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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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, or the ability to proliferate in response to Ag in
vitro. In individuals with latent EBV infection, the frequencies of A2
BMLF-1- or B7 EBNA-3A-specific CD8+ T cells that expressed
CD45RA, CD45RO, CD62 ligand, CCR7, and perforin were stable over
time. However, the expression of CD62 ligand and CCR7 was significantly
higher among EBNA-3A-specific CD8+ T cells than among
BMLF-1-specific CD8+ T cells. Further work is necessary to
understand how phenotypic and functional differences between EBV
epitope-specific CD8+ T cells are related to the biology of
the virus and to the equilibrium between the virus and the host during
persistent infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several lines of evidence suggest that memory EBV-specific CD8+ T cells play an important role in controlling viral replication or preventing disease in established (or latent) infection, and EBV is an important source of morbidity and mortality in individuals with compromised cell-mediated immunity. For this reason several groups, including our own, have thought it important to characterize the development and maintenance of these CD8+ T cell responses over the course of infection.
Adolescents and adults with acute EBV infection frequently present with acute infectious mononucleosis (AIM)3 syndrome and may be easily distinguished by the detection of heterophile Abs in peripheral blood. It has long been recognized that CD8+ T cells are markedly expanded and highly activated during primary infection. Callan et al. (3) were among the first to report that the majority of these CD8+ T cells are EBV-specific. They and others (4, 5) have documented preferential recognition of lytic as opposed to latent epitopes early in acute EBV infection. EBV lytic and latent epitope-specific memory CD8+ T cell responses have also been persistently detected in established (or latent) infection (4, 6, 7).
Characterization of the evolution of the CD8+ T cell repertoire and effector functions should help us to better understand virus-host interactions that contribute to persistent EBV infection. Murine models of lymphocytic choriomeningitis virus (LCMV) infection (a nonpersistent viral infection) suggest that the memory CD8+ T cell repertoire largely reflects the repertoire generated during acute infection, but less is known regarding the relationship between acute and memory epitope-specific CD8+ T cells responses in persistent human viral infections. We have recently reported that the epitope specificity of EBV-specific memory CD8+ T cells varies over the course of infection, and is not simply reflective of CD8+ T cell responses detected during primary infection (5). This suggests that the generation of memory EBV epitope-specific CD8+ T cell responses does not fit the stochastic model proposed on the basis of murine models of viral infection.
The use of HLA class I peptide tetramers together with mAb staining to cell surface Ags and functional (cytokine secretion or cytolysis) assays has allowed examination of the phenotype and function of epitope-specific CD8+ T cells at the single cell level. Using this approach, several investigators have proposed that differential cell surface expression of the CD45 isoforms (RA vs RO), CD27, CD28, and CCR7 delineates a maturational pathway for CD8+ T cell responses (8, 9). Although these studies provide provocative evidence for the heterogeneity of memory CD8+ T cell surface phenotype, strict correlation of these phenotypes with function has been called into question. Recently, Hislop and colleagues (10) have reported a relationship between memory CD8+ T cell phenotype and EBV epitope specificity (lytic vs latent) in individuals with latent infection. EBV latent epitope-specific CD8+ T cells were polarized toward a CD45ROhighCD45RA-CD28+ phenotype, whereas EBV lytic epitope-specific CD8+ T cell responses were CD45RA+ or CD45RO+ and were more commonly CD28-. These phenotypic differences did not correlate with cytokine secretion or cytolytic activity. Lytic and latent epitope-specific CD8+ T cells were heterogeneous for CCR7 expression, but there was a trend toward greater CCR7 expression in the latent epitope-specific CD8+ T cells. Interestingly, effector cell function appeared to be limited to the CCR7- subset of cells.
In our prior studies evaluating EBV epitope-specific CD8+ T cell responses, most of the changes in epitope specificity and epitope-specific CD8+ T cell activation occurred over the first year following presentation with symptoms of AIM. Therefore, we felt it important to begin to evaluate epitope-specific CD8+ T cell phenotype and function at presentation with AIM through at least 1 year following infection. Using an HLA-A2 tetramer made with an epitope derived from BMLF-1 (an early lytic gene; Refs. 1, 11 and 12) and an HLA-B7 tetramer made with an epitope derived from the EBV-encoded nuclear Ag-3A latent protein (13, 14), we sequentially characterized EBV epitope-specific CD8+ T cell responses in HLA-A2 and HLA-B7 individuals presenting with AIM and compared them with memory CD8+ T cell responses detected in HLA-matched individuals with established infection. Phenotypic and functional differences were observed over time within and between these two epitope-specific populations. The potential implications for the establishment of persistent EBV infection are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These studies were conducted in adolescents (aged 17–24 years old) presenting to the clinic at the University of Massachusetts/Amherst Student Health Service (Amherst, MA) with clinical symptoms consistent with AIM (fever, rash, fatigue, and hepatosplenomegaly). Following informed consent, students 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 year (V-5) following presentation. Entry to the study was based on a positive Monospot test and presence of atypical lymphocytes. Acute EBV infection was confirmed through the detection of IgM for the EBV viral capsid Ag.
All participants were analyzed using HLA class I tetramers upon blood receipt (within 4 h of blood draw) and participants with tetramer-binding CD8+ T cells were further analyzed using a panel of activation and adhesion markers (see below). The HLA type of all study participants was confirmed through molecular HLA class I typing, and there was a 100% correspondence between tetramer-binding CD8+ T cells and HLA type.
Healthy, EBV seropositive adults >30 years of age were also studied.
Non-HLA A2 and HLA B7, EBV seropositive individuals served as negative
controls in all studies. Prior EBV infection was confirmed through the
detection of EBV capsid Ag-specific IgG Abs in the peripheral blood.
All of these individuals likely experienced EBV infection a minimum of
10 years before study; none had prior history of AIM. Following
informed consent, study participants 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 (Worchester, MA).
Molecular HLA class I typing
Molecular HLA class I typing was done on all study participants by Microdiagnostic (Nashville, TN).
Generation of HLA class I tetramers
HLA class I tetramers with EBV peptides (A2 BMLF-1/GLCTLVAML, B7 EBNA-3A/RPPIFIRLL, and A2 LMP-2/CLGGLLTMV were made as previously described; Ref. 15). Refolded monomers were mixed with streptavidin-allophycocyanin conjugate (BD PharMingen, San Diego, CA) at a molar ratio of 4:1 to form tetramers. These tetramers stained non-HLA-A2 or non-HLA-B7 individuals with acute EBV infection and HLA-A2 and/or HLA-B7 EBV seronegative individuals at 0.02 ± 0.01%. Tetramer staining greater than the mean of the background plus three SD, or 0.05% of CD8+ T cells, was considered significant.
HLA-peptide tetramer staining to enumerate and characterize EBV epitope-specific CD8+ T cells
All four-color flow cytometry analysis was performed using fresh, whole EDTA-anticoagulated blood. Samples were analyzed on a BD Biosciences FACSort with an added laser and FACSCaliber software (BD Biosciences, San Jose, CA). A tetramer screen was performed using the A2 BMLF-1-specific and B7 EBNA-3A-specific tetramers on all patients’ whole blood within 4 h of blood draw, and activation marker studies were performed within 14 h of blood draw. Patients with tetramer-binding CD8+ T cells were analyzed for activation marker expression immediately pending confirmation of HLA type. HLA class I tetramers can bind nonspecifically to non-T cell populations, thus cells were first gated through both CD3 and CD8 to insure that the tetramer-positive cells were all CD8+ T cells. Cells were analyzed with a tetramer conjugated to allophycocyanin, CD8 FITC (Sigma Aldrich, St. Louis, MO), CD3 PerCP (BD Biosciences), and one of the following Abs: HLA-DRPE, CD45ROPE, CD45RAPE, CD69PE, CD62 ligand (CD62L)PE, CD28PE, CD27PE, and CD38PE (BD PharMingen). Isotope controls IgG1 FITC, IgG2b PerCP, and one of each IgG1PE, IgG2aPE, and IgG2bPE were done on each patient at each time point. Permeabilization for staining with perforin (BD PharMingen) was done after tetramer staining and lysis of the RBC (FACSlyse, BD Biosciences). Permeabilized isotope control Abs were run alongside each analysis for perforin; 100,000 lymphocyte events were collected per sample.
Detection of cytokine-secreting CD8+ T cells following in vitro stimulation with EBV peptides
Fresh whole blood (0.3 ml/test, heparin) was incubated for
1 h at 37°C with 0.5–10 µM EBV or control HIV-1 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 HLA-A2 latent donors used in
this study was HIV-1 infected, so the HIV-1 gag (SLYNTVATL) peptide was
used as a negative control. After a 1-h incubation with peptide,
Golgiplug (BD PharMingen) was added to the cells and they were
incubated an additional 5 h. After incubation the cells were
incubated with 2 µM EDTA for 15 min with vigorous vortexing every few
minutes. Cells to be analyzed by tetramers were then stained with the
allophycocyanin-A2 BMLF-1, allophycocyanin-B7 EBNA-3A,
allophycocyanin-A2 latent membrane protein-2, or an
allophycocyanin-HIV-1 gag tetramer for 20 min. After tetramer staining,
the cells were lysed with BD lyse (BD Biosciences), fixed, and
permeabilized using Cytofix/Cytoperm solution (BD Biosciences). The
cells were stained with combinations of the following Abs for tetramer
staining: CD8 CyChrome, IFN- FITC, CD69 PE, CD69 FITC, CD28 PE,
CD62L PE, CD45RA PE, and CD45RO PE (BD PharMingen). For cells not
stained with tetramers, after the permeabilization step the cells were
stained with IFN- allophycocyanin, CD3 PerCP, CD69PE, and CD8 FITC.
The cells were incubated at room temperature for 30 min, and then
washed with 1% FBS in PBS. Samples were analyzed immediately by
four-color flow cytometry. Appropriate isotope-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 three
SD, or 0.05% of CD8+ T cells.
In vitro culture and generation of cell lines
Cell lines were generated from Ficoll separated, and in some cases sorted PBMC and cultured in RPMI with 10% FCS (R-10) and 20 U/ml of IL-2 by adding 5 µM EBV-specific peptide or control HIV-1 gag peptide. These cell lines were fed twice weekly and maintained for 1 mo. Alternatively, Ficoll-separated and in some cases sorted PBMC were incubated with EBV peptide or control peptide-pulsed, psoralen-fixed autologous B lymphoblastoid cell lines (BLCL) at a ratio of 1 BLCL:5 PBMC. These cell lines were also fed twice weekly with 20 U/ml of IL-2 in R-10 and restimulated with peptide-pulsed BLCL once every 2–3 wk. These cell lines were maintained for up to 3 mo.
Statistical analysis
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). A total of 18 (10 HLA-A2, 4
HLA-B7, and 4 HLA-A2 and HLA-B7) individuals with AIM were studied. For
comparison, 16 (9 HLA-A2, 2 HLA-B7, and 5 HLA-A2 and HLA-B7) healthy
EBV seropositive individuals were studied.
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Analyses of individuals with acute EBV infection showed that all
HLA-A2-restricted, BMLF-1-specific CD8+ T cells
coexpressed CD45RO at presentation with AIM. By 2 wk postpresentation,
3–15% of the A2 BMLF-1 tetramer-binding CD8+ T
cells expressed CD45RA and were CD45RO negative. There were significant
differences in the expression of CD45RO and CD45RA by BMLF-1-specific
CD8+ T cells at presentation, and at 6 mo or 1
year following acute infection (RO, p = 0.001; RA,
p = 0.001; Fig. 2, a and b). By 6 mo and 1 year, there was no
statistically significant difference between the acute patients and
healthy EBV seropositive donors in the percentages of A2 BMLF-1
tetramer-binding CD8+ T cells that expressed
CD45RA vs CD45RO. Indeed, varying percentages of
CD45RA+A2 BMLF-1 tetramer-binding cells were
detected in all of the EBV seropositive donors (range 14.8–76.8%;
Fig. 2b). None of the BMLF-1 tetramer-binding
CD8+ T cells in the long-term EBV seropositive
donors coexpressed both CD45RA and CD45RO (data not shown). The
percentages of CD45RO+ and
CD45RA+A2 BMLF-1-specific
CD8+ T cells remained stable in individuals with
latent infection over four measurements repeated during an 18-mo time
period.
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Because the peptides used were not only derived from different proteins, but also restricted by different HLA molecules, we wanted to determine whether CD8+ T cells that bound to HLA-A2 peptide tetramers made with latent protein epitopes also expressed only the CD45RO isoform. Analyses of HLA-A2 latent donors with an A2 LMP-2 tetramer revealed that 100% of the CD8+ cell populations binding to these tetramers also expressed only CD45RO (n = 8; data not shown). These data demonstrate that for two latent epitopes restricted by distinct HLA alleles, the coexpression of the CD45RO isoform predominated.
Differential expression of cell surface homing molecules on A2 BMLF-1 and B7 EBNA-3A tetramer-binding CD8+ T cells
The lymphocyte cell surface molecule CD62L has been shown to play
a role in the specific homing of T cells to peripheral lymph nodes
(16). CD62L expression on A2 BMLF-1 tetramer-binding cells
varied from individual to individual during acute EBV infection (Fig. 3a). Significant differences
were not discerned in the percentage of A2 BMLF-1-specific
CD8+ T cells that coexpressed CD62L over time.
Higher frequencies of B7 EBNA-3A tetramer-binding
CD8+ T cells that coexpressed CD62L were detected
at the 6-mo time point (71.8 ± 17.8%; p < 0.01)
and the 1-year time point (78.8 ± 8.2%; p <
0.001) when compared with A2 BMLF-1 tetramer-binding
CD8+ T cells at the 6-mo (43.3 ± 11.6%)
and one-year (44.8 ± 14.2%) time points (Fig. 3b).
The frequencies of CD62L+B7 EBNA-3A
tetramer-binding CD8+ T cells (74.7 ±
11.1%) detected in the long-term EBV seropositive population were also
significantly higher (p < 0.05) than the
frequencies of CD62L+A2 BMLF-1 tetramer-binding
cells (34.4 ± 10.4%; Fig. 3c) in the long-term EBV
seropositive population.
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d
demonstrates that B7 EBNA-3A tetramer-binding
CD8+ T cells in latent individuals more commonly
expressed CCR7 (54 ± 24% CCR7+) than A2
BMLF-1 tetramer-binding CD8+ T cells (18 ±
10% CCR7+; p < 0.001). For each
individual, the percentage of CCR7+
tetramer-binding CD8+ T cells was always less
than the percentage of CD62L-positive tetramer-binding
CD8+ T cells. For HLA-A2 individuals, the small
percentages of CCR7-positive tetramer-binding
CD8+ T cells were distributed between both the
CD45RA and CD45RO populations.
No differences in IFN- production by A2 BMLF-1 or B7 EBNA-3A
tetramer-binding CD8+ T cells
Because of the observed differences of A2 BMLF-1 and B7 EBNA-3A
with respect to the expression of the CD45RO isoform over time, we
wanted to determine whether these phenotypic differences corresponded
with the ability to produce IFN- during AIM and latent EBV
infection. AIM study participants and long-term EBV seropositive
individuals were studied by tetramer staining combined with the in
vitro stimulation assay for cytokine production to determine the
percentage of tetramer-binding cells expressing CD69 and IFN-.
IFN- production by A2 BMLF-1 tetramer-binding
CD8+ T cells was lowest at presentation with
symptoms (V-1; 33 ± 12%), and was significantly different
(p < 0.05) from 6 mo postpresentation (V-4,
48 ± 11%) and from the long-term latent individuals (65 ±
17%; p < 0.001; Fig. 4a). There was also a
significant difference (p < 0.05) in IFN-
production between the 6-mo time point and the long-term latent
individuals. Although all IFN- producing A2 BMLF-1
CD8+ T cells coexpressed CD69, many CD69-positive
T cells did not produce IFN- (Table I).
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production by B7 EBNA-3A
tetramer-binding CD8+ T cells was lowest at
presentation with symptoms (V-1; 41 ± 12%), increased by 6 mo
postpresentation (V-4, 66 ± 17%; not significant) and was
significantly different from the long-term latent individuals (82
± 17%; p < 0.01; Fig. 4b). There was no
significant difference between the 6-mo time point and the long-term
latent individuals for B7 EBNA-3A tetramer-binding
CD8+ T cells. There were also no significant
differences in the percentage of IFN- producing B7 EBNA-3A and A2
BMLF-1 CD8+ T cells when compared at similar time
points.
As shown in Fig. 2, we detected populations of A2 BMLF-1-specific
CD8+ T cells that coexpressed either CD45RA or
CD45RO, but only populations of CD45RO+B7
EBNA-3A-specific CD8+ T cells. Because we
determined that not all tetramer-binding CD8+ T
cells produced IFN-, we wanted to determine which subpopulations of
epitope-specific CD8+ T cells did not produce
IFN-, or express CD69. Fig. 5 shows a
representative analysis (n = 8) to evaluate the ability
of A2 BMLF-1 tetramer-staining cell subsets to produce IFN-. This
figure clearly demonstrates that both CD45RO+
(Fig. 5a) and CD45RA+ (Fig. 5b) BMLF-1 tetramer-binding CD8+ T
cells were able to produce cytokines in the short-term assay at the 2
µM concentration of peptide.
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production by CD62L+ cells could not be
determined by this method. The perforin content Of A2 BMLF-1 and B7 EBNA-3A tetramer-binding CD8+ T cells does not correlate with cell surface expression of the CD45 isoform
We were not able to determine significant differences in the
abilities of the A2 BMLF-1 and B7 EBNA-3A tetramer-binding
CD8+ T cells to produce IFN-, nor were we able
to detect differences in IFN- production by the subpopulations of
CD45RA+, CD45RO+, or
CD28+ positive tetramer-binding
CD8+ T cells. Thus, we wanted to determine
whether differences in perforin content existed between A2 BMLF-1 and
B7 EBNA-3A tetramer-binding CD8+ T cells. Fig. 6a demonstrates that A2 BMLF-1
tetramer-binding CD8+ T cells had variable levels
of perforin at presentation (V-1) and 6 mo post EBV infection. Perforin
was detected in up to 50% of tetramer-binding cells through 1-year
post EBV infection and in latent individuals (Fig. 6a). No
statistically significant differences between the perforin content of
A2 BMLF-1 and B7 EBNA-3A tetramer-binding CD8+ T
cells were detected during presentation (V-1) through 6 mo (V-4).
However, HLA-B7 EBNA-3A tetramer-binding cells containing perforin were
uncommonly detected after 6 mo and the overall percentage of perforin
containing B7 EBNA-3A tetramer-binding CD8+ T
cells was significantly lower (1.9 ± 3.2%; p <
0.05; Fig. 6b) than the percentage of perforin containing A2
BMLF-1 tetramer-binding CD8+ T cells (19.1
± 17.8%; Fig. 6a) at 1 year. Additionally, the percentage
of perforin containing B7 EBNA-3A CD8+ T cells in
long-term EBV seropositive individuals (5.8 ± 8.7%; Fig. 6b) was significantly lower (p <
0.05) than the percentage of perforin containing A2 BMLF-1
tetramer-binding CD8+ T cells in long-term EBV
seropositive individuals (17.7 ± 12.9%; Fig. 6a).
Interestingly, the percentage of perforin containing A2 BMLF-1 or B7
EBNA-3A CD8+ T cells remained stable over three
repeated measurements over a 1-year time period for all of our
long-term EBV seropositive donors.
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shows an example of FACS analysis of
two long-term EBV seropositive individuals (both HLA-A2, B7) and the
perforin content of A2 BMLF-1 (donor 001) or B7 EBNA-3A (donor 006)
tetramer-binding CD8+ T cells within the
CD45RA+ and CD45RO+
subsets. Table II demonstrates the
variability in the percentages of perforin containing A2 BMLF-1
CD8+ T cells for 14 HLA-A2 long-term EBV
seropositive individuals tested, and like Fig. 7, shows that for this
epitope the majority of perforin-containing cells were within the
CD45RA+CD62L- populations
of CD8+ T cells. Thirteen of 14 had detectable
perforin in their BMLF-1 tetramer-binding CD8+ T
cells (range 0–34%). Only donor 007 had more
CD45RO+ than CD45RA+ BMLF-1
tetramer-binding CD8+ T cells containing
perforin. Table III demonstrates low
percentages of perforin-containing cells in HLA-B7 EBNA-3A
tetramer-staining cells; four of seven HLA-B7 long-term EBV
seropositive donors had no detectable perforin.
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Because we could not distinguish functional differences
between A2 BMLF-1 and B7 EBNA-3A-specific CD8+ T
cells directly ex vivo, we derived cell lines from individuals who were
both HLA-A2+ and HLA-B7+ to
determine whether differences existed between the abilities of
CD45RA+ and CD45O+ T cells
to proliferate in vitro. CD3+ T cells from three
HLA-A2+B7+ individuals were
sorted into CD45RA+ or
CD45RO+ populations (Fig. 8, a, e, and
i; representative donor 003). The sorted cells were then
stimulated with either peptide-pulsed autologous BLCL or peptide only
and propagated in vitro. Fig. 8, b–d, shows that after only
4 days in culture the small population of
CD45RA+BMLF-1 tetramer-binding
CD8+ T cells present after sorting (Fig. 8a) had expanded and switched their CD45 isoform expression
to CD45RO (Fig. 8, b and c). The expansion with
peptide only was smaller, but still resulted in the expression of
CD45RO by BMLF-1 tetramer-binding CD8+ T cells
(Fig. 8d). Because BLCL express 10 latent proteins from EBV,
we detected a switch in isoform expression for many
non-tetramer-binding CD8+ T cells which were
cultured with BLCL (Fig. 8, b and c), but not for
many non-tetramer-binding CD8+ T cells when the
cultures were just stimulated with peptide (Fig. 8d).
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e shows the
percentage of tetramer-binding CD8+ T cells after
sorting, and Fig. 8, f and g, shows after culture
with A2 BMLF-1 peptide-pulsed BLCL. Of note, none of these cells
switched their isoform to CD45RA in culture.
Culture of the sorted CD45RO+ (Fig. 8i) or CD45RA+ T cells with B7
EBNA-3A-pulsed BLCL (Fig. 8, j and k) or with B7
EBNA-3A peptide only (Fig. 8l) showed expansion only in
cultures derived from the CD45RO+ T cells, as
expected. Similarly, the CD45RO+ B7
EBNA-3A-specific CD8+ T cells expanded quite
readily in vitro and all retained their CD45RO+
phenotype (Fig. 8, j and k).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many groups have begun to evaluate the cell surface phenotype of
Ag-specific CD8+ T cells. Champagne and
colleagues (9) have reported a correlation between cell
surface phenotype and function in HIV-1-specific
CD8+ T cells, and from this have proposed a
differentiation model for CD8+ T cells in which
fully differentiated effector cells are of the
CD45RA+, CD27-,
CD28-, and CCR7-
phenotype. The present study does not support this model for EBV
infection. At presentation with AIM, 100% of lytic and latent
epitope-specific CD8+ T cells expressed only the
CD45RO isoform (Fig. 2); as acute infection resolved, the
BMLF-1-specific CD8+ T cells coexpressed either
the RA or the RO isoform, while the EBNA-3A- and LMP2-specific
CD8+ T cells continued to express only the RO
isoform. Others have shown that lytic protein-specific
CD8+ T cells (HLA A2-restricted, BMLF-1-specific
CD8+ T cells and HLA B8-restricted,
BZLF-1-specific CD8+ T cells from
individuals with established infection can express either the CD45RA or
CD45RO isoforms; Refs. 3, 6). The
CD45RO+B7 EBNA-3A-specific
CD8+ T cells were capable of IFN- production
and contained perforin. The
CD45RA+BMLF-1-specific CD8+
T cells were clearly not end stage differentiated effector cells, as
has been suggested, because they were easily expanded in culture
(8, 9).
Lytic epitope-specific CD8+ T cell responses are detected earlier in AIM than latent epitope-specific CD8+ T cell responses and are generally higher in magnitude (5). The differences in CD45 isoform expression on memory CD8+ T cells might simply reflect differences in the magnitude of the epitope-specific CD8+ T cell response generated during acute infection. However, the frequencies of BMLF-1-specific CD8+ T cell responses measured 1–2 wk following presentation with AIM varied 100-fold among individuals (5), and there was no discernible relationship between the magnitude of the epitope-specific response measured during acute infection and expression of CD45RA during convalescence.
Coexpression of either the CD45RA or RO isoforms by epitope-specific
CD8+ T cells has also been reported in
individuals with established cytomegalovirus infection
(19). High early viral replication concurrent with the
generation of the epitope-specific response might contribute to the
observed differences in CD45 isoform expression. In acute EBV
infection, up to 25% of circulating B cells may be infected with EBV
(2). In this regard, it is interesting to note that the
CD45RA+ A2 BMLF-1-specific
CD8+ T cells reverted to CD45RO (Fig. 8, a–d) following reexposure to Ag in vitro.
The production of IFN- by BMLF-1 and EBNA-3A-specific
CD8+ T cells was low in early infection but
increased with time. Variability was noted in IFN- production by
epitope-specific CD8+ T cells in latently
infected individuals. In mice infected with low doses of LCMV, IFN-
production appears to correlate well with the number of
tetramer-binding CD8+ T cells detected during
acute infection (20, 21, 22). However, in mice infected with
high doses of LCMV, with resultant persistent infection, this
concordance is lost (23). This same discordance between
tetramer-staining cells and the ability to produce IFN- has been
shown in acute SIV infection (24) and acute hepatitis C
virus infection (25), and has been proposed as a
mechanism for the establishment of persistent infection.
It is possible that the in vitro assay might not accurately reflect the
in vivo functional capabilities of the EBV epitope-specific
CD8+ T cells. During acute EBV infection,
Ag-specific CD8+ T cells are already highly
activated (5) and either may not be capable of producing
IFN- or may enter apoptotic pathways following reexposure to Ag in
vitro. As the viral load diminishes, the cells detected during latent
infection are not highly activated and may be more capable of producing
IFN- in the in vitro assay.
Cell surface CD62L and CCR7 expression was significantly higher on EBNA-3A-specific CD8+ T cells, than on BMLF-1-specific CD8+ T cells from convalescent or latently infected individuals. These data are compatible with data from Hislop et al. (10), who also reported differences in CCR7 and CD62L expression between lytic and latent epitopic-specific CD84 T cells. Murine models have shown that there are phenotypic differences between memory CD8+ T cells in the lamina propria and other tissues (26), and this suggests that subpopulations of memory cells will express receptors which allow them to interact with APCs and/or a specific microenvironment. The ligands for CD62L (L-selectin), peripheral lymph node addressin, are expressed at very high levels in tonsillar tissue (27). It could be that the B7 EBNA-3A-specific CD8+ T cell populations retain CD62L and CCR7 expression to gain re-entry into this site. Other recently published studies also suggest that CCR7 expression on T cells (and its ligand, EBV-induced molecule 1 ligand chemokine, expression on lymph node and tonsillar tissue) are important for the trafficking of lymphocytes to these lymphoid sites (28, 29).
While both of the epitope-specific CD8+ T cell populations we studied demostrated in vitro function, EBV is never eliminated. One important caveat of persistent infections in humans is that the virus may persist for years with limited gene expression. Thus, even though the host has functional virus-specific CD8+ T cells, virus-infected cells might be unapparent to immune surveillance.
In individuals with latent EBV infection, the percentages of A2 BMLF-1 or B7 EBNA-3A-specific CD8+ T cells remain remarkably stable over time (5). Interestingly, analysis of the perforin content of A2 BMLF-1 and B7 EBNA-3A-specific CD8+ T cells in long-term EBV seropositive donors showed that similar percentages of perforin-containing epitope-specific CD8+ T cells were always found within the same individual over a period of 1 year. This is similar to the consistency in the overall percentages of tetramer-binding CD8+ T cells coexpressing CD45 RA or RO in these individuals. Work by Khan et al. (30), has elegantly shown that the peripheral blood EBV load remains constant in individuals over time periods of several years. Thus, for the two populations of EBV-epitope-specific CD8+ T cells studied, the expression of CD45RA, CD45RO, CD62L, CCR7, and perforin appears to be stable over time, reflecting equilibrium between the virus and the host immune system. Further work is necessary to understand how perturbations in the EBV-specific CD8+ T cell repertoire relate to disease manifestations in individuals with compromised cell-mediated immunity.
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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: AIM, acute infectious mononucleosis; LCMV, lymphocytic choriomeningitis virus; BLCL, B lymphoblastoid cell lines; CD62L, CD62 ligand.
Received for publication November 9, 2001. Accepted for publication February 1, 2002.
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