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Division of Viral Immunology, Center for AIDS Research, Kumamoto University, Kumamoto, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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CD27+CD28-CD45RA-CD27-CD28-CD45RA-.
This was supported by the observation that the frequency of
CCR5+ cells and CCR7+ cells decreased during
this sequence. Analysis of CCR5 and CCR7 expression in the
CD27+CD28+ memory cell subset demonstrated the
presence of three CCR5/CCR7 populations:
CCR5-CCR7+,
CCR5+CCR7+, and
CCR5+CCR7-. These findings suggested the
following differentiation pathway:
CD27+CD28+CD45RA-
(CCR5-CCR7+CCR5+CCR7+CCR5+CCR7-)CD27+CD28-CD45RA-CD27-CD28-CD45RA-.
The presence of a CD27-CD28+ subset with a
CCR5+CCR7- phenotype implies a specialized
role for this subset in the differentiation of CD8+ T
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD27 and CD28 can be used as cell surface markers to discriminate naive and memory CD8+ T cells from effector CD8+ T cells (6, 7, 8). These molecules are well known as T cell costimulatory molecules. CD28 interacts with CD80(B7-1) and CD86(B7-2), which are expressed on APCs (9, 10), and has a dual role as an adhesion and a signaling molecule. Coengagement of CD28 with the TCR is required for T cell activation (11). CD28 is down-regulated after TCR stimulation (12). CD27 is less well characterized than CD28. Its expression is up-regulated upon TCR stimulation (13, 14, 15). The CD27 ligand CD70 is transiently up-regulated by Ag receptor stimulation on both T and B cells (16). CD27 ligation enhances TCR-induced expansion of both CD4+ and CD8+ T cells (17, 18). A recent study suggested that CD27 is required for generation of T cell memory rather than for differentiation of CD8+ T cells to effector cytotoxic T cells (19). CD45RA, which is one of two CD45 isoforms, can be also used as a marker of naive CD8+ T cells (20). Recent studies using virus epitope-specific HLA tetramers demonstrated that CD45RA+CD8+ T cells include effector T cells (21, 22), suggesting that naive CD8+ T cells cannot be identified by the presence of CD45RA alone.
Previous studies using human CD8+ T cells costained with an anti-CD45RA mAb and an anti-CD27 or anti-CD28 showed that CD8+ T cells with a CD28+CD45RA- or CD27+CD45RA- phenotype express no or only a low level of perforin but have the ability to produce cytokines, while CD8+ T cells with a CD28-CD45RA+ or CD27-CD45RA+ phenotype express a high level of perforin but have a limited ability to produce cytokines (8, 23). These observations strongly suggest that human CD8+ T cells with a CD28+CD45RA- or CD27+CD45RA- phenotype are memory cells and that those with a CD28-CD45RA+ or CD27-D45RA+ phenotype are effector cells. As CD8+ T cells with a CD28-CD45RA- or CD27-CD45RA- phenotype express a medium level of perforin, these cells are thought to be intermediate between memory and effector T cells, i.e., to be memory/effector T cells (23, 24).
CCR7 is a chemokine receptor that functions as a homing receptor in the
migration of naive and memory CD8+ T cells to
secondary lymphoid tissues. A role for CCR7 in
CD8+ T cells has been demonstrated through the
identification of
CCR7+CD45RA+ naive
CD8+ T cells and
CCR7+CD45RA- and
CCR7-CD45RA- memory
CD8+ T cells (25). Furthermore, a
recent study suggested the following differentiation lineage for
Ag-specific CD8+ T cells:
CCR7+CD45RA+CCR7+CD45RA-CCR7-CD45RA-CCR7-CD45RA+
(26). This study suggested that
CCR7+CD45RA+CD8+
and
CCR7+CD45RA-CD8+
T cells are precursor memory T cells while
CCR7-CD45RA-CD8+
and
CCR7-CD45RA+CD8+
T cells are preterminally differentiated and terminally differentiated
cells, respectively. Thus, analysis of chemokine receptor expression
may be useful for discriminating among naive, memory, and effector
CD8+ T cells.
In this study, we attempted to clarify the differentiation pathway of memory CD8+ T cells to memory/effector CD8+ T cells. We used HLA class I tetramers and flow cytometry to detect EBV- or human CMV (HCMV)4-specific CD8+ T cells in healthy individuals. Tetramer+CD8+ T cells were analyzed for CD27, CD28, CD45RA, and perforin expression by four-color flow cytometric analysis. In addition, the surface expression of two chemokine receptors, CCR5 and CCR7, and the function (cytotoxic activity and cytokine production) of different CD27/CD28 subsets of CD45RA-CD8+ T cells were examined. These analyses enabled us to propose a pathway for the differentiation of memory CD8+ T cells to memory/effector CD8+ T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood samples were taken from 18 HCMV-seropositive and
EBV-seropositive healthy adult individuals (see Table II
).
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The HLA-A*0201-restricted HCMV-specific CTL epitope pp65 495–503 (NLVPMVATV; HCMV-1) (27) and two HLA-A*1101-restricted EBV-specific CTL epitopes, EBV-1 (EBV 3B 416–424; IVTDFSVIK) and EBV-2 (EBV 3B 399–408; AVFDRKSDAK) (28), were previously identified. These peptides were generated using an automated peptide synthesizer (model PSSM-8; Shimazu, Kyoto, Japan) with F-moc strategy followed by cleavage. The purity of the synthesized peptides was examined by mass spectrometry.
Cells
C1R cells expressing HLA-A*1101 (C1R-A*1101) or HLA-A*0201 (C1R-A*0201) were previously generated (29) and were maintained in RPMI 1640 medium supplemented with 10% FCS and 0.15 mg/ml hygromycin B.
Monoclonal Abs
Anti-CD27-FITC mAb, anti-CD28-FITC mAb, anti-CD28-PE mAb, anti-CD28-CyChrome mAb, anti-CD28-allophycocyanin mAb, anti-CD45RA-CyChrome mAb, anti-CCR5-FITC mAb, anti-CCR7 mAb, and anti-perforin-PE mAb were obtained from BD PharMingen (San Diego, CA). Anti-CD8-FITC mAb and anti-CD8-allophycocyanin mAb were obtained from DAKO (Glostrup, Denmark).
HLA class I/EBV or HCMV peptide tetramers
HLA class I-peptide tetrameric complexes were synthesized
as previously described (30). Briefly, recombinant HLA
class I proteins (HLA-A*0201 and HLA-A*1101) and human
2-microglobulin were produced in
Escherichia coli cells transformed with the relevant
expression plasmids. The H chain was modified by deletion of the
transmembrane cytosolic tail and COOH-terminal addition of a sequence
containing the BirA biotinylation site. Two EBV CTL epitopes (EBV-1 and
EBV-2) and one HCMV CTL epitope (HCMV-1) were used for refolding of HLA
class I molecules. The HLA class I-peptide complexes were refolded in
vitro. The 45-kDa complexes were isolated using gel filtration on a
Superdex G75 column (Amersham Pharmacia Biotech, Little Chalfont,
U.K.). Purified complexes were biotinylated with BirA enzyme
(Avidity, Denver, CO). The biotinylated complexes were purified using
gel filtration first on a Superdex G75 column and then on a MonoQ
column (Amersham Pharmacia Biotech). HLA class I/peptide tetrameric
complexes (tetramers) were mixed with allophycocyanin-labeled
streptavidin (BD PharMingen) at a molar ratio of 4:1.
Flow cytometric analysis
CD8+ T cells were purified from cryopreserved or fresh PBMC using anti-CD8-coated magnetic beads (MACS CD8 Microbeads; Miltenyi Biotec, Bergisch Gladbach, Germany). The percentage of CD8+ T cells in purified cells was >98%. A total of 1 x 106 CD8+ T cells were mixed with tetramers at a concentration of 0.02–0.04 mg/ml. After incubation at 37°C for 30 min, the cells were washed once with RPMI/10% FCS, and then anti-CD27 mAb, anti-CD28 mAb, and anti-CD45RA mAb were added to the cell suspension. The cells were incubated at 4°C for 30 min, and then the cells were washed two times with PBS/10% FCS.
To determine intracellular perforin expression in CD27/CD28 subsets of both tetramer-positive cells and total CD8+ T cells, cells stained with anti-CD27 mAb, anti-CD28 mAb, and anti-CD45RA mAb were fixed with 4% paraformaldehyde at 4°C for 20 min, then permeabilized with PBS supplemented with 0.1% saponin containing 20% FCS (permeabilizing buffer) at 4°C for 10 min. Cells were washed with permeabilizing buffer and then resuspended in 100 µl of the same buffer. Anti-perforin mAb was added, the cell suspension was incubated at 4°C for 30 min, and the cells were washed three times in permeabilizing buffer at 4°C. PE-labeled mouse IgG1 was used as negative control.
The cells were finally resuspended in PBS containing 2% paraformaldehyde and then were analyzed using a FACSCalibur with CellQuest software (BD Biosciences, San Jose, CA). Between 70,000 and 200,000 gated events were acquired for analysis of intracellular perforin. Intracellular perforin expression is represented as the normalized mean fluorescence intensity (NMFI), which is the mean fluorescence intensity (MFI) of cellsstained with anti-perforin-PE mAb divided by the MFI of cells stained with the control mouse IgG1-PE mAb.
CTL assay
CTL activity was measured by a standard 51Cr release assay as follows. Target cells (2 x 105) were incubated for 60 min with 100 µCi Na2 51Cr in saline and then washed three times with RPMI 1640 medium containing 10% NCS. Labeled target cells (2 x 103/well) were added into U-bottom 96-well plates with the indicated amount of peptide. After incubation for 1 h, CD8+ T cells purified by anti-CD8 mAb-coated beads or CD27-CD28-CD45RA-CD8+ and CD27+CD28-CD45RA-CD8+ T cells purified by a cell sorter (FACSVantage SE; BD Biosciences) were added and the mixtures were incubated for 6 h at 37°C. The supernatants were collected and analyzed with a gamma counter. Spontaneous 51Cr release was determined by measuring the cpm in supernatants from wells containing only target cells (cpm spn). Maximum 51Cr release was determined by measuring the cpm in supernatants from wells containing target cells in the presence of 2.5% Triton X-100 (cpm max). Specific lysis = ((cpm exp - cpm spn)/(cpm max - cpm spn)) x 100, where cpm exp is the cpm in supernatants of wells containing both target and effector cells.
Measurement of cytokine-producing cells by flow cytometry
To measure cytokine production in CD27/CD28 subsets of EBV- or
HCMV-specific CD8+ T cells, cells were purified
from fresh PBMC isolated from healthy individuals who have EBV- or
HCMV-specific CD8+ T cells using
anti-CD8-coated magnetic beads (MACS CD8 Microbeads; Miltenyi
Biotec). CD8+ T cells (2 x
106) were dispensed into a U-bottom 96-well plate
with or without 1 µM of a EBV or HCMV peptide and incubated for
6 h. After the first 2 h of incubation, brefeldin A (10
µg/ml) was added to each well. The cells were then stained with a
mixture of anti-CD27 mAb, anti-CD28 mAb, and anti-CD45RA
mAb, fixed with 4% paraformaldehyde at 4°C for 20 min, and then
permeabilized with PBS supplemented with 0.1% saponin containing 20%
NCS (permeabilizing buffer) at 4°C for 10 min. Cells were resuspended
in permeabilizing buffer and then stained with anti-IFN-
mAb,
anti-TNF-
mAb, or anti-macrophage-inflammatory protein
(MIP)-1 mAb. The cells were finally resuspended in PBS
containing 2% paraformaldehyde and then the percentage of
intracellular IFN--, MIP-1-, or TNF--positive cells was
analyzed by flow cytometry. To measure the number of EBV- or
HCMV-specific CD8+ T cells in each CD27/CD28
subset, CD8+ T cells were stained with an EBV or
HCMV tetramer and a mixture of anti-CD27 mAb, anti-CD28 mAb,
and anti-CD45RA mAb. The percentage of cytokine-positive cells in
tetramer-positive cells in each CD27/CD28 subset was
determined.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HLA-A*1101 is a common allele in the Japanese population, and
IVTDFSVIK (EBV-1) and AVFDRKSDAK (EBV-2) are known to be
HLA-A*1101-restricted immunodominant epitopes. Therefore, we used these
peptides to generate tetramers to analyze EBV-specific
CD8+ T cells in PBMC from EBV-seropositive
healthy Japanese individuals with HLA-A*1101. Ex vivo flow cytometry
analysis demonstrated that in 5 of the 10 individuals studied >0.1%
of total CD8+ T cells were EBV-1
tetramer+, and that in 6 of the 10 individuals
>0.1% of total CD8+ T cells were EBV-2
tetramer+ (Table I). Eight individuals had
tetramer+CD8+ T cells for
at least one of the EBV epitopes. Analysis of CD28 and CD45RA
expression on the
tetramer+CD8+ T cells
demonstrated that most individuals had a high number of
tetramer+CD8+ T cells with
a CD28+CD45RA- memory
phenotype but only a low number with a
CD28-CD45RA-
memory/effector phenotype (Fig. 1 and
Table I). Perforin expression was much lower in the
tetramer+CD8+ T cells than
in total CD8+ T cells and HCMV-specific
CD8+ T cells (data not shown). These results
indicate that most EBV-specific CD8+ T cells in
healthy individuals are memory CD8+ T cells.
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CD45RA-CD8+ T cells have four CD27/CD28 subsets
Previous studies suggested that CD8+ T cells
with a CD28+CD45RA- or
CD27+CD45RA- phenotype are
memory T cells, whereas those with a
CD28-CD45RA- or
CD27-CD45RA- phenotype
are memory/effector T cells. This classification is based on perforin
expression: cells with the former phenotypes have no or only a low
level of perforin while the latter have a higher level of perforin, but
a lower level than that of
CD28-CD45RA+CD8+
T cells (23). To clarify the differentiation pathway of
memory (CD28+CD45RA- or
CD27+CD45RA-)
CD8+ T cells to memory/effector
(CD28-CD45RA- or
CD27-CD45RA-)
CD8+ T cells, we analyzed the expression of both
CD27 and CD28 in total
CD45RA-CD8+ T cells in
PBMC isolated from 18 healthy individuals. In the majority of these
individuals, cells with a
CD27+CD28+ phenotype formed
a major population of total
CD45RA-CD8+ T cells, while
three other phenotypes,
CD27+CD28-,
CD27-CD28+, and
CD27-CD28-, formed a
minor population (Fig. 2A and Table II
). Exceptions were observed in three of
the 18 individuals (A16, U2, and U9), where the
CD27-CD28- subset was the
major population (Table II). Perforin expression was lowest in the
CD27+CD28+ subset, highest
in the CD27-CD28- subset,
and intermediate in the
CD27+CD28- and the
CD27-CD28+ subsets (Fig. 2B). This suggests that
CD27+CD28+CD45RA-CD8+
cells are memory T cells and that this subset may differentiate via the
CD27+CD28- subset and/or
the CD27-CD28+ subset to
the CD27-CD28- subset,
which from its perforin expression level appears to have
characteristics more typical of cytotoxic effector T cells than the
other subsets.
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), and individual
M30, who exclusively had HCMV-specific
CD45RA-CD8+ T cells. CD27
and CD28 expression was analyzed on the EBV
tetramer+CD45RA- fraction
of CD8+ T cells isolated from individuals M10 and
M19. The percentage of
CD27+CD28+,
CD27+CD28-, and
CD27-CD28- cells in
EBV-specific CD8+ T cells was similar to that in
total CD8+ T cells, but the percentage of
CD27-CD28+ cells in
EBV-specific CD8+ T cells was much lower than
that in total CD8+ T cells (Fig. 3A). Similarly,
CD27+CD28+,
CD27+CD28-, and
CD27-CD28- subsets were
observed in HCMV-specific CD8+ T cells from
individual M30, while only a small number of
CD27-CD28+ cells was
detected (Fig. 3A). Perforin expression in each EBV-specific
and HCMV-specific
CD45RA-CD8+ T cell subset
was similar to that observed in each total
CD45RA-CD8+ T cell subset,
although expression in the
CD27+CD28- subset appeared
to be higher in EBV- and HCMV-specific CD8+ T
cells than in total CD8+ T cells (Figs. 2B and 3B). These results support the idea that
the CD27+CD28+ and
CD27-CD28- subsets are
memory and memory/effector CD8+ T cells,
respectively, and that the
CD27+CD28- subset is an
intermediate in the differentiation pathway from
CD27+CD28+ to
CD27-CD28-.
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We evaluated the ex vivo cytotoxic activity of EBV-specific
CD45RA-CD8+ T cells with
different CD27/CD28 phenotypes for target cells (C1R-A*1101 or
autologous T cell blast) pulsed with EBV-1 or EBV-2.
CD8+ T cells were isolated from PBMCs from two
EBV-seropositive, healthy individuals (E1 and U5) with HLA-A*1101.
Approximately 98 and 81% of EBV-specific CD8+ T
cells from E1 and U5, respectively, had a
CD27+CD28+CD45RA-
phenotype (Tables I and II). These CD8+ T cells
exhibited no cytotoxic activity for HLA-A*1101+
target cells pulsed with EBV-1 and EBV-2 peptides at a
tetramer+CD8+ T cell:target
ratio of 1:1 (Fig. 4A). These
results indicate that
CD27+CD28+CD45RA-CD8+
T cells are not effector cytotoxic T cells.
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A), because PBMCs containing
both EBV-specific CD8+ T cell subsets were not
available for this experiment. Both the
CD27+CD28- and
CD27-CD28- subsets
effectively killed target cells pulsed with an HCMV-1 peptide (Fig. 4B). These results are consistent with the perforin
expression levels observed in these subsets. Cytokine production in different CD27/CD28 subsets of CD45RA-CD8+ T cells
Cytokine production in
CD27+CD28+,
CD27+CD28-, and
CD27-CD28- subsets of
CD45RA-CD8+ T cells was
investigated using two EBV epitope peptides and an HCMV-1 peptide.
Purified CD8+ T cells from individuals U2 and M19
were analyzed for IFN--, TNF--, and MIP-1-producing cells
6 h after stimulation with the appropriate peptide(s). All three
subsets from both individuals effectively produced these three
cytokines, although cytokine-producing cells seemed to be more
prevalent in the
CD27-CD28- subset than in
other subsets (Fig. 5). These results
indicate that these three subsets of HCMV-specific and EBV-specific
CD8+ T cells are able to effectively produce
cytokines.
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Our recent study showed that
CD28+CD45RA-CD8+
T cells express a high level of CCR5, and that this expression is
decreased in
CD28-CD45RA-CD8+
T cells (31). To further investigate CCR5 expression in
different CD27/CD28 subsets of
CD45RA-CD8+ T cells, we
stained total CD8+ T cells from three individuals
(M10, M19, and M30) with anti-CCR5, anti-CD27, anti-CD28,
and anti-CD45RA mAb (Fig. 6).
Approximately 60–70% of the
CD27+CD28+ subset and
10–30% of the
CD27-CD28- subset were
CCR5+. The
CD27+CD28- subset showed
an intermediate number of CCR5+ cells that was
much lower than that of the
CD27+CD28+ subset. These
results support the sequence
CD27+CD28+CD27+CD28-CD27-CD28-
for the differentiation of memory CD8+ T cells to
memory/effector CD8+ T cells suggested by the
observed perforin expression levels in these subsets. However, the
CD27-CD28+ subset had the
highest number of CCR5+ cells of the four subsets
(Fig. 6), suggesting that this subset may have characteristics more
similar to memory T cells.
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), with tetramers and
anti-CD27, anti-CD28, and anti-CCR5 mAbs. CCR5 expression
was similar to that observed in total CD8+ T
cells, although the percentage of CCR5+ cells in
each subset was higher in
tetramer+CD8+ T cells than
in total CD8+ T cells (Fig. 7). These results indicate that the
number of CCR5+ cells decreased during
differentiation from memory
(CD27+CD28+CD45RA-)
CD8+ T cells to memory/effector
(CD27-CD28-CD45RA-)
CD8+ T cells, and support the idea that the
CD27+CD28- subset is
intermediate between the
CD27+CD28+ and
CD27-CD28- subsets in the
differentiation pathway.
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A recent study showed that CCR7 is expressed on
CD27+CD45RA+ and
CD27+CD45RA- but not on
CD27-CD45RA+CD8+
T cells (32), suggesting that CCR7 is expressed only on
naive and memory CD8+ T cells. In addition, our
recent study showed that
CD28+CD45RA-CD8+
T cells have three CCR5/CCR7 subsets:
CCR5-CCR7+,
CCR5+CCR7+, and
CCR5+CCR7-
(31). Because most
CD28+CD45RA- cells in
total CD8+ T cells express CD27 (Table II), it is
likely that
CD27+CD28+CD45RA-CD8+
T cells contain both CCR7+ and
CCR7- populations. Indeed, flow cytometry
analysis of different CD27/CD28 subsets of total
CD45RA-CD8+ T cells showed
that the CD27+CD28+
subset contains both CCR7+ and
CCR7- populations (Fig. 8A). In contrast, most cells
in both the CD27+CD28- and
CD27-CD28- subsets were
CCR7-. These results, together with those of
CCR5 expression in these subsets of
CD45RA-CD8+ T cells,
suggest that
CD27+CD28+CD45RA-CD8+
T cells have three subsets,
CCR5-CCR7+,
CCR5+CCR7+, and
CCR5+CCR7-, while
CD27+CD28-CD45RA-CD8+
and
CD27-CD28-CD45RA-CD8+
T cells have two subsets,
CCR5+CCR7- and
CCR5-CCR7-.
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B). A higher number of CCR7+ cells
was observed in individual M10 compared with M19, suggesting that the
CD27+CD28+ subset of
EBV-specific CD8+ T cells is more differentiated
in individual M19 than M10. Only 10–15% of
tetramer+CD8+ cells in M10
were
CD27+CD28-CD45RA-
or
CD27-CD28-CD45RA-,
while 
30% of
tetramer+CD8+ T cells in
M19 expressed these phenotypes (Figs. 3, 7A, and
8B), also suggesting that EBV-specific
CD8+ T cells are more differentiated in M19 than
in M10.
The CD27-CD28+ subset does
not express CCR7 (Fig. 8A), while most of this subset
expresses CCR5 (Fig. 7). This suggests that this subset is more
differentiated than
CD27+CD28+CD45RA-CD8+
T cells but less differentiated than
CD27+CD28-CD45RA-CD8+
and
CD27-CD28-CD45RA-CD8+
T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table II). The same results
were found in HCMV-specific CD8+ T cells from
other healthy individuals (our unpublished observation). A recent study
showed that most growing populations of EBV-specific
CD8+ T cells in PBMC from patients with acute EBV
infection express both a
CD27+CD28-CD45RO+
and
CD27-CD28-CD45RO+
phenotype (33). These findings suggest the following
hypotheses: 1) the
CD27-CD28+ subset is an
intermediate in a subdominant pathway of differentiation from
CD27+CD28+ to
CD27-CD28- subsets (this
subset might be resting memory T cells); and 2) the
CD27-CD28+ subset is an
intermediate in the differentiation pathway of
CD8+ T cells specific for particular Ags from
CD27+CD28+ to
CD27-CD28-.
Perforin expression in the
CD27+CD28- subset of total
CD45RA-CD8+ T cells seems
to vary among individuals. The level of perforin expression in the
CD27+CD28- subset was
similar to that of the
CD27-CD28- subset in
individual U2, but similar to that of the
CD27+CD28+ subset in
individuals U5, U7, U10, and U11 (Fig. 2). Similar findings were
observed in EBV-specific CD8+ T cells. The
CD27+CD28- subset of
EBV-specific CD8+ T cells from individual
M19 expressed a lower level of perforin than the same subset from
individual M30 (Fig. 3). As the sorted
CD27+CD28- subset of
HCMV-specific CD8+ T cells from M30, which
expressed a relatively higher level of perforin, effectively killed
target cells, this subset should include effector cytotoxic T cells. A
recent study also showed that the
CD27+CD28-CD45RO+
subset of EBV-specific CD8+ T cells has cytotoxic
activity (33). Thus, it is likely that this subset
contains memory/effector CD8+ T cells which can
kill target cells but may include a wide range of subpopulations
between memory and memory/effector phenotypes or various combinations
of memory and memory/effector cells in different individuals.
A previous study showed that
CD27+CD45RA-CD8+
and
CD27-CD45RA-CD8+
T cells effectively produce IFN- and TNF- after strong and
nonspecific stimulation with PMA and ionomycin
(8). Recent studies showed effective production of IFN-
from both CD28+ and CD28-
fractions and from both CD45RA+ and
CD45RA- fractions in EBV-specific
CD8+ T cells after epitope peptide stimulation
(34). In addition, a recent study showed that perforin
expression and cytokine production segregated into subpopulations of
CD8+ T cells: perforin-positive cells express
CD45RA but neither CD27 nor CD28, while IFN--producing cells have
both CD28(CD27)+ and
CD28(CD27)- phenotypes and a
CD45RA- phenotype (35). These
studies imply that the
CD27+CD28+,
CD27+CD28-, and
CD27-CD28- subsets of
CD45RA-CD8+ T cells can
effectively produce cytokines. Indeed, we showed in this work that
these subsets effectively produced IFN-, TNF-, and MIP-1 after
stimulation with epitope peptides. Thus, the
CD27+CD28- and
CD27-CD28- subsets have
cytotoxic activity and can produce cytokines, whereas the
CD27+CD28+ subset can only
produce cytokines.
Our recent study (31) showed that
CD28+CD45RA-CD8+
T cells include three subsets,
CCR5-CCR7+,
CCR5+CCR7+, and
CCR5+CCR7-, and suggested
that these subsets differentiate in the following sequence:
CCR5-CCR7+CCR5+CCR7+CCR5+CCR7-.
The present study further clarified CCR5/CCR7 expression in
CD27+CD28+CD45RA-CD8+
T cells. The CD27+CD28+
subset, which is memory CD8+ T cells, has both
positive and negative populations for CCR5 and CCR7 expression. These
findings suggest that
CD27+CD28+CD45RA-CD8+
T cells also have three CCR5/CCR7 subsets that differentiate in the
following sequence:
CCR5-CCR7+CCR5+CCR7+CCR5+CCR7-
(Fig. 9).
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). The fact that the
CD27-CD28+ subset
expresses a lower level of perforin than the
CD27+CD28- subset also
supports the idea that the
CD27-CD28+ subset has
characteristics close to memory CD8+ T cells.
The CD27+CD28- and
CD27-CD28- subsets fail
to express CCR7, while 25–45% of the former subset and 15–30% of
the latter subset were CCR5+ (Fig. 6). This
indicates that both subsets have two populations,
CCR5+CCR7- and
CCR5-CCR7-, and that the
number of CCR5+CCR7- cells
decreases during differentiation from the
CD27+CD28- subset to the
CD27-CD28- subset. The
number of CCR5+CCR7- cells
further decreased in the
CD28-CD45RA+ effector
CD8+ T cell population (31). Because
CCR5 is a receptor for RANTES, MIP-1, and MIP-1
(36, 37, 38, 39), it may play a role in the migration of memory
CD8+ T cells to secondary lymph nodes and
inflammatory sites. Indeed, recent studies showed accumulation of
CCR5+ T cells at inflammatory sites in
inflammatory kidney diseases (40), inflammatory bowel
diseases (41), multiple sclerosis (42, 43),
and rheumatoid arthritis (44). In contrast, binding of
CCR5 ligands and CD3/CD28 costimulations causes down-regulation of CCR5
surface expression (45, 46, 47). These findings suggest that
some CCR5+ T cells may stop expressing CCR5
following migration to inflammatory sites and recognition of epitopes
presented by MHC molecules.
A recent study demonstrated that both the
CD27+CD28- and
CD27-CD28- subsets of
CD45RO+CD8+ T cells expand
in acute EBV-infected individuals and that both subsets have cytotoxic
activity (34). This study also showed that, in a CD3
mAb-mediated, redirected cytotoxicity assay, a
CD27+CD28+CD45RO+
subset from a healthy individual had no cytotoxic activity while this
subset from a patient with acute EBV infection had cytotoxic activity.
The present study showed that this subset in EBV-specific
CD8+ T cells from EBV-seropositive healthy
individuals showed low perforin expression and no cytotoxic activity
(Figs. 3 and 4). These findings suggest that this subset of
EBV-specific CD8+ T cells in patients with acute
EBV infection may contain populations with cytotoxic effector function
that are difficult to discriminate by analysis of CD27/CD28/CD45RA
phenotypes.
Previous studies have analyzed the expression of surface
molecules on EBV-specific CD8+ T cells from
EBV-seropositive healthy individuals by flow cytometry using HLA class
I tetramers and mAb against these molecules (48, 49).
These studies showed that these cells express CD27, CD28, CD45RO, and
CD62L, although the percentage of EBV-specific
CD8+ T cells expressing these molecules
varied among individuals. Using HLA-A*1101 latent epitope tetramers,
the present study showed that most EBV-specific
CD8+ T cells from EBV-seropositive healthy
individuals have a
CD27+CD28+CD45RA-
memory phenotype. A recent study revealed that EBV lytic
epitope-specific CD8+ T cells heterogeneously
express CD28 and CD45RA/RO, whereas most EBV latent epitope-specific
CD8+ T cells express CD28 and CD45RO
(34). This suggests that EBV latent epitope-specific
CD8+ T cells are memory T cells while EBV lytic
epitope-specific CD8+ T cells include both memory
and memory/effector or effector T cells. The use of two HLA-A*1101
latent epitope tetramers in the present study also showed that 80%
of EBV-specific CD8+ T cells express a
(CD27+)CD28+CD45RA-
memory phenotype in EBV-seropositive healthy individuals. However, this
varied from 60 to 98% among individuals (Table I).
In summary, we investigated the expression of perforin and two chemokine receptors as well as the function (cytotoxicity and cytokine production) of CD27/CD28 subsets of CD45RA-CD8+ T cells. The data suggested a differentiation pathway for memory CD8+ T cells into memory/effector CD8+ T cells. Further detailed analysis of each step in the differentiation pathway of memory CD8+ T cells is important for our understanding of immune memory in CD8+ T cells.
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Acknowledgments |
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Footnotes |
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2 H.T. and T.M. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Masafumi Takiguchi, Division of Viral Immunology, Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan. E-mail address: masafumi{at}kaiju.medic.kumamoto-u.ac.jp
4 Abbreviations used in this paper: HCMV, human CMV; MFI, mean fluorescence intensity; NMFI, normalized MFI; MIP, macrophage-inflammatory protein.
Received for publication December 31, 2001. Accepted for publication March 28, 2002.
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) and HLA-A*1101-restricted, EBV-1-specific CTL lines for
C1R-A*1101 cells pulsed with EBV-1 and EBV-2 peptides (
) was
measured at an effector (tetramer+ cells):target ratio of
1:1. B, Cytotoxic activity of two CD27/CD28 subsets of
HCMV-specific CD45RA-CD8+ T cells. Left
panel,
CD27+CD28-CD45RA-CD8+
and
CD27-CD28-CD45RA-CD8+
T cells were isolated from the PBMC of individual M30 using a cell
sorter. The percentage of HLA-A*0201-restricted, HCMV-1-specific
CD8+ T cells in each subset was measured using
HCMV-1-A*0201 tetramer. Right panel, The cytotoxic
activity of sorted cells for target cells pulsed with or without HCMV-1
peptide was analyzed. The relative cytotoxic activity of
CD27+CD28-CD45RA-CD8+
T cells (
and 
) for C1R-A*0201 (
