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EBV-Specific CD8+ T Cells from
Asymptomatic Pediatric Thoracic Transplant
Patients Carrying Chronic High EBV Loads
Display Contrasting Features: Activated
Phenotype and Exhausted Function
Camila Macedo, Steven A. Webber, Albert D. Donnenberg,
Iulia Popescu, Yun Hua, Michael Green, David Rowe,
Louise Smith, Maria M. Brooks and Diana Metes
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2011 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2011; 186:5854-5862; Prepublished online 1
April 2011;
doi: 10.4049/jimmunol.1001024
http://www.jimmunol.org/content/186/10/5854
The Journal of Immunology
EBV-Specific CD8+ T Cells from Asymptomatic Pediatric
Thoracic Transplant Patients Carrying Chronic High EBV
Loads Display Contrasting Features: Activated Phenotype
and Exhausted Function
Camila Macedo,*,† Steven A. Webber,‡ Albert D. Donnenberg,x Iulia Popescu,*,†
Yun Hua,*,† Michael Green,‡ David Rowe,{ Louise Smith,‡ Maria M. Brooks,{
and Diana Metes*,†,‖
pstein–Barr virus is a g-herpes virus that infects .90% of
healthy adults worldwide (1, 2). The virus persists in the
B lymphocyte pool as a lifelong asymptomatic latent
infection that can undergo occasional reactivations into the lytic
state with production of infectious virions (viral recrudescence) (1,
2). EBV infection triggers effective memory type 1 (IFN-g, TNFa, cytotoxicity) T cell responses specific for both EBV-lytic and
-latent Ags that maintain the virus under tight immunologic
control with no detectable viral load in the peripheral circulation
in most healthy individuals (3, 4). Although CD4+ T cells provide
“help” during priming and memory reactivation of antiviral CD8+
T cells, the latter compartment plays the pivotal protective role in
E
*Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center,
Pittsburgh, PA 15213; †Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213; ‡Children’s Hospital of Pittsburgh, University of Pittsburgh
Medical Center, Pittsburgh, PA 15213; xDepartment of Medicine, University of Pittsburgh, Pittsburgh, PA 15213; {Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15213; and ‖ Department of Immunology, University of
Pittsburgh, Pittsburgh, PA 15213
Received for publication April 1, 2010. Accepted for publication March 4, 2011.
This work was supported by an SCCOR grant awarded by the National Heart, Lung,
and Blood Institute (5 P50 HL074732-03).
Address correspondence and reprint requests to Dr. Diana Metes, Thomas E. Starzl
Transplantation Institute, E 1549 Thomas E. Starzl Biomedical Tower, 200 Lothrop
Street, Pittsburgh, PA 15213. E-mail address: [email protected]
Abbreviations used in this article: HC, healthy control; HVL, high viral load; LVL,
low viral load; PD-1, programmed death 1; PTLD, posttransplant lymphoma; TCM,
central memory T cell; TEM, effector memory T cell; TEMRA, terminally differentiated effector memory T cell; TMR, tetramer; Tx, transplant; UVL, undetectable viral
load.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001024
EBV immunosurveillance against episodic lytic viral reactivation,
as well as for latent infection control (5–8).
In acquired immunodeficiencies, such as posttransplant (postTx) immunosuppression, EBV T cellular immune surveillance
is impaired, resulting in increased incidence of EBV-associated
posttransplant lymphomas (PTLDs) with high mortality (9–13).
Pediatric patients are at greatest risk for EBV complications
among Tx patients, especially when they are EBV2 pre-Tx (lacking EBV memory T cells) and become EBV+ post-Tx under immunosuppression (11, 13). Serial long-term EBV monitoring by
PCR in the peripheral blood of pediatric thoracic Tx patients
who EBV seroconverted after transplantation has identified three
groups of clinically asymptomatic children: ∼30% who exhibit
undetectable (,100 copies/ml) EBV loads (UVL), resembling
“normal” EBV latency; approximately 50% display persistent low
(100–16,000 copies/ml) viral loads (LVL), whereas ∼20% show
persistent, dangerously high (.16,000 copies/ml) EBV loads
(HVL) in peripheral blood for months to years after primary
post-Tx EBV infection, a status not encountered in immunocompetent subjects (14, 15). We have recently shown that asymptomatic HVL carriers had a 45% risk for progression to aggressive
late-onset PTLD, including diffuse large B cell, Hodgkin’s, and
Burkitt’s lymphomas (16). Characterization of CD8+ T cells
profiles and of their dynamics in asymptomatic pediatric Tx
patients should clarify, at least in part, the immunopathogenesis
that associates with the shift from EBV latency to the persistent
HVL carrier state, which may help clinicians develop better prophylactic strategies.
Hierarchical and progressive loss of type 1 CD8+ T cell function
is a hallmark of T cell exhaustion during chronic viral infections
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Serial EBV load monitoring of clinically asymptomatic pediatric thoracic organ transplant patients has identified three groups of
children who exhibit undetectable (,100 copies/ml), chronic low (100–16,000 copies/ml), or chronic high (>16,000 copies/ml) EBV
loads in peripheral blood. Chronic high EBV load patients have a 45% rate of progression to late-onset posttransplant lymphoproliferative disorders. In this article, we report that asymptomatic patients carrying EBV loads (low and high) expressed
increased frequencies of EBV-specific CD8+ T cells, as compared with patients with undetectable EBV loads. Although patients
with low viral load displayed EBV-specific CD8+ T cells with moderate signs of activation (CD38+/2/CD127+/2), programmed
death 1 upregulation and effective IFN-g secretion, high EBV load carriers showed significant CD38+ upregulation, features of
cellular exhaustion (programmed death 1+/CD1272) accompanied by a decline in IFN-g release. Immunopolarization of EBVspecific CD8+ T cells was skewed from the expected type 1 (IFN-g) toward type 0 (IFN-g/IL-5) in patients, and Tr1 (IL-10) in high
load carriers. These results indicate the importance of chronic EBV load and of the levels of antigenic pressure in shaping EBVspecific memory CD8+ T cells. Concomitant phenotypic and functional EBV monitoring is critical for identifying the complex
“functional” versus “exhausted” signature of EBV-specific CD8+ T cells, with implications for immunologic monitoring in the
clinic. The Journal of Immunology, 2011, 186: 5854–5862.
The Journal of Immunology
Materials and Methods
Human subjects and PBMC isolation
Forty-four asymptomatic pediatric thoracic Tx patients and 11 healthy
control (HC) volunteers were consented under Institutional Review Boardapproved protocols at the University of Pittsburgh and analyzed in a crosssectional study with additional longitudinal observations in 70% of patients
over a period of 3 y. Patient demographics and immunosuppressive regimens are shown in Table I. All patients were EBV+ at the time of blood
donation, as confirmed by serology, and were also positive for HLA-A02
and/or HLA-B08 allele. Patients were divided into three groups according
to their peripheral blood EBV loads determined by PCR, regardless of their
pre-Tx EBV status (see below). Maintenance immunosuppression was
comparable for all three cohorts of patients, and consisted of a combination
of a calcineurin inhibitor (tacrolimus or cyclosporine) and an antiproliferative agent (mycophenolate mofetil) with or without the use of lowdose prednisone. Nine patients received induction therapy with polyclonal
anti-T cell Abs (Thymoglobulin or ATGAM). Five to ten milliliters of
heparinized whole blood was collected from each subject and used directly
in flow cytometry analysis, whereas PBMCs were isolated by density gradient centrifugation (32) and banked frozen for functional assays.
Definition of EBV load groups
Patients were categorized into three groups according to their peripheral
whole blood EBV load levels as follows: 1) UVL carriers with no EBV load
detected by PCR in .80% of determinations including the time of analysis;
2) chronic LVL carriers with EBV loads ranging between 100 and 16,000
genomic copies/ml blood, detected in .20% of measurements, including
the time of analysis; and 3) chronic HVL carriers with EBV loads .16,000
genomic copies/ml blood on at least 50% of determinations, and over
a period of at least 6 mo before the current immunologic analyses (33).
Synthetic peptides and HLA class I/peptide TMRs
EBV-derived peptides were synthesized at the Peptide Synthesis Facility at
University of Pittsburgh: 1) BMLF1 (GLCTLVAML)-lytic cycle and LMP2a
(CLGGLLTMV)-latent cycle peptides restricted by HLA-A02+; and 2)
BZLF1 (RAKFKQLL)-lytic cycle and EBNA3A (FLRGRAYGL)-latent
cycle peptides restricted by HLA-B08+. These peptides were used to
stimulate EBV-specific CD8+ T cells in ELISPOT assays at a final concentration of 10 mg/ml. (PE)-HLA-A02+ TMRs incorporating HLA-A02–
restricted peptides were purchased from Beckman Coulter (Fullerton, CA),
whereas (PE)-HLA-B08+ TMRs incorporating HLA-B08–restricted peptides were generated at the National Institute of Allergy and Infectious
Diseases MHC-Tetramer Core Facility (Emory University, Atlanta, GA).
Media and reagents
RPMI 1640 was supplemented with 2 mM L-glutamine, 10 mM HEPES,
100 IU/ml penicillin/streptomycin, and 10% heat-inactivated FCS (all from
Life Technologies BRL, Grand Island, NJ) and referred to as complete
media. BSA, PMA, and ionomycin were purchased from Sigma (St. Louis,
MO).
Flow cytometry
Whole blood (100 ml/tube) was incubated with a mixture of mAbs including CD62L-FITC (Beckman Coulter), CD45RO-FITC, CD45RA-allophycocyanin, CD8-PerCp, CD38-PE-Cy7, CD152-allophycocyanin (BD,
San Jose, CA), CD127-Pacific blue, PD-1–FITC (eBioscience, San Diego,
CA), and a given EBV-specific TMR for 30 min in the dark at room temperature. Isotype controls and a negative TMR (Beckman Coulter) were used
as negative control. Cells were then incubated with 2 ml/tube of 1X lysing
buffer (BD) for an additional 10 min at room temperature to allow RBCs
lysis. Tubes were washed twice and fixed with 1% paraformaldehyde. All
events were collected using an LSR II (BD) flow cytometer and analyzed
with Diva software (BD) or FlowJo (Tree Star, Ashland, OR).
IFN-g and IL-5 ELISPOT assays
ELISPOT assays were performed as previously described (34). In brief, 96well plates (Millipore, Bedford, MA) were precoated with anti-human
IFN-g (10 mg/ml; Mabtech, Sweden) or anti-human IL-5 (10 mg/ml; BD
Pharmingen) mAbs in PBS (Cellgro, Herndon, VA) overnight at 4˚C.
Plates were washed and PBMCs were seeded at 3 3 105/well with correspondent EBV-peptide (10 mg/ml) at 37˚C and 5% CO2 for 24 (IFN-g)
and 48 h (IL-5). PMA (5 ng/ml) plus ionomycin (100 ng/ml) was used as
positive control (1 3 105cells/well). The wells were washed, and a secondary biotinylated anti-human IFN-g mAb (2 mg/ml; Mabtech) or biotinylated anti-human IL-5 (2 mg/ml; BD Pharmingen) mAbs were added
for an additional 2 or 4 h, respectively, at 37˚C. The reaction was developed with 3-amino-9-ethylcarbazole substrate (Sigma) for IFN-g and
tetramethylbenzidine substrate (KPL Laboratories, Gaithersburg, MD) for
IL-5. Spots were counted with an ELISPOT plate reader (Immunospot;
Cellular Technology, Cleveland, OH).
Determination of IL-10 levels by ELISA
Culture supernatants from ELISPOT assays were collected and levels of IL10 quantified by ELISA. Primary and secondary mAbs, and the recombinant
cytokine for IL-10 ELISA were purchased from Pierce Endogen (Rockford,
IL). The lower limits of detection for this assay were 60 pg/ml.
Statistical analysis
The distributions of all laboratory variables were examined, and the logarithm transformation was applied when data were approximately lognormally distributed. For analyses involving one observation per patient,
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in mice and humans, with IFN-g production being the last function lost (17, 18). The programmed death 1 (PD-1) molecule,
a member of the CD28/B7 family, has been shown to critically
regulate T cell activation by delivering inhibitory signals that
suppress TCR signaling and function (19). During chronic LCMV
infection in mice and HIV and HCV infections in humans, PD-1 is
upregulated on CD8+ T cells and is instrumental in inhibiting
virus-specific CD8+ T cell proliferation, cytokine production, and
cytolytic activity, leading to cellular exhaustion (20–23). However, recent studies in HIV and HCV patients have concluded that
a PD-1 upregulation on CD8+ T cells can be either a sign of
physiologic T cell activation during viral infection control or can
associate with T cell exhaustion (24, 25). Therefore, other molecules in addition to PD-1 should be monitored to accurately
identify the Ag-specific CD8+ T cell status. For example, CD127
(IL-7Ra) is an important receptor for T cell survival and memory
differentiation, and is shown to significantly downmodulate during
persistent HVL infections and to inversely correlate with the state
of CD8+ T cell exhaustion (26–28). CTLA-4 is yet another inhibitory molecule that can be upregulated on T cells during
chronic viral infections, but its contribution to CD8+ T cell exhaustion is less well understood (21, 29).
Although the expression of PD-1 has been documented on EBVspecific CD8+ T cells from subjects undergoing acute infectious
mononucleosis and during resolution (30), as well as from HIVand HCV-infected subjects (23, 31), no studies were performed on
EBV-specific CD8+ T cells from asymptomatic pediatric thoracic
Tx patients to clarify the significance of chronic EBV load, PD-1
upregulation, and CD8+ T cell functional state. In addition, most
of the previous immunologic studies in Tx patients monitored few
parameters at a time (7, 8), which may be insufficient for an accurate understanding of the complex Ag-specific CD8+ T cell
differentiation state at a given time. In this study, we have used
tetramer (TMR) technology to detect EBV-specific CD8+ T cells,
concomitantly incorporating several memory (CD45RA, CD62L)
and activation/differentiation (CD38, CD127, PD-1) markers in
flow cytometry. We have also monitored the EBV-specific functional (IFN-g, IL-5, and IL-10) parameters for a comprehensive
definition of immunologic “signatures” in these asymptomatic
pediatric Tx patients in relation to their EBV load levels.
Our results indicate that chronic EBV-Ag–specific stimulation
(LVLs and HVLs) is associated with increased CD8+ T cell frequencies that display heterogeneous phenotypic and functional
features with direct implications for immunological monitoring in
the clinical setting. In addition, we have found that EBV-specific
CD8+ T cells are alternatively polarized (decreased IFN-g/IL-5
ratio) in children, a state that may contribute, in addition to immunosuppression and other yet unknown factors, to EBV reactivation and viral load accumulation in pediatric Tx patients.
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two-tailed Student t tests were used to compare mean values between viral
load groups. When multiple observations were available per patient,
generalized estimating equations models with empirical SEs were used to
estimate mean levels for Ag measures by group controlling for the correlation among measures from the same patient. The significance (p values) of the global hypothesis test for any differences among all viral load
groups and the significance of each pairwise test were derived from these
models. Generalized estimating equations linear regression models with
empirical SE estimates were also used to analyze the association between
viral load (coded as a continuous variable), lytic and latent Ag levels, and
functional outcome measures controlling for within patient correlation of
observations. The p values #0.05 were considered statistically significant.
Results
Characterization of memory CD8+ T cell phenotypes in
peripheral blood of pediatric thoracic organ Tx patients
CD8+ T cells as compared with UVL patients (Fig. 1B). In addition, CD8+ T cells from HVL carriers also expressed the highest
levels of CD38 and PD-1 expression with concomitant downregulation of CD127 (Fig. 1C). CTLA-4 was not selectively upregulated on CD8+ T cells from HVL carriers (Fig. 1C). Taken
together, these results suggest that high EBV antigenic burden
present in the asymptomatic HVL pediatric Tx recipients has
triggered a bystander increase in the frequency of CD8+ T cells
that show signs of T cell activation (CD38+/CD45RA2/CD62L2)
and exhaustion (PD-1+/CD1272). Conversely, the lack of constant
EBV-Ag challenge seen with UVL patients resulted in CD8+
T cells with quiescent phenotypes. LVL patients and HCs displayed intermediate signs of CD8+ T cell activation.
Frequency of EBV-specific CD8+ T cells in peripheral blood of
asymptomatic pediatric thoracic organ Tx patients
We next investigated the overall frequency of EBV-specific CD8+
T cells from peripheral blood of asymptomatic Tx patients carrying EBV loads (LVL and HVL) as compared with UVL and
HCs. Fig. 2A shows the gating strategy for EBV-specific CD8+
T cell identification. We have used flow cytometry with TMR
probes specific for EBV-lytic (BMLF1, BZLF1) or -latent (LMP2a,
EBNA3A) epitopes presented on HLA-A02+ (Fig. 2B) or HLAB08+ allele (Fig. 2C) to identify the frequencies (%) and absolute
numbers of EBV-specific CD8+ T cells detected from each subject.
Although HLA-B08+ allele is less frequent (∼10%) in the general
population as compared with HLA-A02 (∼45%), the purpose of
analyzing HLA-A02+ and HLA-B08+ patients was to demonstrate
whether the patterns of lytic- or latent-specific CD8+ T cell
responses are similar across MHC class I restriction elements.
Overall, both LVL and HVL pediatric Tx patients displayed significantly larger populations (% and absolute numbers) of circulating EBV-lytic and -latent specific CD8+ T cells as compared
with UVL carriers, in whom the TMR+ cells were barely
FIGURE 1. Frequency and activation/memory phenotypes of CD8+ T cells in peripheral blood. A, Frequency (%) and absolute numbers of CD8+ T cells
in PBLs assessed from pediatric Tx patients and HCs. B, Dot plots presenting the CD8+ TN (naive), TCM, TEMRA, and TEM, and total percentage of CD8+
T cells with CD45RA2CD62L2 (TEM) phenotype analyzed in PBLs from pediatric Tx patients and HCs. C, CD8+ T cell expression of CD38, PD-1,
CD127, and CTLA-4 in PBLs from pediatric Tx patients and HCs. *p # 0.05, one-tailed t test.
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Chronic antigenic stimulation by latent viruses has profound effects on the phenotype and function of memory CD8+ T cells (35).
Therefore, we decided to investigate the overall frequency,
memory, and activation phenotypes of CD8+ T cells from our
patients. We considered UVL patients as controls for pediatric
LVL and HVL Tx patients, who were similar for sex, age, and
immunosuppression, but did not carry EBV loads in peripheral
blood. We also considered latently infected HC volunteers as
controls, to further illustrate differences between memory responses to EBV in children and adults. Fig. 1A shows the overall
frequency (%) and absolute numbers of CD8+ T cells in peripheral
blood for each cohort. Patients carrying an EBV load (LVL and
HVL) presented significantly higher frequencies (LVL: 26 6 8;
HVL: 33 6 12%) and absolute numbers (LVL: 500 6 200 cells/
ml; HVL: 700 6 500 cells/ml) of circulating CD8+ T cells as
compared with UVL (frequency, 19 6 9%; absolute number, 300 6
200 cells/ml). HVL patients displayed the highest percentage of
CD8+ T cells among groups, and presented with a significantly
greater proportion of T effector memory (TEM: CD45RA2CD62L2)
CONTRASTING FEATURES OF EBV-SPECIFIC CD8+ T CELLS
The Journal of Immunology
5857
detectable by flow cytometry (Fig. 2B, 2C). This finding supports
the role of Ag stimulation in triggering the Ag-specific T cell
clonal expansion. Interestingly, EBV lytic-specific TMR+ cells
were more frequent than the latent-specific TMR+ cells for all
groups tested, suggesting a more active lytic viral stimulation than
latent antigenic challenge (Fig. 2B, 2C). A significant direct correlation between the EBV load and percentage of EBV-lytic
TMR+ cells was found over time in HVL patients (Fig. 2D). In
addition, LVL and HVL carriers displayed comparable high levels
of EBV-lytic–specific CD8+ T cells to HCs. We speculate that HCs
displayed high EBV-lytic–specific CD8+ T cell frequencies, despite undetectable circulating EBV loads at the time of analysis,
because of progressive CD8+ T cell clonal inflation and accu-
mulation in response to occasional Ag challenge throughout life
(36). In addition, .70% of participants were analyzed longitudinally over the period of this study to determine the stability of
EBV-specific CD8+ T cell frequencies. Results showed that TMR
frequencies remained consistent for each EBV-epitope over the
study period, for all asymptomatic patients carrying a stable EBV
load (Fig. 2E).
Activation/differentiation memory phenotypes of EBV-specific
CD8+ T cells in peripheral blood of asymptomatic pediatric
thoracic organ Tx patients
We next characterized the memory phenotypes of EBV-specific
(TMR+) CD8+ T cells from different cohorts of asymptomatic
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FIGURE 2. Frequency of EBV-specific CD8+ T cells in peripheral blood. A, Gating strategy to identify the EBV (TMR)-specific CD8+ T cells in
peripheral blood. B, Frequency (%) and absolute counts of HLA-A02–restricted, CD8+ T cells specific for lytic (BMLF1) and latent (LMP2a) epitopes. C,
Frequency (%) and absolute counts of HLA-B08–restricted, CD8+ T cells specific for lytic (BZLF1) and latent (EBNA3A) epitopes. Data in B and C are
represented as Tukey box plots, where the boxes indicate median and quartile values. Each dot represents the mean value of the frequencies of all EBVspecific CD8+ T cells determinations for a given subject. D, Correlation between EBV loads and the frequency of HLA-A02–restricted EBV lytic
(BMLF1)-specific CD8+ T cells. Each dot represents the value obtained from one determination in one patient at a time. E, Follow-up analysis of the EBV
load and the frequency of EBV-lytic (BMLF1)-specific CD8+ T cells in peripheral blood of representative HLA-A02+ LVL (top graph) and HVL (bottom
graph) pediatric Tx patients. In each case, the measurements were performed at 6 mo intervals of each other. *p # 0.05, one-tailed t test.
5858
upregulated on EBV-specific CD8+ T cells from patients or HCs
(Fig. 3C). No data could be recorded for EBV-specific CD8+
T cells from UVL patients, because the frequencies of TMR+ cells
in this cohort were at the limit of detection of the technique. Taken
together, these results show that asymptomatic pediatric Tx
patients that carry an EBV load are phenotypically heterogeneous,
and that the level of EBV antigenic pressure is important for
shaping EBV-specific memory/activation and differentiation CD8+
T cell status.
Frequency of functional (IFN-g+) EBV-specific memory CD8+
T cells in peripheral blood
We next inquired whether the differences in the levels of EBV
load have functional consequences. The overall frequency of IFNg–producing cells in response to nonspecific PMA+ionomycin stimulation (Fig. 4A) and to EBV-specific peptide stimulation (Fig.
4B, 4C) was significantly higher in LVL pediatric Tx patients than
in UVL. Although the EBV-specific CD8+ T cell absolute numbers
in LVL and HVL were comparable (Fig. 2B, 2C), HVL carriers
showed significantly lower levels of IFN-g–producing cells in
response to PMA+ionomycin (Fig. 4A), and a decline in IFN-g
responses against EBV lytic- and latent-specific stimulation, as
compared with LVL carriers (Fig. 4B, 4C). These data confirm
the phenotypic findings and suggest CD8+ T cell functional “exhaustion” in HVL carriers. In addition, HCs displayed strong IFNg responses to nonspecific and to EBV-specific stimulation, comparable with LVL pediatric Tx patients, and significant higher than
HVL (Fig. 4A–4C), confirming their functionality. There was a
highly significant direct correlation between the percentage of
FIGURE 3. Activation/memory phenotypes of EBV-specific memory CD8+ T cells in peripheral blood. Peripheral blood from representative HLA-A02+
pediatric Tx patients and HCs was stained to detect either EBV-lytic–specific CD8+ (A) T cells or EBV-latent–specific (B) CD8+ T cells, and further
analyzed for their memory phenotype distribution and for expression of CD38, CD127, and PD-1. C, Overall expression of CD38, PD-1, CD127, and
CTLA-4 on EBV-lytic (BMLF1)-specific CD8+ T cells. D, Overall expression of CD38, PD-1, and CD127 on EBV-latent (LMP2a)–specific CD8+ T cells.
*p # 0.05, one-tailed t test.
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Tx patients and HCs. As illustrated in Fig. 3, there were significant
differences in the levels and patterns of memory subsets and of
activation/differentiation status, depending on the level of EBV
load (LVL versus HVL) and on the source of EBV-Ag (lytic versus
latent). Representative staining patterns of EBV-specific CD8+
T cells are shown in Fig. 3A (lytic) and 3B (latent), whereas
overall data are shown in Fig. 3C (lytic) and 3D (latent). For
most patients, the EBV-lytic–specific CD8+ T cells were divided
between TEM and terminally differentiated effector memory
(TEMRA: CD45RA+CD62L2 ) (Fig. 3A), whereas EBV-latent–
specific CD8+ T cells were divided between central memory
(TCM: CD45RA2 CD62L+) and TEM (Fig. 3B). These patterns
were also detected in HCs (Fig. 3A, 3B), and confirm the previously described relation between EBV-epitope source and EBVspecific (TMR+) CD8+ T cell’s distinct ability to home in the
periphery (TEM, TEMRA) or in the secondary lymphatic organs
(TCM), and to function (TEM versus TCM) (3, 8, 34). In addition,
CD38 and CD127 molecules were expressed at intermediate levels
on both EBV lytic- and latent-specific CD8+ T cells from LVL
patients (and HCs), a reflection of their moderate EBV-antigenic
challenge and quiescent status (Fig. 3C, 3D). EBV-specific CD8+
T cells from HVL carriers expressed significant greater levels of
CD38 as compared with LVL patients and HCs, significantly increased PD-1, and significantly lower levels of CD127 as compared with HCs (Fig. 3C, 3D). Moreover, some HVL pediatric Tx
patients displayed uniformly EBV lytic- and latent-specific CD8+
T cells with effector (CD45RA2CD62L2) phenotypes, CD38+,
PD-1+, and CD1272, indicative of acute signs of EBV-antigenic
challenge (Fig. 3A, 3B). Interestingly, CTLA-4 was not selectively
CONTRASTING FEATURES OF EBV-SPECIFIC CD8+ T CELLS
The Journal of Immunology
5859
EBV-lytic–specific TMR+ cells and EBV-lytic–specific IFN-g+
cells from LVL patients and HCs, whereas the correlation was less
significant for HVL carriers (Fig. 4D). No correlation was observed in UVL carriers. In addition, the ratios between the absolute numbers of lytic-specific TMR+ to absolute numbers of lyticspecific IFN-g+ were higher for HVL carriers (266 6 502) compared with LVL carriers (43 6 56) (Fig. 4E), indicating less
functional (IFN-g+) cells among TMR+ CD8+ T cells for HVL
carriers.
Immunopolarization of memory EBV-specific CD8+ T cell in
peripheral blood of asymptomatic pediatric thoracic organ Tx
patients
It is well established that type 1 (IFN-g) CD8+ T cells are essential
for an adequate anti-EBV control, whereas type 2 (IL-4, IL-5) or
Tr1 (IL-10) CD8+ T cells may be supportive of viral complications. Therefore, we assessed the immunopolarization of circulating CD8+ T cells from asymptomatic pediatric Tx patients and
HCs by calculating the ratio between IFN-g– and IL-5–producing
cells, and by measuring IL-10 production. After a short in vitro
exposure to PMA+ionomycin (Fig. 5A), memory CD8+ T cells
from asymptomatic pediatric Tx patients displayed a true type 1
immunopolarization with a high IFN-g/IL-5 ratio (5:1), regardless
of their EBV load, and similar to the polarization of memory
CD8+ T cells from HCs who are known to display type 1 polarization (7, 8). Interestingly, however, after in vitro exposure to
EBV-lytic– or EBV-latent–specific peptide stimulation, memory
CD8+ T cells from asymptomatic pediatric Tx patients displayed
a skewed type 0 immunopolarization (IFN-g/IL-5 ratio: 2.5:1),
whereas HVL carriers also showed increased IL-10 production
(Fig. 5B, 5C). These results were significantly different from those
obtained with HCs who maintained a strong type 1 polarization
(IFN-g/IL-5 ratio: 5:1) and low IL-10 in response to EBV stimulation (Fig. 5B, 5C). Of note, all three cohorts of Tx patients
were on similar immunosuppressive regimens (Table I). Together,
these results indicate a biased immunopolarization of EBV-specific
CD8+ T cells in pediatric Tx patients as compared with type 1
immunopolarization seen in HCs, which may be detrimental for
effective EBV control. In addition, increased IL-10 production by
EBV-specific CD8+ T cells in response to EBV-peptide stimulation
seen with HVL suggests induced Tr1 as a mechanism of cellular
exhaustion.
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FIGURE 4. Frequency of functional (IFN-g+) EBV-specific memory CD8+ T cells in peripheral blood. A, The number of IFN-g–secreting cells/105 PBMCs
in response of PMA+ionomycin stimulation from pediatric Tx patients and HCs. B, The number of IFN-g–secreting cells/3 3 105 PBMCs in response HLAA02–restricted EBV-lytic– (BMLF1) and -latent (LMP2a)-specific peptides, or (C) HLA-B08–restricted EBV-lytic (BZLF1) and -latent (EBNA3A)-specific
peptide stimulation. D, Correlation between EBV-lytic (BMLF1)-specific CD8+ T cells and the frequency of IFN-g–producing EBV-lytic (BMLF1)-specific
CD8+ T cells in response to peptide stimulation in peripheral blood of pediatric Tx patients and HCs. E, Ratio between absolute numbers of EBV-lytic (BMLF1)
TMR+ and IFN-g+. Each dot in B–E represents the mean value of the determinations for a given subject. *p # 0.05, one-tailed t test.
CONTRASTING FEATURES OF EBV-SPECIFIC CD8+ T CELLS
5860
Discussion
Our results have identified critical differences in the CD8+ T cell
phenotype and function among asymptomatic pediatric thoracic
Tx patients. LVL and HVL patients displayed significant expansion of bystander CD8+ T cells and of EBV-specific CD8+ T cells
as compared with UVL carriers. We also found that EBV-lytic–
specific CD8+ T cell responses were more frequent than EBVlatent–specific responses in both LVL and HVL carriers; this was
confirmed for both HLA-A02+ and HLA-B08+ patients. Moreover, EBV load directly correlated with EBV-lytic–specific CD8+
T cells in HVL patients. Although our previous published data
highlighted the accumulation of EBV latently infected B cells
with high viral DNA copy number in HVL carriers (37, 38), our
results in this study indicate for the first time, to our knowledge,
that asymptomatic chronic HVL carrier state after pediatric thoracic organ transplantation may entail active lytic viral replication
(identified by TMR staining) in addition to the accumulation of
EBV latently infected cells in peripheral blood. Thus, future
studies are required to clarify the nature of the life cycle of the
virus in immunosuppressed children. A puzzling situation was
offered by the UVL patients (∼30% of asymptomatic pediatric Tx
patients) who carried EBV infection in its latent form (no detectable viral loads) and did not present EBV-specific TMR+ cells
in the peripheral blood. The lack of the chronic EBV load in this
cohort may be independent of T cell control, but possibly depend
on innate immune surveillance, an inherent lack of host B cell
permissiveness for increased EBV replication, or other factors
(e.g., genetic, virus related).
We have further identified differences in the activation/differentiation status of memory CD8+ T cells in patients carrying a viral load (Fig. 3) (26–28). EBV-specific CD8+ T cells from
both LVL and HVL carriers upregulated PD-1, and this is in good
agreement with previously published data that illustrate PD-1 upregulation on either physiologic Ag stimulation (24) or chronic
antigenic stimulation leading to functional exhaustion (21). EBVspecific CD8+ T cells from LVL displayed overall intermediate
levels of CD127 and CD38, and retained potent IFN-g, whereas
HVL carriers showed significant CD38 upregulation, exhausted
phenotypes (PD-1+/CD1272), and a decline in IFN-g production
(Figs. 3, 4). These data confirm Lang et al.’s (27) results describing
Table I. Patient demographics
Type of Tx
Mean time post-Tx 6 SD, y
Mean age 6 SD, y
Sex, M/F
EBV load, genome copies/ml (range)
HLA
Induction therapy
EBV status pre-Txa
IS regimen
UVL (n = 9)
Chronic LVL (n = 21)
Chronic HVL (n = 14)
HC (n = 11)
9 heart
5.4 6 4.5
8.3 6 5.2
4/5
Undetectable
-A02(8)–B08(2)
11%
89% seronegative
44% CNI
22% CNI/MMF
33% CNI/MMF/Pred
20 heart, 1 lung
8.2 6 3.9
13.4 6 4.2
12/9
100–13,000
-A02(17)–B08(7)
5%
52% seronegative
29% CNI
29% CNI/MMF
18% CNI/Pred
24% CNI/MMF/Pred
9 heart, 4 lung, 1 heart/lung
4.2 6 3.0
10.8 6 5.9
7/7
22,000–510,000
-A02(14)–B08(3)
50%
86% seronegative
28% CNI
29% CNI/MMF
7% CNI/Pred
36% CNI/MMF/Pred
NA
NA
42.8 6 7.0
4/7
Undetectable
-A02(9)–B08(3)
NA
NA
a
All patients were EBV seropositive at time of analysis.
CNI, calcineurin inhibitor; IS, immunosuppressive; MMF, mycophenolate mofetil; NA, nonapplicable; Pred, prednisone.
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FIGURE 5. Immunopolarization of memory EBV-specific CD8+ T cells in peripheral blood. A, The ratio between the number of IFN-g– and of IL-5–
secreting cells from PBMCs in response to PMA+ionomycin stimulation from pediatric Tx patients and HCs. B, Ratio between the numbers of IFN-g– and
IL-5–secreting cells from PBMCs in response to EBV-lytic (BMLF1) or EBV-latent (LMP2a) stimulation from HLA-A02+ pediatric Tx patients and HCs.
C, IL-10 secretion measured by ELISA in the supernatants harvested from IL-5 ELISPOT in response to EBV-lytic (BMLF1) stimulation from HLA-A02+
pediatric Tx patients and HCs.
The Journal of Immunology
and a significant inverse correlation between EBV load and CD4+
T cell ATP release in the HVL group, indicating the importance of
CD4+ T cell help for the long-term maintenance of CD8+ T cell
memory (54). Our findings provide a rational for future prospective multicenter studies focusing on asymptomatic HVL
carriers and their long-term CD8+ and CD4+ T cell follow-up, and
to study potential correlations among viral load, immunologic parameters, and their predictive role for development of PTLD.
Acknowledgments
We thank Dr. Angus W. Thomson for valuable review of the manuscript.
We also acknowledge the National Institutes of Health Tetramer Facility
(Emory University, Atlanta, GA) for generating the PE-HLA-B08-BZLF1
and -EBNA3A tetramers.
Disclosures
The authors have no financial conflicts of interest.
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