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Department of Pediatrics and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The use of HLA class I tetramers in the lymphocytic choriomeningitis virus (LCMV)3 mouse model has shown that very high levels of Ag-specific CD8+ T cells are generated and maintained into memory (9). The size of the CD8+ T cell memory pool is reflective of the initial clonal burst size during the acute LCMV response (10). The TCR repertoire of the primary antiviral CD8 T cell response to LCMV was shown to be similar to that of the memory pool (11). Altogether, these data suggest a stochastic selection of memory cells from the pool of CD8+ T cells activated during primary infection.
Similar conclusions were reached by Callan et al. (12) in a study of CD8+ T cell responses from acute through chronic EBV infection. However, recent evidence from human HIV-1 infection has shown that an HLA-A2-restricted response to an HIV-1 gag epitope found in a majority of HLA-A2 individuals with chronic HIV-1 infection was not present in 11 HLA-A2 individuals during acute HIV-1 infection (13). This suggests that for persistent viral infections the memory/effector pool may not be reflective of the initial pool of CD8+ T cells and that the generation of CD8+ T cells may not fit a stochastic model.
To examine the generation and maintenance of CD8+ T cell responses during a persistent human viral infection, we analyzed a cohort of individuals experiencing acute EBV infection using several EBV peptide epitopes restricted by common HLA alleles: HLA-A*0201 (A2); HLA-B*0702 (B7); and HLA-A*0301 (A3). By studying individuals with acute EBV infection, we were able to determine the frequencies of CD8+ T cell responses to previously defined lytic and latent epitopes during acute infection. We then followed these same individuals into latent EBV infection to characterize the evolution of EBV epitope-specific CD8+ T cell responses over time.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These studies were conducted in adolescents (17–24 yr old) presenting to the clinic at the University of Massachusetts Amherst Student Health Service (Amherst, MA) with clinical symptoms consistent with acute EBV infection (fever, rash, fatigue, hepatosplenomegaly). After informed consent, students presenting with acute-like symptoms gave five blood samples (50 ml each) at the following time points: at presentation with symptoms (V-1); and 1 wk (V-2), 2 wk (V-3), 6 mo (V-4); and 1 yr (V-5) after presentation. Inclusion in the study was based on a positive Monospot test and the presence of atypical lymphocytes. Acute EBV infection was confirmed through the detection of IgM for the EBV viral capsid Ag.
Healthy EBV-seropositive adults >30 yr of age were recruited for these studies from the UMMS research community. All of these individuals likely experienced EBV infection for a minimum of 10 yr before study. Prior EBV infection was confirmed through the detection of EBV capsid Ag-specific IgG Abs in the peripheral blood. After informed consent, study subjects provided blood samples (50 ml) at entry and every 3 mo thereafter. These studies were approved by the Human Studies Committee at the University of Massachusetts Medical School (Worcester, MA).
Molecular HLA class I typing
Molecular HLA class I typing was done on all study participants by Microdiagnostic (Nashville, TN).
Detection of cytokine-secreting CD8+ T cells after in vitro stimulation with EBV peptide
A modification of the method by Kern et al. (14)
was used for the detection of cytokine-secreting
CD8+ T cells. Whole blood (0.3 ml/test, heparin)
was incubated for 1 h at 37°C with 2 µM EBV peptides. Because
staphylococcal enterotoxin B (Toxin Technology, Sarasota, FL) is able
to nonspecifically stimulate T cells to secrete IFN-
, it was used as
a positive control. None of the donors used in this study was HIV-1
infected; therefore, the HLA-A2-binding, HIV-1 gag SLYNTVATL peptide
was used as a negative control. After 1 h incubation with peptide,
Golgiplug (BD PharMingen, San Diego, CA) was added to the cells, and
they were incubated for an additional 5 h. After incubation, the
cells were incubated with 2 µM EDTA for 15 min with vigorous
vortexing every few minutes. The cells were stained with combinations
of the following Abs: IFN- APC, CD3 PerCP, CD69 PE, and CD8 FITC.
The cells were incubated at room temperature for 30 min and then washed
with 1% FBS in PBS; 100,000 events gated on the lymphocyte population
were collected and analyzed immediately by 4-color flow cytometry.
Appropriate isotype, negative, and positive controls were used to
define positive and negative cell populations. Background IFN-
production in this assay was 0.02 ± 0.01% for stimulation of
individuals with either a peptide derived from HIV-1- or EBV-derived
non-HLA-binding peptides. Significant IFN- production was considered
to be greater than the mean of the background plus 3 SD, or 0.05% of
CD8+ T cells.
Generation of HLA class I tetramers
HLA-A*0201 H chain (residues 1–275) and human
2-microglobulin (2m)
in the prokaryotic expression system pET R&D were obtained from D.
Garboczi (National Institutes of Health, Bethesda, MD). The 3' end of
the HLA-A*0201 H chain was modified with a BIR A biotinylation site as
published (15). HLA-B*0702 was a gift of Dr. E. Pamer
(Memorial Sloan-Kettering Cancer Center, New York, NY). As previously
described (16), HLA class I H chain constructs or
2m were grown to midlog phase and induced with
0.5 M isopropyl--thiogalactosidase. Inclusion bodies were purified
and solubilized in 6 M guanidine-HCl, pH 8.2.
The H chain, 2m, and peptide were refolded by
dilution as described (16). The A2 BMLF-1 peptide,
GLCTLVAML, and the B7 EBNA-3A peptide, RPPIFIRRL, were synthesized by
the University of Massachusetts Medical Center peptide core facility
and purified before usage. Briefly, 1 µM H chain, 2 µM
2m, and 30 µM peptide were refolded by
dilution (0.4 M L-arginine, 0.1 M Tris (pH 8.0), 0.002 M
EDTA, 0.005 M reduced glutathione, 0.5 M oxidized glutathione, and 0.5
mM PMSF (pH 8.3)) and incubated for 24 h at 10°C with gentle
stirring. The refolded monomer was concentrated using an Amicon Stirred
Cell (Amicon, Beverly, MA). The 45-kDa refolded product was isolated on
an ACTA fast protein liquid chromatograph using a Superdex 200 column
(Amersham-Pharmacia, Piscataway, NJ). After gel filtration, the
refolded monomer was biotinylated with biotin ligase (birA; Avidity,
Denver, CO) and washed to remove excess biotin. The resulting
biotinylated monomer was mixed with streptavidin-APC conjugate (BD
PharMingen) at a molar ratio of 4:1 to form tetramers
(15). To stain 100 µl whole blood, 100 ng tetramer were
used. Percentages of tetramer-binding cells were expressed as a
percentage of total CD8+ T cells. These tetramers
stained non-A2 or non-B7 individuals with acute EBV infection and A2,
B7 individuals who were EBV seronegative at 0.02 ± 0.01%.
Tetramer staining greater than the mean of the background plus 3 SD, or
0.05% of CD8+ T cells, was considered
significant. A total of 100,000 events gated on the lymphocyte
population were collected and analyzed.
Four color flow cytometry for Ki-67 and annexin V expression
Four-color flow cytometric analysis was performed using whole EDTA blood for Ki-67 analysis and heparin preserved blood for annexin V analysis. Because annexin V binding is Ca2+ dependent, EDTA blood stained with annexin V was run alongside as a negative control. Samples were analyzed on a BD Biosciences FACSort with an added laser and FACSCaliber software (BD Biosciences, San Jose, CA). HLA class I tetramers can bind nonspecifically to non-T cell populations; therefore cells were first gated through both CD3 and CD8 to ensure that the tetramer positive cells were all CD8+ T cells. Cells were analyzed with a tetramer conjugated to APC, CD8 FITC (Sigma, St. Louis, MO), CD3 PerCP (BD Biosciences), and Ki-67 PE or annexin V PE (BD PharMingen, San Diego, CA). Permeabilization for staining with Ki-67 was done after tetramer staining and lysing of the cells. Permeabilized isotype controls were run alongside each analysis for Ki-67.
Statistics
Means and SDs were calculated and compared with each other using the Student t test to determine p values. The Wilcoxon signed rank test, a nonparametric paired t test, was used to determine p values for the differences in the range of tetramer-staining CD8+ T cells between study time points for the group with acute EBV infection. The Mann-Whitney U test was used to determine p values between the range of tetramer staining cells between study time points for the acute EBV infection group and the long term latently infected group. To determine whether differences in the percentage of tetramer staining cells were significant, tetramer staining was performed on 12 identical samples for 14 individuals, and the means ± SD for those 12 samples were determined. These were then compiled for the 14 individuals to determine normal variations in tetramer staining. Differences >0.2 ± 0.2% were considered to represent a significant change in magnitude.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first examined the frequency and magnitude of
CD8+ T cell responses to previously defined EBV
lytic protein epitopes (Table I
) during
acute and latent EBV infection using an in vitro assay that detects
single-cell production of IFN- (Fig. 1). The three most prevalent HLA types in
our acute population were HLA-A2+ (45%,
n = 25), HLA-A3+ (16%,
n = 10), and HLA-B7+ (23%,
n = 12); 67% of our acute population had one or more
of these alleles. We therefore prioritized evaluation of responses to
these epitopes. Because HLA typing results were not available at
baseline testing, we used all of the peptides listed in Table I during
presentation with symptoms (V-1); once HLA typing was completed,
peptide panels were individualized for later visits. We then followed
these individuals with acute EBV infection into latent EBV infection
and evaluated their CD8+ T cell responses up to 1
yr postpresentation (V-5). Fresh whole blood was used for all assays to
avoid the possible loss of Ag-specific CD8+ T
cells during Ficoll (Amersham Pharmacia Biotech, Piscataway, NJ)
separation and cell cryopreservation.
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CD8+ T cell responses to an
HLA-A2+-restricted BMLF-1 epitope were detected
in the majority of HLA-A2+ individuals during
acute and latent infection. An HLA-A2+-restricted
gp85 epitope (gp85SV) was also recognized during the acute and the
latent stages of infection. By contrast, CD8+ T
cell responses to the six other lytic epitopes were more commonly
detected during acute infection than during latent infection.
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in response to stimulation with the A2 BMLF-1 peptide
(range, 0.08–12%; median, 2.3%; Fig. 2
). The majority (25 of 27
= 93%) of latently infected individuals also produced IFN- in
response to this peptide at frequencies ranging from 0.06 to 1.6%
(median, 0.56%). Responses to the A2 BZLF-1 epitope were commonly seen
during acute EBV infection (5 of 8 = 63% of individuals; range of
IFN- production, 0–1.25%) but rarely detected during latent
infection (1 of 8 = 12.5%; range of IFN- production,
0–0.15%). Eight of ten (80%) HLA-B7+
individuals responded to the B7 BZLF-1 peptide (range, 0–1.4%) during
acute infection; the response to the B7 BZLF-1 peptide was detected in
only 25% of individuals (2 of 8; range, 0–0.34%) during latent
infection. Similar to the pattern seen for
HLA-A2+- and
HLA-B7+-restricted lytic epitopes, responses were
also more frequently detected to the A3 BRLF-1 epitope during acute EBV
infection (8 of 10 = 80%; range, 0–3.2%), than during
latent EBV infection (1 of 8 = 13%; range, 0–0.03%).
We also examined the frequency and magnitude of
CD8+ T cell responses to previously defined EBV
latent protein epitopes (Table I) in acute and latent EBV infection
using the in vitro assay for the production of IFN- (Fig. 3). Latent epitope responses were rarely
detected in individuals presenting with acute EBV infection. Only 2 of
20 (10%) HLA-A2+ individuals responded to the A2
EBNA-3A epitope during acute EBV infection (range, 0.05–0.06%), but
14 of 20 (70%) latently infected HLA-A2+
individuals responded to the A2 EBNA-3A epitope (range, 0.05–0.41%);
3 of 20 (13.6%) latently infected HLA-A2+
individuals also responded to the A2 LMP-1 epitope (range,
0.09–0.5%). Whereas only 3 of 12 (25%) acutely infected
HLA-B7+ individuals responded to the B7 EBNA-3A
epitope (range, 0–0.44%), all 10 latently infected individuals
responded to the B7 EBNA-3A epitope (range, 0.08–1.2%). A similar
pattern was detected for the B7 EBNA-3C epitope. Only one
HLA-A3+ individual (n = 8;
0.23%) responded to the A3 EBNA-3A epitope during acute EBV infection,
and this response was not maintained into latent EBV infection.
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We detected IFN- responses to the A2 BMLF-1 peptide in all
individuals with acute EBV infection and in all of our acute study
individuals 1 yr post-EBV infection. Because IFN- production can
underestimate the frequency of these responses, we developed HLA class
I tetramers to more precisely determine the magnitude of the A2
BMLF-1-specific response (Fig. 4a). At presentation with
acute EBV infection, A2 BMLF-1 (lytic EBV gene) tetramer-binding
CD8+ T cells were detected in all
HLA-A2+ individuals (V-1, n = 25;
range, 0.1–47% of CD8+ T cells; median, 2.31%
(Fig. 4b). One week postpresentation (n =
19), the percentage of A2 BMLF-1-specific T cells ranged from 0.2 to
15.9% (median, 2.35%); 7 of 19 (36%) HLA-A2+
individuals at this time point had increases in their percentages of A2
BMLF-1 tetramer-binding CD8+ T cells. The median
frequency of A2 BMLF-1 tetramer-binding CD8+ T
cells then decreased at 2 wk (V-3; n = 19; range,
0.14–9.76%; median, 1.08%), 6 mo (V-4; n = 11;
range, 0.2–1.93%; median, 1.19%) and 1 yr postpresentation (V-5;
n = 7; range, 0.08–1.4%; median, 0.23%).
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B7 EBNA-3A-specific responses were detected in only three of eight of
the HLA-B7+ individuals with acute EBV infection
but in all EBV latently infected individuals. We developed a B7 EBNA-3A
tetramer to more precisely determine the magnitude of
epitope-specific CD8+ T cells during acute
and latent EBV infection (Fig. 5a). B7 EBNA-3A
tetramer-binding CD8+ T cells were detected
rarely (3 of 9 = 33%) and at low frequencies in
HLA-B7+-positive individuals with acute EBV
infection at multiple time points within 2 wk of symptom onset (V-1,
V-2, or V-3; range, 0–0.44%; Fig. 5b). In the three
individuals with detectable B7 EBNA-3A tetramer-binding
CD8+ T cells during V-1, the changes in magnitude
between presentation and 2 wk were not statistically different. B7
EBNA-3A tetramer-binding cells have been detected in all individuals 6
mo after entry into the study (V4; 8 of 8; range, 0.08–1.2%; median,
0.18%) and 1 yr after entry to the study (V5; 5 of 5; range,
0.16–0.46%; median, 0.24%). B7 EBNA-3A tetramer-staining cells have
also been detected in all 6 HLA-B7+ EBV-long
term-seropositive individuals studied thus far (range, 0.08–1.95%;
median, 0.39%).
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b). By
contrast, there was very little change in the percentages of B7 EBNA-3A
CD8+ T cells for the three individuals analyzed
(Fig. 5b). We wanted to determine whether the initial burst
size of CD8+ T cells determined the final
magnitude of response detected 1 yr post-EBV infection. Comparison of
the percentages A2 BMLF-1-specific CD8+ T cells
in early acute infection with the percentage of A2 BMLF-1-specific
CD8+ T cells at 1 yr for the same individual
revealed no correlation between the initial and final magnitude of the
response (Fig. 4b and data not shown). Additionally, there
appeared to be no detectable burst for the B7 EBNA-3A tetramer-binding
CD8+ T cells; there were no significant
differences between the initial and final magnitude of the B7 EBNA-3A
response in the 3 HLA-B7+ individuals studied
(Fig. 5b). Changes in magnitude of epitope-specific CD8+ T cell responses from acute through chronic infection
Because the early A2 BMLF-1-specific CD8+ T
cell frequencies did not appear to predict CD8+ T
cell frequencies at 6 mo or 1 yr, we wanted to determine the dynamics
of loss or gain of CD8+ T cells during the year.
Tables II and III show the fold loss (or increase
represented by a number <1) over time in the frequencies of A2 BMLF-1
or B7 EBNA3A tetramer-staining cells. Large changes in the frequencies
of A2 BMLF-1 tetramer-binding CD8+ T cells were
observed; in most (6 of 10) individuals, the largest changes occurred
between 2 wk and 6 mo postpresentation. By contrast, the frequencies of
the B7 EBNA-3A tetramer-binding CD8+ T cells did
not change much over the course of our study. All of the
HLA-B7+ individuals who did not have detectable
B7 EBNA-3A tetramer-binding CD8+ T cells during
the first 2 wk of our study developed them between 2 wk and 6 mo of the
study.
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shows analyses of the
expansion of epitope-specific cells using Ki-67, an Ag expressed during
all active stages of the cell cycle. At presentation with symptoms,
60 ± 12% of the A2 BMLF-1 tetramer-binding
CD8+ T cells coexpressed Ki-67. A significant
reduction in Ki-67 coexpression was noted at 1 (26 ± 16) and 2 wk
postpresentation (5 ± 4%) and persisted through 6 mo to 1 yr
postinfection (Fig. 6a). Analyses of Ki-67 expression for 3
HLA-B7+ individuals with detectable B7
EBNA-3A-specific CD8+ T cells at V-1–V-3 showed
similar Ki-67 expression (data not shown). The coexpression of Ki-67 on
the A2 BMLF-1 tetramer-binding CD8+ T cells was
not significantly different from the coexpression of Ki-67 in the whole
CD8+ T cell population at all time points.
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b). The percentage of CD8+ T cells
coexpressing annexin V appeared to decrease at 1 wk (3.59 ±
4.42%) and 2 wk (2.04 ± 2.47%) after presentation, but these
differences were not statistically significant. Similar patterns of
expression of annexin V were detected in B7 EBNA-3A tetramer-binding
CD8+ T cells. No annexin V expression was
detected in the A2 BMLF-1 or B7 EBNA-3A tetramer-binding
CD8+ T cells from the 6-mo or 1-yr postacute EBV
infection patients or for the long term-seropositive individuals. EBV-specific memory populations remain stable over time in latently infected individuals
EBV is a chronic viral infection in which Ag persists during the
lifetime of the host, and the effect of this Ag on the immune system
over time is unknown. Selin et al. (27) have shown that
the frequency of virus-specific CD8+ T cells can
change after infection with heterologous viruses. Humans do not live in
germfree environments, and we would expect our long term
EBV-seropositive donors to come into contact with many different types
of pathogens during a period of 2 yr. To determine the stability of the
EBV-specific CD8+ T cell populations over time,
we monitored eight long term EBV-seropositive individuals several times
during a period of 20 mo to determine their change in frequency of A2
BMLF-1- or B7 EBNA-3A-specific CD8+ T cell
responses. Fig. 7 demonstrates that the
frequency of both A2 BMLF-1- and B7 EBNA-3A-specific
CD8+ T cell responses were stable over
time.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of the eight early or lytic protein epitopes evaluated, all but one (an
HLA A2-restricted gp350 epitope) were recognized by the majority of
individuals evaluated during acute EBV infection. By contrast,
responses to eight previously defined EBV latent protein epitopes were
infrequently detected during acute infection. Of the eight EBV latent
protein epitopes evaluated, only one A2- and two B7-restricted EBNA-3
epitopes were well recognized during latency. When detected, the
CD8+ T cell responses to EBV latent protein
epitopes were of lower frequency (
1% of CD8+ T
cells) than CD8+ T cell responses to the lytic
epitopes and remained stable during the 1 yr of follow-up. These
results suggest the predominance of lytic epitope-specific
CD8+ T cell responses during acute EBV infection
and are in agreement with other recently published data from fewer
individuals (23, 28, 29, 30). Although it has been suggested
that the differential response of CD8+ T cells to
lytic and latent Ags is due to different stages of the EBV lifecycle
(13), all of the latent gene products are expressed during
the lytic cycle (31). Lytic proteins also appear to be
expressed periodically during latent EBV infection (32, 33). Recently, it has been reported that although the EBNA-3A
protein is expressed by cultured BLCL lines in vitro, EBNA-3A
expression was not detected in either the peripheral blood or tonsillar
cells of latently infected healthy, EBV-seropositive donors
(34). Although these data do not exclude intermittent or
ongoing low level expression of EBNA-3A (particularly at a site outside
the blood), they suggest that the differential expression of viral
proteins does not fully account for the response to these Ags.
Of interest is that Callan et al. (12, 28, 29) have reported that HLA-B8+ individuals generate and maintain an HLA-B8+-restricted CD8+ T cell response to a BRLF-1 epitope. In all but one of our HLA-A3+ individuals, BRLF-1 epitope-specific immune responses were readily detectable during acute EBV infection, but not at 1 yr post-EBV infection. If the presence of a specific CD8+ T cell response were simply dependent on the presence of Ag, we would expect to see maintenance of BRLF-1 responses to both the A3 and the B8 epitopes. Our data demonstrating loss of an HLA-A3+-restricted CD8+ T cell response to BRLF-1 along with the data of Callan et al. demonstrating the maintenance of an HLA-B8-restricted response to BRLF-1 suggest that maintenance of the response to this epitope may be determined in a large part by the HLA type of the individual. Alternatively, cross-reactive responses present before antigenic challenge may lead to preferential expansion and maintenance of some responses compared with others. This has been demonstrated in the mouse model of acute LCMV infection, in which pre-existing responses to LCMV resulted in increased CD8+ T cell frequencies to heterologous viral epitopes when compared with naive mice (35).
Study of the dynamics of the generation and maintenance of BMLF-1 (lytic) and EBNA-3A (latent) epitope-specific CD8+ T cells using HLA class I tetramers showed that the early frequencies of CD8+ T cell responses to these epitopes did not correlate with their maintenance into the pool of memory CD8+ T cells. Previous studies in the mouse have shown a correlation between the initial burst size and the final magnitude of the response (10). Our results demonstrated a massive expansion of A2 BMLF-1-specific CD8+ T cells, but large expansion of B7 EBNA-3A-specific CD8+ T cells, or any of the other latent EBV-specific CD8+ T cells were not detected during any of the time points of this study. These EBNA-3A-specific CD8+ T cell responses were maintained at percentages similar to those of the A2 BMLF-1-specific CD8+ T cells in all of the HLA-A2+B7+ individuals. These data suggest that a large burst size is not necessary to provide the host with both effector and long term memory CD8+T cells and that the generation of memory CD8+ T cells in a persistent viral infection is not simply a stochastic process.
In contrast to the dynamic nature of epitope-specific CD8+ T cell responses during acute infection, the frequency of A2 BMLF-1- and B7 EBNA-3A-specific CD8+ T cells remained constant in all chronically infected individuals studied during 2 yr. Although these individuals likely experienced many immune challenges during the 2 yr of study, there were only minor perturbations in their frequencies over time. Data generated by Khan et al. (36) demonstrated that EBV viral DNA loads are stable in individuals over time periods spanning several years. Periodic reactivations of the EBV virus in the peripheral blood have also been demonstrated, suggesting that the stability DNA load over time is not that way because the virus is quiescent (32, 33). Our data demonstrating that CD8+ T cell frequencies to two different EBV epitopes are also stable over time suggest that during persistent viral infections a long term homeostasis exists between the virus and the immune system.
In summary, the study of EBV as a model for the generation and maintenance of epitope-specific CD8+ T cells in a human persistent viral infection has shown that the memory pool is not reflective of the initial pool of CD8+ T cells and that the generation of CD8+ T cell responses may not fit a stochastic model. The maintenance of epitope-specific CD8+ T cells to a persistent viral infection over time reflects a homeostasis reached between virus and the immune system. Perturbations in this homeostasis may in part explain EBV-associated diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Katherine Luzuriaga, Pediatrics/Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Suite 318, Worcester, MA 01605. E-mail address: katherine.luzuriaga{at}umassmed.edu
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; A2, HLA-A*0201; B7, HLA-B*0702; A3, HLA-A*0301; 2m, 2-microglobulin.
Received for publication April 10, 2001. Accepted for publication August 14, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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D. Hochberg, T. Souza, M. Catalina, J. L. Sullivan, K. Luzuriaga, and D. A. Thorley-Lawson Acute Infection with Epstein-Barr Virus Targets and Overwhelms the Peripheral Memory B-Cell Compartment with Resting, Latently Infected Cells J. Virol., May 15, 2004; 78(10): 5194 - 5204. [Abstract] [Full Text] [PDF]
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L. Gibson, G. Piccinini, D. Lilleri, M. G. Revello, Z. Wang, S. Markel, D. J. Diamond, and K. Luzuriaga Human Cytomegalovirus Proteins pp65 and Immediate Early Protein 1 Are Common Targets for CD8+ T Cell Responses in Children with Congenital or Postnatal Human Cytomegalovirus Infection J. Immunol., February 15, 2004; 172(4): 2256 - 2264. [Abstract] [Full Text] [PDF]
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E. Amyes, C. Hatton, D. Montamat-Sicotte, N. Gudgeon, A. B. Rickinson, A. J. McMichael, and M. F.C. Callan Characterization of the CD4+ T Cell Response to Epstein-Barr Virus during Primary and Persistent Infection J. Exp. Med., September 15, 2003; 198(6): 903 - 911. [Abstract] [Full Text] [PDF]
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L. Santodonato, G. D'Agostino, R. Nisini, S. Mariotti, D. M. Monque, M. Spada, L. Lattanzi, M. Paola Perrone, M. Andreotti, F. Belardelli, et al. Monocyte-Derived Dendritic Cells Generated After a Short-Term Culture with IFN-{alpha} and Granulocyte-Macrophage Colony-Stimulating Factor Stimulate a Potent Epstein-Barr Virus-Specific CD8+ T Cell Response J. Immunol., May 15, 2003; 170(10): 5195 - 5202. [Abstract] [Full Text] [PDF]
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M. Terajima, J. Cruz, G. Raines, E. D. Kilpatrick, J. S. Kennedy, A. L. Rothman, and F. A. Ennis Quantitation of CD8+ T Cell Responses to Newly Identified HLA-A*0201-restricted T Cell Epitopes Conserved Among Vaccinia and Variola (Smallpox) Viruses J. Exp. Med., April 7, 2003; 197(7): 927 - 932. [Abstract] [Full Text] [PDF]
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M. L. Precopio, J. L. Sullivan, C. Willard, M. Somasundaran, and K. Luzuriaga Differential Kinetics and Specificity of EBV-Specific CD4+ and CD8+ T Cells During Primary Infection J. Immunol., March 1, 2003; 170(5): 2590 - 2598. [Abstract] [Full Text] [PDF]
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U. Karrer, S. Sierro, M. Wagner, A. Oxenius, H. Hengel, U. H. Koszinowski, R. E. Phillips, and P. Klenerman Memory Inflation: Continuous Accumulation of Antiviral CD8+ T Cells Over Time J. Immunol., February 15, 2003; 170(4): 2022 - 2029. [Abstract] [Full Text] [PDF]
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 M. Sester, U. Sester, B. C. Gartner, M. Girndt, A. Meyerhans, and H. Kohler Dominance of Virus-Specific CD8 T Cells in Human Primary Cytomegalovirus Infection J. Am. Soc. Nephrol., October 1, 2002; 13(10): 2577 - 2584. [Abstract] [Full Text] [PDF]
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M. D. Catalina, J. L. Sullivan, R. M. Brody, and K. Luzuriaga Phenotypic and Functional Heterogeneity of EBV Epitope-Specific CD8+ T Cells J. Immunol., April 15, 2002; 168(8): 4184 - 4191. [Abstract] [Full Text] [PDF]
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A. D. Hislop, N. E. Annels, N. H. Gudgeon, A. M. Leese, and A. B. Rickinson Epitope-specific Evolution of Human CD8+ T Cell Responses from Primary to Persistent Phases of Epstein-Barr Virus Infection J. Exp. Med., April 1, 2002; 195(7): 893 - 905. [Abstract] [Full Text] [PDF]
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) or B7 EBNA-3A (
)
tetramer-binding CD8 T cells during 20 mo for 3 A2/B7 and 5 A2 long
term EBV-seropositive donors.