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IMMUNOBIOLOGY
From the Department of Hematology; Department of
Clinical Viro-Immunology, CLB, and Laboratory for Experimental and
Clinical Immunology; and Department of Human Retrovirology, Academic
Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
Molecular Immunology Group, Institute of Molecular Medicine, Oxford,
United Kingdom; and Department of Virology, University Hospital
Rotterdam, Rotterdam, The Netherlands.
Acquired immunodeficiency syndrome-related non-Hodgkin lymphomas
(AIDS-NHL) are thought to arise because of loss of Epstein-Barr Virus
(EBV)-specific cellular immunity. Here, an investigation was done to
determine whether cellular immunity to EBV is lost because of physical
loss or dysfunction of EBV-specific cytotoxic T cells. Data on
EBV-specific cellular immunity were correlated with EBV load. For
comparison, individuals who progressed to AIDS with opportunistic
infections (AIDS-OI) and long-term asymptomatics (LTAs) were studied.
The number of virus-specific T cells was detected using tetrameric
HLA-EBV-peptide complexes; function of these EBV-specific T cells was
determined using the interferon- Epstein-Barr virus (EBV) is a widespread human
gamma herpesvirus. Primary infection with EBV usually occurs
asymptomatically,1 whereafter the virus persists for life
in a latent form in B lymphocytes.2 The initial expansion
and reactivation of these latently infected B lymphocytes is controlled
by specific CD8+ major histocompatibility complex (MHC)
class I-restricted cytotoxic T-lymphocyte (CTL)
responses.3 Because reactivation may result in lytic
antigen expression,4 CTL control during this stage of
reactivation may involve lytic antigen-specific effector T cells.5
During immunodeficiency, a higher rate of reactivation of EBV infection
may lead to uncontrolled lymphoproliferation.6 In human
immunodeficiency virus (HIV)-infected individuals, the incidence of
non-Hodgkin lymphomas (NHLs) is considerably increased (5%-10%), and
most of these lymphomas (75%) are EBV+.7
These acquired immunodeficiency syndrome (AIDS)-related diffuse large
cell NHLs are therefore thought to arise because of loss of
EBV-specific T-cell immunity.8,9
Until now, only few cross-sectional studies have been performed to
study EBV-specific immunity in AIDS-NHL patients. Some studies show
decreased EBV-specific cytotoxic T-cell activity in patients with AIDS
and AIDS-related complex.10 In contrast, other studies by
Carmichael et al and Geretti et al comparing EBV- with HIV-specific
T-cell responses showed sustained EBV-specific CTL responses with
declining HIV-1-specific CTL in advanced HIV-1 infection, suggesting
selective loss of HIV-1-specific CTL.11,12 We previously
reported a longitudinal study into the kinetics of HIV-1- and
EBV-specific CTL responses in HIV-infected individuals using limiting
dilution analysis to determine the number of CTL precursors (CTLp).
This study revealed that loss of HIV-specific CTLs is not necessarily
paralleled by loss of EBV-specific T-cell responses. In patients with
AIDS-NHL, diagnosis of NHL was found to be preceded by a decrease in
EBV CTLp and an increase in the number of infected B cells as measured
in an in vitro EBV transformation assay.8
The underlying cause for this loss in EBV-specific CTLp is unknown.
Recently, new techniques using MHC class I-peptide tetrameric complexes have been developed for enumerating antigen-specific CD8+ T cells.13 This method demonstrated a
much higher frequency of antigen-specific circulating T cells than
previously estimated by limiting dilution analysis. Moreover,
interferon (IFN)- Study population
We analyzed longitudinal PBMC samples from 5 HIV-1 infected individuals
progressing to AIDS-related diffuse large cell NHL (NHL6006, NHL0118,
NHL6118, NHL0139, NHL0308), starting at or soon after HIV-1
seroconversion. For comparison, we studied PBMC samples from 3 HIV-1-infected individuals progressing to AIDS (classification of the
Centers for Disease Control, 1993) with opportunistic infections
(PROG0232, PROG0341, PROG0642) and 3 HIV-infected LTA (LTA0036,
LTA1160, LTA0057) individuals with CD4+ T-cell counts above
500/µL during more than 8 years of asymptomatic follow-up.
Characteristics of the HIV-1-infected individuals are summarized in
Table 1. In addition, PBMC samples from 6 HIV Flow cytometry and tetramer staining MHC class I tetramers complexed with EBV peptides were produced as previously described.13 The peptides used (synthesized by solid-phase methods using an automated multiple-peptide synthesizer and Fmoc chemistry) were 2 immunodominant epitopes from EBV lytic cycle proteins, the HLA-A2-restricted epitope GLCTLVAML (A2-GLC) from BMLF-115 and the HLA-B8-restricted epitope RAKFKQLL (B8-RAK) from BZLF-1,16 and 1 immunodominant epitope from the latent antigen EBNA-3A, the HLA-B8-restricted epitope FLRGRAYGL (B8-FLR).17 Biotinylated class I peptide complexes were tetramerized by addition of allophycocyanin or phycoerythrin-conjugated streptavidin.Two-color fluorescence analysis was performed as previously described.18 Briefly, PBMCs were thawed, and 1.5 × 106 cells were stained in phosphate-buffered saline (PBS) supplemented with 0.5% (vol/vol) bovine serum albumin (BSA) with MHC class I tetramers and peridinin chlorophyll protein (PerCP)-conjugated monoclonal antibody (mAb) CD8 (Becton Dickinson, San Jose, CA). After staining, cells were washed with PBS/BSA and fixed in PBS/1% paraformaldehyde, and at least 250 000 events were acquired using a FACSCalibur flow cytometer (Becton Dickinson). The tetramer staining was very reproducible because multiple stainings on PBMCs from the same donor gave similar results. To determine the percentage of dead cells in each sample, a propidium iodide staining was performed. Lymphocytes were gated by forward and side scatter. Data were analyzed using the software program Cell Quest (Becton Dickinson). T-lymphocyte immunophenotyping for CD4 and CD8 membrane markers was performed in real time by flow cytometry. Elispot assay for single-cell IFN-  -producing antigen-specific T cells were enumerated
using IFN--specific Elispot assays as previously
described.14 A total of 96-well nylon-backed plates (Nunc,
Roskilde, Denmark) were coated overnight with 50 µL of 15 µg/mL
anti-IFN- mAb, 1-DIK (Mabtech, Stockholm, Sweden), in 0.1 M
carbonate/bicarbonate buffer, pH 9.6. After 6 wash steps with culture
medium (RPMI 1640, Gibco, Life Technologies, Breda, The Netherlands) to
remove unbound antibody, plates were blocked for 1 hour with RPMI 1640 supplemented with 10% fetal calf serum. Subsequently, PBMCs were added
in triplicate wells at 1 × 105 cells per well in case of
HLA-B8-restricted responses or 2 × 105 cells per well
in case of HLA-A2-restricted responses in the absence or presence of 2 µM peptide. As a positive control to test the capacity of PBMCs to
produce IFN- in general, phytohemagglutinin (Murex Diagnostics,
Dartford, United Kingdom) was added. Cultures were incubated overnight
at 37°C in 5% CO2. The next day, cells were removed by
washing with PBS/0.05% Tween 20, and the second biotinylated
anti-IFN- mAb, 7-B6-1 biotin (Mabtech), was added at 1 µg/mL in
PBS and left for 3 hours at room temperature, followed by
streptavidin-conjugated alkaline phosphatase (Mabtech) for an
additional 2 hours. Individual cytokine-producing cells were detected
as dark purple spots after a 10-minute reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (BCIP/NBT, Sigma, St Louis, MO). Reactions were stopped by extensive washing in water. Nylon membranes were dried, and spots were counted after computerized visualization by a scanner (Hewlett-Packard, Boise,
ID). The number of specific T-cell responders per 106 PBMCs
were calculated after subtracting negative control values. Because the
percentage of dead cells and the percentage of CD8+ T
cells were assessed in the same samples, the number of specific T-cell
responders per 106 living CD8+ T cells could be
calculated. This assay was very reproducible when performed on multiple
samples from EBV+ donors, detecting as low as 1 positive cell per 1 × 105 PBMCs (0.001%).
Intracellular IFN- production in most potential cells.
Stronger stimulation protocols (10 µg peptide per milliliter for 6 hours) did not substantially increase the number of IFN--producing
cells. After incubation, cells were washed and stained in PBS
supplemented with 0.5% (vol/vol) BSA for 15 minutes with HLA-B8-RAK
tetramers (APC) and PerCP-conjugated mAb CD8 (Becton Dickinson). After
membrane staining, cells were washed with PBS/BSA and fixed with 4%
paraformaldehyde, permeabilized (permeabilization kit, Becton
Dickinson), and stained intracellularly with IFN--PE (Becton
Dickinson) for 30 minutes at 4°C. At least 200 000 events in the
lymphogate were acquired using a FACSCalibur flow cytometer
(Becton Dickinson).
DNA extraction and real-time quantitative TaqMan assay PBMCs (1 × 106) were lysed by addition of L6-lysis buffer.20 Genomic DNA was extracted by precipitation with isopropanol, and DNA from 2 × 105 cells was amplified using PCR primers selective for the EBV DNA genome encoding the nonglycosylated membrane protein BNRF1 p143.21,22 PCR amplification was performed as previously described23 using EBV/p143 forward and reverse primers resulting in a 74-base pair DNA product. In the PCR reaction, a fluorigenic EBV/p143-specific probe was added with a FAM reported molecule attached to the 5' end and a TAMRA quencher linked at the 3' end to detect amplified DNA. Amplification and detection was performed with an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). Real-time measurements were taken, and a threshold cycle value was calculated for each sample by determining the point at which the fluorescence exceeded a threshold limit of 0.04. Each run contained several negative controls (no template or EBV DNA), a positive control (a known amount of EBV
copies), and a standard dilution of plasmid DNA containing the PCR
product as insert, which was calibrated with an EBV-quantified standard (Advanced Biotechnologies, Epsom, United Kingdom). The
analyzed sensitivity of the assay was between 50 and
5 × 106 copies per milliliter. All reactions were
performed in duplicate and only considered positive when both
replications were above the threshold limit. The variation between
duplicates was as low as 7.5%.
Statistical analysis To compare EBV-specific tetramer+ T cells, IFN--producing CD8+ T cells, and EBV load early and
late in HIV infection, Wilcoxon tests were performed. To compare all
variables early or late between groups, Mann-Whitney tests were
performed using the software program SPSS 7.5 (SPSS, Chicago, IL). To
test the relation between CD4+ T-cell numbers and
EBV-specific functional CD8+ T cells,
EBV-tetramer+ T cells, or total CD8+ T cells,
regression analyses (mixed linear model) were performed. Regression
analysis and multivariate analysis to search for predictors of the
number of functional T cells were performed after cube root
transformation of all variables. To correct for a possible correlation
between multiple observations from one person, compound symmetry was
used as correlation structure using the Proc Mixed procedure of the
software program SAS.
Direct visualization and functional analysis of EBV-specific CD8+ T lymphocytes To investigate the presence and function of EBV-specific CD8+ T cells, we studied CD8+ T cells specific for 2 epitopes derived from lytic antigens, the HLA-A2-restricted epitope GLCTLVAML (A2-GLC) and the HLA-B8-restricted epitope RAKFKQLL (B8-RAK), and for 1 epitope derived from a latent antigen, the HLA-B8-restricted epitope FLRGRAYGL (B8-FLR). To detect the presence of antigen-specific CD8+ T cells, we stained antigen-specific T cells using tetrameric HLA-peptide complexes. To assess the function of antigen-specific CD8+ T cells, we used the IFN- Elispot assay, which shows the number of
IFN--producing T cells after peptide stimulation. To determine the
sensitivity of both assays, we applied them both to an HIV-specific T-cell clone that was selected in vitro to respond to one specific (HIV
RT) peptide.24 In Figure 1
(left panel), tetramer staining of this T-cell clone is shown,
revealing that 87% of the T-cell clone was specific for the tetramer
containing the specific peptide. IFN- Elispot revealed that
virtually all tetramer+ T cells from the T-cell clone
produced IFN- upon stimulation with the specific peptide (Figure 1A,
right panel), because almost every (93%) tetramer+ T cell
could be accounted for in the Elispot assay. In addition, IFN-
production was similar in both Elispot assay and intracellular FACS
staining (manuscript in preparation). Tetramer staining of PBMCs from a
healthy EBV-carrying individual showed that approximately 3% of the
CD8+ T cells were specific for an EBV-peptide (Figure 1B,
left panel), half of which produced IFN- after peptide stimulation
in a direct ex vivo assay using intracellular staining (Figure 1B,
right panel) or Elispot assay (data not shown). Furthermore, both
assays were reproducible and sensitive on both fresh and frozen
material (data not shown).
In preliminary studies using IFN- Lower numbers of functional EBV-specific CD8+ T cells
in HIV+ versus HIV and HIV+ individuals in the course of
HIV-1 infection. For comparisons, both the total number of circulating
(tetramer+ as percentage of CD8+) and the total
number of functional (IFN--producing as number per 106
CD8+) EBV-specific CD8+ T cells was
calculated from the individual peptide-specific CD8+ T
cells. Combining these 2 parameters, the proportion of
IFN-+ tetramer+ T cells could be determined.
In this study population, 0.2% to 5% of the CD8+ T cells
were EBV-specific. (Figure 2A). No
difference in the percentage of tetramer+ EBV-specific
CD8+ T cells was observed between HIV
Progressive loss of function of EBV-specific CD8+ T lymphocytes in AIDS-NHL patients To study the cause of the defective EBV-immune surveillance in AIDS-NHL patients, we investigated both the number and function of EBV-specific CD8+ T cells in the course of HIV-1 infection in AIDS-NHL patients. For comparison, progressors to AIDS-OI and LTA individuals were studied. Detailed results from 2 study participants (1 LTA, 1 AIDS-NHL) are shown in Figure 3. Most EBV-specific CD8+ T cells were directed against the lytic epitope B8-RAK (0.7%-3.6% in Figure 3C), whereas fewer T cells were directed against the latent epitope B8-FLR (0.1%-1.1% in Figure 3B). In HLA-A2+ individuals, 0.2% to 1.2% of the T cells were directed against the lytic epitope A2-GLC (0.6%-0.8% in Figure 3D).
For all individuals, the total percentage of tetramer+ and
number of IFN-
In most individuals no change in the percentage of
tetramer+ T cells was observed in the course of HIV-1
infection (Figure 4A). In AIDS-NHL patients, the function of these
EBV-specific T cells significantly decreased in the course of HIV-1
infection (P = .04, Wilcoxon test) (Figure 4B). The
decrease in IFN- The decrease in IFN- Furthermore, the decline in IFN- Progressors to AIDS-OI showed lower but stable percentages of
tetramer+ T cells (Figure 4A) and IFN- Comparison of the number of IFN- staining after peptide stimulation (B8-RAK) of
PBMCs from an AIDS-NHL patient at an early time point (Figure 7, left panel) and PBMCs from an LTA
individual at 2 time points (Figure 7, right panel). At an early time
point in NHL0308, the percentage of RAK-specific IFN--producing T
cells as assessed by intracellular staining (0.19%, or 1900 per
106 CD8) (Figure 7B) is of the same magnitude as the number
of IFN- producers found by Elispot assay (~1900, Figure 7A, dotted
line). Similar to the Elispot results, the number of CD8+ T
cells with intracellular IFN- staining increased in the course of
infection for the LTA individual (from 0.16% to 0.3%) after stimulation, which correlated well with IFN- production in Elispot (Figure 7A). Moreover, the proportion of IFN--producing
tetramer+ T cells for the AIDS-NHL patient early in
infection was 9% (Figure 7C), which corresponded well with the
percentages observed using tetramer staining in combination with
IFN- Elispot assay (around 8% early in HIV-1 infection)
(Figure 4C).
EBV-specific CD8+ T-lymphocyte function is dependent on CD4+ T cells Next, we investigated whether there was a possible relation between CD4+ T-cell numbers and the number of EBV-specific CD8+ T cells. In AIDS-NHL patients, CD4+ T-cell numbers decreased significantly in the course of HIV-infection (P = .04, Wilcoxon test). In 4 of 5 AIDS-NHL patients, loss of functional EBV-specific CD8+ T cells (Figure 5B,C) was related in time to a drop in CD4+ T-cell counts below 200/µL (Figure 5A). Furthermore, when CD4+ T-cell counts increased again after antiretroviral treatment (dideoxyinosine, or DDI) in patient NHL0308, a parallel increase in functional EBV-specific CD8+ T cells was observed (Figures 3 and 5B).In LTAs and progressors to AIDS-OI, who did not show a functional loss of EBV-specific CD8+ T cells (Figure 6B), CD4+ T-cell numbers were stable during most time points and no significant decrease in CD4+ T-cell numbers was observed (P = .11, Wilcoxon test). In LTAs and progressor P0232, CD4+ T-cell numbers never dropped below 200 CD4+ T cells per microliter (Figure 6A). Indeed, when CD4+ T-cell numbers measured at all time
points were plotted against the number of EBV-specific CD8+
T cells that produced IFN-
Functional loss of EBV-specific CD8+ T lymphocytes is paralleled by an increase in EBV load To investigate whether loss of functional T cells is associated with an increase in EBV load and whether this precedes the development of AIDS-NHL, EBV load was determined using a sensitive and specific real-time quantitative PCR. As shown in Figure 4, loss of EBV-specific CD8+ T-cell function (Figure 4B) was associated with an increase in EBV load in AIDS-NHL patients (P = 0.08, Wilcoxon test) (Figure 4D), although the absolute number of EBV copies was not different from absolute EBV load observed in LTAs and other progressors to AIDS (D.v.B. et al, manuscript submitted). Interestingly, in patient NHL0308 after the start of DDI treatment, EBV-specific CD8+ T cells slightly increased and a small reduction in EBV load was observed (Figure 5C).In contrast, in LTAs no increase and, in progressors to AIDS-OI, even a
decrease in EBV load was observed (Figure 4D, middle and right panels).
The increase in load ( To study whether EBV load is able to drive the number of
IFN-
To investigate the cause of decreasing numbers of CTLp in AIDS-NHL
patients, which is believed to lead to defective EBV-immune control, we
studied number (using MHC class I tetramers) and function (using
IFN- The observed correlation between loss of function of EBV-specific CD8+ T cells and lower CD4+ T-cell numbers indicates an important role for CD4+ T cells in maintaining the functional capacity of CD8+ T cells. Our data are in good agreement with studies on T-helper dependence of chronic lymphocytic choriomeningitis virus (LCMV)-specific CTL in mouse models.25-27 A critical role for CD4+ T cells has also been shown during immunization,28 and progressive loss of CTL in the absence of adequate helper cell function has been demonstrated for several murine viral infections.29-31 Furthermore, CD4+ T cells also appear to be essential for long-term persistence of adoptively transferred virus-specific CTL in humans.27,32,33 In the natural course of HIV infection, it has been shown that progressors to AIDS lose CD8+ CTLs when functional HIV-specific CD4+ T cells disappear. In contrast, in nonprogressors, who have stable CD4+ T-cell numbers, HIV-specific CTL responses can be sustained for long periods of time,34-37 indicating that sustained HIV-specific helper activity is required for maintenance of functional CD8+ T-cell responses.34,38,39 The fact that most EBV-specific CD8+ T cells were directed against lytic epitopes suggests that these lytic antigen-specific T cells play a role not only during acute infection40 but also in controlling EBV reactivation by eliminating virus-producing cells at an early stage. Loss of functional lytic antigen-specific CD8+ T cells could therefore lead to an increase in EBV DNA, as we indeed observed. As the pool of EBV-infected B cells grows, there is an increased risk of subsequent genetic hits resulting in malignant outgrowth of EBV-infected B cells. Because functional CD8+ T cells specific for latent antigens appear to be lost as well, newly developed tumor cells will not be destroyed. Our data suggest that loss of both lytic and latent antigen-specific CD8+ T cells may contribute to the risk for development of AIDS-NHL. In HIV infection, 0.2% to 5% of the CD8+ T cells were
found to be EBV-specific, of which 13% were shown to produce IFN- The observed low percentage of IFN- The low percentage of IFN- The phenomenon of antigen-specific CD8+ T cell dysfunction
has also recently been shown for hepatitis C virus45
during a period of acute infection, for tumor-specific T cells in
melanoma patients,46 and for HIV-specific T
cells.47 This state of dysfunction has been shown to occur
both at the level of IFN- In AIDS-NHL patients the observed loss of function of EBV-specific CD8+ T cells was accompanied by an increase in EBV load, although absolute EBV load did not differ between groups (data not shown). This increase in EBV load cannot be attributed to technical variation, because we have low variation in duplicate measurements (7.5%) and correction for the quantity of input DNA did not change the observed patterns. In addition, increases in EBV load are not due to increases in the number of total B cells, because correction for the number of B cells in PBMC samples did not lead to altered patterns (data not shown). Thus, finally, in AIDS-NHL patients immune control over EBV seemed to be lost. Surprisingly, in LTAs enormous transient bursts of EBV load were observed. These peaks in viral load seemed to be paralleled by expansions of functional CD8+ T cells specific for EBV. Because EBV load subsequently decreased, these cells apparently were able to control EBV viremia. Indeed, multivariate analysis of AIDS-OI patients and LTAs showed that EBV load, besides the number of CD4+ T cells, could predict the number of functional T cells, indicating that EBV load is indeed able to drive EBV-specific T cells when sufficient CD4+ T cells are present. In progressors to AIDS-OI, EBV-specific CD8+ T cells on total were lower, suggestive of physical loss or lack of expansion of these T cells. In these patients, the relatively low number of EBV-specific T cells did, however, not lead to an increase in EBV load, suggesting that there is adequate control. Alternatively, it could be that these individuals eventually would have developed AIDS-NHL had they not developed AIDS-OI. In conclusion, our data suggest that the development of AIDS-NHL is a multifactorial process involving at least virologic and immunologic parameters. Thus, both determinants are required to obtain a complete picture of the virus-host balance. We show that not so much the total number of circulating EBV-specific CD8+ T cells but, mainly, the number of functional EBV-specific CD8+ T cells is important in keeping EBV infection under control. When EBV-specific CD8+ T cells start to lose function, in most cases as a consequence of a decrease in CD4+ T cells, this is paralleled by an increase in EBV load. To be able to predict the occurrence of an AIDS-NHL, all these factors should be taken into account.
This study was part of the Amsterdam Cohort Studies on AIDS and HIV-1 infection, a collaboration of the Municipal Health Service, the Academic Medical Center, and Central Laboratory of the Blood Transfusion Service (CLB). We thank Dr M. Roos and collaborators for T-lymphocyte immunophenotyping, and N. Dukers for statistical analysis.
Submitted September 7, 2000; accepted March 1, 2001.
Supported by the Dutch Cancer Society (grant 96-1168), the Dutch AIDS Fund (grant 1007), and the Dutch Organization for Scientific Research (NWO).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Debbie van Baarle, Dept of Clinical Viro-Immunology, CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands; e-mail: d_van_baarle{at}clb.nl.
1. Miller G. Epstein-Barr virus: biology, pathogenesis and medical aspects. In: Fields BN,Knipe DM, eds. Virology. 2nd ed. New York, NY: Raven Press; 1990:1921-1958. 2. Sixbey JW, Nedrud JG, Raab-Traub N, Hanes RA, Pagano JS. Epstein-Barr virus replication in oropharyngeal epithelial cells. N Engl J Med. 1984;310:1225-1230[Abstract]. 3. Rickinson AB. Cellular immunological responses to the virus infection. In: Epstein MA,Achong BG, eds. The Epstein-Barr Virus: Recent Advances. Philadelphia, PA: Lippincott-Raven; 1990:75-115. 4. Speck SH, Chatila T, Flemington E. Reactivation of Epstein-Barr virus: regulation and function of the BZLF1 gene. Trends Microbiol. 1997;5:399-405[CrossRef][Medline] [Order article via Infotrieve]. 5. Rickinson AB, Moss DJ. Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection. Annu Rev Immunol. 1997;15:405-431[CrossRef][Medline] [Order article via Infotrieve]. 6. Cohen JI. Epstein-Barr virus lymphoproliferative disease associated with acquired immunodeficiency. Medicine. 1991;70:137-160[CrossRef][Medline] [Order article via Infotrieve]. 7. Kersten MJ, Van Gorp J, Pals ST, Boon F, Van Oers MHJ. Expression of Epstein-Barr virus latent genes and adhesion molecules in AIDS- related non-Hodgkin's lymphomas: correlation with histology and CD4-cell number. Leuk Lymphoma. 1998;30:515-524[Medline] [Order article via Infotrieve]. 8. Kersten MJ, Klein MR, Holwerda AM, Miedema F, Van Oers MHJ. EBV-specific cytotoxic T cell responses in HIV-1 infection: different kinetics in patients progressing to opportunistic infection or non-Hodgkin's lymphoma. J Clin Invest. 1997;99:1525-1533[Medline] [Order article via Infotrieve].
9.
Shibata D, Weiss LM, Hernandez AM, Nathwani BN, Bernstein L, Levine AM.
Epstein-Barr virus-associated non-Hodgkin's lymphoma in patients infected with the human immunodeficiency virus.
Blood.
1993;81:2102-2109 10. Blumberg RS, Paradis T, Byington R, Henle W, Hirsch MS, Schooley RT. Effects of human immunodeficiency virus on the cellular immune response to Epstein-Barr virus in homosexual men: characterization of the cytotoxic response and lymphokine production. J Infect Dis. 1987;155:877-890[Medline] [Order article via Infotrieve].
11.
Carmichael A, Jin X, Sissons P, Borysiewicz L.
Quantitative analysis of the human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocyte response at different stages of HIV-1 infection: differential CTL responses to HIV-1 and Epstein-Barr virus in late disease.
J Exp Med.
1993;177:249-256 12. Geretti AM, Dings MEM, Van Els CACM, et al. Human immunodeficiency virus type 1 (HIV-1)- and Epstein-Barr virus-specific cytotoxic T lymphocyte precursors exhibit different kinetics in HIV-1-infected persons. J Infect Dis. 1996;174:34-45[Medline] [Order article via Infotrieve].
13.
Altman JD, Moss PAH, Goulder PJR, et al.
Phenotypic analysis of antigen-specific T lymphocytes.
Science.
1996;274:94-96
14.
Lalvani A, Brookes R, Hambleton S, Britton WJ, Hill AVS, McMichael AJ.
Rapid effector function in CD8+ memory T cells.
J Exp Med.
1997;186:859-865 15. Bodegain C, Wolf H, Modrow S, Stuber G, Jilg W. Specific cytotoxic T-lymphocytes recognize the immediate-early transactivator ZTA of Epstein-Barr virus. J Virol. 1995;69:4872-4879[Abstract].
16.
Steven NM, Annels NE, Kumar A, Leese AM, Kurilla MG, Rickinson AB.
Immediate early and early lytic cycle proteins are frequent targets for the Epstein-Barr virus-induced cytotoxic T cell response.
J Exp Med.
1997;185:1605-1617
17.
Burrows SR, Sculley TB, Misko IS, Schmidt C, Moss DJ.
An Epstein-Barr virus-specific cytotoxic T cell epitope in EBNA3.
J Exp Med.
1990;171:345-350
18.
Ogg G, Kostense S, Klein MR, et al.
Longitudinal phenotypic analysis of human immunodeficiency virus type 1-specific T lymphocytes: correlation with disease progression.
J Virol.
1999;73:9153-9160 19. Kostense S, Ogg GS, Manting EH, et al. High viral burden in the presence of major HIV-specific CD8+ T cell expansions: evidence for impaired T cell effector function. Eur J Immunol. 2001;31:677-686[CrossRef][Medline] [Order article via Infotrieve].
20.
Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noorda J.
A rapid and simple method for purification of nucleic acids.
J Clin Microbiol.
1990;28:495-503
21.
Cameron KR, Stamminger T, Craxton M, Bodemer W, Honess RW, Fleckenstein B.
The 160,000-Mr virion protein encoded at the right end of the herpesvirus saimiri genome is homologous to the 140,000-Mr membrane antigen encoded at the left end of the Epstein-Barr virus genome.
J Virol.
1987;61:2063-2070
22.
Meyohas MC, Marechal V, Desire N, Bouillie J, Frottier J, Nicolas JC.
Study of mother-to-child Epstein-Barr virus transmission by means of nested PCRs.
J Virol.
1996;70:6816-6819
23.
Niesters HGM, van Esser J, Fries E, Wolthers KC, Cornelissen J, Osterhaus ADME.
The development of a high-throughput real-time quantitative assay for Epstein-Barr virus detection.
J Clin Microbiol.
2000;38:712-715 24. Klein MR, Van der Burg SH, Hovenkamp E, et al. Characterization of HLA-B57-restricted human immunodeficiency virus type 1 Gag- and RT-specific cytotoxic T lymphocyte responses. J Gen Virol. 1998;79:2191-2201[Abstract].
25.
Matloubian M, Concepcion RJ, Ahmed R.
CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection.
J Virol.
1994;68:8056-8063
26.
Battegay M, Moskophidis D, Rahemtulla A, Hengartner H, Mak TW, Zinkernagel RM.
Enhanced establishment of a virus carrier state in adult CD4+ T-cell deficient mice.
J Virol.
1994;68:4700-4704
27.
Zajac AJ, Blattman JN, Murali-Krishna K, et al.
Viral immune evasion due to persistence of activated T cells without effector function.
J Exp Med.
1998;188:2205-2213 28. von Herrath MG, Yokoyama M, Dockter J, Oldstone MBA, Whitton JL. CD4-deficient mice have reduced levels of memory cytotoxic T lymphocytes after immunization and show diminished resistance to subsequent virus challenge. J Virol. 1996;70:1072-1079[Abstract].
29.
Cardin RD, Brooks JW, Sarawar SR, Doherty PC.
Progressive loss of the CD8+ T cell-mediated control of a g-Herpesvirus in the absence of CD4+ T cells.
J Exp Med.
1996;184:863-871
30.
Hasenkrug KJ, Brooks DM, Dittmer U.
Critical role for CD4(+) T cells in controlling retrovirus replication and spread in persistently infected mice.
J Virol.
1998;72:6559-6564
31.
Walter EA, Greenberg PD, Gilbert MJ, et al.
Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.
N Engl J Med.
1995;333:1038-1044
32.
Rooney CM, Smith CA, Ng CY, et al.
Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients.
Blood.
1998;92:1549-1555 33. Brodie SJ, Lewinsohn DA, Patterson BK, et al. In vivo migration and function of transferred HIV-1-specific cytotoxic T cells. Nat Med. 1999;5:34-41[CrossRef][Medline] [Order article via Infotrieve].
34.
Rosenberg ES, Billingsley JM, Caliendo AM, et al.
Vigorous HIV-1 specific CD4+ T cell responses associated with control of viremia.
Science.
1997;278:1447-1450 35. Pontesilli O, Carotenuto P, Kerkhof-Garde SR, et al. Lymphoproliferative response to HIV-1 p24 in long-term survivors of HIV-1 infection is predictive of persistent AIDS-free infection. AIDS Res Hum Retroviruses. 1999;15:973-981[CrossRef][Medline] [Order article via Infotrieve].
36.
Klein MR, Van Baalen CA, Holwerda AM, et al.
Kinetics of Gag-specific CTL responses during the clinical course of HIV-1 infection: a longitudinal analysis of rapid progressors and long-term asymptomatics.
J Exp Med.
1995;181:1365-1372 37. Rinaldo CR, Huang XL, Fan Z, et al. High levels of antihuman immunodeficiency virus type 1 (HIV-1) memory cytotoxic T-lymphocyte activity and low viral load are associated with lack of disease in HIV-1 infected long-term nonprogressors. J Virol. 1995;69:5838-5842[Abstract]. 38. Pitcher CJ, Quittner C, Peterson DM, et al. HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression. Nat Med. 1999;5:518-525[CrossRef][Medline] [Order article via Infotrieve].
39.
Kalams SA, Buchbinder SP, Rosenberg ES, et al.
Association between virus-specific cytotoxic T-lymphocyte and helper responses in human immunodeficiency virus type 1 infection.
J Virol.
1999;73:6715-6720
40.
Callan MFC, Tan L, Annels N, et al.
Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus in vivo.
J Exp Med.
1999;187:1395-1402
41.
Tan LC, Gudgeon N, Annels NE, et al.
A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers.
J Immunol.
1999;162:1827-1835
42.
Meyaard L, Otto SA, Jonker RR, Mijnster MJ, Keet RPM, Miedema F.
Programmed death of T cells in HIV-1 infection.
Science.
1992;257:217-219 43. Meyaard L, Otto SA, Keet IP, Roos MT, Miedema F. Programmed death of T cells in HIV-1 infection: no correlation with progression to disease. J Clin Invest. 1994;93:982-988. 44. Slifka MK, Rodriguez F, Whitton JL. Rapid on/off cycling of cytokine production by virus-specific CD8+ T cells. Nature. 1999;401:76-79[CrossRef][Medline] [Order article via Infotrieve].
45.
Lechner F, Wong DK, Dunbar PR, et al.
Analysis of successful immune responses in persons infected with hepatitis C virus.
J Exp Med.
2000;191:1499-1512 46. Lee PP, Yee C, Savage PA, et al. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat Med. 1999;5:677-685[CrossRef][Medline] [Order article via Infotrieve].
47.
Appay V, Nixon DF, Donahoe SM, et al.
HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function.
J Exp Med.
2000;192:63-76
© 2001 by The American Society of Hematology.
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  J. N. Blattman, E. J. Wherry, S.-J. Ha, R. G. van der Most, and R. Ahmed Impact of Epitope Escape on PD-1 Expression and CD8 T-Cell Exhaustion during Chronic Infection J. Virol., May 1, 2009; 83(9): 4386 - 4394. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
 E. Piriou, K. van Dort, N. M. Nanlohy, M. H. J. van Oers, F. Miedema, and D. van Baarle Loss of EBNA1-specific memory CD4+ and CD8+ T cells in HIV-infected patients progressing to AIDS-related non-Hodgkin lymphoma Blood, November 1, 2005; 106(9): 3166 - 3174. [Abstract] [Full Text] [PDF]
|
|
|
|
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|
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 |
|
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|
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|
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|
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|
|
|
|
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|
 A. T.C. Chan, Q. Tao, K. D. Robertson, I. W. Flinn, R. B. Mann, B. Klencke, W. H. Kwan, T. W.-T. Leung, P. J. Johnson, and R. F. Ambinder Azacitidine Induces Demethylation of the Epstein-Barr Virus Genome in Tumors J. Clin. Oncol., April 15, 2004; 22(8): 1373 - 1381. [Abstract] [Full Text] [PDF]
|
|
|
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|
|
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|
|
|
|
|
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|
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|
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Top
Abstract
Introduction
) indicates CD4; (
), CD8.
(B) Longitudinal analysis of B8-FLR-specific CD8+ T
lymphocytes as assessed by tetramer staining (percentage of
CD8+ T cells, solid line) and IFN-
IFN-
29% and
IFN-
= 0.076), which was highly significant (P < .037), whereas no
correlation was found between CD4+ T-cell numbers and the
number of tetramer+ T cells (
), AIDS-OI.