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IMMUNOBIOLOGY
Direct killing of Epstein-Barr virus (EBV)–infected B cells by CD4 T cells
directed against the EBV lytic protein BHRF1
Elise Landais, Xavier Saulquin, Emmanuel Scotet, Lydie Trautmann, Marie-Alix Peyrat, John L. Yates, William W. Kwok,
Marc Bonneville, and Elisabeth Houssaint
Due to their low frequency, CD4 T-cell
responses to Epstein-Barr virus (EBV)
lytic antigens are, so far, poorly characterized. Human peptide major histocompatibility complex (MHC) class II multimers
provide a means to detect and characterize such rare T cells. Along a screening of
T-cell responses to lytic or latent EBV
antigens within peripheral blood leukocyte (PBL)– or synovial-derived CD4 Tcell lines, we identified an human leukocyte antigen–DR*0401 (HLA-DR*0401)–
restricted epitope derived from BHRF1
(BamHI fragment H rightward open reading frame 1), a viral protein produced
during the early stages of the lytic cycle.
We show here that T-cell responses to
this particular BHRF1 epitope are shared
by most EBV-infected DR*0401ⴙ individuals, as BHRF1-specific CD4 T cells could
be sorted out from all the DRB*0401 T-cell
lines analyzed, using magnetic beads
coated with recombinant BHRF1/DR*0401
complexes. Sorting with these peptide
MHC class II multimers was very efficient,
as the yield of recovery of BHRF1-specific T cells was nearly 100%. Functional
analysis of a large number of clones
responding to BHRF1/DR*0401 demonstrated their cytolytic action against autologous and allogeneic DR*0401ⴙ EBVtransformed B-lymphoblastoid cell lines
(B-LCLs), with 40% to 80% killing efficiency and potent interferon ␥ production, thus suggesting that this CD4 T-cell
population contributes to the control of
EBV replication. B-LCL lysis by these
T-cell clones was DR*0401 dependent,
EBV dependent, and was not merely due
to bystander killing. Taken together, these
data provide the first demonstration that
a lytic antigen can induce a direct cytolytic response against EBV-infected cells.
(Blood. 2004;103:1408-1416)
© 2004 by The American Society of Hematology
Introduction
Epstein-Barr virus (EBV) is a human gamma herpesvirus that
establishes in 95% of the adult population a lifelong latent infection
in resting memory B cells. The EBV carrier state (latency) is
characterized by expression of a limited set of EBV genes and
sporadic reactivation of the lytic cycle in latently infected cells,
leading to viral replication. Upon reactivation in tonsillar B
lymphocytes, EBV can productively infect oropharyngeal epithelium, resulting in infectious virus production and transmission.1-3
During childhood, patients’primary infection by EBV is usually
asymptomatic and leads to a lifelong persistence of the virus. Host
immune responses are thought to be of central importance both in
limiting the primary infection and in controlling the lifelong virus
carrier state. The high frequency of EBV-associated lymphoproliferative diseases or lymphomas in immunocompromised individuals strongly supports a role for anti-EBV T cells in containing EBV
infection.4
The CD8 T-cell response has been the matter of intensive
investigations. Large CD8⫹ clonal expansions have been described
in infectious mononucleosis patients and there is some evidence
that clones detected in the memory cytotoxic T-lymphocyte (CTL)
response are selected during the acute phase and may persist at high
circulating frequencies,5 contributing to the CD8⫹ expansion seen
in healthy adults.6 Recently, the use of human leukocyte antigen
(HLA) class I peptide tetramers has highlighted the large proportion of CD8 T cells directed to EBV antigens.7,8 These CD8⫹ CTL
responses are preferentially directed toward the early lytic proteins,
BZLF1 (BamH fragment Z left frame 1) and BMLF1 (BamHI-M
leftward reading frame 1)9-12 and to a lesser extent, toward the
latent nuclear antigens Epstein-Barr virus nuclear antigen 3A
(EBNA3A), EBNA3B, and EBNA3C.12-14
Much less is known about the CD4⫹ T-cell responses to EBV,
although there is an increasing awareness of their key role in (1)
supporting high-affinity antibody (Ab) production; (2) initiating
and, particularly, maintaining CTL numbers and function; and (3)
performing direct effector activity. Analysis of CD4 T-cell responses has been greatly hampered by the small size of the CD4
compartment. No CD4 T-cell expansions have been detected using
highly sensitive heteroduplex techniques, even during the early
stages of EBV acute infection.15 The EBV specificity of some
CD4⫹ T-cell clones has been previously reported.16-19 In more
recent systematic analysis, the nuclear antigen EBNA1 was found
to be a main EBV latency antigen for CD4⫹ T cells.20-22
Here we describe the identification and characterization of a
dominant CD4 T-cell response to BHRF1 (BamHI fragment H
From the INSERM U463, Institut de Biologie, Nantes, France; Faculté des
Sciences de Nantes, Nantes, France; Virginia Mason Research Center,
Seattle, WA; and Roswell Park Center Institute, Buffalo, NY.
E.L. and X.S. contributed equally to this work.
Submitted March 27, 2003; accepted October 2, 2003. Prepublished online as Blood
First Edition Paper, October 16, 2003; DOI 10.1182/blood-2003-03-0930.
Supported by grants from the Association pour la Recherche sur la Polyarthrite
(ARP), Ligue Nationale Contre le Cancer (LNCC), Centre Hospitalier
Universitaire de Nantes, European Community (grant QLK2-CT-2001-01205)
and by institutional grants from INSERM.
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Reprints: Elisabeth Houssaint, INSERM U463, Institut de Biologie, 9 quai
Moncousu, 44035 Nantes Cedex 01, France; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology
BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
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BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
CYTOTOXIC EBV-SPECIFIC CD4 T CELLS
rightward open reading frame 1) in DR*0401 individuals. We
screened for anti-EBV CD4 T cells in peripheral blood leukocyte
(PBL)–derived T-cell lines from healthy donors or kidney recipients and in synovial fluid (SF)–derived T-cell lines from arthritis
patients, previously shown to be greatly enriched for EBV-specific
CD8 T cells.11 Along this screening, an HLA-DR*0401–restricted
BHRF1 epitope, an early antigen of the lytic cycle, was identified.
Taking advantage of the recent availability of major histocompatibility complex (MHC) class II tetramers,23 we set up an efficient
sorting strategy to isolate BHRF1-specific cells, using multimers of
HLA-DR*0401/BHRF1 peptide complexes coated on immunomagnetic beads. BHRF1-specific T cells were sorted out from all
DR*0401 T-cell lines studied with nearly 100% efficiency. Moreover, the direct contribution of BHRF1-specific T cells to the
control of EBV replication was suggested by their ability to kill
autologous B-lymphoblastoid cells (B-LCLs) and to produce
interferon ␥ (IFN-␥).
Patients, materials, and methods
Donors and HLA-DR typing
All T-cell samples were from HLA-DR4⫹ and EBV⫹ donors11,12 (Table 1).
PBLs were obtained from 2 healthy virus carriers (D3 and D5) and 2 kidney
graft recipients (KRs). SF-derived lymphocytes were obtained for clinical
indications from patients suffering from various forms of acute or chronic
arthritis: 9 patients with rheumatoid arthritis (RA), 1 patient with ankylosing spondylitis (AS), and 1 patient with Reiter syndrome (RS). Mononuclear cells from peripheral blood (PBMCs) and from synovial fluid were
isolated by Ficoll/Hypaque density gradient centrifugation. HLA-DRB1
typing was performed by using standard molecular typing techniques at the
Etablissement de Transfusion Sanguine (Nantes, France). This study was
approved by the Centre Hospitalier Universitaire de Nantes institutional
review board.
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T-cell lines and T-cell clones
PBL- or SF-derived lymphocytes were maintained in RPMI 1640 supplemented with 10% pooled human serum, 1 mM L-glutamine, and 150 IU/mL
recombinant interleukin 2 (IL-2). Positive selection for CD4⫹ PBMCs was
performed by immunomagnetic sorting using an anti-CD4–specific monoclonal Ab (mAb) as previously described.11 CD4 purity (⬎95%) was
confirmed by staining with anti-CD8 and anti-CD4 antibodies, followed by
flow cytometric analysis. CD4⫹ polyclonal T cells were then expanded in
vitro in IL-2 culture medium supplemented with purified leukoagglutinin
(0.5 ␮g/mL) and irradiated (30 Gy) allogeneic feeder cells (PBLs and
B-LCLs at a 10:1 ratio). Clones were obtained by seeding synovial T cells at
0.3 cells/well in IL-2/CM, leukoagglutinin (0.5 ␮g/mL), and irradiated
allogeneic feeder cells (5 ⫻ 104 PBLs and 5 ⫻ 103 B lymphoblastoid
cells/well).
Generation of EBV B-LCLs
B-LCLs were generated for each donor by culturing 2 ⫻ 106 PBMCs in 100
␮L of RPMI and 10% fetal calf serum (FCS) with 500 ␮L of EBVcontaining supernatant from the virus-producing B95.8 marmoset cell line.
Cultures were performed in the presence of 1 ␮g/mL cyclosporin A. After
24 hours, 2 mL of RPMI 1640 containing 10% FCS, 1mM L-glutamine, and
50 ␮g/mL gentamicin was added to each well.
A DR*0401⫹ B-LCL transformed with an EBV mutant, in which
BHRF1 was deleted (BHRF1–knockout [KO]), was generated by transforming 105 CD3-depleted PBMCs from donor D3 (DR*0401⫹) with supernatant (104 colony-forming units) from B95/B531a cells, containing in
roughly equal amounts B95.8 virus and a B95.8-derived mutant, B531a, in
which cytomegalovirus–immediate early (CMVIE) neo construct replaces
most of BHRF1 gene (B95/B531a).24 Two days later, G418 (GIBCO BRL,
Carlsbad, CA) was added to the medium at 700 ␮g/mL (active drug
concentration). G418-resistant LCL clones were obtained and the absence
of BHRF1 was confirmed by polymerase chain reaction (PCR).
Recombinant vaccinia-EBV viruses
Recombinant vaccinia virus (rVV) vectors coding for each of the EBV
latent proteins (Epstein-Barr nuclear antigens [EBNAs] 1, 2, 3A, 3B, 3C,
and LP, latent membrane protein 1 [LMP1], and LMP2) or some EBV lytic
Table 1. Sorting of BHRF1-specific CD4 T cells with DR*0401/BHRF1 multimers
Sorting with DR4/PYY multimers
Donors
TNF production upon stimulation
with PYY peptide, pg/mL
% of DR4/PYY tetramer ⴙ cells
HLA-DR4
subtype
Presorted cells
Postsorted cells
Presorted cells
Postsorted cells
95.7
PBL-derived T-cell lines
D3
DR*0401
⬍1
⬎ 200
0.7
D5
DR*0401
NT
⬎ 200
NT
NT
KR1
DR*0401
⬍1
⬎ 200
NT
NT
KR7
DR*0407
⬍1
⬍1
NT
NT
SF-derived T-cell lines
RA1
DR*0401
85
⬎ 200
2.2
99
RA3
DR*0401
⬍1
⬎ 200
0.26
40
RA5
DR*0401
15
⬎ 200
0.22
99.2
RA6
DR*0401
⬍1
60
NT
NT
RA14
DR*0401
150
⬎ 200
NT
97.5
RS2
DR*0401
12
⬎ 200
0.26
97
RA2
DR*0404
⬍1
⬍1
NT
NT
RA7
DR*0404
⬍1
⬍1
NT
NT
RA9
DR*0404
⬍1
⬍1
NT
NT
RA19
DR*0404
⬍1
⬍1
NT
NT
AS1
DR*0404
⬍1
⬍1
NT
NT
The efficiency of sorting of BHRF1-specific CD4 T cells using DR*0401/BHRF1 monomers coated on magnetic beads was evaluated (1) by staining presorted and
postsorted cells with the DR4/BHRF1 tetramer, and (2) by measuring in presorted cells and in postsorted cells, the TNF production (pg/mL) upon stimulation with the
BHRF1122-133 peptide in an autopresentation assay.
NT indicates not tested.
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BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
LANDAIS et al
proteins (BZLF1, BRLF1, BHRF1, BMLF1, BamHI-M rightward open
reading frame 1 [BMRF1], BamHI left frame 1 [BHLF1], BALF2, BALF5,
BCRF1, and BLLF1) have been previously described.13,14 B-LCLs were
incubated with individual rVV (10 plaque-forming units [PFUs] per cell)
overnight, and then effector T cells were added to infected B cells at a 10:1
effector-target (E/T) ratio. Activation of T cells was evaluated either in a
tumor necrosis factor ␣ (TNF-␣) or in a 51chromium release assay.
Peptides
A set of 23 residue peptides, overlapping by 12 amino acids and spanning
the whole BHRF1 protein (Chiron Mimotopes, Victoria, Australia), was
used for the characterization of the BHRF1/DR*0401 epitope. The 12–
amino acid peptide BHRF1122-133 (PYYVVDLSVRGM, designated PYY)
was obtained from Genosys (The Woodlands, TX). Peptide stock solutions (20 mg/mL in dimethyl sulfoxide [DMSO]) were diluted first to
2 mg/mL in acetic acid (1%) and second to the final concentration in RPMI
1640 culture medium.
TNF assay
Production of TNF-␣ by activated lymphocytes was estimated in an
autopresentation assay. To trigger TNF-␣ release by responding T cells,
5 ⫻ 103 clonal T cells or 5 ⫻ 104 polyclonal T cells were incubated with
peptides at various concentrations. After 6 hours, the supernatant was
collected and its TNF content was determined by testing its cytotoxic effect
on Walter and Eliza Hall Institute (WEHI) 164 clone 13 cells in an MTT
(3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay.25
Generation of HLA-DR*0401ⴚ peptide tetramers
Peptide-loaded HLA-DR0401 tetramers were produced as described previously.23 Briefly, recombinant DRA1*0101 and DRB1*0401, in which the
transmembrane domain was replaced by a leucine zipper, were produced in
Drosophila S2 cells. At the end of the ␤-chain cDNA is a biotinylation
sequence that allows site-specific biotinylation using the BirA enzyme. The
resulting biotynilated heterodimers were loaded with peptides for 3 days at
37°C. Phycoerythrin (PE)–labeled streptavidin was used to produce fluorescent peptide–loaded DR*0401 tetramers. Peptide used corresponded to the
sequence of the BHRF1 protein residues 122-133 (peptide PYY).
DRA1*0101/DRB1*0401 tetramers loaded with the HA307-319 peptide
(hemagglutinin) were used as a control.23
Immunomagnetic cell sorting
Immunomagnetic sorting of DRB1*0401/BHRF1-specific T cells was
performed as previously described,26 using beads coated with HLADR*0401/BHRF1 peptide monomers. For coating immunomagnetic beads,
1 ␮g HLA-DR4 peptide monomers diluted in 30 ␮L of phosphate-buffered
saline–bovine serum albumin (PBS-BSA) 0.1% were incubated for 1 hour
at room temperature with 5 ⫻ 106 streptavidin-coated beads (Dynabeads
M-280 streptavidin; DYNAL, Compiègne, France), followed by several
washes. PBL- or SF-derived lymphocytes (5 ⫻ 106) were rotated for 4
hours at room temperature with monomer-coated beads. After 9 washes,
bead-coated cells were expanded by polyclonal activation as described
in “T-cell lines and T-cell clones” and then cultured for 2 to 3 weeks
before testing.
Sequence analysis of T-cell receptor (TCR) transcripts
RNA from 5 ⫻ 106 T-cell clones was extracted with TRIZol reagent
(Invitrogen, Cergy Pontoise, France) according to the supplier’s instructions and dissolved in 15 ␮L of water. Reverse transcriptions, PCR
amplifications, and sequencing were performed as described.11
Flow cytometric determination of cell surface and intracellular
markers
Staining with DR4/PYY tetramers was performed by incubating CTL
clones or T-cell lines (2.5 ⫻ 105) for 1 hour at 37°C with 1 ␮g of each
PE-labeled tetramer in 50 ␮L of culture medium. Cells were then washed in
PBS containing 1% FCS and 0.1% NaN3 and stained with fluorescein
isothiocyanate (FITC)–conjugated monoclonal antibody against CD4
(PharMingen, San Diego, CA). After a 30-minute incubation, cells were
washed again and analyzed using a Becton Dickinson FACSCan flow
cytometer (San Jose, CA). T-cell lines and T-cell clones were phenotyped
by indirect immunofluorescence with mAb against TCR V␤ regions.
T-cell helper 1 (Th1)/Th2 profiling was performed through intracellular
detection of IFN-␥ and IL-4. T cells were resuspended in RPMI 10% FCS
supplemented with phorbol myristate acetate (PMA; 10⫺7 M; Sigma, St
Louis, MO) and ionomycin (0.5 ␮g/mL; Sigma) and incubated at 37°C.
After 2 hours, monensin (2 nM; Sigma) was added and the cells were
incubated further for 3 hours. The cells were then fixed for 20 minutes in
2% paraformaldehyde, permeabilized using a 0.5% saponin buffer, and
stained with the following antibodies: FITC-conjugated anti–IFN-␥ (Pharmingen) and PE-conjugated anti–IL-4 (Pharmingen). The stained cells were
then analyzed on the flow cytometer. A human T-cell line expressing both
IFN-␥ and IL-4 was included as a control to assess for the specificity of the
intracellular cytokine detection assay.
Cytolytic assays
Cytolytic assays were performed in a 4-hour chromium release assay. The
targets used were unloaded B-LCLs or B-LCLs loaded with peptide at
various concentrations for 30 minutes. In the mAb blocking experiments,
targets were incubated with mAbs for 30 minutes at room temperature
before adding effector cells. In bystander killing, HLA-DR*0401⫹ B-LCLs
were 51Cr labeled while HLA-DR*0401⫺ B-LCLs were left unlabeled.
Assays were performed in triplicate and results were expressed as percent
specific 51Cr release.
Results
Identification of a DR*0401/BHRF1 epitope.
In an attempt to characterize CD4 T-cell responses to EBV, we
started from T-cell lines derived from the synovial fluid of arthritic
patients, within which we previously described an enrichment for
CD8 T cells responding to EBV antigens.11 CD4⫹ T-cell clones
were obtained from SF-derived T-cell lines. Four CD4⫹ T-cell
clones from patient RA1, which proliferated when cocultured with
the autologous B-LCL, were selected for further analysis of antigen
specificity. The autologous B-LCL was infected with rVV encoding
10 lytic EBV proteins and all latent proteins. One of these clones
(clone 4.19 from patient RA1) was activated by the autologous
B-LCL infected with rVV encoding the BHRF1 gene product, as
documented by TNF-␣ production (Figure 1A).
To identify the BHRF1 epitope recognized by clone 4.19, a set
of synthetic peptides of 23 amino acids overlapping by 12 amino
acids, and covering the entire BHRF1 protein sequence, was
synthesized. Autologous EBV-B cells pulsed with each of these peptides were tested in a cytotoxic assay for recognition by clone 4.19.
Two overlapping peptides scored positive, namely BHRF1111-133
and BHRF1122-144 (Figure 1B). The 12–amino acid peptide
BHRF1122-133 (PYYVVDLSVRGM, designated PYY), corresponding to the overlap, was recognized by clone 4.19 (Figure 1C). The
sequence suggested that tyrosine at position 123 or 124 was the
peptide 1 (P1) anchor residue for MHC binding, conforming to the
HLA-DR4 binding motif.27 Recognition by clone 4.19 of autologous EBV-B cells loaded with peptide PYY was inhibited by an
anti–HLA-DR mAb (Figure 2A). Donor RA1 was typed DR1 and
DR4 (subtype DRB1*0401). Peptide PYY was loaded onto a panel
of allogeneic LCLs, sharing DR1 or DR4 with donor RA1. Clone
4.19 recognized efficiently DR*0401 (5 cell lines tested) and
DR*0404 B-LCLs loaded with PYY. Peptide-loaded DR*0406
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BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
CYTOTOXIC EBV-SPECIFIC CD4 T CELLS
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Figure 1. Identification of a DR*0401/BHRF1 epitope recognized by the CD4 T-cell clone 4.19. (A) Recognition of the lytic EBV protein BHRF1 by clone 4.19. CTL cells
were tested in a 4-hour TNF-␣ release assay on autologous B-LCLs expressing individual latent or lytic EBV proteins from vaccinial viral vectors. Results are shown as ␮g/mL.
TNF release was observed at an effector-target ratio of 10:1. (B) Identification of the target epitope of BHRF1-specific clone 4.19 using a panel of peptides (23-mers,
overlapping by 12 amino acids [aa’s]) spanning the BHRF1 protein. These peptides were used as targets in cytotoxic assays by incubating them with 51Cr-labeled autologous
EBV-B cells for 1 hour at 37°C. Clone 4.19 was added at an E/T ratio of 10:1 and Cr release was measured after 4 hours. Note the significant lysis background observed with
unloaded autologous B-LCL (No pep). (C) Stimulation of clone 4.19 by the BHRF1 122-133 peptide (PYY). Cytotoxicity of clone 4.19 to Cr-labeled autologous EBV-B cells
loaded with various concentrations of peptides. Clone 4.19 was added at an E/T ratio of 10:1 and Cr release was measured after 4 hours. Data obtained with the 23-mer
BHRF1111-133 peptide are shown as a positive control. Data obtained with an irrelevant peptide (irr peptide) are shown as a negative control.
B-LCL was weakly recognized, whereas peptide-loaded DR*0402,
DR*0405, and DR*0407 B-LCLs were not recognized (Figure 2B).
Staining of BHRF1122-133–specific CD4 T cells with
DR*0401/PYY tetramers
DRA1*0101/DRB1*0401 tetramers loaded with BHRF1122-133 peptide were produced and their staining specificity was studied using
several CD4⫹ T-cell clones reactive, or not, against this peptide
MHC complex. The BHRF1/DR*0401-specific T-cell clone 4.19
was strongly stained by DRA1*0101/DRB1*0401 tetramers loaded
with BHRF1122-133 peptide but not by tetramers loaded with
irrelevant peptides (Figure 3A). In contrast, clone 4.4 (from donor
RA1), which was not activated by the BHRF1 vaccine, was not
stained by the BHRF1122-133 tetramer (Figure 3A). Although the
PYY peptide was recognized by clone 4.19 when loaded on
HLA-DR*0401⫹ as well as DR*0404⫹ or DR*0406⫹ B-LCLs
(Figure 2B), this clone 4.19 was not stained by DRA1*0101/
DRB1*0404 tetramers loaded with PYY peptide (not shown).
We then tested the ability of the BHRF1122-133 tetramers to
detect antigen-specific cells within polyclonal CD4 T-cell lines,
either PBL derived or SF derived. About 0.7% of cells in the
PBL-derived CD4 T-cell line from the healthy donor D3 were
stained by DR4/BHRF1 tetramers, whereas 2.2% of cells in the
SF-derived CD4 T-cell line from patient RA1 scored positive
Figure 2. Recognition of the BHRF1 122-133 epitope by the CD4
T-cell clone 4.19 is HLA-DR*0401-restricted. (A) Clone 4.19 cells
were cocultured with autologous B-LCL loaded with PYY peptide at 10
␮M. Inhibition with anti-DR (L243), anti-DP (B7.21), or anti–class I
(W6.32) antibodies was performed by addition of mAb at 3 different
concentrations (0.5 to 50 ␮g/mL) during the coculture. Percentage
specific lysis was determined in a standard chromium release assay
at an effector-to-target ratio of 10:1. Recognition of the autologous
B-LCL was blocked by an anti-DR Ab but not by anti-DP or anti–class I
Ab. (B) Autologous and allogeneic DR4⫹ B-LCLs were precoated with
peptide PYYVVDLSVRGM (10 ␮M final) and then exposed to clone
4.19. Clone 4.19 recognized efficiently DR*0401 and DR*0404 BLCLs loaded with PYY. A DR*0406 B-LCL was a weakly recognized
peptide but DR*0402, DR*0405, and DR*0407 B-LCLs were not.
(Figure 3B; Table 1). By contrast, the percentage of specific cells
was below the detection threshold for other T-cell lines (Table 1).
TNF assay also indicated that only some of the CD4 polyclonal
T-cell lines released a detectable amount of TNF-␣ upon stimulation with the PYY peptide (Table 1).
Sorting of BHRF1122-133–specific CD4 T cells by using
DR*0401/PYY multimers coated on magnetic beads
To investigate the presence of rare BHRF1122-133–specific T cells
within polyclonal cell lines derived from EBV-seropositive HLADR4 donors, we performed immunomagnetic sorting using streptavidin beads coated with biotinylated DRB1*0401/BHRF1122-133
monomers (hereafter referred to as multimers), according to a
multimer-based approach allowing sorting of CD8 T cells specific
to defined HLA-A*0201/peptide complexes.26 After a 2-week
culture under polyclonal activation, reactivity of sorted cells to the
BHRF1122-133 peptide was tested both by staining with the
DRB1*0401/BHRF1 tetramer and by measuring TNF production
in a 6-hour self-presentation assay. As attested by TNF production
upon stimulation with the PYY peptide, successful sorting of
BHRF1-specific cells was achieved for all T-cell lines derived from
DRB1*0401 individuals (3 PBL-derived and 6 SF-derived T-cell
lines) but for none of the T-cell lines derived from DRB1*0404 or
DRB1*0407 donors (5 SF-derived 1 PBL-derived T-cell lines). The
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Figure 3. Staining and sorting of CD4 T cells by
DR*0401/BHRF1 multimers. (A) Clone 4.19 (right) was
stained by the DR*0401/BHRF1 tetramer (bottom) but
not by the irrelevant DR*0401/HA tetramer (top). Another
clone from donor RA1, clone 4.4 (left), that did not
recognize BHRF1 in the EBV vaccinia assay, was not
stained by the DR*0401/BHRF1 tetramer. Cells were
double-stained with 20 ␮g/mL of DR*0401 tetramers and
with 10 ␮g/mL monoclonal antibody against CD4. (B)
Yield of sorting of BHRF1-specific cells by using DR*0401/
BHRF1 monomers coated on magnetic beads. Synovial
CD4 T cells from patient RA1 and CD4 PBLs from healthy
donor D3 were stained by DR*0401/BHRF1 tetramers
before (left) and after (right) sorting with DR*0401/
BHRF1 monomers coated on magnetic beads. Sorted
cells were expanded for 3 weeks under polyclonal stimulation before being stained with DR*0401/BHRF1 tetramers. Percentage of cells staining with tetramers is indicated. Bar indicates the range of fluorescence where the
cells were scored positive.
frequency of BHRF1-specific cells after multimer sorting was in
most cases above 95%, as indicated by staining with the BHRF1
tetramer (Table 1; Figure 3B). The BHRF1 sorting efficiency was
confirmed by clonal analysis. Cloning was performed on DR4/
BHRF1 sorted cells for 3 donors (RA1, RA14, D3). Thirty clones
were isolated from donor RA1, 11 from donor RA14, and 37 from
donor D3. All of them released a high level of TNF-␣ upon
incubation with the PYY peptide in an autopresentation assay (not
shown). For patient RA6, sorted cells showed significant TNF
production upon stimulation with the PYY peptide, although they
were not stained by the BHRF1 tetramer. This result probably
reflects a low frequency of DR4/BHRF1-specific T cells in this
line, as also suggested by the nonsaturating TNF response observed
after in vitro exposure of these cells to the PYY peptide (Table 1).
5/6 clones), V␣6s1 usage (by 3/6 clones), and V␤13s1 usage (by
3/6 clones). Some of these clones were selected for further
functional studies.
BHRF1122-133–specific CD4 T cells are Th1-like
Cytokine production was analyzed in 6 clones responding to
BHRF1, under unspecific stimulation with PMA/ionomycin (3
clones from the healthy donor D3: LP15, LP18, LP45; and 3 clones
from the patient RA14: G4, G13, G22). All of them were Th1, as
they secreted IFN-␥, but only low amounts of IL-4 (Figure 4).
DR*0401-dependent killing of B-LCLs by CD4 T-cell clones
specific to BHRF1/DR*0401
Along the characterization of the BHRF1/DR*0401 epitope, we
noticed that the CD4 clone 4.19 lysed unloaded autologous
B-LCLs with a 20% to 40% lysis efficiency but failed to recognize
DR*0404 or DR*0406 B-LCLs (Figures 1-2). To determine
whether the spontaneous killing of DR*0401⫹ B-LCLs was a
general feature of DR*0401-restricted BHRF1-specific CD4 T-cell
clones, 5 clones isolated from 3 DR*0401⫹ donors were tested
against DR*0401 or DR*0404 B-LCLs. Among these clones, 2
were derived from PBLs of the healthy virus carrier D3 (LP15 and
LP45, with different V␤ sequences; Table 2) and 3 were SF-derived
(clone A22 from patient RA1 and clones G4 and G22 from patient
RA14). All clones significantly killed unloaded DR*0401⫹ BLCLs. Percentage of specific lysis was slightly increased when the
DR*0401 B-LCL was loaded with the PYY peptide at 10 ␮M
(Figure 5A and data not shown). In contrast, these clones did not
BHRF1122-133–specific CD4 T cells have a restricted repertoire
We assessed the clonal diversity of BHRF1/DR4-specific T-cell
lines through analysis of their TCR repertoire. TCR V␤ usage by
BHRF1-specific CD4 T cells derived from 6 donors was first
studied by flow cytometry using a panel of 22 TCR V␤–specific
mAbs. Each T-cell line used a predominant V␤ region, which
differed from one T-cell line to another (Table 2). TCR␣ and
␤ chain sequencing of several CD4 T-cell clones indicated a
highly restricted T-cell repertoire, as most clones derived from
a given cell line expressing identical TCR␣ and ␤ chains
(Table 2). Despite the absence of “public” T-cell clones (ie,
expressing TCR sequences shared by distinct individuals),
several recurrent TCR features were noticed within the 6 distinct
clones with fully characterized TCR, such as J␤1s1 usage (by
Table 2. Amino acid sequence of TCR␣ and ␤-chain junction of BHRF1-specific CD4 T-cell clones
TCR ␣-chain sequence
Donor
D3
RA1
RA5
% V␤
V␣
CDR3 alpha
TCR ␤-chain sequence
J␣
V␤
CDR3
J␤
No. of
clones
71% Vb17
V␣6s1
CAM REIQGAQKL VFG
J␣54
V␤17s1
CAS SPADGTMNTEA FFG
J␤1s1
2/2 (LP15)
25% Vb3
V␣6s1
CAM RGITGANSKL TFG
J␣56
V␤3s1
CAS TLQTGAMNTEA FFG
J␤1s1
2/2 (LP45)
V␤13s1
CAS SPGTGTYTEA FFG
J␤1s1
2/2 (A22)
V␤3s1
CAS SQSTGTMNTEA FFG
J␤1s1
2/2
8% Vb9
V␣14s1
CAY RRTGANSKL TFG
J␣56
?
V␣1s4
CAV VLRTGANNL FFG
J␣36
5% Vb3
V␣6s1
CAM REIYNFNKF YFG
J␣21
30% Vb17
16% Vb20
RA14
?
V␣16s1
CAH RRDTGFQKL VFG
J␣8
V␤3s1
CAS SLSRGDWGSPL HFG
J␤1s6
2/2
RA3
100% Vb9
V␣23s1
CAV VNAGGTSYGKL TFG
J␣52
V␤9s1
CAS SLRQGGTEA FFG
J␤1s1
2/2
BHRF1-specific CD4 T-cell clones were derived from BHRF1-specific T-cell lines sorted with BHRF1/DR*0401 multimers.
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BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
CYTOTOXIC EBV-SPECIFIC CD4 T CELLS
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DR4⫺ B-LCLs. All clones killed all the DR*0401⫹ but not the
DR*0404⫹ nor the DR4⫺ B-LCLs (see representative data
obtained with clone A22 in Figure 5B). Altogether these results
indicated that all BHRF1/DR4-specific T-cell clones killed
autologous B-LCLs in a DR*0401-dependent fashion. Accordingly, lysis of peptide-unloaded autologous B-LCLs was blocked
by anti–HLA-DR–specific mAb (data not shown).
Killing of B-LCLs by CD4 T-cell clones specific to
BHRF1/DR*0401 is EBV dependent
Figure 4. BHRF1-specific CD4 T-cell clones show a Th1-like cytokines profile.
Two SF-derived clones (G4 and G14) from patient RA14 and 2 PBL-derived clones
(LP15 and LP18) from the healthy donor D3 (LP15 and LP18) were tested for
cytokine production. IFN-␥ and IL-4 production was detected after stimulation with
PMA and ionomycin. The percentage of cytokine-producing cells is indicated in the
quadrant. One representative experiment of 3 is shown. FSC-H indicates forward
scatter height.
kill an unloaded DR*0404 B-LCL. The SF-derived, but not the
PBL-derived clones, killed DR*0404 B-LCLs loaded with PYY
peptide at 10 ␮M (Figure 5A). This suggested lower affinity of
the DR*0401-restricted clones for PYY/DR*0404 than for
PYY/DR*0401 complexes. Accordingly while SF-derived clones
killed DR*0401 B-LCLs loaded with as low as 1 nM of the PYY
peptide, significant killing of DR*0404 B-LCLs was observed
after loading with at least 10 ␮M of this peptide (not shown).
Spontaneous killing of DR*0401 B-LCLs by BHRF1-specific
clones was confirmed by testing the lytic activity of these 5
clones against a panel of 5 DR*0401⫹, 2 DR*0404⫹, and 3
Figure 5. DR*0401-dependent killing of B-LCLs by BHRF1specific CD4 T-cell clones. (A) Two PBL-derived clones from the
healthy donor D3 (LP15 and LP45) and 2 SF-derived clones (A22 and
G4) killed a DR*0401 B-LCL, either unloaded or loaded with PYY
peptide, while they did not kill an unloaded DR*0404 B-LCL. The 2
SF-derived clones, but not the PBL-derived clones, killed the DR*0404
B-LCL after loading with PYY peptide. Results are expressed as
percent specific lysis at E/T ratio 10:1. (B) The CD4 T-cell clone A22
was tested against a panel of 5 DR*0401⫹ (black symbols) and 3
DR4⫺ (open symbols) unloaded B-LCLs. All DR*0401⫹ B-LCLs were
killed, while DR4⫺ B-LCLs were not.
Owing to the low frequency of cells in lytic cycle within a B-LCL
(classically below 5%), efficient lysis of peptide-unloaded B-LCL
by T-cell clones directed against an EBV lytic epitope was
unexpected and led us to further investigate this phenomenon. To
investigate whether lysis of DR*0401⫹ B-LCLs was due to
bystander killing rather than through direct recognition of BHRF1
in the DR4 context, we studied the cytotoxicity of BHRF1-specific
CTLs toward labeled DR4⫺ B-LCLs mixed with cold DR4⫹
B-LCLs. None of the mismatched LCLs were killed in the presence
of DR4-matched target cells, thus ruling out a mere “bystander”
killing effect (not shown).
We then tested the capacity of DR*0401-restricted, BHRF1specific CD4 T-cell clones to recognize, or not, EBV⫺ DR*0401⫹presenting cells. As shown in Figure 6A, an EBV⫺ DR*0401⫹activated CD8 T-cell clone (which was brightly stained by DRspecific mAb) was not killed by DR4/BHRF1-specific T-cell
clones, unless loaded with the relevant peptide (Figure 6A).
In another series of experiments, we used a DR*0401⫹ B-LCL
transformed with an EBV mutant, in which BHRF1 was disrupted
by insertional mutagenesis (BHRF1-KO).24 While BHRF1-specific
T-cell clones efficiently recognized (both in terms of cytolysis and
TNF production) DR*0401⫹ B-LCLs transformed with wild-type
EBV, they neither killed nor produced TNF (data not shown) when
incubated with the BHRF1-KO B-LCL from the same donor. The
experiment was done twice with 3 BHRF1-specific clones originating from 3 different donors. A representative experiment is shown
in Figure 6B. In contrast, the BHRF1-KO B-LCL (DR4⫹, DR16⫹)
could activate a CD4 T-cell clone from donor RA17 (clone P4.2)
that recognizes the EBV latent protein EBNA3C in the HLA-DR16
context, thus indicating efficient EBV infection of the BHRF1-KO
B-LCL. As shown in Figure 6C, this clone did not exhibit
spontaneous lysis toward DR16⫹ B-LCLs, but it killed DR16⫹
B-LCLs once loaded with the EBNA3C100-111 peptide. The
BHRF1-KO B-LCL was killed, as well as the autologous B-LCL
from donor RA17. Together with the HLA-DR4–dependent killing
of autologous B-LCLs (Figure 5), these results strongly suggest
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1414
LANDAIS et al
BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
Figure 6. Cytolytic activity of the DR*0401-restricted BHRF1-specific CD4 T-cell clone A22 toward DR*0401ⴙ B-LCL is EBV dependent. (A) Cytolytic activity of clone
A22 toward the autologous B-LCL (EBV⫹, DR*0401⫹) or toward the CD8 T-cell clone A2.10 directed against BMLF1/HLA-A*0201 (EBV⫺, DR*0401⫹). Unlike the autologous
B-LCL (left), clone A2.10 (right) was not killed by the BHRF1-specific CD4 T-cell clone A.22, unless it was loaded with PYY peptide. (B) The CTL clone A22 recognized the
DR*0401⫹ B-LCL from donor D3 transformed with the wild-type EBV (WT) but not the same B-LCL transformed with a viral mutant with a KO for BHRF1 gene (KO). The 2
B-LCLs were recognized after loading with PYY peptide. The DR*0401⫹ B-LCL from donor RA1 and the DR4⫺ B-LCL from donor RA17 were included as positive or negative
control, respectively. (C) Clone P4.2 (donor RA17), responding to EBNA3C in the HLA-DR16 context, was used as a control to verify that the BHRF1-KO B-LCL from donor D3
was able to present an epitope from the latent protein EBNA3C. Clone P4.2 did not exhibit spontaneous lysis toward DR16⫹ B-LCL, but it killed its autologous B-LCL, as well as
the WT and the BHRF1-KO B-LCLs from donor D3 (DR4⫹, DR16⫹), once loaded with the EBNA3C100-111 peptide (PHD). Results are expressed as percent specific lysis.
that the spontaneous killing of autologous B-LCLs by DR4/BHRF1specific CTLs is linked to recognition of an HLA-DR–restricted
BHRF1 epitope.
Discussion
Because of the importance of CD4⫹ T cells in antiviral immunity,
much effort is now directed toward identifying MHC class
II–restricted viral antigens. The data presented in this paper
uncover a CD4 T-cell response to the early lytic EBV protein
BHRF1 in the HLA-DR*0401 context (epitope BHRF1122-133). We
show that the response to BHRF1/DR*0401 is recurrent as it was
found in all T-cell lines tested (9 at the total). This response was
found in peripheral blood of healthy EBV carriers and elevated in
synovial fluid of arthritis patients. DRB1*0401/BHRF1122-133 tetramers stained a small number of cells in only 4 DR*0401⫹ T-cell
lines out of the 9 that were tested. It should be underlined that in
contrast to most, if not all, previous studies aiming at isolating
antiviral or antitumoral CD4 T cells, we did not perform any in
vitro antigen (Ag) stimulation prior to testing. BHRF1-specific
cells were efficiently sorted out through immunomagnetic sorting,
using DRB1*0401/BHRF1122-133 monomers coated on magnetic
beads, and these cells could be rapidly expanded under nonspecific
stimulation. The expanded CD4 T cells were nearly 100% specific
to BHRF1, in 4 of 5 cases, thus underlining the efficiency of this
sorting approach with DR4 multimers.
The DR*0401-restricted BHRF1-specific clones, described in
this study, exhibit a strict specificity for DR*0401. BHRF1/
DR*0401-specific clones were not stained by BHRF1/DR*0404
tetramers and they did not kill DR*0404 B-LCLs. This is in
accordance with previous data indicating that although DRA1*0101/
DRB1*0401 and DRA1*0101/DRB1*0404 are structurally very
similar, the limited polymorphism between these alleles at codons
86 and 71 of the DRB1 chain may dictate unique binding patterns.28
The response to BHRF1 appears to be not specifically linked to
arthritis, as this CTL response was also found in peripheral blood of
healthy EBV carriers. A number of synovial-derived CD4 T-cell
lines were included in our screening on the basis of our previous
observation that synovial fluid–derived T-cell lines were enriched
for CD8 T cells responding to EBV antigens in the synovial fluid of
RA patients.11 As a greater number of Ag-specific CD4⫹ T cells are
expected to be found in the tissues affected by autoimmune
diseases or in an infection, we used these synovial fluid–derived
T-cell lines to screen for CD4 T-cell responses to EBV. In fact, one
of the difficulties in analyzing CD4 T-cell responses is inherent to
the rare occurrence of memory CD4 T cells. CD4⫹ T cells of a
given antigen specificity seem to be less abundant than their CD8⫹
counterparts. This may be because peptides from some antigens are
generated at low efficiency, which results in low density on
antigen-presenting cells, or it might reflect localized tissue-specific
expression. In this regard, differential regulation of antiviral T-cell
immunity results in stable CD8⫹, but declining CD4⫹ T-cell
memory.29 Thus, the frequency of virus-specific CD4 T cells is at
least 35-fold lower than CD8 cells during acute and memory phases
of lymphocytic choriomeningitis virus infection.30 This explains
why the few published studies on human class II–restricted
responses using HLA-peptide tetramers required prior in vitro
antigenic selection to detect CD4 T cells specific for an influenza or
herpes simplex virus 2 (HSV-2) epitope in chronic virus carriers.23,31 This also explains why until recently, the CD4 T-cell
response to EBV has been poorly characterized.
The specificity of a few CD4 T-cell clones reacting to lytic or
latent proteins has been described previously.16-19 Moreover, a
systematic analysis of CD4 T-cell responses to latent antigens was
performed by 2 different groups, who reported highly recurrent
responses to EBNA1 and EBNA3C epitopes in most donors,
though at a very low frequency.20,21 The magnitude of responses
specific to EBNA1-derived CD4 epitopes ranged from 60 to 350
IFN-␥–producing cells per 106 CD8-depleted PBMCs,21 but these
responses could be efficiently reactivated with autologous dendritic
cells transduced with EBNA1-vaccinia constructs.20 The response
to BHRF1 described in this paper was found, at least in 2 donors, at
rather high frequencies (0.2% of the CD4 T cells among the PBLs
of an healthy virus carrier and 2.2% of the CD4 synovial T cells of
an arthritic patient). A recent analysis of CD4⫹ and CD8⫹ T-cell
responses during primary infection showed that lytic and latent
EBV protein–specific CD4⫹ T cells were readily detected at
presentation with acute infectious mononucleosis and declined
rapidly thereafter.32
BHRF1-specific clones, be they PBL- or SF-derived, spontaneously killed autologous or HLA-matched B-LCLs (ie, without
further addition of the BHRF1 peptide). Preliminary data indicate
that these clones express perforin and granzyme A. Some,22 but not
all,17 EBNA1-specific CD4 T-cell clones have also been shown to
kill the autologous B-LCLs. However, while EBNA-1 is expressed
during latency, BHRF1 is supposed to be expressed during the lytic
cycle only. The consistent killing of B-LCLs by BHRF1-specific
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BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
CYTOTOXIC EBV-SPECIFIC CD4 T CELLS
CD4 T-cell clones was rather unexpected. In fact, the percentage of
spontaneous killing (30%-60%) far exceeds the small percentage of
B cells, within a B-LCL, that enter lytic infection (eg, 5%). The
underlying mechanism of the spontaneous killing is clearly beyond
the scope of the present study and will need further investigation.
One mechanism could be that most cells take up BHRF1 or its
fragments from the medium after its release from a few lytic cells.
Another possibility is that a small amount of BHRF1 is expressed
during latent infection and presented directly. While it is clear that
BHRF1 is expressed at high levels during early lytic infection, its
expression during latency remains a controversial issue. Latently
spliced BHRF1 cDNAs have been reported,33 but attempts to detect
BHRF1 protein expression in latently infected cells have failed.1
Nevertheless, the lack of recognition of BHRF1-KO B-LCLs by
BHRF1-specific CD4 T-cell clones demonstrates that the spontaneous killing of autologous B-LCLs by DR4/BHRF1-specific CTLs
is linked to direct recognition of an epitope of the lytic protein
BHRF1. DR2-restricted BHRF1-specific CD4 T-cell clones had
previously been isolated from patients with infectious mononucleosis and it was shown that they killed the autologous B-LCLs.19
These BHRF1-specific cells showed a Th1 cytokine profile,
consistent with their cytotoxic potential. Therefore they could
directly take part in the control of EBV replication. Along this line,
the importance of CD4 T cells in the resistance to persistent viral
infections is supported by several observations.34-38 Adoptively
1415
transferred CD8 CMV-specific T-cell clones do not persist in the
long-term without endogenous recovery of CD4 T cells.35 In HIV
infection, a minority of patients are long-term nonprogressors and
this correlates with high CD4 T-cell counts and vigorous CD4
T-cell proliferative responses to HIV proteins.36 Similar observations were made in a mouse model of lymphocytic choriomeningitis virus.37,38 CD4 T cells are believed to be instrumental in the
initiation of CD8 T-cell expansion via stimulation of dendritic
cells,39,40 but their role in maintaining adequate numbers and
function of specific CD8 T cells is less understood. Zajac and
colleagues38 observed that in CD4⫺/⫺ mice, effector functions
rather than frequency of lymphocytic choriomeningitis virus–
specific CD8 T cells were impaired.
In summary, the data presented here show that it is possible to
efficiently sort out and expand CD4 T cells specific to defined
epitopes. Such peptide/MHC class II multimer sorting strategy
could be of potential therapeutic interest for the immunotherapy of
viral infections or cancers.
Acknowledgments
We thank Drs A. Rickinson and M. Kurilla for the gift of
recombinant vaccinia constructs. We also thank Dr H. Vié for
critically reading the manuscript.
References
1. Kieff E. Epstein-Barr virus and its replication. In:
BN Fields, DM Knipe, PM Howley, eds. Fields
Virology. Philadelphia, PA: Lippincott-Raven Publishers; 1996:2343-2396.
2. Rickinson AB, Kieff E. Epstein-Barr virus. In:
BN Fields, DM Knipe, PM Howley, RM
Chanock, JL Melnick, TP Monath, B Roizman,
SE Strauss, eds. Fields Virology. 4th ed. Philadelphia, PA: Lippincott Williams & Wilkins;
2001:2575-2627.
3. Thorley-Lawson DA. Epstein-Barr virus: exploiting the immune system. Nat Rev Immunol. 2001;
1:75-82.
4. Yao QY, Tierney RJ, Croom-Carter D, et al. Frequency of multiple Epstein-Barr virus infections in
T-cell-immunocompromised individuals. J Virol.
1996;70:4884-4894.
5. Callan MFC, Annels N, Steven N, et al. Cell selection during the evolution of CD8⫹ T cell
memory in vivo. Eur J Immunol. 1998;28:43824390.
6. Silins SL, Cross SM, Krauer KG, Moss DJ,
Schmidt CW, Misko IS. A functional link for TCR
expansions in healthy adults caused by persistent
Epstein-Barr infection. J Clin Invest. 1998;102:
1551-1558.
7. Callan MFC, Tan L, Annels N, et al. Direct visualization of antigen-specific CD8⫹ T cells during the
primary immune response to Epstein-Barr virus in
vivo. J Exp Med. 1998;187:1395-1402.
8. Tan LC, Gudgeon N, Annels NE, et al. A re-evaluation of the frequency of CD8⫹ T cells specific for
EBV in healthy virus carriers. J Immunol. 1999;
162:1827-1835.
9. Scotet E, David-Ameline J, Peyrat MA, et al. T
cell response to Epstein-Barr virus transactivators in chronic rheumatoid arthritis. J Exp Med.
1996;184:1791-1800.
10. Steven NM, Annels N, Kumar A, Leese A, Kurilla
MG, Rickinson A. Immediate early and early lytic
cycle proteins are frequent targets of the EpsteinBarr virus-induced cytotoxic T cell response. J
Exp Med. 1997;185:1605-1617.
11. Scotet E, Peyrat MA, Saulquin X, et al. Frequent enrichment for CD8 T cells reactive
against common herpes viruses in chronic inflammatory lesions: towards a reassessment of
the physiopathological significance of T cell
clonal expansions found in autoimmune inflammatory processes. Eur J Immunol. 1999;29:
973-985.
12. Saulquin X, Ibisch C, Peyrat MA, et al. A global
appraisal of immunodominant CD8 T cell responses to Epstein-Barr virus and cytomegalovirus by bulk screening. Eur J Immunol. 2000;30:
2531-2539.
13. Khanna R, Burrows SR, Kurilla MG, et al. Localisation of Epstein-Barr virus cytotoxic T cell
epitopes using recombinant vaccinia: implications
for vaccine development. J Exp Med. 1992;176:
169-178.
14. Murray RJ, Kurilla MG, Brookes JM, et al. Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus
(EBV): implications for the immune control of
EBV-positive malignancies. J Exp Med. 1992;
176:157-168.
15. Maini MK, Gudgeon N, Wedderburn LR, Rickinson A, Beverley PCL. Clonal expansions in
acute EBV infection are detectable in the CD8
and not the CD4 subset and persist with a variable CD45 phenotype. J Immunol. 2000;165:
5729-5737.
16. Wallace LE, Wright J, Ulaeto DO, Morga AJ, Rickinson AB. Identification of two T-cell epitopes on
the candidate Epstein-Barr virus vaccine gycoprotein gp340 recognized by CD4⫹ T cell clones.
J Virol. 1991;65:3821-3828.
17. Khanna R, Burrows SR, Steigerwald-Mullen PM,
Thonson SA, Kurilla MG, Moss DJ. Isolation of
cytotoxic T lymphocytes from heathy seropositive
individuals specific for peptide epitopes from Epstein-Barr virus nuclear antigen 1: implications for
viral persistence and tumor surveillance. Virology.
1995;214:633-637.
18. Khanna R, Burrows SR, Thonson SA, et al. Class
I processing-defective Burkitt’s lymphoma cells
are recognized efficiently by CD4⫹ EBV-specific
CTLs. J Immunol. 1997;158:3619-3625.
19. White CA, Cross SM, Kurilla MG, et al. Recruit-
ment during infectious mononucleosis of CD3⫹
CD4⫹ CD8⫹ virus-specific cytotoxic T cells which
recognize Epstein-Barr virus lytic antigen BHRF1.
Virology. 1996;219:489-492.
20. Münz C, Bickham KL, Subklewe M, et al. Human
CD4⫹ T lymphocytes consistently respond to the
latent Epstein-Barr virus nuclear antigen EBNA1.
J Exp Med. 2000;191:1649-1660.
21. Leen A, Meij P, Redchenko I, et al. Differential
immunogenecity of Epstein-Barr virus latent-cycle
proteins for human CD4⫹ T-helper 1 responses.
J Virol. 2001;75:8649-8659.
22. Paludan C, Bickham K, Nikiforow S, et al. Epstein-Barr nuclear antigen 1-specific CD4⫹ Th1
cells kill Burkitt’s lymphoma cells. J Immunol.
2002;169:1593-1603.
23. Novak EJ, Liu AW, Nepom GT, Kwok WW. MHC
class II tetramers identify peptide-specific human
CD4⫹ T cells proliferating in response to influenza
A antigen. J Clin Invest. 1999;104:R63-R67.
24. Lee MA, Yates JL. BHRF1 of Epstein-Barr virus,
which is homologous to human proto-oncogene
bcl2, is not essential for transformation of B cells
or for virus replication in vitro. J Virol. 1992;66:
1899-1906.
25. Espevik T, Nissen-Meyer JA. A highly sensitive
cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human
monocytes. J Immunol Methods. 1986;95:99105.
26. Bodinier M, Peyrat MA, Tournay C, et al. Efficient
detection and sorting of specific T cells using
MHC class I/peptide multimers with reduced CD8
binding. Nat Med. 2000;6:707-710.
27. Rammensee HG, Friede T, Stevanovic S. MHC
ligands and peptides: first listing. Immunogenetics. 1995;41:178-228.
28. Novak EJ, Liu AW, Gebe JA, et al. Tetramerguided epitope mapping: rapid identification and
characterization of immunodominant CD4⫹ T cell
epitopes from complex antigens. J Immunol.
2001;166:6665-6670.
29. Homann D, Teyton L, Oldstone MBA. Differential
regulation of anti-viral T-cell immunity results in
From www.bloodjournal.org by guest on April 30, 2017. For personal use only.
1416
LANDAIS et al
stable CD8⫹ but declining CD4⫹ T-cell memory.
Nat Med. 2001;7:913-919.
30. Whitmire JK, Ahmed R. Costimulation in antiviral
immunity: differential requirements for CD4⫹ and
CD8⫹ T cell responses. Curr Opin Immunol.
2000;12:448-455.
31. Kwok WW, Liu AW, Novak EJ, et al. HLA-DQ
tetramers identify epitope-specific T cells in peripheral blood of Herpes Simplex virus type 2-infected individuals: direct detection of immunodominant antigen-responsive cells. J Immunol.
2000;164:4244-4249.
32. Precopio ML, Sullivan JL, Willard C, Somasundaran M, Luzuriaga K. Differential kinetics and
specificity of EBV-specific CD4⫹ and CD8⫹ T
cells during primary infection. J Immunol. 2003;
170:2590-2598.
BLOOD, 15 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 4
33. Austin PJ, Flemington E, Yandava CN,
Strominger JL, Speck SH. Complex transcription
of the Epstein-Barr virus BamHI fragment H rightward open reading frame 1 (BHRF1) in latently
and lytically infected B lymphocytes. Proc Natl
Acad Sci U S A. 1988;85:3678-3682.
34. Cardin RD, Brooks JW, Sarawar SR, Doherty PC.
Progressive loss of CD8⫹ T cell-mediated control
of gamma-herpesvirus in the absence of CD4⫹ T
cells. J Exp Med. 1996;184:863-871.
37. Kalams SA, Walker BD. The critical need for CD4
help in maintaining effective cytotoxic T lymphocyte responses. J Exp Med. 1998;188:21992204.
38. Zajac AJ, Blattman JN, Murali-Krishna K, et al.
Viral immune evasion to persistence of activated
T cells without effector function. J Exp Med. 1998;
88:2205-2213.
35. Greenberg PD, Riddell SR. Deficient cellular immunity: finding and fixing the defects. Science.
1999;285:546-551.
39. Ridge JP, Di Rosa F, Matzinger P. A conditioned
dendritic cell can be a temporal bridge between a
CD4⫹ T-helper and a T-killer cell. Nature. 1998;
393:474-478.
36. Rosenberg ES, Billingsley JM, Caliendo AM, et
al. Vigorous HIV-1-specific CD4⫹ T cell responses associated with control of viremia. Science. 1997;278:1447-1450.
40. Schoenberger SP, Toes RE, van der Voort E, Offringa R, Melief CJ. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature. 1998;393:480-483.
From www.bloodjournal.org by guest on April 30, 2017. For personal use only.
2004 103: 1408-1416
doi:10.1182/blood-2003-03-0930 originally published online
October 16, 2003
Direct killing of Epstein-Barr virus (EBV)−infected B cells by CD4 T cells
directed against the EBV lytic protein BHRF1
Elise Landais, Xavier Saulquin, Emmanuel Scotet, Lydie Trautmann, Marie-Alix Peyrat, John L.
Yates, William W. Kwok, Marc Bonneville and Elisabeth Houssaint
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