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Evolution of Epitope-Specific Memory CD4+
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J Immunol 2002; 169:2210-2214; ;
doi: 10.4049/jimmunol.169.4.2210
http://www.jimmunol.org/content/169/4/2210
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Copyright © 2002 by The American Association of
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References
Andrew J. Godkin, Howard C. Thomas and Peter J.
Openshaw
The Journal of Immunology
Evolution of Epitope-Specific Memory CD4ⴙ T Cells After
Clearance of Hepatitis C Virus
Andrew J. Godkin,1*† Howard C. Thomas,* and Peter J. Openshaw*
he CD4⫹ T cell response is thought to be of critical importance in determining the fate of many infections, including the noncytopathic viruses such as hepatitis C virus (HCV)2 (reviewed in Refs. 1 and 2). After clearance of a
pathogen, the characterization of the memory CD4⫹ T cell response has relied mainly on mouse studies (3–5) and is poorly
understood in humans (6 – 8). HCV is an RNA virus that often
causes chronic infection in humans without treatment (9, 10).
There are several lines of evidence that support a key role for
CD4⫹ T cells in orchestrating clearance of HCV, including the
association of viral clearance with the class II major histocompatibility Ag HLA-DR11 (A1*0101, B1*1101) (reviewed in Ref. 11),
the correlation of proliferative responses during the acute infection
with a favorable outcome (12), and loss of these proliferative responses with recurrence of the virus (13). A recent report on the
long-term follow-up of a cohort of patients who had been infected
with the same strain of HCV and subsequently cleared the virus
revealed a persistent CD4⫹ proliferative T cell responses to one or
more viral proteins in the majority of patients, although the serological response waned (14).
The generation of memory lymphocytes is one of the hallmarks
of the specific immune response (reviewed in Ref. 8). It enables a
quantitatively and qualitatively different response on subsequent
rechallenge by the same pathogen. The peripheral repertoire of T
cells consists of fixed pools of naive and memory cells. The function and maintenance of naive and memory T cells appears to be
quite independent of each other (6, 7). When naive cells encounter
pathogenic epitopes expressed by MHC proteins in lymphoid tissue they undergo profound changes, multiplying severalfold and
differentiating into effector cells. After clearance of the pathogen,
T
*Imperial College of Science, Technology, and Medicine, St. Mary’s Hospital, Paddington, London, United Kingdom; and †Department of Integrated Medicine, University Hospital of Wales, Cardiff, United Kingdom
the majority of the expanded pool of T cells undergoes activationinduced cell death, but a percentage will survive long term as
memory cells. The selection of epitope-specific cells, with the
most favorable TCR-MHC interaction, leads to the preservation of
the same specificities in the memory population (4, 15). Apart
from their enhanced responses on antigenic rechallenge, these
memory cells have a variety of features that distinguishes them
from naive cells. Memory cells do not seem to require the continual interaction with self MHC molecules to survive, in contrast to
peripheral naive T cells. Adoptive transfer experiments show that
memory cells, but not naive cells, can survive in knockout mice
that do not express any class I or class II molecules (16, 17).
Memory T cells also develop particular surface markers such as
CD45 isoforms and express a novel range of adhesion molecules
and chemokine receptors facilitating their passage through different tissues (reviewed in Ref. 18).
However, the factors that govern the generation and maintenance of memory cells remains largely a mystery. The CD4⫹ T
cell response is of critical importance in maintaining long-term
protective immunity after clearing many infections (1, 2). The
presence of long term Ab-mediated protection is well documented,
but the development and maintenance of CD4⫹ T cell memory
have been very difficult to study in man. In mice, CD4⫹ T cells
have been found to be of central importance in the maintenance of
protective immunity (2). We have recently used an epitope prediction program (19) that allowed the accurate identification of
HLA-DR11-restricted HCV epitopes (20). Using highly sensitive
ELISPOT assays to identify T cells recognizing these epitopes, we
have detected and counted distinct populations of CD4⫹ memory
cells in HLA-DR11⫹ patients who have cleared HCV infection.
The study of the evolution of these T cell populations offers a
novel insight into CD4⫹ T cell memory development in humans
after recovery from a natural virus infection.
Received for publication May 14, 2002. Accepted for publication June 14, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Materials and Methods
1
Patients were diagnosed with past HCV infection by the clinical history,
the presence of anti-HCV Abs, at least three negative HCV PCRs (separated by 6 –12 mo), and normal liver function tests. The HLA typing was
performed using allele-specific primer PCR to obtain the genotype. These
Address correspondence and reprint requests to Dr. Andrew J. Godkin, Department
of Integrated Medicine, Ward A7, University Hospital of Wales, Heath Park, Cardiff
CF14 4XW, U.K. E-mail address: [email protected]
2
Abbreviation used in this paper: HCV, hepatitis C virus.
Copyright © 2002 by The American Association of Immunologists, Inc.
Study population
0022-1767/02/$02.00
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The generation of memory lymphocytes is one of the hallmarks of the specific immune response. The CD4ⴙ T cell response is of
critical importance in maintaining long-term protective immunity after clearing many infections. However, accurate characterization of these memory CD4ⴙ T cells has relied mainly on mouse studies and is poorly understood in humans. We have detected
and counted epitope-specific populations of CD4ⴙ memory cells in patients who have cleared hepatitis C virus. The kinetics of the
recall response and the expression of the chemokine receptor CCR7 suggested the presence of distinct populations. A population
of memory cells measured in an ex vivo IFN-␥ ELISPOT assay steadily declined after viral clearance. However, memory CD4ⴙ
T cells only characterized after short-term culture with Ag and IL-2, and, recognizing the same epitopes, developed into a
long-term stable population. Depletion of CCR7ⴙ cells from PBMCs markedly reduced the responses in the culture-positive
population while having little effect on the ex vivo responses. The demonstration of these key memory subsets in man opens the
way to defining their role in protective immune responses. The Journal of Immunology, 2002, 169: 2210 –2214.
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0
0
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IFN-␣
IFN-␣ ribavirin
IFN-␣
IFN-␣ amantadine
IFN-␣
IFN-␣
Nil
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0
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Strong
Weak
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Strong
Weak
Nil
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Treatment
Core31–45
Ex Vivo
Responses
70/male
32/female
42/male
47/male
48/female
50/male
45/female
Subjects
1
2
3
4
5
6
7
A previously validated prediction program for identifying
HLA-DR epitopes (19, 21, 24) has enabled us to identify HLADR11-restricted T cell epitopes (24). Using a series of epitopes
derived from four different viral proteins (core, NS3, NS4, NS5),
we screened HLA-DR11⫹ patients who had successfully cleared
HCV for cognate CD4⫹ memory T cells using IFN-␥ ELISPOT
assays comparing ex vivo analyses with short-term cultures. The
results are shown in Table I. All the patients showed positive responses to one or more epitopes after short-term culture; although
the overall range of epitopes recognized and the strength of re-
Years of
Nonviremia
Screening of HLA-DR11⫹ nonviremic HCV patients for cognate
memory CD4⫹ T cells
Age (years)/
Gender
Results
Table I. Memory CD4⫹ T cell responses in nonviremic HLA-DR11⫹ subjectsa
Magnetic cell sorting of CD8⫹ T cells was performed using MACS CD8
MicroBeads and MS⫹ Separation Columns according to the manufacturer’s instructions (Miltenyi Biotec, Bisley, U.K.). The non-neutralizing
mouse IgM anti-CCR7 Ab derived from clone 2H4 (purchased from BD
PharMingen, Oxford, U.K.) was used for depletion of CCR7⫹ T cells.
PBMCs were purified as described above and washed in cold PBS containing 1% human albumin. The cells were incubated on ice for 30 min
with 30 ␮g of Ab/107 cells. After washing twice, rat anti-mouse IgM MicroBeads were added (50 ␮l of beads/107 cells) and incubated at 4°C for
20 min. The cells were then washed and sorted as above. FACS analysis
revealed the purified population was ⬎99% CCR7⫹ cells (data not shown).
Core141–155
A total of 2 ⫻ 105 PBMCs at a concentration of 2 ⫻ 106 cells/ml were
grown in 96-well round-bottom plates with 10 ␮g/ml peptide using R-10
(RPMI plus 10% human AB serum plus antibiotics/L-glutamine supplement) and incubated in a humidified atmosphere containing 5% CO2 at
37°C. On days 3, 6, and 9 the medium was supplemented with IL-2 (10%
Lymphocult T; Biotest Diagnostics, Denville, NJ) and fresh R-10 on days
6 and 9. On day 12 the lines were washed three times in RPMI and assayed
as described above. To confirm presentation by HLA-DR11, matched and
mismatched EBV-transformed B cell lines were used along with antiHLA-DR mAb L243 (22) and anti-DQ 1a3 (23) as described before (24).
Depletion of CCR7⫹ and CD8⫹ T cells
NS41910–1924
NS31207–1221
Cultured Responses to Individual Peptidesb
Short-term propagation of epitope-specific CD4⫹ T cells
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The biological effect of peptides on T cells was studied in either a direct ex
vivo assay or on short-term cultured T cells using single-cell cytokine
release as a measure of Ag-specific effector function in a highly sensitive
ELISPOT assay (anticytokine Abs and streptavidin-alkaline phosphatase
obtained from Mabtech, Nacka, Sweden). The concentrations of Abs used
and washing steps were according to the manufacturer’s instructions. The
method has been previously described (21). For direct ex vivo analysis,
4 ⫻ 105 PBMCs (extracted from heparinized whole blood by centrifugation over Lymphoprep (Nycomed, Oslo, Norway)) were added per well of
a special polymer-backed 96-well filtration plate (Millipore, Moslheim,
France). These cells were incubated at 37°C with 5% CO2 for 18 h. Previous titration experiments revealed ex vivo culture from 6 to 18 h yielded
the same results (data not shown). For measuring responses in short-term
lines 2.5 ⫻ 104 cultured T cells (grown as described below) in R-5 (RPMI
with 5% heat-treated FCS) were added to each well. The peptides were
tested at a final concentration of 10 ␮g/ml and compared with control
wells with no peptide. The plates were either assessed by an independent
observer blinded to the well contents and/or read using an automated
ELISPOT reader (Autoimmun Diagnostika, Strasburg, Germany). Epitopespecific spot-forming cells per well were calculated by subtracting background values from wells with no peptide (majority of background values,
0 –10 spots per well).
NS31245–1259
IFN-␥ ELISPOT assays
NS31581–1595
NS52268–2282
The peptides were designed according to the sequence of viral genotype 1a
based on Simmond’s classification. The candidate peptides 15 or 16 aa in
length were synthesized commercially (ECHAZ Micro Collections, Tubingen, Germany). The sequence and purity of selected peptides was confirmed by HPLC and tandem mass spectrometry analysis.
2
2
2
3.5
4
5
⬎10
Peptide synthesis
0
0
⫹
⫹⫹⫹
⫹⫹
0
⫹
NS52798–2802
tests were performed by the hospital reference laboratories. Ethical committee approval (St. Mary’s National Health Service Trust) was granted for
obtaining blood samples from patients.
a
Epitope-specific T cell responses were measured by IFN-␥ release using ELISPOT assays. The responses to individual HCV peptides were measured ex vivo and after 12-day cultures with IL-2 and peptide. For the cultured responses, initially
5–10 short-term lines were assayed: positive line defined as an increase of more than two times background. ⫹, 10 –33% positive lines; ⫹⫹, ⬎33– 66% positive lines; ⫹⫹⫹, ⬎66% positive lines. The ex vivo responses were measured in an 18-h
IFN-␥ ELISPOT assay; strong positive responses more than three times background, weak response one and one-half to three times background.
b
Underlining indicates an ex vivo response.
2211
⫹⫹
nt
nt
⫹⫹⫹
nt
nt
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The Journal of Immunology
2212
sponse to each epitope were unique to each subject, peptides
core31– 45, core141–155, NS31245–1259, and NS52268 –2282 were recognized in the majority of individuals. However, the ex vivo responses were not present in all individuals and appeared to decline
with time since viral clearance. Furthermore, there was not a direct
correlation between an ex vivo response to a particular epitope and
the cultured response to the same epitope; e.g., subject 1 had strong
ex vivo responses to core31– 45, core141–155, and NS52268 –2282 yet
showed positive cultured responses to core31– 45, NS31207–1221, and
NS52798 –2802. These results suggest that these ex vivo and cultured
responses are measuring different populations of memory cells.
Evolution of the ex vivo and cultured memory response
The overall pattern in Table I suggests the cultured responses develop and persist after clearance of the virus but that the ex vivo
MEMORY CD4⫹ T CELLS AFTER CLEARANCE OF HCV
responses eventually decline. To explore this notion further, two
individuals who demonstrated robust ex vivo responses (subjects 1
and 4) were studied prospectively. The immediate ex vivo (Fig. 1,
A and C) and cultured (Fig. 1, B and D) memory CD4⫹ T cell
responses in these individuals were measured to three epitopes
over a 24-mo period. Both subjects revealed the same pattern.
Initially there was a powerful ex vivo response, with frequencies of
cognate epitope-specific T cells reaching 150 ⫻ 106 PBMCs. For
both individuals these responses to all three epitopes steadily declined. However, the frequencies of epitope-specific T cells detected after culture gradually increased for all three epitopes. The
changes in subject 1 were particularly striking: initially the cultured responses were almost totally absent to all three epitopes, but
over the study period powerful robust responses evolved. The decline in an ex vivo epitope-specific population of memory T cells
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FIGURE 1. The evolution of ex vivo and cultured epitope-specific CD4⫹ memory T cell changes over time. A–D, The changes in frequencies of cognate
T cell responses measured by IFN-␥ ELISPOTS to three HCV epitopes were recorded over 24 mo in two subjects. For both individuals there was a decline
in the ex vivo responses (A and C) but a gradual increase during the same period in cultured responses to the same epitopes (B and D). Experiments 1– 4
were conducted at ⬃6- to 8-mo intervals. E, Direct comparison of the ex vivo response against the frequencies of the cultured response to the same epitope.
The collective data outlined were analyzed. The two populations reveal an inverse relationship (Pearson’s correlation ⫺0.334.
The Journal of Immunology
2213
entering a reasonably stable population. Repeat experiments over
2 years on these subjects confirmed the persistence of these central
CD4⫹ T cells. Furthermore, the relative contribution of each
epitope to the memory pool appeared to be stable over time. A
representative example with subject 7 is given in Fig. 2A. The
epitope NS31245–1259 producing persistently higher frequency
responses (proportion of total responses originally: 74%
NS31245–1259 and 26% core141–155; proportion of total responses
when repeated after 24 mo: 72 and 28%, respectively). It seems
both the range of epitope-specific responses and the hierarchy indicated by frequency are reasonably stable over time. Subject 7
had been nonviremic for the longest period of time and had no ex
vivo responses. The CCR7⫹ cells were magnetically depleted and
the epitope-specific responses to NS31245–1259 were measured after
culture (Fig. 2B). The depletion reduced the response to 46% of the
unsorted PBMCs, but addition of the retained CCR7⫹ cells from
the column restored in part the response to 76% of the original.
FIGURE 2. Stability of the central memory CD4⫹ T cells over time and
contribution of CCR7⫹ T cells to this population. A, Representative experiment from subject 7, who had no ex vivo responses to these HCV
epitopes, having cleared the virus ⬎10 years previously. The cultured responses were repeated after 2 years. The same pattern emerged with
epitope NS31245–1259 dominating over core141–155. B, The responses to
NS31245–1259 were repeated with depletion of CCR7⫹ cells. This markedly
reduced the magnitude of the cultured response. Furthermore, addition of
the retained CCR7⫹ cells from the depletion column to the cultures restored in part the responses.
with the accompanying development of culture-positive memory
cells to the same epitope specificities suggests that in vivo these
latter cells may be developing sequentially from the immediate
effectors and, hence, there is a negative correlation between these
two populations of cells (Fig. 1E).
The kinetics of these changes are different for particular individuals. However, the presence of robust central memory responses many years after viral clearance suggests these cells are
FIGURE 3. The contribution of
CCR7⫹ T cells to ex vivo and cultured
responses. PBMCs were depleted of
CCR7⫹ T cells and then assayed both
ex vivo and after short-term culture.
Depletion of CD8⫹ cells was used as a
control. The results are standardized to
a percentage of the control experiment
(i.e., whole PBMCs). Depletion of the
CCR7⫹ population mainly affected the
cultured responses.
It has been suggested that expression of the lymphotropic chemokine receptor CCR7 on memory T cells acts as a marker for central
memory cells (25). These cells require Ag restimulation/IL-2 to
develop effector functions, whereas the CCR7⫺ cells have the
characteristics of immediate effector memory cells. To examine
the characteristics of HCV epitope-specific memory cells further,
CCR7⫹ T cells were depleted from PBMCs of another two subjects who had both ex vivo and cultured responses (Fig. 3). CCR7
depletion reduced the culture-positive epitope-specific responses
by a similar degree (34 and 33% of unsorted PBMCs), suggesting
a significant proportion of these cells share the characteristics of
the previously described CCR7⫹CD4⫹ central memory cells. In
contrast, the ex vivo responses were only marginally affected by
CCR7⫹ cell depletion (responses 78 and 94% of original), indicating these cells are mainly CCR7⫺. Depletion of CD8⫹ cells, as
a control, did not diminish the responses and confirmed we were
monitoring IFN-␥ production from CD4⫹ T cells.
Discussion
Recently, it has been suggested that memory T cells may reveal
distinct characteristics indicated by particular function, location,
and phenotype (18, 25–28). These populations of cells can be differentiated in part by the presence or absence of the lymphotropic
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CCR7⫹CD4⫹ T cells contribute substantially to the HCV
cultured memory cells but not to ex vivo responses
2214
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chemokine receptor CCR7. Memory CD4⫹ T cells residing in
lymph nodes tend to require a period of restimulation (e.g., Ag and
IL-2) before acquiring an effector function and have been termed
by some as central memory T cells (25). In contrast, CCR7⫺ memory cells respond rapidly to Ag (demonstrating ex vivo cytotoxicity or IFN-␥ release), may localize in peripheral nonlymphoid tissues, and have been termed effector memory T cells (25). It seems
these nonviremic HCV patients may also develop separate populations of memory CD4⫹ T cells that share some of these characteristics with the measured ex vivo responses corresponding to
effector memory T cells and the measured cultured responses to
central memory T cells.
HCV offers a useful model to study these memory cells, because
the infection can be eradicated by treatment and re-exposure to the
pathogen is unlikely in the study population. HLA-DR11 has been
associated with clearance of HCV in several studies (11). Having
recently identified HCV-derived HLA-DR11-restricted epitopes
(24), we were able to track memory cells in HLA-DR11⫹ patients
using highly sensitive ELISPOT assays. The presence of a strong
ex vivo response to particular epitope(s) did not necessarily correlate to a positive cultured response and, vice versa, ex vivo responses to these epitopes were absent in cases that were positive
after culture, offering support to the concept that there are functionally distinct epitope-specific CD4⫹ memory T cell populations. The CCR7⫹ cells appear to contribute mainly to these cultured “central” memory HCV-specific T cells. Furthermore, there
appears to be an inverse relationship between these two populations, with the evolution of a long-term culture-positive population
of memory T cells at the expense of the ex vivo population. The
relationship and interdependence between these two categories of
memory T cells is unclear. Whether long-term central memory
cells develop from primary effector cells in a sequential fashion or
in parallel is not known (29). Recently it has been proposed that in
humans immediate effectors may develop from central memory
cells (25). While this may be the case in vitro, the evolution of the
responses measured prospectively suggests that the converse may
apply in vivo after clearance of HCV.
There are contrasting results regarding the stability of CD4⫹
memory T cells after clearance of a pathogen (4, 30). However, the
presence of robust central memory responses to HCV epitopes
many years after viral clearance suggests these cells are entering a
stable population. The range of HCV epitope-specific responses in
these central memory cells, as well as the hierarchy indicated by
frequency, also appear to be reasonably stable over time in this
study.
A key question is whether one or both of these populations can
confer protective immunity, and what factors are involved in their
maintenance. It has been demonstrated for LCMV infection that
activated memory cells are necessary for protection in peripheral
sites, and that these cells require persistent exposure to Ag (31). It
is possible that a reservoir of HCV Ag persists after viral clearance, and that this Ag maintains the effector memory population.
The steady decline in the effector memory T cells and the development of the central T cells may mirror a diminishing supply of
Ag. There is also indirect evidence in humans that persistent Ag
aids the maintenance of long-term protective immunity against intracellular pathogens (32). Now that we have identified these populations of memory cells to a human pathogen, the challenge in the
future is to identify whether these central memory cells confer
long-term protection to a parenterally acquired infection like HCV.
MEMORY CD4⫹ T CELLS AFTER CLEARANCE OF HCV