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*
University of Birmingham/Medical Research Council Centre for Immune Regulation, University of Birmingham, Birmingham, United Kingdom;
Millennium Pharmaceuticals, Cambridge, MA 02139; and
Department of Clinical Immunology, Royal Free Hospital, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, while naive CD8+ cells produce only
IL-2. The chemokine receptor profile and migratory potential of
CD45RA+ memory cells is very similar to CD45RO+
cells but different to naive CD8 cells. In accord with this,
CD45RA+ memory cells were significantly underrepresented in
lymph nodes, but account for virtually all
CD8+CD45RA+ T cells in peripheral tissues of
the same individuals. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3
years of the proliferative cycle (6). This phenomenon is
crucial for the maintenance of a homeostatic balance to T cell
populations, but also defines a limit to memory maintained by
continuously cycling cells. Several lines of evidence have suggested
that a population of memory cells exists in the stable
CD45RA+ state (7, 8, 9, 10), but the lack
of markers available to discriminate them from naive cells has
prevented this hypothesis from being tested. Primed rat T cells that
lack the CD45RC isoform re-express it when introduced into nude rats,
suggesting the potential for reversion (7). In humans,
CD4+CD45RA+ T cells contain
a high frequency of recall Ag-responsive cells in primed individuals,
which can be revealed when adequate costimulation of T cells is
provided (8). The concept of a stable, noncycling
population of memory T cells is attractive because the naive pool is
rapidly depopulated following initial Ag challenge; clonal senescence
induced by the progressive increase in susceptibility to apoptosis
should consequently lead to loss of response within a few years. The ability to identify individual Ag-specific CD8+ T cells by staining with MHC class I-peptide tetramers (11, 12) provides a novel way to address these issues. We have used such reagents to track human CD8+ T cell responses to two common herpes viruses: EBV and CMV. These are genetically stable agents that persist at a low level in the healthy immunocompetent host and therefore provide a chronic stimulus to the immune system. Furthermore, primary EBV infection is clinically recognizable as infectious mononucleosis (IM),4 allowing the CD8+ response to be followed in individual patients from the acute primary phase to memory.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Samples were collected from nine subjects (two HLA-A2, six HLA-B8, one both HLA-A2 and -B8) with recent onset acute IM as soon as the diagnosis was confirmed. Chronic-phase samples were collected from the same patients 12 mo later. In three cases, cryopreserved samples were tested from IM patients (HLA-B8) obtained at diagnosis and 1 and 10 years after resolution of symptoms. Samples were also collected from five healthy subjects (two HLA-A2, three HLA-B7) that had tested positive for CMV IgG by latex agglutination at least 3 years previously and from three healthy subjects that had been EBV seropositive for at least 10 years (one HLA-A2, two HLA-B8). Peripheral blood, thoracic lymph nodes, and liver tissue was obtained from healthy organ donors and from individuals suffering from hepatitis C infection.
Cell preparation
Mononuclear cells were isolated from fresh heparinized venous or
umbilical cord blood samples using Ficoll-Hypaque density gradient
centrifugation (Pharmacia, St. Albans U.K.), according to the
manufacturer’s instructions. Samples for flow cytometry were
immediately cryopreserved. CD8+ lymphocytes were
purified from freshly isolated PBMC by negative selection with magnetic
Dynabeads (Dynal, Bromborough, Wirral, U.K.) as previously described
(13). Briefly, adherent cells were depleted by two rounds
of incubation on human serum-coated petri dishes at 37°C for 45 min.
An Ab cocktail consisting of RFB7 (CD37), OKM1 (CD11b), RFT4 (CD4)
(Royal Free Hospital, London, U.K.), RM052 (CD14), 3G8 (CD16), IMMU510
(TCR
) (Beckman Coulter, High Wycombe, U.K.), and GA-R2/HIR2
(glycophorin A) (BD PharMingen, Cowley, Oxford, U.K.) was used to
enrich for CD8+ cells. A single round of Dynabead
depletion routinely generated samples of 60–80%
CD8+ cells. These cells were used for
transmigration assays or further purified to
CD8+CD45RO+ or
CD8+CD45RA+ populations by
two rounds of bead depletion with the addition of SN130 (CD45RA) or
UCHL1 (CD45RO) (Royal Free Hospital). Purities were routinely
>95%.
Liver-infiltrating lymphocytes were isolated from tissue removed during routine transplant operations from inflamed (five samples) and noninflamed (three samples) liver tissue as previously described (14). Blood and lymph node cells were isolated from the same individuals; expression of chemokine receptors was not affected by these procedures (14). Approval for these procedures was granted by the University Hospital Trust Local Ethics Committee.
MHC class I tetramers
Soluble PE-labeled peptide-MHC class I tetramers were constructed as previously described (11). EBV tetramers consisted of HLA-A2 with the peptide GLCTLVAML (BMLF1, aa 190–197) (15) or YVLDHLIVV (BRLF1, aa 109–117) (N. Annels, unpublished data), and HLA-B8 with RAKFKQLL (BZLF1, aa 280–288) (15). CMV tetramers consisted of HLA-A2 with NLVPMVATV (pp65, aa 495–503) (16) and HLA-B7 with TPRVTGGGAM (pp65, aa 417–426) (16).
Flow cytometry
Aliquots of PBMC were analyzed by four-color flow cytometry
using a panel of surface molecule-specific Abs as follows: CD40L FITC
(Ancell, Bayport, MA), CD16 FITC, HLA-DR FITC (Dako, Cambridge, U.K.),
CD62L, CD29 FITC (Beckman Coulter), CCR1, CCR5, CCR6, CXCR3, CXCR5,
CD49e, CD57 FITC, CD58 FITC, (Serotec, Oxford, U.K.), CD11a FITC, CD18
FITC, CD49d (HP2/1), CD27 FITC, CD28 FITC, CD29, CD61, CD69 FITC (BD
PharMingen), CCR2, CXCR1, CXCR4, CD31 (9G11) (R&D Systems, Abingdon,
U.K.), CCR7 (Millennium Pharmaceuticals, Cambridge, MA),
4
7 (ACT-1) (a gift
from Dr. M. Robinson, Celltech, Slough, U.K.), CD49f (4F10) (Imperial
Cancer Research Fund, London, U.K.). Indirect labeling was achieved
using an anti-mouse IgG FITC-conjugated Ab (Southern Biotechnology
Associates, Birmingham, AL). Free binding sites were blocked with
normal mouse serum (Serotec). Cells were colabeled with anti-CD8
phycoerythrin-Texas Red (ECD) (Beckman Coulter) and anti-CD45RA
PE-Cy5 (Serotec) and either the appropriate PE-conjugated MHC class I
tetramer or anti-CD11a PE (Beckman Coulter).
Intracellular protein expression was detected in PBMC labeled initially with anti-CD11a PE and anti-CD8 ECD Abs. Cells were resuspended in Permeafix (Ortho Diagnostic Systems, Amersham, U.K.) for 40 min at room temperature, before labeling with anti-CD45RA PE-Cy5 and either anti-perforin FITC (Ancell), anti-Bcl-2 FITC (Dako), or anti-Bcl-xL (Santa Cruz Biotechnology, Santa Cruz, CA). Bcl-xL binding was detected with a goat anti-rabbit IgG FITC Ab (Southern Biotechnology Associates). Samples were analyzed on a Beckman Coulter Epics XL benchtop flow cytometer (Beckman Coulter).
Telomere length analysis
Purified adult CD8+CD45RA+ lymphocytes were labeled with Ab-38-biotin (anti-CD11a) (a gift from Prof. N. Hogg, Imperial Cancer Research Fund, London, U.K.), followed by streptavidin Cy5 (Jackson ImmunoResearch, West Grove, PA). Adult CD8+CD45RA+ cells expressing high or low levels of CD11a were sorted to >95% purity using a Coulter EPICS Elite flow cytometer (Beckman Coulter), as previously described (6). Cord blood mononuclear cells, purified adult CD8+CD45RO+, and sorted adult CD8+CD45RA+ CD11ahigh and CD11alow cells were labeled with a FITC-conjugated peptide nucleic acid probe specific for telomeric repeat sequences (FITC-OO-(CCCTAA)3) (PE Biosystems, Framingham, MA) as previously described (17). Samples were analyzed on the EPICS XL flow cytometer.
Cell cycle analysis
Freshly isolated PBMC were surface labeled with anti-CD11a PE, anti-CD8 ECD, and anti-CD45RA PE-Cy5 Abs before fixing in 70% ethanol for at least 4 h at -20°C. Cells were stained with control IgG1 FITC (Dako) or the Ki-67-specific Ab MIB FITC (Beckman Coulter) for 30 min before analysis on the EPICS XL flow cytometer.
Intracellular cytokine assay
Purified CD8+CD45RA+
and CD8+CD45RO+ lymphocytes
were labeled with anti-CD11a biotin, followed by streptavidin
RED670 (Life Technologies, Paisley, U.K.). Cells were stimulated for
6 h at 37°C, 5% CO2 in the presence of
200 ng/ml PMA and 500 ng/ml calcium ionophore A23187 (Sigma, Poole,
U.K.). Brefeldin A (5 µg/ml, Sigma) was added after 1 h of
culture. Cells were labeled with anti-CD8 ECD before incubation in
Permeafix, and finally labeled with optimal concentrations of
anti-IFN- FITC and anti-IL-2 PE (BD PharMingen).
Transmigration assays
Migration of CD8+ lymphocytes was assessed
using 6.5-mm diameter, 5.0-µm pore size Transwell inserts (Corning
Costar, Cambridge, MA) as previously described (18).
Optimal doses (determined by prior titration) of the chemokines
macrophage inflammatory protein (MIP)-1, IL-8, (PeproTech, Rocky
Hill, NJ), Epstein-Barr virus induced molecule 1 chemokine, secondary
lymphoid tissue chemokine stromal cell-derived factor-1 (SDF-1),
RANTES, MIP-1, or monocyte chemoattractant protein-1
(MCP-1) (R&D Systems) were prepared in 0.5% fraction V BSA (Sigma) in
RPMI 1640 (Sigma) supplemented with L-glutamine,
benzylpenicillin, and streptomycin (Life Technologies). Chemokines were
warmed to 37°C, and 600 µl was added to the lower Transwell
chamber. A total of 5 x 105
CD8+ lymphocytes in 100 µl were added to the
upper Transwell chamber. After incubation for 18 h at 37°C, 5%
CO2, cells were carefully resuspended from the
upper and lower chambers into 250 µl of 0.5% fraction V BSA/RPMI
1640. Control wells containing no chemokine or 100 ng/ml IL-15
(PeproTech) were included in each assay.
Cell counts for transmigration assays were determined by flow cytometry using an aliquot of recovered cells resuspended in a fixed volume of 25 µg/ml propidium iodide (Sigma), 25% FCS in PBS. The number of viable cells present in each sample was determined on the basis of cell size and exclusion of propidium iodide. A further aliquot of cells was labeled with anti-CD11a FITC, anti-CD45RA PE (Beckman Coulter) and anti-CD8 Tricolor (Caltag, Burlingame, CA) for phenotype analysis by flow cytometry. The migration of each subset of cells was determined as follows. Specific cell migration = (number of cells of phenotype in lower chamber)/(total number of cells of phenotype in upper and lower chambers).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We constructed tetramers specific for three EBV lytic cycle
epitopes, GLC and YVL restricted through HLA A2.01 and RAK restricted
through HLA B8. In each case, donors with the appropriate HLA alleles,
but EBV uninfected as defined by seronegativity in standard Ab
screening assays, never showed significant levels of tetramer staining
of CD8+ T cells (Fig. 1
a). By contrast, in acute IM
patients with the appropriate HLA alleles the frequency of cells
binding to these lytic epitopes was extremely high; 8–40% of
CD8+ T cells were tetramer positive (Fig. 1b) but fell to lower values (0.5–10% of
CD8+ cells) over the 12 mo following resolution
of IM symptoms and establishment of the EBV carrier state. At the time
of primary infection, all three EBV epitope-specific responses were
largely composed of CD45RO+ cells with few if any
cells in the CD45RA+ subset, consistent with
their in vivo activation at this time. After resolution of the
infection, the distribution of tetramer-positive cells for all three
epitopes shifted significantly to include separate
CD45RO+ and CD45RA+
subpopulations. Fig. 1, b and c shows results
from a single patient during acute and postconvalescent phases, and
Fig. 1d summarizes the data from all nine patients studied.
We then studied healthy long-term carriers of EBV using the above three
tetramers and also healthy carriers of CMV using tetramers specific for
two immunodominant epitopes, both derived from the pp65 virion protein
and restricted through HLA-A2.01 or -B7. In each case, tetramer
staining was restricted to individuals with the relevant HLA allele,
and the stained cells were distributed between the
CD8+ T cells subpopulations expressing CD45RA and
CD45RO. CD8+ T cells that have recently been
recruited from the naive pool and then revert to rest can show
expression of both CD45RA and CD45RO (7, 9, 10). While a
small proportion of tetramer-bearing cells were observed to express low
levels of both isoforms, CD45RAbright cells were
invariably CD45RO-; this population was gated
for subsequent experiments (Fig. 1, e and f).
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CD8+CD45RA+ memory cells express high levels of LFA-1
Memory T cells exhibit distinct patterns of migration compared
with naive T cells, and these are determined by their expression of
specific adhesion molecules and chemokine receptors. For example, naive
T cells express high levels of L-selectin and CCR7 that promote their
recruitment via high endothelial venule (HEV) into lymph node, whereas
memory cells lose these two receptors and instead express high levels
of integrins and chemokine receptors that promote recruitment into
tissue. Therefore, we used the EBV and CMV epitope tetramers to
identify memory cells within the circulating
CD8+CD45RA+ T cell pool and
analyzed their profile of cell surface integrin and chemokine receptor
expression compared with that of the
CD8+CD45RA+ T cell pool as
a whole. As shown in Fig. 2 by
representative data based on the EBV RAK/B8 epitope, all
tetramer-staining cells within the CD45RA+
population showed high levels of expression of the LFA-1 adhesion
molecule assayed by staining for its constituent chains: CD11a and
CD18. In this regard, the tetramer-binding
CD45RA+ population was identical with
tetramer-binding cells within the
CD45RO+ population. However, the total
CD8+CD45RA+ pool was
biphasic with respect to LFA-1 levels, clearly containing
LFA-1high and LFA-1low
fractions; this was in contrast to the total
CD8+CD45RO+ pool, which was
uniformly LFA-1high. Studies with all five
tetramers showed the same pattern of results; all tetramer-positive
cells showed high levels of LFA-1, whether in the
CD45RA+ or the CD45RO+
population and regardless of the time after infection.
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). The
CD62LlowCD29highCD49ehigh
phenotype was identical with that of the CD45RO population. That
CD45RA+ tetramer-binding cells are
LFA-1high/CD49ehigh and
CD62Llow has major implications for their
migratory pathways. The LFA-1high phenotype of tetramer-bearing memory T cells is stable over time
In three patients with IM, we were able to study cryopreserved
samples taken at diagnosis (1 mo after infection) and 1 and 10 years
after infection. In all three cases, at all time points, the
tetramer-binding cells were exclusively CD11ahigh
and CD18high (Fig. 3a), irrespective of their
CD45 status. This was also true for asymptomatic individuals infected
over 20 years previously (data not shown).
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These data suggest that all CD45RA+ memory
cells are LFA-1high. Intriguingly, this
population of
CD45RA+LFA-1high cells does
not exist in neonates (Fig. 3b), and previous reports
suggest that the frequency of
CD8+CD45RA+LFA-1high
cells increases with age (19), providing indirect evidence
that the LFA-1high population reflects
accumulated memory cells of diverse specificities. Although it is not
possible to test this by direct observation of Ag binding, a clear
prediction of the hypothesis is that
CD8+CD45RA+LFA-1high
cells should have undergone significantly more cell divisions than
CD8+CD45RA+LFA-1low
cells. We tested this prediction by measuring the length of telomeres
using an in situ hybridization flow cytometry assay (17)
(Fig. 3c). Telomeres consist of repeating units at the end
of chromosomes that are shortened progressively with each cycle of
division. The enzyme telomerase can replace lost telomeres in stem cell
populations, but in T cells, telomere length is a good index of the
number of divisions that cells have undergone. The population of
CD45RA+LFA-1high cells had
significantly shorter telomeres and had therefore undergone many more
cycles of division than
CD45RA+LFA-1low cells.
Furthermore, the telomere length in primed
(CD45RO+) cells was indistinguishable from
CD45RA+LFA-1high cells.
These data strongly support the proposal that
CD45RA+LFA-1high cells
represent a population of memory CD8 cells.
CD45RA+ memory cells express a CCR7lowCCR5high phenotype
It has been assumed that the majority of CD45RA+ T cells are naive and thus migrate between blood and lymph nodes via the HEV. However, the low levels of CD62L would hinder recruitment of the CD45RA+ tetramer-binding population into lymph nodes. High levels of LFA-1 would promote migration across inflamed endothelium, and high levels of CD49e (the fibronectin receptor) would allow them to interact with extracellular matrix. Taken together, these changes suggest CD45RA+ tetramer-binding cells are similar to CD45RO+ cells and are programmed to migrate into extralymphoid tissues.
We tested this hypothesis further by studying the expression of a range
of chemokine receptors on CD8 T cell subsets
(CD45RA+ and CD45RO+) in
comparison with expression of the -chain of LFA-1 (CD11a) (Fig. 4). We found marked differences in the
expression of several chemokine receptors between the
CD45RA+LFA-1high (memory)
and CD45RA+LFA-1low (naive)
populations.
CD45RA+LFA-1high memory
cells were CCR5high,
CXCR4low, CCR7low, and
contained a population of CXCR1high cells. In
contrast CD45RA+LFA-1low
cells were CXCR1low,
CCR5low, CXCR4high, and
CCR7high. Expression of CCR7 by
CD45RA+ cells proved to be as effective as LFA-1
expression at discriminating these populations (Fig. 4). This was
confirmed by substituting tetramer for the Ab to CD11a in the analysis
for the four discriminatory chemokines (Fig. 5). Tetramer-positive cells, both in the
CD45RA+ and CD45RO+
populations, were predominantly
CXCR4lowCCR7low.
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) with three exceptions: CCR6 showed a biphasic distribution in
primed cells, but all
CD45RA+LFA-1high cells were
negative. CXCR3 was expressed by a significant proportion of
CD45RO+ cells, whereas very few
CD45RA+LFA-1high memory
cells expressed it. CCR7 was also expressed by a small population of
primed (CD45RO+) cells, though not as a
distinctly separate population. In contrast, all
CD45RA+LFA-1high cells were
negative for CCR7 expression. The expression of CCR7 has previously
been shown to be a prerequisite for entry into lymph nodes across HEV,
while CCR5 is associated with entry into inflamed tissues
(20), and the clear discordance in expression of these
receptors between the naive and memory CD45RA+
populations suggests different migratory potential. The population of
CD45RO+-primed cells that coexpress CCR7 has
recently been proposed as a "central memory" reservoir population
by Sallusto et al. (21), who suggested that such cells may
migrate to secondary lymphoid tissue. In vitro migration of CD8+ T cell subsets
The close similarity in the chemokine receptor profiles of
CD45RA+LFA-1high memory
cells and CD45RO+-primed cells suggested that the
migratory potential of
CD45RA+LFA-1high memory
cells may resemble that of primed T cells rather than naive CD8 cells.
To assess whether the differential chemokine receptor profile induces
functional effects on the migration of these subsets, we performed
chemotaxis assays using a variety of chemokines. The number of cells
from each subset that migrated was expressed as a proportion of the
number of those cells in the original population. In all of the
specific chemokine migration assays, the results obtained for migration
of CD45RA+LFA-1high memory
cells were very similar to those for the
CD45RO+-primed population. Thus both populations
migrated effectively to RANTES, MIP-1, and MIP-1, all of which
are ligands for CCR5, which is expressed by both populations (Fig. 6a). Primed
(CD45RO+) and
CD45RA+LFA-1high memory
cells also migrated in response to IL-8, which binds to CXCR1, again
expressed by cells in both populations. However, substantial migration
of naive cells was only observed with the CCR7 ligands Epstein-Barr
virus-induced molecule 1 ligand chemokine and secondary lymphoid tissue
chemokine and with stromal cell-derived factor-1, which is the
only known ligand for CXCR4 (Fig. 6a). These results closely
accord with the chemokine receptor expression (Fig. 4) and suggest that
naive cells will preferentially enter lymph nodes via HEV, whereas
CD45RA+LFA-1high memory
cells are able to respond to inflammatory signals from tissue.
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To test whether the CD45RA+ memory
population does indeed migrate to different locations in vivo, compared
with naive cells, we studied the expression of CD11a and CCR7 on
CD8+CD45RA+ T cells derived
from peripheral blood, thoracic lymph nodes, and liver tissue in each
of eight patients (Fig. 6b). In each case, the peripheral
blood population displayed the expected biphasic distribution of CD11a,
which showed an inverse association with CCR7. In contrast, the lymph
nodes tested contained almost entirely
CD11alowCCR7high cells,
while the liver tissue contained almost exclusively
CD11ahighCCR7low cells.
This was the case in five inflamed samples (hepatitis C) and three
healthy donors’ liver tissue.
Stability and function of CD8+ T cell subsets
The close relationship between
CD45RA+LFA-1high cells and
CD45RO+ cells in terms of adhesion molecule
expression, chemokine receptor expression, and migration patterns
suggests that the function of these cells is likely to be different to
CD45RA+LFA-1low naive
cells. Primed T cells are known to show a small population in cycle at
any given time and also to have increased susceptibility to apoptosis.
CD45RO+ cells showed a significant population of
Bcl-2low cells in each of the five individuals
tested. In contrast, both
CD45RA+LFA-1high and
CD45RA+LFA-1low cells
showed no evidence of a Bcl-2 low population. Intriguingly,
CD45RA+LFA-1high cells
consistently showed slightly higher levels of
Bcl-xL expression (between 145 and 230% of
median fluorescence intensity in each case) than either naive
CD45RA+LFA-1low cells or
CD45RO+ cells (Fig. 7a), suggesting low
susceptibility to apoptosis. Failure to express Ki-67 in all
individuals tested indicates that
CD45RA+LFA-1high cells are
not actively cycling, in contrast to the CD45RO+
population. Intriguingly,
CD45RA+LFA-1high cells
express high levels of the cytotoxin perforin and when stimulated
produce IFN-; both of these characteristics were shared with
CD45RO+ cells, but not
CD45RA+LFA-1low cells (Fig. 7b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on stimulation, characteristics of a
secondary response.
CD8+CD45RA+CD11ahigh
cells expressed high levels of Bcl-2 and Bcl-xL,
virtually indistinguishable from naive
CD8+CD45RA+CD11alow
cells.
A population of
CD8+CD45RA+LFA-1high
cells has been described previously, though they have not been shown to
reflect an accumulated memory population (22, 23).
High-level expression of perforin and production of IFN- led to the
suggestion that these cells are recently activated effector cells
(22, 23, 24). However, their lack of cell cycle markers and
high levels of antiapoptosis markers argues against this. The origin of
CD8+CD45RA+LFA-1high
cells is not entirely clear. It is possible that these cells represent
an entirely distinct pattern of differentiation from
CD45RA+ naive precursors that have never
expressed CD45RO, but such a differentiation pathway has not been
reported previously (25). Furthermore, the very short
telomeres expressed by this population suggest a very high level of
differentiation, equivalent to the primed CD45RO+
population. Experiments in rats by Bell and Sparshott (7)
support the concept of primed T cell reversion, showing that purified
primed CD8+ T cells (expressing low m.w. CD45
isoforms) do revert to the resting (high m.w. CD45 isoform-expressing)
state in vivo when introduced into either nude or euthymic animals.
In vivo and in vitro studies of viral infection in mouse models have suggested that rapidly proliferating effector CTL precursors are predominantly CD62Llow, while a smaller population of CD62Lhigh cells require extended exposure to Ag before developing effector function (26, 27). The CD8+CD45RA+LFA-1high cells identified as a distinct memory population in the present study were heterogeneous with respect to CD62L expression, suggesting that this may represent a distinct facet of memory discrimination. Recent data suggest that tetramer-binding cells in mouse models similarly express high levels of LFA-1, which is up-regulated on activation and maintained by memory cells thereafter, entirely consistent with the present study (28).
In the present study, we used tetramers specific for two viruses, EBV and CMV, to detect Ag-specific cells in patients with active infections and asymptomatic seropositive individuals. Both of these viruses persist in humans after infection, and previous studies have reported that between 0.1 and 10% of the CD8+ T cell population bind specific tetramers in individuals with persistent viral infections (12, 29, 30, 31). It is likely that such high levels are maintained by continuous exposure to Ag because tetramer-binding cells decline rapidly after clearance of nonpersistent viruses, frequently falling below the level of detection, but increase rapidly again after rechallenge with Ag (32, 33). Infection with HIV leads to high levels of HIV tetramer-binding cells, but a reduction in viral load after starting antiviral therapy is accompanied by a fall in the number of tetramer-binding T cells (34, 35). Thus the population of CD8+CD45RA+CD11ahighCCR7low memory cells we describe may have arisen as an adaptation to control persistent viral infections. However, it is likely that such cells are a facet of memory for all viruses and that the lack of detection in blood in nonpersistent viral infections is because the number of cells involved is much smaller (33). Furthermore, a recent study has shown that stable LFA-1high tetramer-binding memory cells were generated following infection of C57BL/6J mice with the vesicular stomatitis virus (28), which is a transient, nonpersistent virus (36, 37).
Memory cells are believed to recirculate to lymph nodes, where they acquire Ag on subsequent secondary challenge (1). However, the population of cells we describe appears unlikely to enter secondary lymphoid tissue via HEV because they lack the two receptors that are crucial for extravasation across HEV, CCR7 and L-selectin. The tissue homing characteristics of high CCR5 and CXCR1 expression (20) were corroborated by the observation that the CD45RA+ memory population accounts for virtually all of the CD8+CD45RA+ cells seen in normal and hepatitis C-infected liver tissues. It would seem most likely that these cells reenter secondary lymphoid tissue via the afferent lymphatics, like CD45RO+ cells (38). However, compared with CD8+CD45RO+ cells, they were underrepresented in secondary lymphoid tissue (approximately half the expected frequency in each of eight individuals tested).
These data suggest that the stable, noncycling population of CD8+CD45RA+LFA-1high T cells migrates to tissues, rather than lymph nodes. The CD45RO+CCR7+ population described as central memory cells (21) are a primed population, committed to cell division approximately every 2 wk (4). Such central memory cells may represent a precursor population, giving rise either to more highly differentiated effector cells (CD45RO+CCR7-) or to the CD45RA+LFA-1highCCR7- population of stable memory cells. Our data are consistent with the observations of Masopust et al. (28), who showed that LFA-1high (effector) memory cells in vesicular stomatitis virus-infected mice either continuously migrate through or reside in nonlymphoid tissues.
The overwhelming fate of primed T cells in any immune response is death
by apoptosis, which is essential to prevent a rapid and cumulative
increase in the size of the immune system (39). Therefore,
it follows that rescue of T cells from apoptosis under these conditions
is a prerequisite for immune memory. T cell apoptosis can be inhibited
either by IL-2R common -chain cytokines (40) or by
stromal-derived type I IFN (41). Common -chain
cytokines induce cell cycle in addition to survival and are probably
most important during the course of an active immune response. IFNs
inhibit apoptosis but also induce a stable resting
(G0) phenotype, characteristic of memory cells.
Type I IFNs in lymph nodes are thought to play a significant role in
the inhibition of T cell apoptosis leading to memory. Competition for
entry into the stable CD45RA+ population is
likely to be considerable. In a stable adult immune system, this
population cannot expand significantly over time, though it does
develop from nothing to 50% of the total
CD45RA+ population between birth and adulthood.
The data presented here together with recent reports (28, 42) suggest that
CD45RA+LFA-1highCCR7-
memory cells play a significant role in memory for both persistent and
transient viral infections. The processes that induce reversion of
primed CD45RO+ T cells to a stable
CD45RA+LFA-1highCCR7-
memory state are not known, but the crucial homeostatic constraints
suggest the lymph node as the most plausible site and the
CD45RO+CCR7+ "central
memory" population as strong candidates for precursors.
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Acknowledgments |
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Footnotes |
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2 J.M.F., N.E.A., S.J.C., and P.S. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Mike Salmon, Rheumatology Unit, Division of Immunity and Infection, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K. E-mail address: M.Salmon{at}bham.ac.uk
4 Abbreviations used in this paper: IM, infectious mononucleosis; ECD, phycoerythrin-Texas Red; MIP, macrophage inflammatory protein; HEV, high endothelial venule.
Received for publication November 16, 2000. Accepted for publication April 27, 2001.
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