JOURNAL OF VIROLOGY, Apr. 2005, p. 4896–4907
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 8
CD4?T-Cell Responses to Epstein-Barr Virus (EBV) Latent-Cycle
Antigens and the Recognition of EBV-Transformed
Lymphoblastoid Cell Lines
H. M. Long,1T. A. Haigh,1N. H. Gudgeon,1A. M. Leen,1C.-W. Tsang,1J. Brooks,1E. Landais,2
E. Houssaint,2S. P. Lee,1A. B. Rickinson,1* and G. S. Taylor1
CRUK Institute for Cancer Studies, University of Birmingham, Birmingham, United Kingdom,1and INSERM U463,
Institut de Biologie, Nantes, France2
Received 20 August 2004/Accepted 23 November 2004
There is considerable interest in the potential of Epstein-Barr virus (EBV) latent antigen-specific CD4?T
cells to act as direct effectors controlling EBV-induced B lymphoproliferations. Such activity would require
direct CD4?T-cell recognition of latently infected cells through epitopes derived from endogenously expressed
viral proteins and presented on the target cell surface in association with HLA class II molecules. It is therefore
important to know how often these conditions are met. Here we provide CD4?epitope maps for four EBV
nuclear antigens, EBNA1, -2, -3A, and -3C, and establish CD4?T-cell clones against 12 representative
epitopes. For each epitope we identify the relevant HLA class II restricting allele and determine the efficiency
with which epitope-specific effectors recognize the autologous EBV-transformed B-lymphoblastoid cell line
(LCL). The level of recognition measured by gamma interferon release was consistent among clones to the
same epitope but varied between epitopes, with values ranging from 0 to 35% of the maximum seen against the
epitope peptide-loaded LCL. These epitope-specific differences, also apparent in short-term cytotoxicity and
longer-term outgrowth assays on LCL targets, did not relate to the identity of the source antigen and could not
be explained by the different functional avidities of the CD4?clones; rather, they appeared to reflect different
levels of epitope display at the LCL surface. Thus, while CD4?T-cell responses are detectable against many
epitopes in EBV latent proteins, only a minority of these responses are likely to have therapeutic potential as
effectors directly recognizing latently infected target cells.
Epstein-Barr virus (EBV), a herpesvirus with B-cell growth
transforming ability and lymphomagenic potential, provides
one of the most instructive systems in which to study T-cell
responses to viral infection in humans (11, 25). Primary infec-
tion is usually asymptomatic but in some individuals can
present as infectious mononucleosis, a self-limiting lympho-
proliferative disease where the symptoms are coincident with
the appearance of a large reactive T-cell response. Following
primary infection, the virus is carried for life as a latent infec-
tion of the circulating memory B-cell pool (1), with low-level
reactivation from latency into virus productive (lytic) infection
at oropharyngeal sites. Immune T-cell responses clearly play
some role in the maintenance of this virus-host balance since
T-cell-immunocompromised individuals show increased virus
replication in the oropharynx (4) and an increased risk of
EBV-driven B-lymphoproliferative disease (22). One of the
outstanding issues to resolve in this respect is the relative
contribution made by CD4?and CD8?T-cell responses to this
The HLA class I-restricted CD8 response has attracted the
most attention, for two reasons. Firstly, primary T-cell re-
sponses seen in the blood of infectious mononucleosis patients
are largely of CD8?T-cell origin; indeed, many of these in
vivo-primed reactivities have been mapped to epitopes drawn
from EBV lytic- and latent-cycle antigens (28, 29). Secondly,
memory T-cell responses reactivated from immune donors by
in vitro stimulation with the virus-transformed B-lymphoblas-
toid cell line (LCL) are likewise dominated by CD8?effectors.
Many such effectors recognize epitopes drawn from the latent-
cycle proteins that are expressed in all LCLs, namely, nuclear
antigens EBNA1, -2, -3A, -3B, -3C, and -LP and latent mem-
brane proteins LMP1 and -2, with responses to EBNA3A-,
-3B-, and -3C-derived epitopes frequently in the majority (8,
18). Such CD8 responses to EBV latent antigens are of par-
ticular interest because of their ability to recognize and kill
virus-transformed B cells in vitro and their therapeutic poten-
tial against EBV-driven B-lymphoproliferative disease in vivo
The growing interest in HLA class II-restricted CD4?T-cell
responses to the virus reflects not only an appreciation of the
general role that CD4?T cells are thought to play in the
maintenance of effective CD8 immunity (7, 26, 30) but also the
fact that EBV infects target cells in which the HLA class II
pathway of antigen presentation is active (33). This raises the
possibility that virus-specific CD4?T cells are able to recog-
nize infected cells directly and, if they are, could act (like
CD8?T cells) as effectors in their own right. Certainly there
are examples where indicator antigens have been expressed
endogenously within LCLs and appear to have gained direct
intracellular entry into the HLA class II processing pathway (2,
20, 23, 34), in some way bypassing the usual means of HLA
class II presentation involving uptake as exogenously acquired
antigen (33). The first CD4?T-cell clones to EBV latent pro-
* Corresponding author. Mailing address: CR-UK Institute for Can-
cer Studies, The University of Birmingham, Vincent Dr., Edgbaston,
Birmingham B15 2TT, United Kingdom. Phone: 44 121 414 4492. Fax:
44 121 414 4486. E-mail: A.B.Rickinson@bham.ac.uk.
teins, specific for an EBNA1-derived and an EBNA2-derived
epitope, respectively, were identified as rare components of
LCL-reactivated memory T-cell preparations (9, 10). Of these,
only the EBNA2-specific clone appeared to be capable of rec-
ognizing LCLs directly in cytotoxicity assays (10). Since that
time, CD4?recall responses to more latent-cycle epitopes
have been generated by a variety of protocols, many involving
in vitro stimulation with dendritic cells either overexpressing
antigen from vaccinia virus vectors or preloaded with exoge-
nous antigen or with epitope peptides (13, 17, 21, 27, 32).
Although these clones display specificity for antigen in protein
loading assays, there are differing reports of their ability to
recognize LCLs naturally expressing cognate antigen from the
resident EBV genome. It is not clear whether these divergent
results reflect technical differences in the way in which the
clones are generated in vitro or genuine differences in the way
in which either individual antigens or individual epitopes are
processed and presented. The present study (i) provides de-
tailed CD4 epitope maps for four EBV latent-cycle antigens,
EBNA1, -2, -3A, and -3C; (ii) establishes CD4?T-cell clones
to 12 selected epitopes from these antigens; and (iii) charac-
terizes these clones in terms of their HLA class II restriction,
their functional avidity in peptide titration assays, and their
ability to recognize autologous LCL targets in gamma inter-
feron (IFN-?) release, cytotoxicity, and outgrowth assays.
MATERIALS AND METHODS
Cell preparations and cell lines. Peripheral blood mononuclear cells (PBMCs)
were separated from healthy, EBV-immune donors by Ficoll-Hypaque centrifu-
gation into RPMI 1640 medium (Invitrogen) supplemented with 2 mM glu-
tamine, 100 IU of penicillin per ml, 100 ?g of streptomycin per ml, and 5%
autologous serum. Where specified, PBMCs were depleted of CD8?T cells with
CD8 Dynabeads (Dynal) in accordance with the manufacturer’s recommended
protocol. Dendritic cells were prepared as previously described (13), by 6-day
culture of adherent PBMCs in the above medium supplemented with granulo-
cyte-macrophage colony-stimulating factor and interleukin-4, and then matured
for 24 h in 50 ng of tumor necrosis factor alpha per ml. EBV-transformed LCLs
were prepared with prototype 1 strain B95.8 or prototype 2 strain Ag876. All
LCLs were cultured in medium (as described above) containing 10% fetal calf
serum, and all assays involved B95.8 virus-transformed LCLs unless otherwise
Synthetic peptides and protein preparations. Epitope peptides were synthe-
sized by 9-fluorenylmethoxycarbonyl chemistry (Alta Bioscience, University of
Birmingham) and dissolved in dimethyl sulfoxide (DMSO), and their concentra-
tions were determined by biuret assay. The preparation and purification of
baculovirus-expressed EBNA1 protein have been described elsewhere (13).
EBNA2 protein and appropriate control preparations made in baculovirus ex-
pression systems were kindly provided by Friedrich Gra ¨sser, Homburg-Saar,
Germany (6). EBNA3A and EBNA3C protein preparations and controls were
likewise made from appropriate baculovirus vectors by infection of insect cells
and purification of nuclear proteins.
ELISPOT assays. CD8-depleted PBMC preparations were tested in ELISPOT
assays of IFN-? release as previously described (13), with pools of overlapping
peptides (three or four peptides per pool) from the antigen sequence of interest,
followed by assays on individual peptides within positive pools. The EBNA1 and
EBNA2 panels were 20-mer peptides (overlapping by 15 residues), and the
EBNA3A and -3C panels were 15-mer peptides (overlapping by 10 residues), all
based on the B95.8 EBV strain sequence.
In vitro reactivation protocols. CD4?T-cell clones were generated from
cultures of CD8-depleted PBMCs 7 days after in vitro stimulation either with 5
?M epitope peptide directly loaded onto the cells for 1 h or with autologous
dendritic cells loaded either with peptide or with protein (13) or (as for CD8?
T-cell activation) with gamma-irradiated autologous LCLs (18). On day 7, cells
were cloned by limiting dilution at 3 cells per well on autologous gamma-
irradiated LCLs (104/well) loaded with the relevant peptide at 5 ?M and allo-
geneic gamma-irradiated, phytohemagglutinin-treated PBMCs (105/well) in in-
terleukin-2-supplemented medium with 5% autologous serum as previously
described (12). Growing microcultures were screened for peptide reactivity by
IFN-? enzyme-linked immunosorbent assay (ELISA), and selected clones were
expanded as described above with fetal calf serum-supplemented medium.
ELISAs of IFN-? release and MAb blocking. Cloned T cells were incubated in
U-bottom or V-bottom microtest plate wells with standard numbers of autolo-
gous, HLA-matched, or HLA-mismatched LCLs that were either unmanipulated
or prepulsed for 1 h with 5 ?M peptide (or an equivalent concentration of
DMSO solvent as a control) or preexposed in serum-free medium for 2 h to
specific EBV antigen preparations (or to control antigen preparations) and then
washed. The supernatant medium harvested after 18 h was assayed for IFN-? by
ELISA (Endogen) in accordance with the manufacturer’s recommended proto-
col. In blocking assays, LCLs were preincubated with monoclonal antibodies
(MAbs) specific for HLA-DR (L243; ATCC clone HB-55), HLA-DQ (SPV-L3;
Serotec), and HLA-DP (B7.21; kindly provided by A. M. de Jong, Leiden Uni-
versity, Leiden, The Netherlands) for 1 h before addition of T-cells to the assay.
Chromium release assays. CD4?T-cell clones were tested for killing of target
cells at known effector/target ratios in 5- and 18-h chromium release assays, and
results were expressed as percent specific lysis of the target line. Targets were
HLA-matched or HLA-mismatched LCLs preexposed for 1 h to 5 ?M epitope
peptide or to an equivalent concentration of DMSO solvent as a control.
Outgrowth assays. Target LCLs (HLA matched and HLA mismatched, either
unmanipulated or prepulsed with 5 ?M epitope peptide and then washed) were
serially diluted into replicate U-bottom microtest plate wells at 104to 300 per
well, and T cells were added to half of the replicates at 104/well. Plates were
incubated at 37°C in 5% CO2with weekly refeeding, and LCL outgrowth was
scored after 4 weeks. Results are expressed as the minimum seeding of LCLs
required for successful outgrowth.
CD4 epitope mapping of EBV latent-cycle antigens. In initial
epitope mapping experiments, we increased the number of
EBV-seropositive donors screened for CD4?T-cell reactivity
to EBNA1 and EBNA3C peptide panels (13) and extended the
analysis to new peptide panels covering the primary sequences
of EBNA2 and EBNA3A. The results of these assays are sum-
marized in Fig. 1, showing all of the individual peptides against
which CD4?T-cell memory was detected; numbers refer to the
coordinate of the first amino acid in the peptide sequence. For
each peptide, the histogram indicates the overall percentage of
seropositive donors who made a detectable response. These
results, based on the screening of 23 to 32 donors per peptide
panel, confirm that a 200-amino-acid stretch in the C-terminal
half of EBNA1 is a rich source of CD4 epitopes, with 75% of
the donor cells reactive to 1 or more of the 17 epitopes in this
area. In addition 70% of the donor cells also responded to 1 or
more of 11 epitopes in the much larger EBNA3C protein. Of
note are individual epitopes, for example, EBNA1 515 (TSL)
and EBNA3C 386 (SDD), that were recognized by about 30%
of the donors tested. Although almost as large as EBNA3C,
the 944-amino-acid EBNA3A protein proved to be much less
immunogenic to the CD4?T-cell response, with only three
epitope peptides detected and ?25% of the donors reactive to
any one of these epitopes. Screening of EBNA2, a protein with
a unique sequence similar in size to that of EBNA1, revealed
only six epitopes; however, one of these (EBNA2 276, PRS)
was recognized by 40% of the donors tested, and this was
largely responsible for the overall frequency of EBNA2-reac-
tive donors reaching 65%.
Throughout these ELISPOT assays, we consistently found
that the size of CD4 epitope-specific memory populations in
peripheral blood lay between the detection threshold of 30
spot-forming cells (SFC) and a maximum value of 350 SFC per
106CD8-depleted PBMCs. This range is 10-fold smaller than
VOL. 79, 2005CD4?T-CELL RECOGNITION OF EBV-INFECTED B CELLS4897
the equivalent range of EBV latent epitope-specific CD8?
T-cell memory, which, for immunodominant CD8 epitopes,
can reach up to 3,000 SFC/106PBMCs (5, 31). Furthermore,
the size of the CD4 response to any particular epitope peptide
varied between individual donors across the full range, again in
contrast to certain immunodominant CD8 epitopes, which in
donors with the appropriate HLA type consistently produce
large responses in the ELISPOT assay (5, 31; data not shown).
Antigen specificity of CD4?T-cell clones generated by
epitope-peptide stimulation. On the basis of the above assays,
we selected 12 epitopes (4 from EBNA1, 3 from EBNA2, 2
from EBNA3A, and 3 from EBNA3C) against which to gen-
erate CD4?T-cell clones. These epitopes included some that
were recognized by a large proportion of donors, for example,
EBNA1 515 (TSL), EBNA2 276 (PRS), and EBNA3C 386
(SDD), and others that represented relatively rare responses.
Using donors with detectable CD4?T-cell memory to the
individual peptides in ELISPOT assays, we generated CD4?
T-cell clones by limiting-dilution cloning of PBMCs 7 days
after peptide stimulation in vitro. All proliferating cultures
were first screened for peptide reactivity in ELISAs of IFN-?
release, and peptide-specific clones were checked for antigen
specificity by testing on autologous antigen-presenting cells
that had been loaded with an exogenous supply of the relevant
EBV protein preparation. Figure 2 shows representative re-
sults from these antigen specificity assays, with clones raised
against the EBNA1 515 (TSL), EBNA2 276 (PRS), EBNA3A
780 (GPW), and EBNA3C 386 (SDD) epitopes. In each case,
we were able to confirm that the clones recognized not only the
epitope peptide but also processed antigen, whereas there was
no significant response to control epitope or antigen prepara-
tions. Specific recognition of antigen-loaded cells clearly re-
quired processing and could not be ascribed to contamination
of the protein preparation with preformed peptide, since pre-
fixing the presenting cells eliminated the response to antigen
but not that to exogenously loaded peptide (data not shown).
Figure 2 also shows representative MAb blocking assays in
which peptide-loaded autologous cells were used as targets in
the presence of high concentrations of HLA-DP-, -DQ-, and
-DR-specific MAbs. The results show that the TSL-, PRS- and
GPW-specific clones were all restricted through an HLA-DR
allele and the SDD-specific clones were all restricted through
FIG. 1. Mapping of CD4?epitopes within the primary sequences of EBNA1, -2, -3A, and -3C. The position of each epitope is identified by a
number that refers to the coordinate of the first amino acid in the protein as encoded by EBV strain B95.8. The bar above each epitope position
indicates the percentage of all EBV-seropositive donors tested who responded to the relevant epitope peptide in ELISPOT assays. Epitopes against
which CD4?T-cell clones were later derived are also identified by a three-letter code corresponding to the first three amino acids of the epitope
sequence and by an asterisk above the bar. For each protein, the number of unique amino acids (aa) is shown; this excludes the 233-amino-acid
glycine-alanine repeat domain (residues 93 to 325) for EBNA1 and the 42-amino-acid polyproline repeat domain (residues 59 to 100) for EBNA2.
4898LONG ET AL.J. VIROL.
an HLA-DQ allele. In each case, the relevant allele could be
determined by screening partially HLA-matched target cells
for the ability to present the peptide. This identified the re-
stricting alleles as HLA-DR103 for TSL, HLA-DR52 for PRS,
HLA-DR1 for GPW, and HLA-DQ5 for SDD (data not
shown). In this way, restriction was mapped to defined
HLA-DR or HLA-DQ alleles for 10 of the 12 epitopes being
studied and to HLA-DP alleles for the other 2 epitopes (see
LCL recognition by CD4?T-cell clones: IFN-? release as-
says. For the 12 sets of clones reactive to individual epitopes,
we then conducted a series of IFN-? release assays (i) to
determine each clone’s functional avidity, defined in peptide
titration assays as that peptide concentration mediating 50%
maximal recognition, and (ii) to determine the efficiency with
which each clone recognized the unmanipulated LCL, express-
ing this as a percentage of the maximal response seen on the
same targets loaded with an optimal concentration of epitope
peptide. All of the clones generated against the same epitope-
HLA allele combination were functionally similar to one an-
other, and so the data for individual epitopes are illustrated
with a representative clone in each case.
Figure 3 presents the results for three CD4 epitopes in
EBNA1, PQC (529) restricted through HLA-DR14, VFL
(564) restricted through an HLA-DP allele, and TSL (515)
restricted through HLA-DR103. Peptide titrations (top pan-
els) determined the functional avidities as 15 nM for PQC-
specific clones, 30 nM for VFL-specific clones, and 90 nM for
TSL-specific clones. The same clones were then assayed at a
range of T-cell inputs (500 to 5,000 per well) against the au-
tologous LCL and an HLA-mismatched LCL (seeded at 5 ?
104per well) where the LCL targets had either been preex-
posed to the cognate peptide at a 5 ?M concentration and then
washed (middle panels) or left untreated (bottom panels). The
PQC-specific clone showed strong recognition of the peptide-
loaded autologous LCL, with levels of IFN-? release increasing
with increased T-cell input, and not of peptide-loaded control
targets. However, this clone, and several other PQC-specific
clones, showed no detectable recognition of the autologous
LCL without added peptide. This was also true of clones di-
FIG. 2. Antigen (Ag) specificity of CD4?T-cell clones raised against the TSL (EBNA1 515), PRS (EBNA2 276), GPW (EBNA3A 780), and
SDD (EBNA3C 386) epitopes. (Top panels) Clones (500 T cells per well) were stimulated overnight with autologous LCLs that were either
unmanipulated, prepulsed for 1 h with 5 ?M epitope peptide (pep; or an equivalent concentration of control peptide), or preexposed in serum-free
medium for 2 h to specific EBV antigen preparations (or to a control antigen) and then washed before the assay. Responses are expressed as
percentages of the maximum IFN-? induced by peptide-loaded target cells in each case. (Bottom panels) The results shown in the bottom panels
are from a separate experiment in which autologous LCLs were preexposed for 1 h to epitope peptide at a concentration mediating half-maximal
responses, washed, and then incubated for a further hour in the presence of MAb to HLA-DP, HLA-DQ, or HLA-DR or in medium as a control
(No MAb) before the addition of 500 T cells to the cell suspension. Results are expressed as described above. Note that assays conducted with
the PRS-specific clones were carried out with LCLs transformed with the type 2 Ag876 EBV strain, in which the PRS epitope sequence is mutated
(10), thereby reducing background LCL recognition to zero.
VOL. 79, 2005CD4?T-CELL RECOGNITION OF EBV-INFECTED B CELLS4899
rected against a different EBNA1 epitope, NPK (475) (data
not shown). By contrast, clones specific for the VFL epitope
again responded well to peptide-loaded autologous targets but
in this case also showed low-level IFN-? release in response to
the autologous LCL itself. This recognition was reproducible,
titrated against the input T-cell number, and also was blocked
specifically by the same anti-HLA-DP MAb as had blocked the
recognition of exogenously loaded peptide in the earlier assays.
However, comparing the observed levels of release of IFN-?
against peptide-loaded and unloaded targets in the same assay
gave an efficiency of recognition of the unmanipulated LCL of
only 0.2%. Other VFL-specific clones gave similarly low values.
Interestingly, CD4 clones to the third EBNA1 epitope, TSL,
were also capable of recognizing the untreated autologous
LCL, but in this case at higher levels representing around 3%
of that seen on peptide-loaded targets. Recognition again ti-
trated against the input T-cell number and was blocked with
the appropriate antibody, in this case the anti-DR MAb.
The latter result was of particular interest since different
groups have reported either no LCL recognition by TSL-spe-
cific CD4?T-cell clones (9) or recognition that appeared to be
strong, albeit never directly compared with that seen on pep-
tide-loaded cells (17). To determine whether such differences
might have arisen wholly or in part through the use of different
in vitro reactivation protocols, we established TSL-specific
CD4?T-cell clones by standard peptide stimulation and com-
pared these to clones generated from the same donor by stim-
ulation with EBNA1 protein-loaded dendritic cells. We found
that the two sets of clones were very similar in the above types
of assay (data not shown), and indeed their functional avidities
were similar to those reported by others for TSL-specific
clones reactivated by autologous LCL stimulation (9).
Figure 4 shows corresponding data for CD4?T-cell clones
raised against EBNA2 epitope PAQ (301), EBNA3A epitope
GPW (780), and EBNA3C epitope SDD (386). These showed
functional avidities of 100, 30, and 30 nM, respectively, a range
not dissimilar from that seen with the EBNA1 epitope-specific
clones. Again, all three epitope-specific reactivities in Fig. 4
showed strong recognition of peptide-loaded autologous (but
not HLA-mismatched) LCL targets and also recognized the
unmanipulated autologous LCL at levels representing 3% (for
PAQ), 1% (for GPW), and 7% (for SDD) of the optimal levels
seen on peptide-loaded targets. These values were reproduc-
ibly observed both on repeat testing of the particular clones
illustrated and on testing of other clones generated against the
same epitopes. In this case, we also compared SDD-specific
clones generated by standard peptide stimulation against
clones produced by autologous LCL stimulation and found
that the two sets were again functionally very similar (data not
Similar experiments were conducted on a further five
epitopes from the EBNA2, EBNA3A, and EBNA3C proteins,
and we again observed that each individual epitope was asso-
ciated with its own characteristic level of LCL recognition (for
a summary, see Table 1). Of particular interest was EBNA2
epitope PRS (276) because it had been recognized by 40% of
the seropositive donors tested in ELISPOT assays (Fig. 1) and
in an earlier study (21) appeared to be presented in several
different HLA class II contexts. On the basis of our ELISPOT
screening, we selected four PRS-responsive donors with dis-
parate HLA class II types and from each generated PRS-
specific clones that, when tested in MAb blocking assays, were
all found to be restricted through an HLA-DR allele. Since all
individuals possess two DRB1 alleles and, in some cases, also
one or two additional DR alleles (designated DR51, -52a, -52b,
-52c, and -53), we screened each set of clones on a large panel
of fully DR-typed target cells loaded with the PRS peptide.
Figure 5 shows data from a representative range of targets
sufficient to map the HLA restricting allele in each case. We
found that PRS-specific clones from the four different donors
used four different alleles, DR52a, DR52b, DR52c, and DR7.
The four different sets of clones were then tested for func-
tional avidity and for autologous LCL recognition. As shown in
Fig. 6, all clones restricted through DR52 alleles had unusually
high avidities in peptide titration assays, with 50% end points
at 6 nM (DR52a), 3 nM (DR52b), and 7 nM (DR52c), whereas
the value of 30 nM for DR7-restricted clones was in the range
seen earlier for clones restricted through other DRB1 alleles.
Interestingly, all four sets of clones showed detectable recog-
nition of the unmanipulated autologous LCL but at widely
different efficiencies. These ranged from 1% of that seen on
peptide-loaded targets for the DR52a- and DR52c-restricted
clones to 15% for the DR7-restricted clones and to as high as
35% for DR52b-restricted clones. To check the reproducibility
of the latter result, we generated PRS-specific clones from a
second DR52b-positive donor by standard peptide stimulation
and from the same donor by two other in vitro reactivation
protocols, namely, stimulation with peptide-loaded dendritic
TABLE 1. Summary of epitope-specific CD4?T-cell clones
aFunctional avidity is defined as the concentration of epitope peptide medi-
ating 50% maximal IFN-? release in peptide titration assays. Values shown are
the means of results of assays on several epitope-specific clones (except for PHD
and DR52c PRS, where only one clone was available).
bRecognition of the unmanipulated autologous LCL, as measured by IFN-?
release, is expressed as a percentage of the IFN-? release seen in the same assay
against the same LCL optimally loaded with epitope peptide. The value for each
epitope is the mean from assays on several epitope-specific clones (except for
PHD and DR52c PRS, where only one clone was available).
4900 LONG ET AL.J. VIROL.
cells and stimulation with the LCL alone. All epitope-specific
DR52b-restricted clones, regardless of the stimulation proto-
col or individual donor, gave a similar pattern of results.
Table 1 summarizes the data obtained with CD4?T-cell
clones to all 12 of the epitopes studied, including the PRS
epitope in its four different HLA contexts. Epitopes are
grouped in accordance with their source antigen and, within
each group, ordered by the efficiency of LCL recognition
shown by epitope-specific clones; the functional avidity of
these clones is shown alongside.
LCL recognition by CD4?T-cell clones: cytotoxicity and
LCL outgrowth assays. The ability of EBV latent-epitope-
specific CD4?T cells to recognize naturally infected B-cell
targets implies that such T cells might have a direct effector
role in the control of EBV infection. For that reason, we
examined whether the CD4?T-cell recognition of LCLs ob-
served by IFN-? release was also detectable with other func-
tional readouts, namely, short-term cytotoxicity and longer-
term LCL outgrowth assays. This work was conducted with
CD4?T-cell clones to five selected epitopes; these were (in
FIG. 3. Functional analysis of CD4?T-cell clones against the PQC (EBNA1 529), VFL (EBNA1 564), and TSL (EBNA1 515) epitopes. (Top
panels) Clones (100 T cells per well) were stimulated overnight with autologous LCLs (5 ? 104per well) either unmanipulated (neg) or loaded
with epitope peptide at 10?5to 10?10M concentrations. Responses were assayed by IFN-? release and expressed as a percentage of the maximum
peptide-induced response. (Middle panels) Clones (500 to 5,000 T cells per well) were stimulated as described above with the autologous LCLs
(match) or with HLA class II-mismatched LCLs (5 ? 104per well), both previously exposed to 5 ?M epitope peptide and then washed before the
assay. Responses are expressed as IFN-? release in picograms per milliliter. (Bottom panels, upper section) Clones were tested at the same time
as above, on the same autologous and HLA class II-mismatched LCLs but with no exogenous peptide treatment. Responses are expressed as IFN-?
release in picograms per milliliter. The efficiency with which each clone is able to recognize unmanipulated autologous LCL targets is expressed
as a percentage of the maximal response seen on the same targets loaded with peptide (box at upper right of each graph). (Bottom panels, lower
section) The results shown in the lower section of the bottom panels are from a separate experiment in which the responses of 500 T cells to the
autologous LCL (non-peptide loaded) were assayed either alone (no MAb) or in the presence of MAbs to HLA-DP, HLA-DQ, or HLA-DR as
VOL. 79, 2005 CD4?T-CELL RECOGNITION OF EBV-INFECTED B CELLS4901
order of increasing efficiency of LCL recognition by IFN-?
release) clones specific for EBNA1 epitopes PQC and TSL,
EBNA2 epitope PAQ, EBNA3C epitope SDD, and DR52b-
restricted EBNA2 epitope PRS.
Figure 7 presents chromium release assay data showing the
levels of killing observed against HLA class II-matched and
mismatched target LCLs, each tested with and without pre-
loading with the relevant epitope peptide. Because pilot ex-
periments had shown that killing was not always apparent
within the conventional 5-h assay period, we measured specific
lysis after both 5 and 18 h. All of the clones showed killing of
the HLA-matched peptide-loaded LCL, apparent within 5 h
and stronger by 18 h, and no killing of the mismatched peptide-
loaded target. However, the clones differed in the ability to kill
the unmanipulated HLA-matched LCL. The PQC (data not
shown)- and TSL-specific CD4?effectors did not lyse these
targets significantly, even within 18 h. The PAQ-specific clone
gave marginal killing after 18 h only, while the SDD-specific
and PRS-specific clones both gave a hint of killing within 5 h
and clearly detectable lysis at the later time.
We then set up outgrowth assays in which replicate cultures
of HLA-matched and mismatched LCLs, either untreated or
preloaded with the relevant epitope peptide, were seeded into
U-bottom microtest plate wells at doubling dilutions of 104to
300 cells per well; to some wells at each seeding, a standard
number of CD4?T cells (104cells per well) were then added
from the same clones as tested above. The cultures were main-
tained in standard cell culture medium for 3 to 4 weeks and
examined for successful LCL outgrowth. Although these ex-
periments could be accurately scored by microscopic inspec-
tion of the cultures, in several cases we confirmed by CD19
staining that successful outgrowth involved the LCL and not
FIG. 4. Functional analysis of CD4?T-cell clones against the PAQ (EBNA2, 301), GPW (EBNA3A, 780), and SDD (EBNA3C, 386) epitopes.
The experimental design and expression of results are the same as in Fig. 3, except that the range of T-cell numbers used in LCL stimulation
experiments extended from 100 to 2,500 per well.
4902 LONG ET AL.J. VIROL.
surviving T cells. Figure 8 expresses the results of these assays
as the minimum number of each LCL required for successful
outgrowth under the various conditions. The results are con-
sistent with the earlier experiments in that clear evidence of
LCL growth inhibition was limited to T-cell–LCL combina-
tions with the higher levels of LCL recognition as determined
by IFN-? release. Thus, PQC (data not shown)-, TSL-, and
PAQ-specific CD4?T-cell clones, which had shown levels of
unmanipulated LCL recognition of 0 to 3% of that seen
against peptide-loaded targets, markedly inhibited outgrowth
of HLA-matched LCLs if they had been peptide loaded but
had no effect on the corresponding nonloaded cells; as a spec-
ificity control, these same clones had little if any effect on
HLA-mismatched LCLs with or without peptide loading. By
contrast, clones specific for the SDD and PRS epitopes, which
had shown stronger recognition of autologous LCL in IFN-?
assays, clearly were able to inhibit the outgrowth of HLA-
matched (but not mismatched) LCLs even without peptide
The extent to which EBV latent-specific CD4?T cells are
able to recognize naturally infected LCL targets is an impor-
tant in vitro indicator of their likely potential as direct effectors
controlling EBV-driven lymphoproliferations in vivo. Most in
vitro studies to date have focused on CD4?T-cell responses to
just one of the available latent proteins, EBNA1 (9, 13, 17, 27,
32) and, even when studying responses to the same epitope (9,
17), have reported discordant results with respect to LCL rec-
ognition. To address this issue in a more systematic way, the
present work set out to identify CD4 epitopes in a broader
range of latent-cycle antigens, EBNA1, -2, -3A, and -3C, and
then to generate CD4?T-cell clones to a representative panel
of epitopes drawn from these four proteins.
Screening with peptide panels showed that three antigens,
EBNA1, -2, and -3C, are each recognized by a majority (65 to
75%) of EBV-seropositive donors, whereas only a small num-
ber (?25%) respond to EBNA3A. Interestingly certain
epitopes, for example, TSL in EBNA1, PRS in EBNA2, and
SDD in EBNA3C, were recognized by 30 to 40% of the donors
tested. Such a high frequency is explained in the case of the
PRS epitope by its capacity to elicit responses in the context of
several different HLA alleles (Fig. 5) (21). The same also
appears to be true of the TSL epitope because, although we
established epitope-specific clones that were DR103-restricted,
not all responders to TSL in ELISPOT screening assays ex-
press this allele (13; data not shown). However, this was not
the case for the SDD epitope; this was restricted to HLA-DQ5,
a high-incidence allele in Caucasian populations (16), and all
responders to this epitope indeed proved to be HLA-DQ5
positive. It is important to note that, while a high percentage of
immune donors might respond to certain epitopes, these are
not necessarily immunodominant responses in terms of abso-
lute size. Thus, both here and in an earlier study (13), we found
that responses to all CD4 epitopes fell within a rather narrow
size range and the strongest responses did not consistently map
to a particular set of epitopes or to epitopes from a particular
antigen. This contrasts with the CD8 response to EBV latent-
cycle antigens, which is not only much larger than the CD4
response but also tends to focus preferentially on immuno-
dominant epitopes from the EBNA3A, -3B, and -3C proteins
Having established CD4?T-cell clones to 12 representative
epitopes and confirmed their specificity for the relevant EBV
target antigen in protein loading assays, we determined their
functional avidity by peptide titration. There were two impor-
tant findings in this regard. Firstly, all clones produced by
peptide stimulation of PBMCs and specific for the same
FIG. 5. Analysis of HLA class II allele restriction of CD4?T-cell
clones specific for the PRS (EBNA2, 276) epitope. Clones were estab-
lished from four donors whose HLA-DR types are in each case iden-
tified (Auto). These clones were then tested as described in the legend
to Fig. 3 (middle panels) against peptide-loaded cells of the autologous
LCL and of allogeneic LCLs of known HLA-DR type (Allo LCL).
Alleles matched with the autologous cells are identified by shading.
Results are expressed as a percentage of the maximum IFN-? release
observed in the assay.
VOL. 79, 2005CD4?T-CELL RECOGNITION OF EBV-INFECTED B CELLS 4903
epitope-HLA allele combination, whether from the same do-
nor or different donors, gave similar peptide titration curves.
Secondly, in several cases we compared epitope-specific CD4?
T-cell clones that had been generated from individual donors
either by conventional peptide stimulation, by peptide- or an-
tigen-loaded dendritic cells, or by LCL stimulation and, when-
ever we tested them, found no significant differences in func-
tional avidity. We infer that stimulation protocols are not
major sources of artifacts in these experiments and that the
CD4?T-cell clones being used are genuinely representative of
the epitope-specific memory populations present in our EBV-
We then turned to the question of LCL recognition. To
allow comparisons to be made between individual clones to a
single epitope and between clones specific for different
epitopes, in each case we expressed the level of recognition of
the unmanipulated LCL as a percentage of that seen in parallel
against the same LCL preloaded with an optimal concentra-
tion of epitope peptide. The overall findings, summarized in
Table 1, allow a number of conclusions to be drawn. Firstly, the
level of LCL recognition is consistent among clones with the
same epitope specificity but differs markedly, from 0 to 35% of
optimal peptide-loaded values, between clones with different
specificities. Secondly, these interepitope differences are not
obviously related to the antigenic source of the epitope. Thus,
levels of LCL recognition ranged from 0 to 3% of peptide-
loaded values for EBNA1 epitopes, from 1 to 35% for EBNA2
epitopes, from 1 to 4% for EBNA3A epitopes, and from 0 to
7% for EBNA3C epitopes. Thirdly, the differences in LCL
recognition cannot solely be explained by differences in the
functional avidities of the CD4?T-cell clones. For example,
clones to the PQC epitope in EBNA1 had an avidity of 15 nM
and showed no LCL recognition whereas clones to the TSL
epitope also in EBNA1 recognized the LCL at an efficiency of
3% yet required a sixfold higher peptide concentration (90
nM) for half-maximal IFN-? release in peptide titration assays.
Likewise, among EBNA2-specific clones, those against the
GQT epitope showed a 15% efficiency of LCL recognition yet
were 10-fold less avid in peptide titration assays than DR52a-
restricted, PRS-specific clones, which show much less efficient
(1%) LCL recognition. We conclude that, as for CD8?T-cell
clones in this viral system (3, 14, 15), the observed level of LCL
FIG. 6. Functional analysis of CD4?T-cell clones specific for the PRS (EBNA2, 276) epitope derived from different EBV-seropositive donors
and restricted through HLA-DR52a, -DR52b, -DR52c, and -DR7, respectively. The experimental design and expression of results are the same
as in Fig. 4. Note that peptide titration assays involving the DR52b-restricted PRS clones were conducted with the Ag876 virus-transformed LCL
as in Fig. 2.
4904LONG ET AL.J. VIROL.
recognition will be a function both of the inherent avidity of
the CD4?T-cell clones and of the degree of representation of
the epitope-HLA complex on the LCL surface.
The example of the PRS epitope is particularly interesting in
this regard. It was already clear from the literature that this
epitope can elicit responses in the context of several different
HLA class II alleles. Thus, Khanna et al. reported strong kill-
ing of the autologous LCL by a PRS-specific clone reactivated
by LCL stimulation and restricted through an HLA-DQ allele
(10). Subsequently, Omiya and colleagues generated PRS-spe-
cific clones by peptide stimulation from several different do-
nors and mapped their restriction to five different HLA alleles.
Interestingly, all clones recognized the autologous LCL by
IFN-? release, although at levels that were never compared to
the maximal peptide-induced response, whereas only clones
restricted through an unidentified DR52 allele killed LCL tar-
gets in cytotoxicity assays (21). Our work extends this analysis
by accurately quantitating LCL recognition by PRS-specific
clones restricted through four different HLA class II alleles.
Recognition ranged from 1% efficiency for DR52a- and
DR52c-restricted clones through 15% efficiency for DR7-re-
stricted clones to 35% efficiency for DR52b-restricted clones.
Again, these differences cannot be explained by differences in
functional avidity but instead reflect how the level of represen-
tation of the PRS epitope on the LCL surface is critically
influenced by the identity of the restricting allele.
A similar phenomenon may underlie the apparent discrep-
ancy in the literature with respect to CD4?T-cell clones spe-
cific for the TSL epitope in EBNA1. Khanna et al. reported no
LCL recognition in cytotoxicity assays by a DR1-restricted TSL
clone (9). This is reminiscent of the DR103-restricted clones
described in the present work, which again did not kill the LCL
but showed low-level (3%) efficiency of recognition by IFN-?
release. In contrast, the TSL-specific clones with strong LCL
killing described by Munz and colleagues (17) may be re-
stricted through a different HLA class II allele that mediates
more efficient epitope presentation at the LCL surface. With
FIG. 7. Killing of LCL targets by CD4?T-cell clones against the
TSL (EBNA1 515), PAQ (EBNA2 301), SDD (EBNA3C 386), and
PRS (EBNA2 276) epitopes. Five- and 18-h chromium release assays
were conducted with HLA class II-matched and mismatched LCL
targets either unmanipulated or previously exposed to 5 ?M epitope
peptide and then washed before the assay. Results are expressed as
percent specific chromium release from target cells at effector/target
ratios of 5:1 (■) and 2.5:1 (?).
FIG. 8. Inhibition of LCL outgrowth by the epitope-specific CD4?
T-cell clones used in Fig. 7. For each clone, two HLA class II-matched
LCLs and one mismatched LCL were seeded at doubling dilutions of
104to 300 cells per well either alone or with the addition of 104CD4?
T cells. The LCLs were either unmanipulated or previously exposed to
5 ?M epitope peptide and then washed before seeding. Results are
expressed as the minimum LCL seeding required for successful out-
growth in each case. For each clone, the results from LCL–T-cell
cocultures are shown for the unmanipulated LCL (■) and in the
adjacent column for the peptide-loaded LCL (u). These values are in
each case compared with the corresponding results for outgrowth of
the unmanipulated LCL or of the peptide-loaded LCL cultured in the
absence of T cells (dotted lines).
VOL. 79, 2005CD4?T-CELL RECOGNITION OF EBV-INFECTED B CELLS4905
promiscuous epitopes such as PRS and TSL, one can begin to
look for correlations between the level of epitope presentation
on infected cells and immunogenicity of the epitope in vivo.
Clearly in the case of PRS, even low-level presentation in the
context of the HLA-DR52a or -52c allele can elicit a response;
however, we found that only a small number of individuals with
the DR52a or DR52c allele screened actually made a detect-
able response to the peptide in ELISPOT assays. By contrast,
most individuals positive for HLA-DR7 or -DR52b, an allele
that mediates more efficient presentation of the epitope, did
have detectable PRS-specific memory (data not shown). These
questions will be better addressed once the restricting alleles
for other apparently promiscuous EBV epitopes have been
One of our main motivations for this work was to address the
potential importance of EBV latent epitope-specific CD4?T
cells as direct effectors capable of recognizing and eliminating
EBV-driven lymphoproliferations in vivo. Our final series of
experiments, measuring both short-term cytotoxicity and long-
term inhibition of target cell outgrowth in vitro, show firstly
that all of the epitope-specific CD4?T-cell clones tested were
cytotoxic on epitope-loaded LCL targets and could inhibit
their outgrowth in cocultivation assays. However, parallel as-
says on unmanipulated LCL targets split the clones into two
groups. Clones with an efficiency of LCL recognition at or
below 3% in IFN-? assays showed no detectable killing in 18-h
assays and no detectable inhibition of outgrowth against the
unmanipulated LCL, whereas clones with efficiencies of 7% or
greater were active in both situations. These results, and those
of other studies with CD4?T-cell clones against as yet unde-
fined targets on the LCL surface (5), are all consistent with the
view that inhibition of LCL outgrowth correlates strongly with
cytotoxic activity. The present work confirms that CD4?T cells
specific for some EBV latent-cycle epitopes can prevent LCL
outgrowth in vitro (17, 19, 21) and therefore, like their CD8?
T-cell counterparts, could act directly against EBV-driven lym-
phoproliferative lesions in vivo (24). However, our data sug-
gest that this is only true of CD4?T cells against a minority of
epitopes, namely, those epitopes represented on the surface of
latently infected cells above a critical threshold. This is not to
imply that T cells specific for other latent-cycle epitopes do not
play an important role in vivo; for example, they may act to
help CD8?responses but such helper activity is more likely to
be induced by specific recognition of antigen exogenously ac-
quired and presented by dendritic cells rather than of antigen
endogenously expressed by infected cells themselves. From a
therapeutic standpoint, identifying the subset of epitope-HLA
combinations that can mediate direct T-cell recognition and
determining the route whereby these epitopes access the HLA
class II pathway in infected cells represent important priorities
for future work.
This work was supported by Cancer Research UK.
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