
and all were found to retain specific proliferative responses to
HSV-2 antigen (23). The antigenic specificity of subclones, as
determined by IRV mapping or reactivity with HSV glycopro-
teins, has been identical with that of the parent culture in all of
12 clones tested (data not shown). In addition, no cases of
reactivity of HSV-2 type-specific T-cell clones with more than
one nonoverlapping IRV were noted (Table 3).
The lesion CD4+ response appears to be directed against a
variety of viral epitopes. Using lesion-derived HSV-specific
CD4+ T-cell clones from five individuals, we identified at least
10 different epitopes: 1 to 3, epitopes contained within gB, gC,
and gD; 4, a type-specific HSV-2 epitope contained within
amino acids 425 to 444 of VP16; 5, a type-specific HSV-2
epitope or epitopes mapping to approximately 0.30 to 0.46 map
units; 6, a type-specific HSV-2 epitope or epitopes mapping to
approximately 0.59 to 0.67 map units but not gC; 7, a type-
specific HSV-2 epitope (or epitopes) mapping to approxi-
mately 0.67 to 0.73 map units but not VP16; 8, a type-specific
HSV-2 epitope (or epitopes) mapping to approximately 0.82 to
1.0 map units; 9, a type-specific HSV-2 epitope (or epitopes)
not mappable with the current panel of IRV; and 10, a
type-common epitope (or epitopes)
present in recombinant soluble gB and gD.
It is of interest that each of the five patients studied yielded
at least one lesion-derived HSV-2 type-specific CD4+ T-cell
clone reactive with a HSV-2 epitope present within IRV
RS1G31 and thus mapping to the approximate region of 0.67
to 0.73 map units. The maximum left-hand border of HSV-2
sequences contained within IRV RS1G31 is defined by the
presence of the HSV-1 Kpnl-A to -Y' cleavage site at nucle-
otide 103101 (31), within the HSV-1 UL47 coding sequence
(26). The minimum left-hand border of HSV-2 sequences in
RS1G31 has been mapped in the present experiments to be
within or 5' of the DNA encoding amino acids 425 to 444 of
VP16 (UL48) of HSV-2. The minimum right border of the
HSV-2 sequences within RS1G31 is defined by the presence of
the HSV-2EcoRI-L to -H cleavage site (31); this site cannot be
precisely placed with published sequence information. The
maximal right-hand border of HSV-2 sequences within IRV
RS1G31 is defined by the absence of the HSV-2 BglII-I to -H
cleavage site (31). The crossover point must lie to the left of
the 5' end of the HSV-2 UL53 gene, since the HSV-2 BglII-I to
-H cleavage site is not present in the UL53 coding sequence
(15) or the remaining rightward region of UL of HSV-2 (25,
34). IRV RSIG31 may, therefore, contain HSV-2 coding
sequences for some of UL47 of HSV-2, but the HSV-2
sequences do not extend into the coding region of UL53 of
Of the total of eight HSV-2 type-specific clones mapping to
IRV RS1G31, only one of six available for testing reacted with
IRV RP-2 and was thus specific for the UL48 gene product
VP16. The 0.67-0.73 region of the HSV-2 genome contains, in
addition to VP16, the genes for the abundant tegument
proteins UL47 and UL49 and the genes for the probable
membrane glycoprotein UL49.5 or UL49A (4, 5, 16, 54). The
UL50 and UL52 protein products are perhaps less likely
candidates for CD4+ T-cell antigens, since they are present in
small amounts in infected cells (30, 47) and have not been
identified in virions. Little is known about the UL51 gene
product of HSV (3). Study of additional HSV-2 lesions and
finer definition of the T-cell epitope(s) will allow exploration of
the possibility that an immunodominant HSV-2 type-specific
CD4+ antigen may be encoded by a gene in this portion of the
The lesion-derived clone 1A.B.25
in addition to those
is the first published
CD4+ T-cell clone reactive with VP16. VP16 is an abundant
virion tegument protein, present as 400 to 2,000 molecules per
virion (6, 29). Our results are consistent with the hypothesis
that preformed virion proteins, rather than endogenously
synthesized viral proteins, would be expected to recognized by
the CD4+ T-cell response (7). The predicted amino acid
sequences of VP16 of HSV-1 and HSV-2 are 86% identical,
with maximal divergence in the carboxy-terminal activation
subdomain (11). Type-specific T-cell clone 1A.B.25
stricted by HLA DQ2, as determined from data from partially
HLA matched allogeneic APC and inhibition of proliferative
responses to autologous APC by various anti-HLA class II
MAbs (Table 6). Inhibition of the proliferative response to
allogeneic, HLA DQ2-bearing APC plus antigen by anti-HLA
DQ MAb would also be expected; this confirmatory experi-
ment and identification of further HLA restriction elements of
additional T-cell clones are under way. The naturally processed
epitope of VP16 of HSV-2 most likely includes the overlap
region, amino acids 430 to 439, shared by the two antigenic
peptides 425 to 439 and 430 to 444 (Fig. 3). This overlap region
(EEVDMTPADA) shares negatively charged amino acids at
positions 1 and 9 and alanine at position 8 with positions 2, 9,
and 10 of the HLA DQ2-restricted peptide IDVWLGGLAE
NSLP (human thyroid peroxidase positions 632 to 645 [14,
A low proportion (5 of 47 [11%]) of lesion-derived CD4+
T-cell clones were specific for the abundant membrane glyco-
protein gB, gC, or gD. Several factors may have limited
detection of T-cell clones with these specificities. First, the
recombinant gB2 and gD2 molecules used in this study have
C-terminal deletions; the reagents include 79 and 82%, respec-
tively, of the predicted amino acid sequences of the native
proteins after cleavage of N-terminal signal sequences (44, 55).
T-cell clones reactive with whole HSV antigens would be
scored as negative for gB2 or gD2 if they were specific for
epitopes not included in the recombinant soluble glycopro-
teins. Second, only HSV-2 type-specific T-cell clones reacting
with RS1G25 but not RS1G31 antigens were screened for gC2
reactivity; type-common, gC-specific T-cell clones would be
missed by this method. However, the 31% divergence between
the amino acid sequences of gCl and gC2 (41) makes it likely
that most T-cell epitopes of HSV gC will be type specific.
Third, the amount ofglycoprotein antigen used in proliferation
assays may have been suboptimal for some T-cell clones,
although a 2-,ug/ml concentration of HSV gB, gC, and gD has
previously been shown to be optimal for eliciting proliferative
responses from bulk PBMC and PBMC-derived CD4+ T-cell
clones (43, 52). Even allowing for these potential limiting
factors, however, there is still a marked contrast between the
previously reported finding that most PBMC-derived HSV-
specific CD4+ T-cell clones are specific for gB or gD (52) and
the results of the present study with lesion-derived T-cell
The large size of the HSV genome (33) and the diverse
peptides bound to various human HLA class II molecules (9)
lead to the prediction that a large number of human CD4+
epitopes are present with HSV-2. Considerable diversity was
detected among the antigens recognized by CD4+ T-cell clones
recovered from individual patients. For example, a minimum
of six different specificities were detected for patient 5: gB2, at
least one HSV-2 type-specific epitope encoded by HSV-2
DNA present within each of the three IRV (RH1G7, RS1G31,
and R7015), at least one type-common epitope in addition to
gB2, and at least one HSV-2 type-specific epitope encoded by
HSV-2 gene sequence(s) not contained within the panel of
IRV. The overall detection of at least 10 T-cell epitopes
HUMAN LESIONAL HSV-SPECIFIC CD4+ T CELLS
recognized by lesion-infiltrating HSV-specific CD4+ T cells
provides a minimum estimate of the diversity of viral epitopes
because of the small number of patients studied, the limited
availability of purified viral proteins, and, for HSV-2 type-
specific T-cell clones, the incomplete coverage and crude
mapping of the HSV-2 genome provided by the current panel
of IRV. We plan to combine epitope mapping with additional
IRV and determination of individual clonal HILA restriction
elements with consensus motifs for HLA class II-binding
peptides (9) and HSV sequence data to define epitopes at the
The biological roles of lesion-infiltrating antigen-specific
CD4+ T cells in the resolution of recurrent HSV-2 lesions are
not known. Patients with deficiencies of CD4+ cell function,
such as advanced human immunodeficiency virus infection,
have delayed and incomplete resolution of recurrent HSV-2
infection (46). In addition to the gC- and gD-specific clones
described above, only one additional HLA-restricted, HSV-
specific CD4+ T-cell clone with CTL activity was detected
among the 47 T-cell clones described in this study (3/47 = 6%).
In contrast, 30 to 80%, depending on the donor, of PBMC-
derived HSV-specific CD4+ T-cell clones obtained with sec-
ondary in vitro stimulation have had CTL activity (52). The
apparent difference may be related to culture conditions, as
progressive increases in apparent CTL activity with in vitro
restimulation with antigen have been described for human
CD4+ clones specific for other viruses (17). Additional poten-
tial effector functions include secretion of lymphokines with
antiviral or immunomodulatory activity, including gamma in-
terferon (18, 19), and B-cell helper activity (49). Studies of
lymphokines produced in situ in HSV lesions and in vitro by
HSV-specific CD4+ T cells are under way.
In summary, CD4+ T cells present within human recurrent
HSV-2 lesions recognize a diverse set of type-common and
HSV-2 type-specific antigens. T-cell clones specific for gB and
gD have been detected, extending observations with PBMC-
derived T-lymphocyte clones. Human T-cell clones specific for
gC and the virion tegument protein VP16 have been detected
for the first time, and at least five additional HSV-2 type-
specific antigens have been defined by using patterns of
reactivity with HSV-1 x HSV-2 IRV. In one patient for which
two recurrent HSV-2 lesions have been studied, a T-cell clone
reactive with a HSV-2 type-specific antigen mapping to the
same region of the HSV-2 genome (approximately 0.30 to 0.46
map units) was recovered from both lesions. Study of addi-
tional lesions and genetic analysis of the T-cell receptor genes
of T-cell clones derived from sequential HSV lesions may
determine whether clonotypic T cells participate in the host
reaction to serial HSV recurrences within an individual pa-
tient. Continued work on the diversity and antigenic specificity
of the human CD4+ response to HSV may further our
understanding of the processes by which the host controls
human recurrent herpes simplex infection.
Specimens were collected by Gail Barnum, Michael Remington,
Peter Tretheway, and Mary Shaughnessy. Invaluable technical assis-
tance was given by Annette Peck and Hiyam Abbo. HLAtypingwas
performed byBrendaNisperos and John Hansen at the Fred Hutchin-
son Cancer Research Center, Seattle, Wash. Bernard Roizman and
Patricia Spear, University of Chicago, provided recombinant HSV
strains. David B. Lewis and Tom Cotner, University ofWashington,
Seattle, provided MAbs and helpful advice.
This work was supported by National Institutes of Health grants
A131448 and A134616 (D.M.K.),A120381 (L.C.),and AI27323 (S.J.T.)
and by a Junior Faculty Research Award from the American Cancer
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