of June 28, 2018.This information is current as Phenotype and Exhausted Function

Display Contrasting Features: ActivatedPatients Carrying Chronic High EBV Loads

TransplantAsymptomatic Pediatric Thoracic T Cells from+EBV-Specific CD8

Louise Smith, Maria M. Brooks and Diana MetesIulia Popescu, Yun Hua, Michael Green, David Rowe, Camila Macedo, Steven A. Webber, Albert D. Donnenberg,

http://www.jimmunol.org/content/186/10/5854doi: 10.4049/jimmunol.1001024April 2011;

2011; 186:5854-5862; Prepublished online 1J Immunol 

Referenceshttp://www.jimmunol.org/content/186/10/5854.full#ref-list-1

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The Journal of Immunology

EBV-Specific CD8+ T Cells from Asymptomatic PediatricThoracic Transplant Patients Carrying Chronic High EBVLoads Display Contrasting Features: Activated Phenotypeand 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*,†,‖

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 lympho-

proliferative 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 EBV-

specific 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 EBV-

specific 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.

Epstein–Barr virus is a g-herpes virus that infects .90% ofhealthy adults worldwide (1, 2). The virus persists in theB lymphocyte pool as a lifelong asymptomatic latent

infection that can undergo occasional reactivations into the lyticstate with production of infectious virions (viral recrudescence) (1,2). EBV infection triggers effective memory type 1 (IFN-g, TNF-a, cytotoxicity) T cell responses specific for both EBV-lytic and-latent Ags that maintain the virus under tight immunologiccontrol with no detectable viral load in the peripheral circulationin 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

EBV immunosurveillance against episodic lytic viral reactivation,as well as for latent infection control (5–8).In acquired immunodeficiencies, such as posttransplant (post-

Tx) immunosuppression, EBV T cellular immune surveillanceis impaired, resulting in increased incidence of EBV-associatedposttransplant lymphomas (PTLDs) with high mortality (9–13).Pediatric patients are at greatest risk for EBV complicationsamong Tx patients, especially when they are EBV2 pre-Tx (lack-ing EBV memory T cells) and become EBV+ post-Tx under im-munosuppression (11, 13). Serial long-term EBV monitoring byPCR in the peripheral blood of pediatric thoracic Tx patientswho EBV seroconverted after transplantation has identified threegroups of clinically asymptomatic children: ∼30% who exhibitundetectable (,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% showpersistent, dangerously high (.16,000 copies/ml) EBV loads(HVL) in peripheral blood for months to years after primarypost-Tx EBV infection, a status not encountered in immunocom-petent subjects (14, 15). We have recently shown that asymp-tomatic HVL carriers had a 45% risk for progression to aggressivelate-onset PTLD, including diffuse large B cell, Hodgkin’s, andBurkitt’s lymphomas (16). Characterization of CD8+ T cellsprofiles and of their dynamics in asymptomatic pediatric Txpatients should clarify, at least in part, the immunopathogenesisthat associates with the shift from EBV latency to the persistentHVL carrier state, which may help clinicians develop better pro-phylactic strategies.Hierarchical and progressive loss of type 1 CD8+ T cell function

is a hallmark of T cell exhaustion during chronic viral infections

*Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center,Pittsburgh, PA 15213; †Department of Surgery, University of Pittsburgh Medical Cen-ter, Pittsburgh, PA 15213; ‡Children’s Hospital of Pittsburgh, University of PittsburghMedical Center, Pittsburgh, PA 15213; xDepartment of Medicine, University of Pitts-burgh, Pittsburgh, PA 15213; {Graduate School of Public Health, University of Pitts-burgh, Pittsburgh, PA 15213; and ‖Department of Immunology, University ofPittsburgh, 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. StarzlTransplantation Institute, E 1549 Thomas E. Starzl Biomedical Tower, 200 LothropStreet, Pittsburgh, PA 15213. E-mail address: metesdm@upmc.edu

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 differenti-ated effector memory T cell; TMR, tetramer; Tx, transplant; UVL, undetectable viralload.

Copyright� 2011 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/11/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001024

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in mice and humans, with IFN-g production being the last func-tion lost (17, 18). The programmed death 1 (PD-1) molecule,a member of the CD28/B7 family, has been shown to criticallyregulate T cell activation by delivering inhibitory signals thatsuppress TCR signaling and function (19). During chronic LCMVinfection in mice and HIV and HCV infections in humans, PD-1 isupregulated on CD8+ T cells and is instrumental in inhibitingvirus-specific CD8+ T cell proliferation, cytokine production, andcytolytic activity, leading to cellular exhaustion (20–23). How-ever, recent studies in HIV and HCV patients have concluded thata PD-1 upregulation on CD8+ T cells can be either a sign ofphysiologic T cell activation during viral infection control or canassociate with T cell exhaustion (24, 25). Therefore, other mole-cules in addition to PD-1 should be monitored to accuratelyidentify the Ag-specific CD8+ T cell status. For example, CD127(IL-7Ra) is an important receptor for T cell survival and memorydifferentiation, and is shown to significantly downmodulate duringpersistent HVL infections and to inversely correlate with the stateof CD8+ T cell exhaustion (26–28). CTLA-4 is yet another in-hibitory molecule that can be upregulated on T cells duringchronic viral infections, but its contribution to CD8+ T cell ex-haustion is less well understood (21, 29).Although the expression of PD-1 has been documented on EBV-

specific CD8+ T cells from subjects undergoing acute infectiousmononucleosis and during resolution (30), as well as from HIV-and HCV-infected subjects (23, 31), no studies were performed onEBV-specific CD8+ T cells from asymptomatic pediatric thoracicTx patients to clarify the significance of chronic EBV load, PD-1upregulation, and CD8+ T cell functional state. In addition, mostof the previous immunologic studies in Tx patients monitored fewparameters at a time (7, 8), which may be insufficient for an ac-curate understanding of the complex Ag-specific CD8+ T celldifferentiation state at a given time. In this study, we have usedtetramer (TMR) technology to detect EBV-specific CD8+ T cells,concomitantly incorporating several memory (CD45RA, CD62L)and activation/differentiation (CD38, CD127, PD-1) markers inflow cytometry. We have also monitored the EBV-specific func-tional (IFN-g, IL-5, and IL-10) parameters for a comprehensivedefinition of immunologic “signatures” in these asymptomaticpediatric 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 fre-quencies that display heterogeneous phenotypic and functionalfeatures with direct implications for immunological monitoring inthe clinical setting. In addition, we have found that EBV-specificCD8+ T cells are alternatively polarized (decreased IFN-g/IL-5ratio) in children, a state that may contribute, in addition to im-munosuppression and other yet unknown factors, to EBV reac-tivation and viral load accumulation in pediatric Tx patients.

Materials and MethodsHuman subjects and PBMC isolation

Forty-four asymptomatic pediatric thoracic Tx patients and 11 healthycontrol (HC) volunteers were consented under Institutional Review Board-approved protocols at the University of Pittsburgh and analyzed in a cross-sectional study with additional longitudinal observations in 70% of patientsover a period of 3 y. Patient demographics and immunosuppressive regi-mens are shown in Table I. All patients were EBV+ at the time of blooddonation, as confirmed by serology, and were also positive for HLA-A02and/or HLA-B08 allele. Patients were divided into three groups accordingto their peripheral blood EBV loads determined by PCR, regardless of theirpre-Tx EBV status (see below). Maintenance immunosuppression wascomparable for all three cohorts of patients, and consisted of a combinationof a calcineurin inhibitor (tacrolimus or cyclosporine) and an antipro-liferative agent (mycophenolate mofetil) with or without the use of low-dose prednisone. Nine patients received induction therapy with polyclonal

anti-T cell Abs (Thymoglobulin or ATGAM). Five to ten milliliters ofheparinized whole blood was collected from each subject and used directlyin flow cytometry analysis, whereas PBMCs were isolated by density gra-dient centrifugation (32) and banked frozen for functional assays.

Definition of EBV load groups

Patients were categorized into three groups according to their peripheralwhole blood EBV load levels as follows: 1) UVL carriers with no EBV loaddetected by PCR in.80% of determinations including the time of analysis;2) chronic LVL carriers with EBV loads ranging between 100 and 16,000genomic copies/ml blood, detected in .20% of measurements, includingthe time of analysis; and 3) chronic HVL carriers with EBV loads.16,000genomic copies/ml blood on at least 50% of determinations, and overa 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 atUniversity 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)-latentcycle peptides restricted by HLA-B08+. These peptides were used tostimulate EBV-specific CD8+ T cells in ELISPOT assays at a final con-centration 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 pep-tides were generated at the National Institute of Allergy and InfectiousDiseases 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 fromLife Technologies BRL, Grand Island, NJ) and referred to as completemedia. 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 in-cluding CD62L-FITC (Beckman Coulter), CD45RO-FITC, CD45RA-allo-phycocyanin, 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 tem-perature. Isotype controls and a negative TMR (Beckman Coulter) were usedas negative control. Cells were then incubated with 2 ml/tube of 1X lysingbuffer (BD) for an additional 10 min at room temperature to allow RBCslysis. Tubes were washed twice and fixed with 1% paraformaldehyde. Allevents were collected using an LSR II (BD) flow cytometer and analyzedwith 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, 96-well plates (Millipore, Bedford, MA) were precoated with anti-humanIFN-g (10 mg/ml; Mabtech, Sweden) or anti-human IL-5 (10 mg/ml; BDPharmingen) mAbs in PBS (Cellgro, Herndon, VA) overnight at 4˚C.Plates were washed and PBMCs were seeded at 3 3 105/well with cor-respondent 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 aspositive control (1 3 105cells/well). The wells were washed, and a sec-ondary biotinylated anti-human IFN-g mAb (2 mg/ml; Mabtech) or bio-tinylated anti-human IL-5 (2 mg/ml; BD Pharmingen) mAbs were addedfor an additional 2 or 4 h, respectively, at 37˚C. The reaction was de-veloped with 3-amino-9-ethylcarbazole substrate (Sigma) for IFN-g andtetramethylbenzidine substrate (KPL Laboratories, Gaithersburg, MD) forIL-5. Spots were counted with an ELISPOT plate reader (Immunospot;Cellular Technology, Cleveland, OH).

Determination of IL-10 levels by ELISA

Culture supernatants from ELISPOTassays were collected and levels of IL-10 quantified by ELISA. Primary and secondary mAbs, and the recombinantcytokine for IL-10 ELISAwere 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 log-arithm transformation was applied when data were approximately log-normally distributed. For analyses involving one observation per patient,

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two-tailed Student t tests were used to compare mean values between viralload groups. When multiple observations were available per patient,generalized estimating equations models with empirical SEs were used toestimate mean levels for Ag measures by group controlling for the cor-relation among measures from the same patient. The significance (p val-ues) of the global hypothesis test for any differences among all viral loadgroups and the significance of each pairwise test were derived from thesemodels. Generalized estimating equations linear regression models withempirical SE estimates were also used to analyze the association betweenviral load (coded as a continuous variable), lytic and latent Ag levels, andfunctional outcome measures controlling for within patient correlation ofobservations. The p values #0.05 were considered statistically significant.

ResultsCharacterization of memory CD8+ T cell phenotypes inperipheral blood of pediatric thoracic organ Tx patients

Chronic antigenic stimulation by latent viruses has profound ef-fects 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 ourpatients. We considered UVL patients as controls for pediatricLVL and HVL Tx patients, who were similar for sex, age, andimmunosuppression, but did not carry EBV loads in peripheralblood. We also considered latently infected HC volunteers ascontrols, to further illustrate differences between memory re-sponses to EBV in children and adults. Fig. 1A shows the overallfrequency (%) and absolute numbers of CD8+ T cells in peripheralblood for each cohort. Patients carrying an EBV load (LVL andHVL) 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 ascompared with UVL (frequency, 196 9%; absolute number, 3006200 cells/ml). HVL patients displayed the highest percentage ofCD8+ T cells among groups, and presented with a significantlygreater proportion of T effector memory (TEM: CD45RA

2CD62L2)

CD8+ T cells as compared with UVL patients (Fig. 1B). In ad-dition, CD8+ T cells from HVL carriers also expressed the highestlevels of CD38 and PD-1 expression with concomitant downreg-ulation of CD127 (Fig. 1C). CTLA-4 was not selectively upreg-ulated on CD8+ T cells from HVL carriers (Fig. 1C). Takentogether, these results suggest that high EBV antigenic burdenpresent in the asymptomatic HVL pediatric Tx recipients hastriggered a bystander increase in the frequency of CD8+ T cellsthat show signs of T cell activation (CD38+/CD45RA2/CD62L2)and exhaustion (PD-1+/CD1272). Conversely, the lack of constantEBV-Ag challenge seen with UVL patients resulted in CD8+

T cells with quiescent phenotypes. LVL patients and HCs dis-played intermediate signs of CD8+ T cell activation.

Frequency of EBV-specific CD8+ T cells in peripheral blood ofasymptomatic pediatric thoracic organ Tx patients

We next investigated the overall frequency of EBV-specific CD8+

T cells from peripheral blood of asymptomatic Tx patients car-rying EBV loads (LVL and HVL) as compared with UVL andHCs. Fig. 2A shows the gating strategy for EBV-specific CD8+

T cell identification. We have used flow cytometry with TMRprobes specific for EBV-lytic (BMLF1, BZLF1) or -latent (LMP2a,EBNA3A) epitopes presented on HLA-A02+ (Fig. 2B) or HLA-B08+ allele (Fig. 2C) to identify the frequencies (%) and absolutenumbers of EBV-specific CD8+ T cells detected from each subject.Although HLA-B08+ allele is less frequent (∼10%) in the generalpopulation as compared with HLA-A02 (∼45%), the purpose ofanalyzing HLA-A02+ and HLA-B08+ patients was to demonstratewhether the patterns of lytic- or latent-specific CD8+ T cellresponses are similar across MHC class I restriction elements.Overall, both LVL and HVL pediatric Tx patients displayed sig-nificantly larger populations (% and absolute numbers) of circu-lating EBV-lytic and -latent specific CD8+ T cells as comparedwith 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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detectable by flow cytometry (Fig. 2B, 2C). This finding supportsthe role of Ag stimulation in triggering the Ag-specific T cellclonal expansion. Interestingly, EBV lytic-specific TMR+ cellswere more frequent than the latent-specific TMR+ cells for allgroups tested, suggesting a more active lytic viral stimulation thanlatent antigenic challenge (Fig. 2B, 2C). A significant direct cor-relation between the EBV load and percentage of EBV-lyticTMR+ cells was found over time in HVL patients (Fig. 2D). Inaddition, LVL and HVL carriers displayed comparable high levelsof EBV-lytic–specific CD8+ T cells to HCs. We speculate that HCsdisplayed high EBV-lytic–specific CD8+ T cell frequencies, de-spite 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 longitudi-nally over the period of this study to determine the stability ofEBV-specific CD8+ T cell frequencies. Results showed that TMRfrequencies remained consistent for each EBV-epitope over thestudy period, for all asymptomatic patients carrying a stable EBVload (Fig. 2E).

Activation/differentiation memory phenotypes of EBV-specificCD8+ T cells in peripheral blood of asymptomatic pediatricthoracic organ Tx patients

We next characterized the memory phenotypes of EBV-specific(TMR+) CD8+ T cells from different cohorts of asymptomatic

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 EBV-

specific 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.

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Tx patients and HCs. As illustrated in Fig. 3, there were significantdifferences in the levels and patterns of memory subsets and ofactivation/differentiation status, depending on the level of EBVload (LVL versus HVL) and on the source of EBV-Ag (lytic versuslatent). Representative staining patterns of EBV-specific CD8+

T cells are shown in Fig. 3A (lytic) and 3B (latent), whereasoverall data are shown in Fig. 3C (lytic) and 3D (latent). Formost patients, the EBV-lytic–specific CD8+ T cells were dividedbetween TEM and terminally differentiated effector memory(TEMRA: CD45RA

+CD62L2) (Fig. 3A), whereas EBV-latent–specific CD8+ T cells were divided between central memory(TCM: CD45RA

2CD62L+) and TEM (Fig. 3B). These patternswere also detected in HCs (Fig. 3A, 3B), and confirm the pre-viously described relation between EBV-epitope source and EBV-specific (TMR+) CD8+ T cell’s distinct ability to home in theperiphery (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 levelson both EBV lytic- and latent-specific CD8+ T cells from LVLpatients (and HCs), a reflection of their moderate EBV-antigenicchallenge and quiescent status (Fig. 3C, 3D). EBV-specific CD8+

T cells from HVL carriers expressed significant greater levels ofCD38 as compared with LVL patients and HCs, significantly in-creased PD-1, and significantly lower levels of CD127 as com-pared with HCs (Fig. 3C, 3D). Moreover, some HVL pediatric Txpatients 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-antigenicchallenge (Fig. 3A, 3B). Interestingly, CTLA-4 was not selectively

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+ cellsin this cohort were at the limit of detection of the technique. Takentogether, these results show that asymptomatic pediatric Txpatients that carry an EBV load are phenotypically heterogeneous,and that the level of EBV antigenic pressure is important forshaping 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 EBVload have functional consequences. The overall frequency of IFN-g–producing cells in response to nonspecific PMA+ionomycin stim-ulation (Fig. 4A) and to EBV-specific peptide stimulation (Fig.4B, 4C) was significantly higher in LVL pediatric Tx patients thanin UVL. Although the EBV-specific CD8+ T cell absolute numbersin LVL and HVL were comparable (Fig. 2B, 2C), HVL carriersshowed significantly lower levels of IFN-g–producing cells inresponse to PMA+ionomycin (Fig. 4A), and a decline in IFN-gresponses against EBV lytic- and latent-specific stimulation, ascompared with LVL carriers (Fig. 4B, 4C). These data confirmthe phenotypic findings and suggest CD8+ T cell functional “ex-haustion” in HVL carriers. In addition, HCs displayed strong IFN-g responses to nonspecific and to EBV-specific stimulation, com-parable with LVL pediatric Tx patients, and significant higher thanHVL (Fig. 4A–4C), confirming their functionality. There was ahighly 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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EBV-lytic–specific TMR+ cells and EBV-lytic–specific IFN-g+

cells from LVL patients and HCs, whereas the correlation was lesssignificant for HVL carriers (Fig. 4D). No correlation was ob-served in UVL carriers. In addition, the ratios between the abso-lute numbers of lytic-specific TMR+ to absolute numbers of lytic-specific IFN-g+ were higher for HVL carriers (266 6 502) com-pared with LVL carriers (43 6 56) (Fig. 4E), indicating lessfunctional (IFN-g+) cells among TMR+ CD8+ T cells for HVLcarriers.

Immunopolarization of memory EBV-specific CD8+ T cell inperipheral blood of asymptomatic pediatric thoracic organ Txpatients

It is well established that type 1 (IFN-g) CD8+ T cells are essentialfor an adequate anti-EBV control, whereas type 2 (IL-4, IL-5) orTr1 (IL-10) CD8+ T cells may be supportive of viral complica-tions. Therefore, we assessed the immunopolarization of circu-lating CD8+ T cells from asymptomatic pediatric Tx patients andHCs by calculating the ratio between IFN-g– and IL-5–producingcells, and by measuring IL-10 production. After a short in vitroexposure to PMA+ionomycin (Fig. 5A), memory CD8+ T cells

from asymptomatic pediatric Tx patients displayed a true type 1immunopolarization with a high IFN-g/IL-5 ratio (5:1), regardlessof their EBV load, and similar to the polarization of memoryCD8+ T cells from HCs who are known to display type 1 polar-ization (7, 8). Interestingly, however, after in vitro exposure toEBV-lytic– or EBV-latent–specific peptide stimulation, memoryCD8+ T cells from asymptomatic pediatric Tx patients displayeda 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 thoseobtained with HCs who maintained a strong type 1 polarization(IFN-g/IL-5 ratio: 5:1) and low IL-10 in response to EBV stim-ulation (Fig. 5B, 5C). Of note, all three cohorts of Tx patientswere on similar immunosuppressive regimens (Table I). Together,these results indicate a biased immunopolarization of EBV-specificCD8+ T cells in pediatric Tx patients as compared with type 1immunopolarization seen in HCs, which may be detrimental foreffective EBV control. In addition, increased IL-10 production byEBV-specific CD8+ T cells in response to EBV-peptide stimulationseen with HVL suggests induced Tr1 as a mechanism of cellularexhaustion.

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 HLA-

A02–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.

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DiscussionOur results have identified critical differences in the CD8+ T cellphenotype and function among asymptomatic pediatric thoracicTx patients. LVL and HVL patients displayed significant expan-sion of bystander CD8+ T cells and of EBV-specific CD8+ T cellsas compared with UVL carriers. We also found that EBV-lytic–specific CD8+ T cell responses were more frequent than EBV-latent–specific responses in both LVL and HVL carriers; this wasconfirmed for both HLA-A02+ and HLA-B08+ patients. More-over, EBV load directly correlated with EBV-lytic–specific CD8+

T cells in HVL patients. Although our previous published datahighlighted the accumulation of EBV latently infected B cellswith high viral DNA copy number in HVL carriers (37, 38), ourresults in this study indicate for the first time, to our knowledge,that asymptomatic chronic HVL carrier state after pediatric tho-racic organ transplantation may entail active lytic viral replication(identified by TMR staining) in addition to the accumulation ofEBV latently infected cells in peripheral blood. Thus, futurestudies are required to clarify the nature of the life cycle of thevirus in immunosuppressed children. A puzzling situation was

offered by the UVL patients (∼30% of asymptomatic pediatric Txpatients) who carried EBV infection in its latent form (no de-tectable viral loads) and did not present EBV-specific TMR+ cellsin the peripheral blood. The lack of the chronic EBV load in thiscohort may be independent of T cell control, but possibly dependon innate immune surveillance, an inherent lack of host B cellpermissiveness for increased EBV replication, or other factors(e.g., genetic, virus related).We have further identified differences in the activation/dif-

ferentiation status of memory CD8+ T cells in patients carry-ing a viral load (Fig. 3) (26–28). EBV-specific CD8+ T cells fromboth LVL and HVL carriers upregulated PD-1, and this is in goodagreement with previously published data that illustrate PD-1 up-regulation on either physiologic Ag stimulation (24) or chronicantigenic stimulation leading to functional exhaustion (21). EBV-specific CD8+ T cells from LVL displayed overall intermediatelevels of CD127 and CD38, and retained potent IFN-g, whereasHVL carriers showed significant CD38 upregulation, exhaustedphenotypes (PD-1+/CD1272), and a decline in IFN-g production(Figs. 3, 4). These data confirm Lang et al.’s (27) results describing

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.

Table I. Patient demographics

UVL (n = 9) Chronic LVL (n = 21) Chronic HVL (n = 14) HC (n = 11)

Type of Tx 9 heart 20 heart, 1 lung 9 heart, 4 lung, 1 heart/lung NAMean time post-Tx 6 SD, y 5.4 6 4.5 8.2 6 3.9 4.2 6 3.0 NAMean age 6 SD, y 8.3 6 5.2 13.4 6 4.2 10.8 6 5.9 42.8 6 7.0Sex, M/F 4/5 12/9 7/7 4/7EBV load, genome copies/ml (range) Undetectable 100–13,000 22,000–510,000 UndetectableHLA -A02(8)–B08(2) -A02(17)–B08(7) -A02(14)–B08(3) -A02(9)–B08(3)Induction therapy 11% 5% 50% NAEBV status pre-Txa 89% seronegative 52% seronegative 86% seronegative NAIS regimen 44% CNI 29% CNI 28% CNI

22% CNI/MMF 29% CNI/MMF 29% CNI/MMF33% CNI/MMF/Pred 18% CNI/Pred 7% CNI/Pred

24% CNI/MMF/Pred 36% CNI/MMF/Pred

aAll patients were EBV seropositive at time of analysis.CNI, calcineurin inhibitor; IS, immunosuppressive; MMF, mycophenolate mofetil; NA, nonapplicable; Pred, prednisone.

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an inverse correlation between IL-7Ra expression and CD8+ T cellexhaustion during persistent Ag stimulation, and also Celleraiet al.’s (39) data showing that CD127 (re)-expression is linkedwith CD8+ T cells functionality (proliferative capacity). In addi-tion, Paul et al. (40) demonstrated that CD38 upregulation isa strong predictor for virus disease progression in HIV-infectedindividuals, whereas diminishment in CD38 indicates effectiveantiretroviral therapy in viremic HIV patients (41). Because HVLcarriers in our study expressed significantly high levels of CD38,future studies are required to follow-up these patients and de-termine the predictive role of CD38 for progression to symptom-atic disease (PTLD) in our patient population. Interestingly, arecent report by Crough et al. (42) has shown that CD38 upregu-lation is coincident with IFN-g decline in CMV-specific CD8+

T cells from symptomatic CMV recrudescence in solid organ Txpatients (unlike our results, which showed increased CD38 anddecline in IFN-g in asymptomatic HVL carriers), whereas IFN-gwas preserved in asymptomatic CMV Tx patients with LVLs. Webelieve that the differences between our studies may reflect dif-ferences in the following factors: 1) life cycles of CMV versusEBV, 2) age of the patients studied, and 3) clinical/virologic cri-teria of diagnosis.Interestingly, some patients in the LVL or HVL groups did not

express PD-1 on their EBV-specific CD8+ T cells (Fig. 3C, 3D).This suggests either that PD-1 expression may have been transientor that another inhibitory pathway (e.g., CTLA-4, TIM3, 2B4,LAG3) may have been involved in these patients (29, 43–47). Ourresults demonstrate that CTLA-4 expression is not selectivelyupregulated on EBV-specific CD8+ T cells from HVL carriers(Fig. 3C) and endorse future studies that focus on other inhibitorymolecules.Our analyses have further revealed that pediatric patients pre-

sented with skewed EBV-specific immunopolarization from theexpected type 1 (IFN-g) to type 0 (IFN-g/IL-5). Our data confirmpreviously published results that children, although in the processof building “healthy” type 1 immunity (by continuous exposure toviruses and bacteria), display an immature T cell immunopolari-zation (48, 49). The biased immune status of pediatric patients,further burdened by chronic immunosuppression (50), may cre-ate a milieu permissive for impaired antiviral surveillance and,therefore, may place pediatric Tx cohorts at higher risk for per-sistent viral complications. We postulate that the biased EBVimmunopolarization observed specifically in pediatric Tx patientsunder immunosuppressive therapy may represent an independentconfounding factor contributing to this switch from latency tochronic infection. In addition, HVL patients, although on similarimmunosuppressive regimens as UVL and LVL carriers, displayedincreased IL-10 release in response to EBV stimulation, sug-gesting EBV-specific CD8+ regulatory T cell induction as amechanism leading to cellular exhaustion rather than anergy (51).Earlier publications on persistent viral infections have concluded

that, although functionally competent CD8+ T cells are critical foreffective long-term control of viral latency with occasional re-crudescence, they rely on CD4+ T help during priming and sec-ondary expansion of memory CD8+ T cells (5, 6, 52). Conversely,in the absence of CD4+ T help, CD8+ T cells become pro-gressively exhausted, allowing for the viral load to accumulate(35, 53). We have recently examined the CD4+ T cell immuno-competence in the same cohorts of asymptomatic pediatric Txpatients (54). We used the Cylex assay to measure CD4+ T cellATP release in response to PHA stimulation. This test was pre-viously shown to predict the risk for chronic viral complicationsin immunocompromised patients (55–57). Our published resultsrevealed a significant decline in the CD4+ T immunocompetence

and a significant inverse correlation between EBV load and CD4+

T cell ATP release in the HVL group, indicating the importance ofCD4+ T cell help for the long-term maintenance of CD8+ T cellmemory (54). Our findings provide a rational for future pro-spective multicenter studies focusing on asymptomatic HVLcarriers and their long-term CD8+ and CD4+ T cell follow-up, andto study potential correlations among viral load, immunologic pa-rameters, and their predictive role for development of PTLD.

AcknowledgmentsWe 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.

DisclosuresThe authors have no financial conflicts of interest.

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