From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Functional Diversity of the CD8 1 T-Cell Response to Epstein-Barr Virus (EBV):
Implications for the Pathogenesis of EBV-Associated
Lymphoproliferative Disorders
By Rachel A. Nazaruk, Rosemary Rochford, Monte V. Hobbs, and Martin J. Cannon
Epstein-Barr virus (EBV)-specific CD81 cytotoxic T cells are
thought to be critical for the control of EBV, which persists in
healthy individuals as a latent infection of B cells. However,
recent observations have indicated that CD81 T-cell responses are not uniformly cytotoxic and that CD81 T cells
may be subdivided into type 1 and type 2 subsets that
parallel the classically described Th1 and Th2 subsets of
CD41 T cells. Using two-color flow cytometric analysis of
intracellular cytokine expression at the single-cell level, we
have identified two distinct but overlapping subsets of
EBV-specific CD81 T cells, the first of which expressed high
levels of interferon g (IFNg), but little or no interleukin-4
(IL-4), whereas the second subset was IFNg1/IL-41 doublepositive. A significant proportion of EBV-specific CD81 T cells
also expressed IL-13. Subsequent analysis of a panel of 27
EBV-specific CD81 T-cell clones showed inverse relationships
between EBV-specific cytotoxicity and secretion of IL-4,
IL-10, and IFNg, respectively. IL-10 was not secreted by the 11
most strongly cytotoxic clones, suggesting that IL-10 secretion may provide a functional definition of an EBV-specific
type 2 CD81 T-cell subset with reduced EBV-specific cytotoxicity. Finally, we have demonstrated that EBV-specific CD81
T cells that express type 2 cytokines possess the ability to
activate resting B cells. EBV-specific CD81 T cells thus have
the potential to reactivate latent EBV infection in vivo and
may contribute to the development of EBV-associated lymphoproliferative disorders and lymphoma.
r 1998 by The American Society of Hematology.
E
hypersensitivity; and are cytotoxic; whereas type 2 T cells
express IL-4, IL-5, IL-6, IL-10, and IL-13 and provide efficient
help for B cell activation, proliferation, and differentiation.18
These observations prompted us to investigate whether diversity at the levels of cytokine expression and cytotoxic function
was evident in the CD81 response to EBV. In this report,
two-color flow cytometric assays for intracellular cytokine gene
expression at the single-cell level show that EBV-specific CD81
T cells from normal adult donors uniformly express IFNg, but
also demonstrate the presence of IFNg1/IL-41 and IFNg1/IL131 subsets. Subsequent clonal analysis showed significant
inverse relationships between EBV-specific cytotoxicity and
secretion of IL-4, IL-10, and IFNg, respectively. Finally, we
also show that EBV-specific CD81 T cells that secrete type 2
cytokines are efficient activators of normal resting B cells.
PSTEIN-BARR VIRUS (EBV) is a ubiquitous human
gammaherpesvirus that persists throughout life as a latent
infection in small resting B cells. Primary infection in early
childhood is typically asymptomatic, but infection of young
adults may result in infectious mononucleosis, a self-limited
lymphoproliferative disorder characterized by fever, pharyngitis, and cervical lymphadenopathy. During latent infection of
resting B cells in vivo, viral gene expression is limited to
Epstein-Barr nuclear antigen (EBNA) 1 and latent membrane
protein (LMP) 2A.1,2 EBV transforms B cells in vitro, permitting the establishment of immortalized lymphoblastoid cell
lines (LCL). A broader set of viral latent genes is expressed in
LCL, encompassing EBNAs 1 through 6; LMP1, 2A, and 2B;
and two highly expressed but nontranslated small RNA species,
EBER 1 and 2. The EBNAs 2 through 6 and LMPs 1 and 2 are
recognized as targets by virus-specific CD81 cytotoxic T cells
(CTL).3,4 EBNA1 is not recognized by CTL by virtue of its
possession of a series of gly-ala repeat sequences that inhibit the
HLA class I antigen processing pathway.5 Lack of recognition
of EBNA1 by CTL may contribute to the ability of EBV to
persist in resting B cells.
There is a general consensus that CD81 CTL play a critical
immunosurveillance role in the control of persistent EBV
infection. EBV-specific CTL responses are impaired in cyclosporin-immunosuppressed transplant patients,6,7 who are at high
risk of developing EBV-associated posttransplant lymphoproliferative disorders (PTLD) and lymphoma.8-11 In addition, patients with established PTLD have been successfully treated by
transfer of in vitro-stimulated autologous, EBV-specific CTL,12,13
and EBV-specific CTL have been shown to inhibit development
of EBV-associated lymphomas in chimeric SCID/hu mice.14-16
Although the CD81 cytotoxic response to EBV is undoubtedly important in maintaining an asymptomatic host-virus
equilibrium, recent work has shown that the CD81 response
may not be uniformly cytotoxic.17 Furthermore, there is an
increasing body of evidence for the existence of type 1 and type
2 subsets of CD81 T cells similar, but not identical, to the Th1
and Th2 subsets of CD41 T cells.18 Broadly speaking, type 1 T
cells express interleukin-2 (IL-2), interferon g (IFNg), and
tumor necrosis factor a/b (TNFa/b); mediate delayed-type
Blood, Vol 91, No 10 (May 15), 1998: pp 3875-3883
MATERIALS AND METHODS
Human subjects. Venous blood samples were drawn from three
normal individuals, KR (HLA A2, A3, B35, B61), JTC (HLA A1, A28,
B8, B27), and MJC (HLA A1, A2, B8, B27, Cw1). Peripheral blood
lymphocytes (PBL) were separated by centrifugation over Fico/LiteLymphoH (Atlanta Biologicals, Norcross, GA), washed, and cryopreserved.
From the Department of Microbiology and Immunology, University
of Arkansas for Medical Sciences, Little Rock, AR; and the Department
of Epidemiology, University of Michigan, Ann Arbor, MI.
Submitted July 14, 1997; accepted January 8, 1998.
Supported by National Institutes of Health Grants No. CA 63931, CA
73556, and AG 09822 and by a grant from the Arkansas Science and
Technology Authority. R.R. is a Special Fellow of the Leukemia Society
of America.
Address reprint requests to Martin J. Cannon, PhD, Department of
Microbiology and Immunology, Mail Slot 511, University of Arkansas
for Medical Sciences, 4301 W Markham, Little Rock, AR 72205.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9110-0032$3.00/0
3875
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
3876
Stimulator/target cells. LCL were prepared by infection of PBL
from normal individuals with the B95-8 strain of EBV in the presence of
cyclosporin A (1.0 µg/mL). All LCL were subsequently maintained in
RPMI 1640 supplemented with 10% fetal calf serum (FCS), 3 mmol/L
glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 5 3
1025 mol/L 2-mercaptoethanol (RPMI/10). Activated normal B cells
were prepared essentially as described by Banchereau et al.19 Briefly,
PBL were depleted of T cells by panning for 2 hours over OKT3-coated
(50 µg/mL in 2 mL phosphate-buffered saline [PBS], o/n) 6-well Costar
plates (Costar Corp, Cambridge, MA). Nonadherent cells were subsequently cultured in RPMI 1640 supplemented with 10% human AB
serum (Advanced Biotechnologies Inc, Columbia, MD), 3 mmol/L
glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 5 3
1025 mol/L 2-mercaptoethanol (RPMI/10Hu) in 24-well Costar plates
(2 3 105/well) with 2 3 104/well irradiated (7,500 rad) mouse L cells
transfected with human Cdw32 (FcgRII; kindly provided by Kevin
Moore, DNAX, Palo Alto, CA), 100 ng/mL anti-CD40 (monoclonal
antibody [MoAb] 89; Immunotech Inc, Westbrook, ME), and 50 U/mL
recombinant human IL-4 (PharMingen, San Diego, CA). B cells were
fed every 3 to 4 days with fresh RPMI/10Hu plus 50 U/mL IL-4. B cells
were infected o/n with rVV at a multiplicity of injection of 10. The rVV
expressing EBV latent genes LMP2A and EBNAs 2 through 6 were
kindly provided by Mike Kurilla (University of Virginia Health
Sciences Center, Charlottesville, VA) and rVV-TK2 was provided by
Mike Mackett (University of Manchester, Manchester, UK).
T-cell lines and clones. EBV-specific human T-cell lines were
initiated by culturing PBL (2 3 106/mL) with autologous irradiated
(7,500 rad) B95-8 transformed LCL (5 3 104/mL) in RPMI/10Hu. After
9 to 11 days, T cells (2 3 105/mL) were restimulated with irradiated
LCL (2 3 105/mL). Recombinant human IL-2 (50 to 100 U/mL;
provided by the Biological Response Modifiers Program, National
Cancer Institute, Bethesda, MD) was added to the cultures at this time or
3 to 4 days after restimulation. T-cell lines were subsequently maintained by restimulation with irradiated LCL every 14 days, with interim
half-changes of fresh medium plus IL-2 every 3 to 4 days.
T-cell clones were derived by limiting dilution from established
T-cell lines. Briefly, T cells were cultured in 96-well plates (Falcon;
Becton Dickinson, Lincoln Park, NJ) at 100, 30, 10, 3, and 1 cell(s)/well
with autologous irradiated LCL (5 3 104/well) in 150 µL/well
RPMI/10Hu plus 100 U/mL IL-2. After 7 days, cultures received an
additional 100 µL/well RPMI/10Hu plus 100 U/mL IL-2. Positive wells
at limiting dilution (12 wells or fewer positive cultures per 96-well
plate) were restimulated and expanded into 24-well plates (Costar) and
subsequently maintained in 24- or 12-well plates by periodic restimulation/feeding as described above. Phenotype analysis was by flow
cytometry using a FACScan (Becton Dickinson, San Jose, CA) with
MoAbs specific for CD3 (OKT3), CD8 (OKT8), and CD4 (PE-Leu3a;
Becton Dickinson). Anti-CD3 and anti-CD8 were unconjugated MoAbs,
the binding of which was detected with fluorescein isothiocyanate
(FITC) goat antimouse Ig (Sigma, St Louis, MO).
Cytotoxic T-cell assays. Cytotoxicity was tested in a standard
chromium release assay. Targets, which included autologous and HLA
class I-mismatched LCL, NK-sensitive K562 cells, and recombinant
vaccinia virus (rVV)-infected activated normal B cells, were labeled
with Na2[51Cr]O4 for 1 hour and washed three times before incubation
in 96-well round-bottom plates (1 3 104/well; Falcon, Becton Dickinson) with effector T cells at the indicated E:T ratios for 4 to 5 hours.
Released 51Cr in the supernatants was measured with a Cobra AutoGamma counter (Packard, Meriden, CT).
Lymphoproliferation assays. T cells (1 3 104/well) were incubated
with irradiated autologous or HLA class I-mismatched LCL (5 3
104/well) in 96-well flat-bottom plates in 200 µL/well RPMI/10Hu plus
20 U/mL IL-2. After 4 days, 3H-TdR was added (1 µCi/well), and 6
hours later the plates were harvested with a Filtermate 196 cell harvester
NAZARUK ET AL
(Packard) and 3H-TdR incorporation was measured with a Matrix 96
Direct Beta Counter (Packard).
Flow cytometric analysis of intracellular cytokines. This protocol
is adapted from that described by Openshaw et al.20 EBV-specific CD81
T cells were rested for 14 days after antigen stimulation before
activation by phorbol myristate acetate (PMA) and ionomycin. Briefly,
CD81 T cells (7.5 3 105/mL) were incubated at 37°C for 6 hours in
RPMI/10Hu plus 50 ng/mL PMA, 500 ng/mL ionomycin, and 3 µmol/L
monensin (in some experiments, monensin treatment was replaced by
addition of 10 µg/mL Brefeldin A for the final 3 hours of incubation).
Control, nonactivated cultures were incubated in the presence of
monensin or Brefeldin A only. The cells were harvested, washed, and
fixed with 2% paraformaldehyde in PBS for 20 minutes at room
temperature, after which they were washed and stored overnight in PBS
at 4°C. For intracellular staining, the cells were washed and permeabilized by incubation in PBS plus 1% bovine serum albumin (BSA) and
0.5% saponin (S-7900; Sigma) for 10 minutes at room temperature. In
early experiments, activated and control cells were stained with
FITC-anti-IFNg and phycoerythrin (PE)-anti–IL-4 or PE-anti–IL-13
(all from PharMingen). However, later experiments used FITC-antiIFNg, PE-anti–IL-4, and isotype-matched controls (FITC-anti-Igg2a
and PE-anti-Igg1) from Becton Dickinson. To confirm activation, T
cells were also stained with PE-anti-CD69 (Becton Dickinson). After
staining, cells were washed twice with PBS plus 1% BSA and 0.5%
saponin, washed once with PBS plus 0.5% BSA, and fixed a second
time with 2% paraformaldehyde in PBS. Analysis was conducted with a
FACScan, using LYSIS II software and WinMDI 2.5 software (kindly
made available by Joe Trotter, The Scripps Research Institute, La Jolla,
CA).
Assays for cytokine secretion. Supernatants were harvested from T
cells incubated for 48 hours in microwells (1 3 105 T cells/well in 100
µL RPMI/10Hu) precoated with OKT3 (50 µg/mL, o/n). Supernatants
were microfuged for 5 minutes at 12,000g to remove cell debris and
stored at 220°C. Secretion of IFNg, IL-4, and IL-10 by CD81 T cells
was measured by enzyme-linked immunosorbent assay (ELISA), using
commercial assay kits (Biotrak; Amersham Corp, Arlington Heights,
IL). Cytokine levels were quantified against standard curves, in
accordance with the manufacturer’s instructions. The limits of sensitivity of these assays were less than 2 pg/mL for IL-4 and IFNg and less
than 3 pg/mL for IL-10.
Ribonuclease (Rnase) protection assay. Cytokine mRNA expression by EBV-transformed LCL was assessed by multiprobe Rnase
protection assays, essentially as described.21 Probe sets were from
PharMingen.
Measurement of B-cell activation. Purified resting B cells were
isolated from PBL by positive selection with CD19-Dynabeads (Dynal
A.S., Oslo, Norway). Briefly, 7 to 9 3 107 PBL were incubated with 4 3
107 CD19-Dynabeads in 4 mL PBS/2% FCS at 4°C for 60 minutes.
Dynabead-rosetted B cells were magnetically separated, washed five
times with PBS/2% FCS, and subsequently isolated by incubation with
200 µL PBS/2% FCS plus 60 µL CD19-Detachabead reagent (Dynal
A.S.) for 90 minutes at room temperature. Yields were generally 4 to
6 3 106 B cells of 95% to 98% purity by flow cytometric analysis with
FITC-anti-CD3/PE-anti-CD19 (Becton Dickinson). Residual T-cell
contamination was less than 1%. CD81 T-cell lines or clones (5 3
104/well) were irradiated (3,000 rad) and cocultured with B cells (5 3
104/well) in 96-well flat-bottom plates (Falcon) precoated with OKT3
(50 µg/mL in PBS, o/n). B-cell activation and proliferation were
measured by 3H-TdR incorporation after 4 days (as described above).
RESULTS
Characterization of EBV-specific polyclonal T-cell lines.
EBV-specific polyclonal T-cell lines were established from
three normal donors by stimulation of PBL with autologous
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
EBV-SPECIFIC CD81 T-CELL FUNCTION
LCL. The cell lines possessed variable proportions of CD81 T
cells, from 47% (JTC) to greater than 98% (MJC), and all
exhibited strong HLA-restricted cytotoxic function against
autologous LCL (from 34.3% specific lysis at an E:T ratio of
10:1 for JTC T cells to 53.9% lysis at the same E:T ratio for
MJC T cells), with minimal cytotoxicity against HLA class
I-mismatched LCL and NK-sensitive K562 cells. The MJC
T-cell line was further characterized as being HLA A2.1restricted, with specificity for EBV latent gene products LMP2A
(36.7% lysis at an E:T ratio of 10:1), EBNA3 (29.7% lysis), and
EBNA6 (26.5% lysis) in cytotoxicity assays against rVVinfected autologous anti-CD40–activated normal B cells. Cytotoxic specificity for EBNA2, EBNA4, and EBNA5 was not
detected; specificity for EBNA1 and LMP1 was not tested. Of
particular significance, activation of the MJC and KR CD81 T
cells with anti-CD3 (OKT3) or autologous LCL in each case
resulted in secretion of both IFNg and IL-4 detectable by
ELISA (not shown).
Intracellular cytokine expression by EBV-specific polyclonal
CD81 T cells. Whereas ELISAs showed that EBV-specific
CD81 T cells secreted IFNg and IL-4, they could not determine
whether IFNg and IL-4 were coexpressed by CD81 T cells or
whether cytokine expression segregated into discrete IFNg1/
IL-42 and IFNg2/IL-41 CD81 subsets. Recently developed
Fig 1. Two-color flow cytometric analysis of intracellular IFNg and IL-4 expression by EBV-specific
CD81 T cells. T cells were unstimulated (A, C, and E)
or stimulated for 6 hours with PMA and ionomycin
(B, D, and F), as described in Materials and Methods.
T-cell lines from three healthy adult donors were
tested: JTC (A and B), KR (C and D), and MJC (E
and F).
3877
flow cytometric techniques for detection of intracellular cytokine expression at the single-cell level enabled us to address this
question.
CD81 T cells from the JTC and KR lines were purified by
magnetic bead separation, yielding CD81 T-cell preparations
that were greater than 98% pure. The MJC T-cell line was
greater than 98% CD81 and was not subject to further
enrichment. The KR and MJC CD81 T-cell lines had been
subjected to 8 antigen-driven restimulation cycles of 14 days,
and the JTC CD81 T cells had been through 5 restimulation
cycles at the time of these assays. Subsequent clonal analysis
(see below) confirmed that a large majority of the T cells within
the lines were antigen-specific at this point. Two-color flow
cytometric analysis of intracellular IFNg and IL-4 expression
by EBV-specific activated CD81 T cells showed the presence of
two distinct but overlapping subsets: one subset that secreted
IFNg but little or no IL-4 and a second subset that secreted both
IFNg and IL-4 (Fig 1B, D, and F). Furthermore, IFNg1/IL-41
double-positive cells constituted the major subset of CD81 T
cells from donors JTC (58%) and MJC (59%). The finding that
EBV-specific CD81 T cells from all three normal donors
include significant populations of IL-4–secreting cells strongly
suggests that this phenotype is not exceptional. Nonactivated
(ie, resting) CD81 T cells from all three donors failed to stain
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
3878
NAZARUK ET AL
Fig 2. Two-color flow cytometric analysis of intracellular IFNg and IL-13 expression by EBV-specific
CD81 T cells from donor JTC. T cells were unstimulated (A) or stimulated for 6 hours with PMA and
ionomycin (B), as described in Materials and Methods.
for IFNg or IL-4, except for a small proportion (,5%) of MJC
CD81 T cells, which expressed IL-4 and may represent residual
activated cells from earlier antigen stimulation (Fig 1A, C, and
E). Similarly, FITC-anti-IgG2a and PE-anti-IgG1 isotype controls did not stain either activated or nonactivated CD81 T cells
(not shown).
Further analysis showed that EBV-specific CD81 T cells
from normal donors expressed IL-13, a type 2 cytokine that
shares many of the functional characteristics of IL-4.22 Twocolor analysis of IFNg and IL-13 expression by activated JTC
CD81 T cells (78% of which expressed intracellular IL-13) is
shown in Fig 2. Single-color analysis showed that IL-13 was
also expressed by 41% of MJC CD81 T cells and 79% of KR
CD81 T cells (not shown). In all cases, nonactivated CD81 T
cells did not express IL-13.
Correlation of CD81 T-cell phenotype with patterns of
cytokine secretion by antigen-presenting autologous LCL. Cytokines secreted by the antigen-presenting cells, in this case
EBV-transformed LCL, can exert a strong influence on the
differentiation of the responding T cells. Notably, IL-12 is a
powerful promoter of type 1 T-cell responses, whereas IL-4 and
IL-10 will inhibit type 1 responses and favor development of
type 2 responses. Using a multiprobe RNase protection assay,
we measured cytokine mRNA levels expressed by the MJC,
KR, and JTC LCL used to stimulate the EBV-specific CD81
T-cell lines. We found that the MJC and JTC LCL expressed the
p35 subunit of IL-12 at barely detectable levels and that the KR
LCL expressed the p40 subunit of IL-12, but none of the LCL
expressed both the p35 and p40 subunits required for functional
IL-12 (Fig 3). The MJC and JTC LCL expressed IL-10 mRNA,
whereas IL-10 expression by KR LCL was almost undetectable
(Fig 3). Expression of IL-10, combined with the failure of the
LCL to express IL-12, may contribute to a permissive environment for the development of CD81 T-cell responses with type 2
characteristics. We did not test the LCL for expression of IL-4, a
type 2-promoting cytokine, because IL-4 is not generally
expressed by B cells, and we have consistently failed to detect
IL-4 mRNA expression in a wide variety of LCL.23
Clonal analysis of cytotoxic function and cytokine secretion.
Functional analysis of 27 EBV-specific CD81 T-cell clones
isolated by limiting dilution from the MJC CD81 T-cell line
showed extensive diversity with regard to patterns of cytokine
secretion and cytotoxic function (summarized in Table 1). All
the clones secreted IFNg at variable but high levels (26 of 27
clones .400 pg/mL by ELISA). In contrast, levels of IL-4
secretion varied over a wide range (4 pg/mL to .400 pg/mL by
ELISA), although all clones were IL-41. This finding was
Fig 3. Ribonuclease protection assay for cytokine mRNA synthesis by EBV-transformed MJC, JTC, and KR LCL. RNA was extracted
from LCL during log-phase growth, and aliquots from 106 cellequivalents were analyzed with the hCK-2 probe set (PharMingen).
Lanes containing untreated probes (P) and mRNA-protected probes
(MJC, JTC, and KR) are shown. Ribosomal L32 and GADPH are cellular
housekeeping mRNAs. The autoradiogram was from 24 hours of
exposure.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
EBV-SPECIFIC CD81 T-CELL FUNCTION
3879
initially surprising, because analysis of intracellular cytokine
expression by the parent MJC CD81 line had indicated the
existence of an IL-42 subset (Fig 1F). Although it is possible
that our cloning conditions favor the selection of IL-4–secreting
T cells, the apparent discrepancy may simply be a reflection of
different levels of sensitivity between ELISA and flow cytometric analysis of cytokine expression. Only 10 of 25 clones
secreted IL-10 at a level detectable by ELISA. A minority of
clones exhibited little or no cytotoxicity against autologous
LCL, but showed HLA-restricted specificity for EBV in proliferation assays against autologous and HLA class I-mismatched
LCL (not shown).
Correlation of clonal cytokine production with cytotoxic
function showed a significant inverse relationship between IL-4
production and cytotoxicity (r 5 2.59, P 5 .001, from
correlation analysis based on simple linear regression; Fig 4A).
However, several highly cytotoxic clones also secreted relatively high levels of IL-4 (eg, clones 3F11 and 3D11; see Table
1). There was also a significant inverse relationship between
IL-10 production and cytotoxicity (r 5 2.63, P 5 .0008; Fig
4B); indeed, none of the top 11 most strongly cytotoxic clones
Table 1. Functional Characterization of
EBV-Specific CD81 T-Cell Clones From Donor MJC
Clone
%
Kill*
IFNg†
IL-4†
IL-10†
3bE10
3F5
3E11
3F9
10F10
3D7
10D1
3H1
3B2
10H11
3G4
3A5
3E7
3C12
3aB4
3G7
3D6
3bA3
3G1
3G2
3F2
3C1
3D11
3F11
3E8
3aH11
3H5
0.1
0.5
1.0
1.4
3.0
3.1
5.2
11.2
17.0
18.9
23.4
34.1
40.9
42.2
43.2
44.9
45.0
46.5
48.2
48.2
48.6
51.4
53.0
53.6
60.3
62.5
64.2
.5,000
.5,000
4,215 (4,150-4,280)
4,812 (4,625-5,000)
.5,000
.5,000
.5,000
.5,000
4,258
.5,000
4,476 (3,970-5,000)
1,723 (1,580-1,870)
143 (140-145)
545 (536-554)
2,528 (2,523-2,533)
884 (880-890)
2,090 (1,858-2,285)
629 (605-654)
1,420
3,047 (2,750-3,380)
403 (385-420)
2,555 (2,393-2,748)
500 (475-525)
1,439 (1,335-1,540)
745 (710-780)
829 (827-831)
498 (439-558)
145 (120-175)
249 (238-260)
61 (43-80)
.400
.400
.400
309 (295-331)
195 (186-205)
114
.400
19 (16-22)
24 (3-61)
197 (196-198)
268 (232-307)
76 (28-124)
132 (64-203)
35 (14-50)
161 (149-171)
46 (23-69)
10 (4-16)
48 (45-52)
14
145 (139-152)
170 (127-212)
94 (70-119)
53 (19-93)
32 (31-33)
156 (150-161)
0
375 (355-395)
95 (75-116)
ND
117 (107-127)
43 (40-46)
46 (7-85)
ND
104 (69-138)
0
39 (38-40)
0
0
59 (48-70)
66 (35-97)
0
0
0
0
0
0
0
0
0
0
0
Abbreviation: ND, not done.
*Percentage of cytotoxicity against autologous LCL in a 5-hour
51Cr-release assay at an effector:target ratio of 4:1.
†Cytokine titers are expressed as picograms per milliliter and are
the mean of two values or, in the case of IL-4, four values from two
independent experiments, measured by ELISA of 48-hour supernatants from anti-CD3–activated T-cell cultures, as described in Materials
and Methods. Values listed as greater than exceeded the maximum
readable ELISA titer.
expressed detectable levels of IL-10. Finally, correlation of
IFNg secretion with cytotoxicity again showed a statistically
highly significant inverse relationship (r 5 2.79, P 5 .0001;
Fig 4C). In this case, the distribution suggested two distinct
subsets, the first of which produced high levels of IFNg but was
relatively noncytotoxic, whereas the second subset was highly
cytotoxic but produced lower levels of IFNg.
Can EBV-specific CD81 T cells activate resting B cells?
The observation that EBV-specific CD81 T cells can express
IL-4, IL-10, and IL-13, all of which are regarded as type 2
cytokines and are involved in B-cell activation and proliferation, suggested that they may be capable of providing help for
B-cell activation. Accordingly, we conducted coculture assays
of purified resting B cells with irradiated EBV-specific CD81 T
cells in the presence or absence of plate-bound anti-CD3 MoAb
(OKT3). The results presented in Fig 5 show that the MJC
CD81 T-cell line and five fully characterized clones derived
therefrom (see Table 1) were all able to activate resting B cells
in 4-day microwell proliferation assays. The most potent helper
CD81 clone was 3bE10 (stimulation index of 75), which was
noncytotoxic and secreted IL-10 in addition to IL-4 (see Table
1). However, we did not observe a correlation between the
magnitude of B-cell activation and the level of IL-4 expression
by the MJC CD81 clones; it is possible that the level of IL-13
expression (which was not assessed) may also make a significant contribution to the level of B-cell activation. Four EBVspecific CD81 T-cell clones from donor JTC were also tested for
their ability to activate resting B cells. These clones exhibited
uniformly low cytotoxic function against autologous JTC LCL
(mean of 6.5% specific lysis at an E:T ratio of 4:1), but mounted
strong HLA-restricted lymphoproliferative responses (not
shown). All four clones, when stimulated with plate-bound
anti-CD3 MoAb, were efficient activators of resting B cells,
furnishing B-cell stimulation indices greater than 100 (not
shown). The cytokine profiles for the JTC CD81 clones were
not determined.
For both the MJC and the JTC CD81 T-cell clones, B-cell
proliferation in the absence of plate-bound anti-CD3 was
minimal, indicating that the response was strongly dependent
on concomitant activation of the T cells. Control wells of
irradiated T cells only, in the presence or absence of anti-CD3,
showed no evidence of 3H-TdR uptake. Supernatants from
48-hour cultures of activated T cells failed to activate resting B
cells, indicating that helper function was contact dependent (not
shown). EBV-specific CD81 T cells express low levels of gp39
(CD40) ligand upon activation by PMA/ionomycin, but the
extent to which B-cell activation is dependent on gp39 expression by CD81 T cells is not known; these studies are in progress.
DISCUSSION
CD81
The
T cell has long been regarded as an MHC class
I-restricted effector cell that secretes IFNg and TNFa/b and is
predominantly cytotoxic, most notably against virus-infected
target cells. However, recent reports have lent substance to the
proposal that CD81 T-cell responses exhibit considerable
functional diversity and may be assigned to type 1 and type 2
subsets that broadly parallel the classical Th1 and Th2 subsets
of CD41 T cells.18,24-27 In this report, we present evidence that
the CD81 T-cell response to EBV is also functionally diverse
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
3880
NAZARUK ET AL
Fig 4. Patterns of EBV-specific cytotoxicity and cytokine secretion
by 27 EBV-specific CD81 T-cell clones from donor MJC. (A) Correlation
of cytotoxicity and IL-4 secretion (r 5 2.59, P F .001). (B) Correlation of
cytotoxicity and IL-10 secretion (r 5 2.63, P F .0008). (C) Correlation of
cytotoxicity and IFNg secretion (r 5 2.79, P F .0001). Cytotoxicity was
measured in a 5-hour 51Cr-release assay at an effector:target ratio of 4:1.
Cytokine secretion was measured by ELISA of 48-hour supernatants
from anti-CD3–activated T-cell cultures, as described in Materials and
Methods.
and that CD81 T-cell effector functions are not limited to
virus-specific cytotoxicity.
Two-color flow cytometric analysis of intracellular cytokine
expression at the single-cell level by EBV-specific polyclonal
CD81 T cells showed that IFNg expression was a common
denominator and further showed the existence of CD81 T-cell
subsets that expressed both IFNg and IL-4. For two of three
donors, the IFNg1/IL-41 subset was the major subset (58% of
JTC CD81 T cells and 59% of MJC CD81 T cells). A high
frequency of IFNg1/IL-41 double-positive CD81 T cells was
associated with expression of IL-10 by the antigen-presenting
LCL, suggesting that cytokine expression by the LCL may, at
least in part, influence the phenotype of the responding T cells.
We also found that EBV-specific CD81 T cells expressed IL-13,
in two instances (JTC and KR) at a higher frequency than IL-4
(78% and 79% IL-131 v 58% and 28% IL-41, respectively).
IL-13 shares many of the characteristics of IL-4, including the
ability to induce proliferation and differentiation of B cells
activated by T-cell gp39 (CD40 ligand) interaction with B-cell
CD40.28 In this context, it is notable that IL-13 expression is
enhanced by cyclosporin A (CsA), whereas IL-4 expression is
inhibited.29 IL-13 expression by EBV-specific CD81 T cells
may thus contribute to B-cell activation and potential EBV
reactivation from latency in CsA-immunosuppressed transplant
patients and may consequently play a role in the development of
EBV-associated oligoclonal and polyclonal posttransplant
lymphoproliferative disorders.
Although analysis of intracellular cytokine expression provided strong evidence for functional diversity within the CD81
T-cell response to EBV, with a significant proportion of
responding cells capable of expressing type 2 cytokines, the
possibility remained that the IL-4– and IL-13–expressing CD81
T cells were no more than nonspecific bystanders and that
specificity for EBV resided solely within the IFNg1/IL-42
subset, which has a classical type 1 phenotype and would be
expected to be strongly cytotoxic. To address this problem and
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
EBV-SPECIFIC CD81 T-CELL FUNCTION
Fig 5. Activation of resting B cells by an EBV-specific CD81 T-cell
line from donor MJC (P10) and five EBV-specific CD81 T-cell clones
(3aH11, 3bE10, 3C12, 3E8, and 3F11) derived from the MJC CD81 T-cell
line. Small resting B cells were purified from peripheral blood by
positive selection with anti-CD19–coupled magnetic beads, as described in Materials and Methods, and cultured for 4 days with
g-irradiated T cells in the presence (7) or absence (h) of plate-bound
anti-CD3. Stimulation indices were calculated as the ratio of 3H-TdR
uptake by B cells cultured with T cells divided by 3H-TdR uptake by B
cells cultured without T cells. Background counts (typically F100
cpm) by control wells of irradiated T cells were subtracted before
calculation of stimulation indices for B-cell proliferation.
to gain a correlation of cytotoxic function with patterns of
cytokine synthesis, we undertook an extensive clonal analysis
of the CD81 T-cell response to EBV. Functional analysis of 27
EBV-specific CD81 T-cell clones showed wide variability in
cytotoxicity against autologous LCL. Those clones that exhibited little or no cytotoxicity were nevertheless found to mount
strong HLA-restricted proliferative responses against autologous LCL, thus confirming the specificity of the response. It is
not known whether these clones fail to lyse autologous LCL by
virtue of low TCR affinity, as described by Hill et al,17 or
whether noncytotoxicity is related to CD81 T-cell type 2 subset
differentiation; the two possibilities are not mutually exclusive.
A significant inverse relationship between cytotoxicity and
the level of IL-4 secretion was observed, although all the clones
secreted IL-4 detectable by ELISA. In contrast, only 10 of 25
clones tested secreted IL-10 upon activation, with the 11 most
strongly cytotoxic clones failing to secrete detectable levels of
IL-10. These results suggest that IL-10 secretion, rather than
IL-4 secretion, may more accurately define a type 2 CD81
T-cell response with reduced cytotoxic function against EBV, a
conclusion that is consistent with the finding that IL-10 is a
powerful modulator of type 1 T-cell responses, through inhibition of IL-12 synthesis by accessory cells.30-32 A similar inverse
relationship between IL-10 secretion and cytotoxic function has
been described for mouse CD81 T cells. Inoue et al33 note that
functionally assigned suppressor CD81 T-cell clones produced
3881
IL-10 upon stimulation with anti-CD3, whereas IL-10 was not
expressed by CD81 cytotoxic T-cell clones.
All EBV-specific CD81 T-cell clones secreted IFNg upon
activation, but we found a strong and statistically highly
significant inverse relationship between IFNg secretion and
cytotoxicity. Two distinct subsets were observed, the first of
which expressed high levels of IFNg but was relatively
noncytotoxic, whereas the second subset was highly cytotoxic
but produced lower levels of IFNg. Although unexpected, in
view of the known cross-regulatory functions of IFNg and
IL-10,32,34 the coincidence of high IFNg and IL-10 secretion by
human CD81 clones has also been described by others.35
Since the discovery of polarized Th1 (type 1) and Th2 (type
2) subsets of CD41 T cells, there has been a long-standing
practice of pigeonholing T-cell responses, and particularly
T-cell clones, as being of a type 1 or a type 2 phenotype.
Although our intracellular cytokine assays on polyclonal EBVspecific CD81 T cells suggest the existence of discrete, but
overlapping, subsets, clonal analysis clearly shows that many
intermediate and apparently contradictory phenotypes exist.
Classical type 1 and type 2 T-cell subsets have previously been
defined by exclusive expression of IFNg or IL-4, respectively,
but simultaneous expression of IL-4 and IFNg by human CD41
and CD81 T cells has been described by several investigators,24,26,36 and all 27 of the MJC CD81 T-cell clones described
in this report secreted both IFNg and IL-4. Furthermore, four of
the five MJC CD81 clones shown to activate resting B cells (a
type 2 T-cell function) were also strongly cytotoxic (a characteristic of type 1 T cells).
The observation that EBV-specific CD81 T cells can provide
help for activation of resting B cells is the major finding of this
report. As the site of EBV latency in vivo is the resting B cell, it
is probable that activation and subsequent differentiation of
latently infected B cells will result in reactivation of EBV lytic
cycle replication.37,38 Experiments in SCID mice engrafted with
human PBL (SCID/hu mice), showing that the presence of T
cells is required for the development of EBV-induced lymphoproliferative disease and tumors,39,40 strongly support the proposal that T-cell help for B-cell activation is a necessary and
critical step in EBV pathogenesis. Furthermore, the work of
Veronese et al39 demonstrated that CD81 T cells were capable of
fulfilling the necessary helper function for development of
EBV-induced tumors in SCID/hu mice, an observation that
supports the proposal that helper, or type 2, CD81 T cells are
capable of playing a significant role in the pathogenesis of
EBV-associated disease. Expression of IL-10 by EBV-specific
CD81 T cells may further contribute to the pathogenesis of
EBV-associated lymphoproliferative disorders. In addition to
IL-4 and IL-13, both of which promote B-cell activation and
proliferation, IL-10 is a potent growth and differentiation factor
for activated B cells41 and has recently been described as a
growth factor for EBV-transformed LCL.42 Moreover, expression of type 2 cytokines, particularly IL-10, may antagonize
type 1 T-cell responses30-32,34 and thus modulate cytotoxic T-cell
control of latent EBV infection.
On the basis of these findings, we would argue that the
conventional view of the role of CD81 T cells in EBV infection
merits reappraisal. In contrast with the consensus that EBVspecific CD81 T cells are primarily cytotoxic and fulfill an
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
3882
NAZARUK ET AL
immunosurveillance role, our results have raised the possibility
that EBV-specific CD81 T cells may also contribute to the
pathogenesis of EBV disease. Because the resting B cell is the
principal site of EBV latency37,38,43 and B-cell activation leads
to viral lytic cycle activation, we propose that EBV-specific type
2 CD81 T-cell responses have the potential to reactivate latent
viral infection and may thus play a major role in the development of EBV-associated lymphoproliferative disorders and
lymphoma in immunocompromised individuals with impaired
EBV-specific cytotoxic T-cell responses. Analysis of the phenotype and function of EBV-specific CD81 T-cell responses in the
various lymphoproliferative diseases and lymphomas associated with EBV may thus contribute to our understanding of the
pathogenesis and immune control of these disorders.
ACKNOWLEDGMENT
The authors thank Melaney Gee and Jim Crouch for skilled technical
assistance. Statistical analysis was performed by Dan Ayers (Biostatistics Office, Arkansas Cancer Research Center, Little Rock, AR).
REFERENCES
1. Qu L, Rowe DT: Epstein-Barr virus latent gene expression in
uncultured peripheral blood lymphocytes. J Virol 66:3715, 1992
2. Tierney RJ, Steven N, Young LS, Rickinson AB: Epstein-Barr
virus latency in blood mononuclear cells: Analysis of viral gene
transcription during primary infection and in the carrier state. J Virol
68:7374, 1994
3. Khanna R, Burrows SR, Moss DJ: Immune regulation in EpsteinBarr virus-associated diseases. Microbiol Rev 59:387, 1995
4. Rickinson AB, Lee SP, Steven NM: Cytotoxic T lymphocyte
responses to Epstein-Barr virus. Curr Opinion Immunol 8:492, 1996
5. Levitskaya J, Coram M, Levitsky V, Imreh S, Steigerwald-Mullen
PM, Klein G, Kurilla MG, Masucci MG: Inhibition of antigen
processing by the internal repeat region of the Epstein-Barr virus
nuclear antigen-1. Nature 375:685, 1995
6. Crawford DH, Sweny P, Edwards JM, Janossy G, Hoffbrand AV:
Long-term T-cell-mediated immunity to Epstein-Barr virus in renalallograft recipients receiving cyclosporin A. Lancet 1:10, 1981
7. Gaston JSH, Rickinson AB, Epstein MA: Epstein-Barr virusspecific T-cell memory in renal-allograft recipients under long-term
immunosuppression. Lancet 1:923, 1982
8. Hanto DW, Frizzera G, Gajl-Peczalska KJ, Simmons RL: EpsteinBarr virus, immunodeficiency, and B cell lymphoproliferation. Transplantation 39:461, 1985
9. Randhawa PS, Yousem SA, Paradis IL, Dauber JA, Griffith BP,
Locker J: The clinical spectrum, pathology, and clonal analysis of
Epstein-Barr virus-associated lymphoproliferative disorders in heartlung transplant patients. Am J Clin Pathol 92:177, 1989
10. Wilkinson AH, Smith JL, Hunsicker LG, Tobacman J, Kapelanski DP, Johnson M, Wright FH, Behrendt DM, Corry RJ: Increased
frequency of posttransplant lymphomas in patients treated with cyclosporine, azathioprine, and prednisone. Transplantation 47:293, 1989
11. Nalesnik M: Posttransplantation lymphoproliferative disorders:
Current perspectives. Semin Thoracic Cardiovasc Surg 8:139, 1996
12. Rooney CM, Smith CA, Ng CYC, Loftin S, Li C, Krance RA,
Brenner MK, Heslop HE: Use of gene-modified virus-specific T
lymphocytes to control Epstein-Barr virus-related lymphoproliferation.
Lancet 345:9, 1995
13. Heslop HE, Ng CYC, Li C, Smith CA, Loftin SK, Krance RA,
Brenner MK, Rooney CM: Long-term restoration of immunity against
Epstein-Barr virus infection by adoptive transfer of gene-modified
virus-specific T lymphocytes. Nat Med 2:551, 1996
14. Boyle TJ, Berend KR, DiMaio JM, Coles RE, Via DF, Lyerly
HK: Adoptive transfer of cytotoxic T lymphocytes for the treatment of
transplant-associated lymphoma. Surgery 114:218, 1993
15. Rencher SD, Slobod KS, Smith FS, Hurwitz JL: Activity of
transplanted CD81 versus CD41 cytotoxic T cells against Epstein-Barr
virus-immortalized B cell tumors in SCID mice. Transplantation
58:629, 1994
16. Lacerda JF, Ladanyi M, Louie DC, Fernandez JM, Papadopoulos
EB, O’Reilly RJ: Human Epstein-Barr virus (EBV)-specific cytotoxic T
lymphocytes home preferentially to and induce selective regressions of
autologous EBV-induced B cell lymphoproliferations in xenografted
C.B.-17 scid/scid mice. J Exp Med 183:1215, 1996
17. Hill AB, Lee SP, Haurum JS, Murray N, Yao Q-Y, Rowe M,
Signoret N, Rickinson AB, McMichael AJ: Class I major histocompatibility complex-restricted cytotoxic T lymphocytes specific for EpsteinBarr virus (EBV) nuclear antigens fail to lyse the EBV-transformed B
lymphoblastoid cell lines against which they were raised. J Exp Med
181:2221, 1995
18. Carter L, Dutton RW: Type 1 and type 2: A fundamental
dichotomy for all T-cell subsets. Curr Opinion Immunol 8:336, 1996
19. Banchereau J, de Paoli P, Valle A, Garcia E, Rousset F:
Long-term human B cell lines dependent on interleukin-4 and antibody
to CD40. Science 251:70, 1991
20. Openshaw PJM, Murphy EE, Hosken NA, Maino V, Davis K,
Murphy K, O’Garra A: Heterogeneity of intracellular cytokine synthesis
at the single cell level in polarized T helper 1 and T helper 2
populations. J Exp Med 182:1357, 1995
21. Hobbs MV, Weigle WO, Noonan DJ, Torbett BE, McEvilly RJ,
Koch RJ, Cardenas GJ, Ernst DN: Patterns of cytokine gene expression
by CD41 T cells from young and old mice. J Immunol 150:3602, 1993
22. De Vries JE: Molecular and biological characteristics of interleukin-13. Chem Immunol 63:204, 1996
23. Rochford R, Cannon MJ, Sabbe RE, Picchio G, Noonan DJ,
Mosier DE, Hobbs MV: Common and idiosyncratic patterns of cytokine
gene expression by Epstein-Barr virus transformed human B cell lines.
Viral Immunol 10:183, 1997
24. Salgame P, Abrams JS, Clayberger C, Goldstein H, Convit J,
Modlin RL, Bloom BR: Differing lymphokine profiles of functional
subsets of human CD4 and CD8 T cell clones. Science 254:279, 1991
25. Maggi E, Giudizi MG, Biagiotti R, Annunziato F, Manetti R,
Piccinni M-P, Parronchi P, Sampognaro S, Giannarini L, Zuccati G,
Romagnani S: Th2-like CD81 T cells showing B cell helper function
and reduced cytolytic activity in human immunodeficiency virus type 1
infection. J Exp Med 180:489, 1994
26. Paganelli R, Scala E, Ansotegui IJ, Ausiello CM, Halapi E,
Fanales-Belasio E, D’Offizi G, Mezzaroma I, Pandolfi F, Fiorilli M,
Cassone A, Aiuti F: CD81 T lymphocytes provide helper activity for
IgE synthesis in human immunodeficiency virus-infected patients with
Hyper-IgE. J Exp Med 181:423, 1995
27. Cronin DC II, Stack R, Fitch FW: IL-4-producing CD81 T cell
clones can provide B cell help. J Immunol 154:3118, 1995
28. Cocks BG, de Waal Malefyt R, Galizzi J-P, de Vries JE, Aversa
G: IL-13 induces proliferation and differentiation of human B cells
activated by the CD40 ligand. Int Immunol 5:657, 1993
29. Van der Pouw Kraan TCTM, Boeije LCM, Troon JTM, Rutschmann SK, Wijdenes J, Aarden LA: Human IL-13 production is
negatively influenced by CD3 engagement. Enhancement of IL-13
production by cyclosporin A. J Immunol 156:1818, 1996
30. Hsieh C, Macatonia SE, Tripp CS, Wolf SF, O’Garra A, Murphy
KM: Development of TH1 CD41 T cells through IL-12 produced by
Listeria-induced macrophages. Science 260:547, 1993
31. D’Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M,
Trinchieri G: Interleukin-10 inhibits human lymphocyte IFN-g production by suppressing natural killer cell stimulatory factor/interleukin-12
synthesis in accessory cells. J Exp Med 178:1041, 1993
32. Trinchieri G: Cytokines acting on or secreted by macrophages
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
EBV-SPECIFIC CD81 T-CELL FUNCTION
during intracellular infection (IL-10, IL-12, IFN-g). Curr Op Immunol
9:17, 1997
33. Inoue T, Asano Y, Matsuoka S, Furutani-Seiki M, Aizawa S,
Nishimura H, Shirai T, Tada T: Distinction of mouse CD81 suppressor
effector T cell clones from cytotoxic T cell clones by cytokine
production and CD45 isoforms. J Immunol 150:2121, 1993
34. Moore KW, O’Garra A, de Waal Malefyt R, Vieira P, Mosman
TR: Interleukin 10. Annu Rev Immunol 11:165, 1993
35. Gerosa F, Paganin C, Peritt D, Paiola F, Scupoli MT, AsteAmezaga M, Frank I, Trinchieri G: Interleukin-12 primes human CD4
and CD8 T cell clones for high production of both interferon-g and
interleukin-10. J Exp Med 183:2559, 1996
36. Paliard X, de Waal Malefijt R, Yssel H, Blanchard D, Chretien I,
Abrams J, de Vries J, Spits H: Simultaneous production of IL-2, IL-4,
and IFN-g by activated human CD41 and CD81 T cell clones. J
Immunol 141:849, 1988
37. Miyashita EM, Yang B, Lam, KMC, Crawford DH, ThorleyLawson DA: A novel form of Epstein-Barr virus latency in normal B
cells in vivo. Cell 80:593, 1995
38. Thorley-Lawson DA, Miyashita EM, Khan G: Epstein-Barr
virus and the B cell: That’s all it takes. Trends Microbiol 4:204, 1996
3883
39. Veronese ML, Veronesi A, D’Andrea E, Del Mistro A, Indraccolo S, Mazza MR, Mion M, Zamarchi R, Menin C, Panozzo M,
Amadori A, Chieco-Bianchi L: Lymphoproliferative disease in human
peripheral blood mononuclear cell-injected SCID mice. I. T lymphocyte
requirement for B cell tumor generation. J Exp Med 176:1763, 1992
40. Coles RE, Boyle TJ, DiMaio JM, Berend KR, Via DF, Lyerly
HK: T cells or active Epstein-Barr virus infection in the development of
lymphoproliferative disease in human B cell-injected severe combined
immunodeficient mice. Ann Surg Oncol 1:405, 1994
41. Rousset F, Garcia E, DeFrance T, Peronne C, Vezzio N, Hsu D,
Kastelstein R, Moore KW, Banchereau J: Interleukin 10 is a potent
growth and differentiation factor for activated human B lymphocytes.
Proc Natl Acad Sci USA 89:1890, 1992
42. Beatty PR, Krams SM, Martinez OM: Involvement of IL-10 in
the autonomous growth of EBV-transformed cell lines. J Immunol
158:4045, 1997
43. Miyashita EM, Yang B, Babcock GJ, Thorley-Lawson DA:
Identification of the site of Epstein-Barr virus persistence in vivo as a
resting B cell. J Virol 71:4882, 1997
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1998 91: 3875-3883
Functional Diversity of the CD8+ T-Cell Response to Epstein-Barr Virus
(EBV): Implications for the Pathogenesis of EBV-Associated
Lymphoproliferative Disorders
Rachel A. Nazaruk, Rosemary Rochford, Monte V. Hobbs and Martin J. Cannon
Updated information and services can be found at:
http://www.bloodjournal.org/content/91/10/3875.full.html
Articles on similar topics can be found in the following Blood collections
Immunobiology (5489 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.