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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2002-01-0160.
IMMUNOBIOLOGY
From the Department of Clinical Immunology, Royal Free
and University College Medical School, London; the Cancer Research
Campaign Institute for Cancer Studies, Birmingham University Medical
School; and the Medical Research Council Centre for Immune Regulation,
Birmingham University Medical School, United Kingdom.
During acute infection, latent and lytic Epstein-Barr virus (EBV)
epitope-specific CD8+ T cells have a CD45RO+
CD45RA The central goal of this study was to identify the
constraints that regulate the persistence of Epstein-Barr virus
(EBV)-specific CD8+ T cells after the resolution of acute
infectious mononucleosis (AIM) to identify how the memory pool
specific for this virus may be maintained. Primary encounter with EBV
induces specific naive CD8+ T cells to proliferate and
express phenotypic markers of priming, such as CD45RO.1,2
Most of this expanded population succumbs to apoptosis after the acute
infection resolves.3,4 The EBV-specific memory pool that
escapes apoptosis consists of a mixture of cells specific for lytic and
latent viral proteins, which are differentially expressed during early
and late stages of the infection, respectively.2,5-7 It is
unclear whether apoptosis remains a constraint on the persistence of
EBV-specific CD8+ T cells after the acute infection
resolves and whether the cells that are specific for different viral
epitopes are equally susceptible to death.
A second limit to the maintenance of memory CD8+ T cells
after the initial infection may be telomere erosion, resulting from excessive proliferation of specific clones.8,9 This may
lead to the development of terminally differentiated end-stage effector cells that are unable to proliferate on restimulation.10
It has been shown, however, that despite the considerable expansion of
EBV-specific CD8+ T cells, telomere loss does not occur
during AIM because of the induction of the enzyme telomerase in these
cells.11,12 Telomere erosion does occur in the
EBV-specific CD8 pool during persistent infection,12
possibly because of frequent restimulation by virus, leading to
progressively reduced telomerase induction that is insufficient to
maintain telomere length in these cells.9 The loss of some
highly expanded clones of EBV-specific CD8+ T cells during
acute infection that are later replaced by other less expanded
populations during chronic infection,56 suggests
indirectly that telomere loss may limit the persistence of
virus-specific CD8+ T cells. However, the critical point at
which telomere loss may lead to the development of a terminally
differentiated senescent population of EBV-specific CD8+ T
cells remains to be determined.
Overlapping markers such as CD45RA, CD45RO, CD28, CD27, and CCR7 have
been used to identify the differentiation state of
EBV13-16 and other virus-specific CD8+ T
cells17-20 that persist after acute infection. A
consistent observation is that during acute infection, cells that are
specific for different viruses are
CD45RO+.1,17 On the resolution of acute
infection, however, a proportion of the specific CD8+ T
cells re-express the CD45RA molecule, and this has been found after
EBV14-16 and cytomegalovirus (CMV)17,20 but
not after influenza virus infections.19 Virus-specific
CD45RA+ and CD45RO+ T-cell compartments found
after the resolution of AIM are derived from the same clones and thus
represent the same cells at different stages of differentiation or
maturation.21,22
It has been suggested that previously primed CD8+ T cells
that re-express CD45RA and down-regulate CCR7 expression may be
approaching end-stage differentiation18,23 and that this
process may be defective in human immunodeficiency-1 (HIV-1)-infected
persons.18,24 We showed previously that EBV lytic
epitope-specific CD8+ T cells that re-express CD45RA
are predominantly CCR7low.14,15 It is
not clear whether these cells represent an end-stage population in
EBV-infected persons or if they can be re-activated to proliferate in
response to specific peptides. The loss of the CD27 marker has also
been associated with terminal differentiation of CD8+ T
cells.25 Although the EBV-specific CD8+
CD45RA+ T-cell pool expresses high levels of
CD27,14,15 the CMV-specific CD8+
CD45RA+ T-cell population,20 shows low
expression of this marker.18,20 This suggests that the
EBV-specific CD8+ CD45RA+ T cells may be less
differentiated than the equivalent subset of CMV-specific T
cells.18 However, it is not known whether either or both
of these populations of virus-specific CD45RA+ T cells have
reached end-stage differentiation.
To clarify the residual replicative potential of EBV-specific
CD8+ T cells after resolution of AIM, we investigated the
characteristics of EBV epitope-specific CD8+ T cells that
re-express CD45RA during and after AIM in the same patients and in
healthy carriers of the virus. Our results indicate that EBV-specific
CD8+ T cells that re-express CD45RA are not a terminally
differentiated end-stage effector population. On the contrary, these
cells have the characteristics of a stable apoptosis-resistant memory
pool that retains functional and substantial replicative capacity.
Sample collection and preparation
Peptide-HLA class 1 tetramers
Tetramers used in this study were highly specific, as demonstrated by the labeling of EBV peptide-specific CD8+ T-cell clones with the appropriate tetramer but not with others of the same HLA-type but directed to other EBV, influenza, or HIV peptide epitopes.5,16 In addition, EBV-seronegative subjects bearing the appropriate HLA type did not react with EBV-specific tetramers, whereas seropositive subjects did not react with HLA-mismatched EBV tetramers.5,15,16 All the batches of EBV-specific tetramers used in this study were checked for specificity as above before use. Flow cytometric analysis Paired acute- and chronic-phase PBMC samples from AIM patients were analyzed by 4-color flow cytometry using PE-conjugated HLA class 1-peptide tetrameric complexes. Specific antibodies directed to other surface markers used included CD8 (Beckman-Coulter, High Wycombe, United Kingdom), CCR7 (Millennium Pharmaceuticals, Cambridge, MA), CD45RA and CD45RO (Serotec, Oxford, United Kingdom), Bcl-2 (DAKO, Cambridge, United Kingdom), and perforin (PharMingen, Oxford, United Kingdom). These antibodies were directly conjugated to fluorescein isothiocyanate (FITC), PE-Cy5, PerCP, allophycocyanin (APC), or PE-Texas Red (ECD). Indirect labeling with the CCR7 antibody was achieved with an anti-mouse IgG FITC-conjugated antibody (Southern Biotechnology Associates, Birmingham, AL). For intracellular proteins, PBMCs were labeled initially with tetramer-PE and anti-CD8-ECD for 30 minutes. Cells were then resuspended in Permeafix (Ortho Diagnostic Systems, Amersham, United Kingdom) for 40 minutes at room temperature, before labeling with anti-CD45RA PE-Cy5 and either an FITC-conjugated isotype control antibody or anti-Bcl-2 FITC. Samples were analyzed on an Epics XL (Beckman-Coulter) or a FACSCalibur (Becton Dickinson) flow cytometer. We also analyzed TCR V usage by EBV-specific
CD8+ T cells in patients who had recovered from AIM using
the same panel of 22 anti-human V chain-specific antibodies
described in detail previously.5
Cell cycle analysis PBMCs were labeled with tetramer-PE, anti-CD8-ECD, and anti-CD45RA-PE-Cy5 antibodies before fixing in 70% ethanol for at least 4 hours at 20°C. Cells were then stained with FITC-labeled
isotype control or anti-Ki-67-FITC antibody for 30 minutes before
analysis on the Epics XL flow cytometer. This antibody detects all
cells in cycle.25
Assessment of telomere length in specific populations using 2-color flow fluorescence in situ hybridization Cells were first stained with biotin-labeled anti-CD4 or anti-CD8 (Beckman-Coulter) followed by streptavidin-Cy5 (Southern Biotech Associates, supplied by Euro-Path, Cornwall, United Kingdom) or with tetramers conjugated to Cy5 as described previously.12 Telomere length was determined using the flow-fluorescence in situ hybridization (FISH) technique.12,26,27 Briefly, PBMCs were washed in PBSA (0.2% BSA) followed by permeabilization using PermeaFix for 30 minutes. Samples were washed twice in PBSA followed by a wash in 1 mL hybridization buffer as described.12 Cells were then resuspended in 200 µL hybridization buffer and incubated with 0.3 µg/mL PNA telomeric (C3TA2)3 or PNA control (alphoid sequences of the X-chromosome, CCCATAACTAAACAC) probe conjugated to FITC as described previously.26-29 PNA probes were obtained from Perseptive Biosystems. Samples were incubated for 20 minutes in the dark, followed by heating at 80°C for 10 minutes and rapid cooling on ice, and then were allowed to hybridize for 2 hours at room temperature in the dark. After washing twice in posthybridization buffer as described previously,12 the samples were analyzed by flow cytometry using a FACSCalibur flow cytometer and Cell Quest Software (Becton Dickinson). Polyfluorescent beads (DAKO FluoroSpheres) were used at the beginning of each experiment to standardize the cytometer.Measurement of virus-specific cell survival in culture PBMCs were isolated from patients who had recovered from AIM up to 20 years earlier and were cultured in 24-well plates at 1 × 106 cells/mL in RPMI 1640 medium (Life Technologies) supplemented with antibiotics and 10% fetal calf serum. Viable cell recovery was assessed at different times by trypan blue dye exclusion. The percentage of CD8+, tetramer-positive cells was determined before and after culture, and this was then multiplied by the viable cell recovery to give the absolute number of tetramer-positive cells that were present. Percentage cell survival was calculated as a fraction of this number relative to the original number of tetramer-positive cells on day 0. In some experiments, the CD8+, tetramer-bearing cells were also examined for CD45RA or Bcl-2 expression before and after culture to determine the relative survival of the CD45RA+ or CD45RA subsets of
these cells.
Restimulation of CD45RA+ and CD45RO+ virus-specific CD8+ T cells with peptide in vitro PBMCs from patients who had recovered from AIM at least 20 years earlier were stimulated with autologous EBV lytic (RAK or GLC) epitope peptide-pulsed PBMCs (0.5 µg/mL/106 cells). Lytic peptide-pulsed PBMCs were incubated with this peptide for 2 hours at 37°C, after which they were irradiated with 120 Gy for 7 minutes using a -irradiator. Responder and peptide-loaded stimulator
cells were cultured in a 1:1 ratio in the presence of 50 U recombinant
IL-2 (R & D Systems, Abingdon, United Kingdom). EBV lytic
epitope-specific activated CD8+ T cells were expanded in
culture by the further addition of IL-2 and fresh culture medium at
3-day intervals. These cells were reactivated with peptide-pulsed PBMCs
at 3-week intervals. The percentage of lytic epitope-specific
CD8+ T cells and their CD45RA or CD45RO reactivity was
determined before and after restimulation. Cytotoxic activity of
expanded virus-specific CD8+ T-cell populations was assayed
in a standard 5-hour chromium release assay using EBV peptide or
dimethyl sulfoxide-pulsed Na2 51CrO4-labeled autologous PHA blasts as
target cells.
Statistics The Student t test was used to determine the significance of the results.
Changes in EBV epitope-specific CD8+ T-cell populations before and after resolution of AIM During AIM, there is a large expansion of CD8+ T cells specific for lytic viral epitopes and a lesser expansion of latent epitope-specific cells (Figure 1). This is followed by a dramatic reduction in lytic epitope-specific cells 1 year after recovery from AIM, though latent epitope-specific CD8+ T-cell populations are not reduced as extensively. Such relative change in lytic versus latent epitope-specific CD8+ T cells is a consistent observation in 12 patients examined during and after the resolution of AIM.
We next investigated the CD45RA and CD45RO expression of lytic and
latent EBV epitope-specific CD8+ T cells before and after
the resolution of AIM. During AIM, almost all virus-specific
CD8+ T cells have been shown to have a CD45RA
To investigate whether the GLC-specific CD8+ T cells that
are CD45RA+ represent the same expanded populations as
their GLC-specific CD45RO+ counterparts, we examined the
V Proliferation in virus-specific CD8+ T cells before and after resolution of AIM We next investigated the extent of proliferation of virus-specific cells before and after AIM, as determined by Ki67 antibody reactivity.14,25 Although there were greater numbers of EBV lytic epitope-specific CD8+ T cells during AIM, there were extremely high levels of cell cycling in lytic and latent epitope-specific populations (Figure 3). This proliferative activity was confined to the CD45RA
(CD45RO+) subset (Figure 3). After the resolution of AIM,
there was a dramatic reduction of cell cycling in total
CD8+ and of latent and lytic virus-specific T cells. The
CD45RA re-expressing populations were totally quiescent, whereas the
small number of cycling tetramer-positive cells that remained were in
the CD45RA populations (Figure 3). Thus, though they were
antigen-experienced, virus-specific CD8+ T cells,
especially those that re-expressed CD45RA after AIM, returned to a
resting state.
Susceptibility to apoptosis of virus-specific CD8+ T cells before and after resolution of AIM Previous studies have shown that the expanded CD8+ T-cell pool present during AIM is susceptible to growth factor deprivation-induced apoptosis.1,3 We now investigated whether this was also true for EBV-specific CD8+ T cells within the total CD8+ pool of patients with AIM (Figure 4). When cultured in the absence of exogenous growth factors, the total CD8+ T cells from patients with AIM perished rapidly by apoptosis, resulting in less than 20% and less than 5% survival after 1 and 5 days of culture, respectively (Figure 4A). During AIM, lytic and latent epitope-specific CD8+ T cells were equally susceptible to rapid apoptosis (only data for lytic epitope-specific cells is shown; Figure 4A). When cells from patients who recovered from AIM were cultured under identical conditions, we found that the total CD8+ T-cell population was now relatively resistant to apoptosis after 5 days of culture (Figure 4B; 65% survival after 5 days of culture). EBV epitope-specific CD8+ T cells present after AIM were more susceptible to death than the total CD8+ population (35% vs 65% survival after 5 days of culture) but were more resistant to death when compared with lytic epitope-specific CD8+ T cells found during AIM (35% vs 5% survival after 5 days; P < .01). Enhanced survival of total CD8+ T cells after recovery from AIM relative to virus-specific populations was probably attributable in part to the presence of apoptosis-resistant naive CD8+ T cells in the former but not in the latter population. Similar observations as to the extent of apoptosis in total CD8+ T cells compared to EBV-specific populations were obtained when healthy EBV carriers who had never had AIM were investigated (data not shown).
We determined the relative survival in culture of EBV-specific CD8+ T cells that do or do not re-express CD45RA (Figure 4C). In these experiments we only investigated lytic epitope-specific populations because there were generally too few CD45RA re-expressing latent virus epitope-specific cells to investigate accurately (Figure 2). We found that in each of 5 experiments performed, EBV-specific cells that re-express CD45RA show better survival than the cells that do not express this molecule (60.8 ± 15.3 vs 30.2 ± 11.7, respectively; P < .012). We investigated why virus-specific CD8+ T cells
re-expressing CD45RA were more resistant to death. Previous studies
using unselected CD8+ T cells during AIM showed that their
susceptibility to apoptosis was linked to reduced expression of the
anti-apoptotic molecule Bcl-2.1 We now demonstrate that
lytic and latent virus epitope-specific CD8+ T cells also
have low Bcl-2 expression during AIM (Figure
5A; acute). However, 1 year after the
resolution of AIM, lytic and latent EBV epitope-specific
CD8+ T cells up-regulate their Bcl-2 expression (Figure 5A;
chronic). The up-regulation of Bcl-2 in virus-specific CD8+
T cells after the resolution of acute viral infection confirms previous
observations that were made in animal models.30 A small population of CD8+ CD45RA+ T cells has high
Bcl-2 expression during AIM (Figure 5A). These cells are
CCR7+ (data not shown) and probably represent naive cells
that are not EBV-specific and have not been stimulated during the
infection.
We next compared the extent to which Bcl-2 increases in
CD45RA+ and CD45RA Telomere length in virus-specific CD8+ CD45RA+ T cells Our observations suggest that the virus-specific cells that re-express CD45RA are a noncycling apoptosis-resistant CD8+ T-cell memory population. One prediction from this would be that the extent of cell cycling over time would be lower in the virus-specific cells that re-express CD45RA than in those that remain CD45RA, which may be reflected in differences in their
relative telomere lengths.
To investigate this, we first purified CD8+
CD45RA+ and CD8+ CD45R0+ T cells
from healthy persons who recovered from AIM and then analyzed the
telomere length of EBV-lytic epitope-specific cells by 2-color
flow-FISH, as described previously (Figure
6).12 In 5 persons tested,
the lytic epitope-specific CD8+ T cells within the CD45RA
population had shorter telomeres than the total CD8+
CD45RA+ T cells, indicating that they had been expanded in
vivo (Figure 6; P < .01). However, in 4 of the 5 persons
tested, the CD45RA-expressing cells within the virus-specific
CD8+ T-cell populations had longer telomeres than
CD45RA
Reactivation of virus-specific CD8+ CD45RA+ T cells by specific peptide To directly test whether EBV-specific CD45RA+ T cells could still replicate, we isolated the CD45RA+ T-cell subset from a person who recovered from AIM 20 years previously. We focused on the lytic epitope (RAK)-specific CD8+ T cells within these CD45RA+ cells before (Figure 7A), 4 days after (Figure 7B), and 7 days after (Figure 7C) stimulation with peptide-pulsed APC. By combining the viable cell recovery and the percentage of virus-specific cells present, we found that there was an 8-fold increase in total virus-specific CD8+ T-cell numbers after 7 days of culture. We also investigated the extent of cycling after peptide stimulation directly in the virus-specific CD8+ T cells by Ki67 antibody staining, before and after culture (Figure 7G-I). Most virus-specific CD8+ T cells were induced to enter cell cycle after specific stimulation. Although 98% of the virus-specific CD8+ T cells expressed CD45RA before culture (Figure 7D), most of these cells coexpressed CD45RO after 4 days of stimulation (Figure 7E). After 7 days of stimulation, 98% of the virus-specific cells expressed CD45RO, and 88% were CD45RA
CD45RO+ (Figure 7F). Activated virus-specific cells that
were originally CD45RA+ also expressed perforin after 4 and
7 days of stimulation, showing that they expressed cytotoxic effector
molecules (not shown). These results collectively demonstrate that
virus-specific CD8+ T cells that re-express CD45RA can be
reactivated to proliferate.
We investigated whether the EBV-specific CD8+ T cells could
be expanded in longer-term culture and whether the perforin expression observed above was directly linked to the cytotoxic activity of the
cells. In the experiment shown (Figure
8), GLC lytic epitope-specific CD45RA and
CD45RO populations were first isolated then stimulated with specific
peptide for 3 weeks. There was a 73- and an 84-fold increase of
CD45RA-enriched and CD45RO-enriched GLC/A2 tetramer staining cells
after 3 weeks of stimulation, respectively (not shown), indicating that
both these populations of epitope-specific cells were capable of
considerable expansion in vitro. On expansion, cells that originally
were CD45RA+ switched to CD45RO expression, whereas cells
that originally were CD45RO retained this phenotype (Figure 8). The
GLC-specific CD8+ T cells that were expanded from the
CD45RA+ and CD45RO+ subsets were highly
cytotoxic. Some of the tetramer-negative cells that originally were
CD45RA+ lost this marker after culture with specific
peptide (Figure 8). This may reflect bystander proliferation or
response to other endogenous peptides that might have been present on
the antigen-presenting cells used. Nevertheless, it is clear that
though most tetramer-positive cells that were expanded from the
CD45RA+ virus-specific subset were CD45RA
In the present study, we identified 2 fundamental changes that occur in EBV lytic and latent epitope-specific CD8+ T cells selected for survival after the resolution of AIM. First, the cells are induced to exit the cell cycle and become quiescent; second, the persisting cells up-regulate Bcl-2 and become relatively resistant to apoptosis. Both these processes are particularly apparent in the CD45RA re-expressing populations, and this may help stabilize the memory CD8+ T-cell pool to this infection. We found that CD45RA+ EBV-specific CD8+ T cells had longer telomeres relative to their CD45RO+ counterparts. However, the EBV-specific CD8+CD45RA+ T cells had consistently shorter telomeres than the total CD8+ CD45RA+ T-cell population, indicating that they had been expanded in vivo. We have also shown previously that most CD45RA-expressing, EBV-specific CD8+ T cells found after the resolution of AIM do not express the CCR7 chemokine receptor, which further supports the possibility that they are antigen experienced14,15 and not naive T cells. Although some EBV-specific CD8+ T-cell clones found during AIM may be lost and replaced by others in memory,5,6 the appearance of CD45RA+ EBV-specific CD8+ T cells is not due to the selection of less expanded populations because the specific clones of cells that do persist after AIM are found in CD45RA and CD45RO compartments.21 Nevertheless, the relative extent of proliferation that has actually occurred in the EBV-specific CD45RA+ and CD45RO+ T cells cannot be determined because the enzyme telomerase can replenish telomeres in proliferating cells.32 Further investigations are in progress to determine whether virus-specific CD8+ cells in CD45RA+ and CD45RO+ compartments up-regulate this enzyme equally after reactivation. The stabilization of lymphocyte populations, by inducing quiescence and resistance to death after episodes of extensive proliferation, is not unique to virus-specific CD8+ T cells after acute infection. During thymic differentiation there is considerable proliferation of CD4+/CD8+ CD45RO+ thymocytes, which occurs in parallel with low Bcl-2 expression and susceptibility of these cells to apoptosis.33,34 However, the selected single-positive CD4+ or CD8+ naive T cells are quiescent, up-regulate Bcl-2, and re-express CD45RA.33,35 Another example is in the B-cell system, where considerable proliferation of germinal center B cells occurs during immune responses.36 These cells have low Bcl-2 expression and are susceptible to death.37 However, when memory cells are selected from these proliferating populations, they are not in cycle, they up-regulate Bcl-2, and they are resistant to death.37 Although the signals associated with stabilizing selected thymocytes and virus-specific CD8+ T cells are not well characterized, there is some evidence that the ligation of certain costimulatory molecules, such as CD40 on germinal center B cells, may be involved.38,39 The functional significance of CD45RA re-expression per se is unclear; however, it may be one of a set of genes induced when cells are triggered by putative stability-inducing signals. Identification of these signals may enable antiviral immunity to be manipulated in immunocompromised persons and in the elderly. Our data indicate that EBV lytic epitope-specific CD45RA+ T
cells found after the resolution of AIM can be activated specifically to proliferate and to express perforin and cytotoxic activity and
CD45RO. This, together with the observations that they are resistant to
apoptosis and have longer telomeres than their CD45RO counterparts,
suggests that they are a stable, noncycling, memory pool of cells and
not end-stage effector cells as has been reported.18 Discrepancies in the ability to reactivate virus-specific
CD8+ T cells that re-express CD45RA between our study and
previous reports may be attributed to the fact that we stimulated
EBV-specific CD8+ T cells with peptide-pulsed APCs, which
exhibit a wide array of costimulatory molecules.13-15 In
contrast, anti-CD3 together with anti-CD28 was previously used for the
stimulation of virus-specific CD45RA+ CD8+ T
cells,18 which might have been suboptimal because a large proportion of these cells have lost CD28 expression.13
Another possibility is that CD45RA re-expressing virus-specific
CD8+ cells found during EBV infection may not be as
terminally differentiated as other populations found during CMV
infection. This is supported by the fact that though the CD45RA
re-expressing EBV-specific cells we studied are largely
CD27+,14,15 large numbers of these
virus-specific cells are CD27 The observation that there is less re-expression of CD45RA by EBV-latent epitope-specific cells suggests that they may not be as stable as those directed to lytic epitopes. The same applies to influenza virus-specific cells that are exclusively CD45RO+ 19 and to HIV-specific cells that may have a deficient capacity to differentiate into a CD45RA re-expressing population.18,24 The finding that more CD45RA re-expression occurs in CMV- than in EBV-specific CD8+ T cells and that CMV induces higher levels of CD8 immunity than EBV has led to the hypothesis that the higher the virus load in vivo, the greater the CD45RA re-expression by virus-specific CD8+ T cells.15,20 If correct, this would suggest that a stable memory CD8+ CD45RA+ T-cell pool would only be established for viral epitopes that are present at high enough levels to induce sufficient specific CD8+ T-cell differentiation. Because EBV-specific CD8+ CD45RA+ T cells are
not a terminally differentiated end-stage effector population, it
suggests that there must be a dynamic balance between the
virus-specific CD45RA and CD45RO compartments of cells during chronic
infection. This is supported by the observation that they have the same
clonality.21 The factors that determine whether the
virus-specific cells persist in a CD45RA or CD45RO state include
stimulatory signals, such as antigen and cytokines, that will
preferentially induce CD45RO expression and cycling of these cells.
However, access to putative signals that confer resistance to apoptosis
and exit from cell cycle will promote the survival of these cells in a
quiescent/stable CD45RA state. Two crucial questions that must be
answered are, first, what exactly is the nature of the signals involved
and, second, do repeated cycles of activation of virus-specific
CD8+ T cells
We would like to thank Professors G. Janossy and P. C. L. Beverley for discussions.
Submitted January 18, 2002; accepted March 22, 2002.
Prepublished online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2002-01-0160.
Supported by grants from Aventis (P.J.D.) and the Medical Research Council (P.J.D.), BBSRC SAGE initiative 77/SAG1002 (F.J.P.), the Arthritis Research Campaign (J.M. Faint), and Programa PRAXIS XXI grant BD/9254/96 (M.V.D.S.). Supported also by a grant from the European Union (Bio-4 Cat CT 98-0214).
P.J.D. and J.M. Faint contributed equally to this work.
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: Arne N. Akbar, Department of Clinical Immunology, Royal Free and University College Medical School, Pond St, Hampstead, London NW3 2QG, United Kingdom; e-mail: aakbar{at}rfc.ucl.ac.uk.
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Top
Abstract
Introduction
followed by induction of quiescence,
stabilization, and CD45RA expression