By
From the Department of Clinical Viro-Immunology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, and Laboratory of Experimental and Clinical Immunology of the University of Amsterdam, 1066 CX Amsterdam, The Netherlands
Human CD8+ memory- and effector-type T cells are poorly defined. We show here that, next
to a naive compartment, two discrete primed subpopulations can be found within the circulating human CD8+ T cell subset. First, CD45RACD45R0+ cells are reminiscent of memory-type T cells in that they express elevated levels of CD95 (Fas) and the integrin family members CD11a, CD18, CD29, CD49d, and CD49e, compared to naive CD8+ T cells, and are able to
secrete not only interleukin (IL) 2 but also interferon
, tumor necrosis factor
, and IL-4. This subset does not exert cytolytic activity without prior in vitro stimulation but does contain virus-specific cytotoxic T lymphocyte (CTL) precursors. A second primed population is characterized by CD45RA expression with concomitant absence of expression of the costimulatory
molecules CD27 and CD28. The CD8+CD45RA+CD27
population contains T cells expressing high levels of CD11a, CD11b, CD18, and CD49d, whereas CD62L (L-selectin) is not
expressed. These T cells do not secrete IL-2 or -4 but can produce IFN-
and TNF-
. In accordance with this finding, cells contained within this subpopulation depend for proliferation
on exogenous growth factors such as IL-2 and -15. Interestingly, CD8+CD45RA+CD27
cells
parallel effector CTLs, as they abundantly express Fas-ligand mRNA, contain perforin and
granzyme B, and have high cytolytic activity without in vitro prestimulation. Based on both
phenotypic and functional properties, we conclude that memory- and effector-type T cells can
be separated as distinct entities within the human CD8+ T cell subset.
Recognition of antigen by the immune system evokes a
coordinate number of changes in lymphocytes and
lymphocyte subsets that allow the system (a) to eliminate or
neutralize potential harmful agents and (b) to respond more
rapidly and appropriately after renewed antigen encounter,
a process referred to as immunological memory. Within
the T cell compartment, unprimed (or naive) T cells (i.e.,
cells which have not yet encountered antigen), effector T
cells (i.e., cells with specialized functions such as cytolysis), and memory T cells can be discerned. Functionally, memory T cells have a number of characteristics that distinguish
them from unprimed cells. Not only do they respond efficiently to recall antigens, but, compared to naive cells,
memory T cells also have less stringent requirements for activation and have the potential to secrete a more extensive
set of cytokines (1). In addition, memory and naive T
cells differ in the expression of several cell surface antigens
(5, 6), although it is questioned whether these markers can
be looked upon as true memory markers or if they reflect cellular activation (7, 8).
Recent studies by Zimmermann et al. (9) have documented phenotypic distinctions between naive, effector,
and memory CD8+ T cells in a murine model of lymphocytic choriomeningitis virus infection and an adoptive
transfer system with CD8+ T cells from TCR transgenic
mice. Their data confirmed previous reports (10) by
demonstrating that compared to naive T cells CTL effectors have strongly downregulated the lymph node homing receptor CD62L (L-selectin) while expression of CD44,
CD11a/CD18, CD11b, and CD49d is enhanced. In contrast to effector T cells, memory T cells are heterogenous
with respect to CD62L expression, have no CD11b on
their surface, and have an intermediate CD49d expression.
Finally, CD45 isoform expression could not be used as a
reliable marker for memory-type T cells (9).
In contrast to the murine T cell subsets, description of
human CD8+ naive, effector, and memory cells is still
rather fragmentary. Cytolytic function of CD8+ cells has
been linked to cells with a CD28 In this study we used simultaneous staining with CD45RA
and CD27 mAbs to separate functionally distinct subpopulations of human CD8+ T cells. Our observations demonstrate that, within the human CD8+ compartment, in accordance with data obtained in experimental animal models,
naive, memory- and effector-type cells can be discriminated with coherent phenotypic and functional properties. This type of CD8+ T cell subset analysis could prove useful in monitoring the immune system in several clinical situations.
Reagents.
The mAbs CLB-T11.1/1, CLB-T11.2/1, CLB-HIK27 (all CD2), CLB-CD3/3 CLB-CD3/4.1, CLB-CD4/1,
CLB-CD8/1, CLB-CD14/1, CLB-FcR gran1 (CD16), CLB-CD19/1, CLB-CD27/1, CLB-CD28, CLB-LFA-1/1 (CD18), CLB-CD49d (VLA-4), and CLB-CD49e (VLA-5), and FITC-conjugated goat anti-mouse Ig, were all produced at the Central
Laboratory of the Netherlands Red Cross Blood Transfusion
Service (CLB; Amsterdam, The Netherlands). CD95 mAb was
provided by Dr. Yonehara (Pharmaceutical Basic Research, Tokyo,
Japan) and CD58 mAb (TS2/9) was a gift from Dr. T.A. Springer
(Center for Blood Research, Boston, MA). CD11b (OKM-1)
mAb was purchased from Ortho Diagnostic Systems (Beerse,
Belgium). FITC-conjugated-CD62L (Leu-8), -CD57, -CD16, -TCR-, a CD11b+, and/or a
CD57+ phenotype (11, 15, 16). Moreover, memory-type
CD8+ CD45RA
CD45R0+ T cell subsets that contain enhanced frequencies of antigen-reactive precursor cells (17),
have low stringent activation requirements, and have an
ability to secrete, apart from IL-2, IFN-
and TNF-
(18),
have been previously described. However, discrimination between naive and memory cells, based solely on CD45
isoform expression, has been proven unreliable, since the
CD45RA+ population contains cells that share a number of
phenotypic features with primed cells, such as low CD62L
(19) and high CD11a/CD18 (20) expression. Moreover,
part of the CD45RA+ cells have downregulated CD27
(21), a feature considered to result from persistent antigenic
stimulation in vivo (22, 23).
/
, and -CD8 mAbs (Leu2a), and PE-conjugated- and
PerCP-labeled CD8 mAb were from Becton Dickinson (San Jose,
CA). CD29 (4B4) and PE-conjugated CD45RA mAbs (2H4) were
obtained from Coulter Immunology (Hialeah, FL). FITC-labeled
CD45RO mAb (UCHL1) was purchased from DAKO (Glostrup, Denmark). Streptavidin red 670 was obtained from GIBCO
BRL (Gaithersburg, MD).
, mouse
IgG1) was obtained from Dr. P. van der Meide (Netherlands Organisation for Applied Sciences TNO, Rijswijk, The Netherlands). Biotinylated anti-human IL-2, FITC-conjugated anti-
human TNF-
, and FITC-conjugated antiperforin mAbs were
purchased from Hölzel Diagnostika (Köln, Germany). Antigranzyme B mAb, provided by Dr. C.E. Hack (CLB), was obtained after immunization of BALB/c mice with native granzyme B.
Cell Preparation and Flow Cytometry.
Human PBMCs were
isolated from buffy coats of healthy donors by Ficoll-Paque density centrifugation (Pharmacia Biotech AB, Uppsala, Sweden).
For phenotypic analysis (Table 1), CD8+ cells (>95% CD8+
CD3+TCR-/
+CD16
) were purified by incubating PBMCs
with saturating amounts of CD4, CD19, CD16, and CD14 mAbs
followed by depletion of positive cells with Dynal beads (Dynal,
Oslo, Norway) as previously described (18). Triple color immunofluorescence analysis was performed as previously described (23).
In brief, purified CD8+ T cells were incubated with an unlabeled
mAb followed by FITC-conjugated goat anti-mouse Ig staining.
After blocking free binding sites on the goat anti-mouse conjugate with 10% normal mouse serum, cells were incubated with
PE-conjugated CD45RA and biotinylated CD27 mAbs. The latter was then stained with Streptavidin red 670.
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Detection of Fas-ligand mRNA.
Total RNA was prepared (TRIzol Reagent; Life Technologies, Paisley, UK) from 2.5 × 106 sorted
T cell subsets (see above). Single-stranded cDNA was synthesized from RNA in a 20-µl reaction mixture containing 500 ng
oligo(dT)12-18 and 200 U reverse transcriptase (RT).1 1 µl of the
reaction mixture was diluted with 11.5 µl of PCR buffer containing 2 mM MgCl2 and 100 pmol of forward and reverse oligonucleotides. Two PCR reactions were set up for each cDNA, corresponding to Fas ligand (forward: 5-GGGTCGACGGGATGTTTCAGCTCTTCCACCTAC - 3
; reverse: 5
-GCTCTAGAACATTCCTCGGTGCCTGTAAC-3
; reference 25) and
HPRT (hypoxanthine-guanine phosphoribosyltransferase; forward: 5
-TATGGACAGGACTGAACGTCTTGC-3
; reverse:
5
-GACACAAACATGATTCAAATCCCTGA-3
; reference 26).
PCR products were resolved on a 1% agarose gel containing ethidium bromide.
Flow Cytometric Detection of Cytokine Production and Intracellular
Staining for Perforin and Granzyme B.
Flow cytometric measurement of cytokine production was performed as previously described (27, 28). In brief, 106 cells/ml were stimulated for 4 h (IL-4,
IFN-, and TNF-
) or 8 h (IL-2) with 1 ng/ml PMA and 1 µM
ionomycin in the presence of the protein-secretion inhibitor
monensin (1 µM; all from Sigma Chemical Co., St. Louis, MO).
This short-term stimulation did not alter the membrane phenotype with respect to CD45 or CD27 expression. After cell surface
staining with PE-conjugated CD45RA and FITC-conjugated
CD27 cells were washed twice with cold PBS and fixated with
PBS/4% paraformaldehyde (at 4°C for 5 min). Fixation was followed by permeabilization with PBS/0.1% saponin (Sigma Chemical Co.)/10% human pooled serum (at 4°C for 10 min).
For all subsequent incubation and washing steps, PBS/0.1% saponin/0.5% BSA was used. Staining of the cytoplasm with biotinylated cytokine mAbs (IL-2, IL-4, and IFN-
; all 5 µg/ml) was
followed by incubation with Streptavidin red 670 (both at 4°C
for 20 min). Biotinylated or FITC-conjugated mouse IgG1 control mAbs (DAKO) were used as negative controls. Data acquisition was performed on a FACScan® and analysis was done using
PC-Lysis software.
Cell Culture and Activation.
All cell culture experiments were
performed in IMDM supplemented with 10% FCS and antibiotics. Sorted CD8+ T cell subsets (5 × 104 cells/well) were stimulated with a combination of three CD2 mAbs (CLB-T11.1/1,
CLB-T11.2/1, and CLB-HIK27, all 5 µg/ml) in the presence or
absence of the following recombinant human cytokines: IL-2 (50 U/ml), IL-4 (10 ng/ml), IL-6 (100 U/ml), IL-10 (100 U/ml), IL-12
(1 ng/ml), IL-15 (10 ng/ml), and IFN- (100 U/ml). Proliferation was measured on day 4 by adding 0.2 µCi/well of [3H]thymidine
(Amersham, Buckinghamshire, UK) during the last 4 h of culture.
All cultures were set up in triplicate.
Cytotoxicity Assay.
CTL activity was determined in a CD3
mAb-mediated cytotoxicity assay as previously described (30). In
brief, FcR-bearing P815 target cells were radiolabeled with
Na51CrO4 (Amersham) for 30 min at 37°C. Purified CD8+ T cell
subsets were incubated with P815 target cells at varying effector/
target ratios in the presence or absence of 5 µg/ml CD3 mAb
(CLB-CD3/4.1). After a 4 h incubation period at 37°C, the supernatants of triplicate cultures were collected and counted in a
gamma counter. Specific cytotoxicity was determined according to the formula: percentage of specific lysis = 100 × [(cpm experimental release cpm spontaneous release)/(cpm maximal release
cpm spontaneous release)]. The standard error of the
mean percentage lysis did not exceed 5%.
Determination of HIV-1-derived Peptide-specific CTL Precursor Frequency.
HIV-1-specific CTL precursors (CTLp) were expanded
in vitro by Ag-specific stimulation and frequencies of RT derived
peptide aa 244-252 (IVLPEKDSW)-specific CTLp were determined as previously described (31, 32). In brief, cryopreserved
PBMCs from an HIV-1 seropositive individual (L090; for case
report see reference 32) were thawed and sorted into CD8+
CD45RA+CD27+ and CD8+CD45RACD27+ subsets. Sorted
cells were cocultivated with stimulator cells (paraformaldehyde
fixated autologous EBV-transformed B lymphoblastoid cell lines
[B-LCL] infected with five multiplicity of infection of recombinant vaccinia virus expressing HIV-1Lai RT) and irradiated feeder
cells (autologous PBMCs; references 31, 32). At days 2 and 9, microcultures were fed with rIL-2 (10 U/ml Proleukin; provided by
Dr. R. Rombouts, Chiron Benelux B.V., Amsterdam, The Netherlands). Cultures were restimulated at day 7 by addition of fixated
autologous stimulator cells in the presence of rIL-2 (10 U/ml). On
day 15, wells were split and tested for cytotoxicity on 51Cr-labeled
autologous B-LCL that were preincubated for 30 min at room
temperature with synthetic peptide at a final concentration of 5 µg/
ml. Lysis of target cells was measured as described above. Wells
were considered positive when 51Cr-release exceeded 10% of
specific lysis. Frequency estimates were made using the "single-hit Poisson model" described by Strijbosch et al. (33).
Helper and Suppressor Activity for Ig Production.
CD8+ T cells
and CD8+ subsets were prepared as described above. The CD8
fraction obtained after purification of CD8+ cells was either depleted of monocytes, NK cells, the remaining CD8+ T cells, and
B cells with CD14, CD16, CD8, and CD19 mAbs and magnetic
beads (Dynal) to generate CD4+ T cells (>95% CD4+) or else
depleted of monocytes, NK cells, and T cells with CD14, CD16,
and CD3 mAbs to prepare B cells (>90% CD19+). To determine
helper capacity, cells of the different T cell subsets (5 × 104/well)
were cultured with autologous B cells (5 × 104/well) in 96-well
flat-bottomed microtiter plates (Greiner, Nürnberg, Germany)
coated with 5 µg/ml CD3 mAb (CLB-CD3/3). Suppressor activity was determined by adding cells of the different CD8+ T cell
subsets (5 × 104/well) to a coculture of autologous CD4+ T and
B cells. All cultures were set up in triplicate and supernatants were
harvested after 14 d and tested for Ig content. IgM and IgG production was measured by ELISA.
As has
been previously noted (21), in contrast to CD4+CD27 T
lymphocytes, which are exclusively CD45RA
, within the
circulating CD8+ compartment CD27
T cells can be
found within both CD45RA
and CD45RA+ fractions
(Fig. 1). These CD8+CD45RA+CD27
cells are, like CD8+
CD45RA
CD45R0+ cells, absent from cord blood (Fig. 1).
Therefore, we hypothesize that in response to antigen two
separate primed CD8+ populations may develop in humans:
(a) a population of CD45RA
T cells, and (b) a population
of CD45RA+CD27
T cells. This study was performed to
analyze whether these phenotypes discriminate between
functionally distinct CD8+ subsets.
In a population of healthy adult donors (n = 30), the distribution of the CD8+ T cell subsets defined by simultaneous CD45RA and CD27 staining was as follows (for definition of the subsets see Fig. 1): (1) CD45RA+CD27+
cells 55 ± 17% (range 28-83%); (2) CD45RACD27+
cells 25 ± 11% (range 9-48%); (3) CD45RA+CD27
cells
13 ± 13% (range 1-50%); and (4) CD45RA
CD27
cells
4 ± 3% (range 0-15%).
To further define the cell surface phenotype of these
subsets, CD8+ T cells were purified from peripheral blood
and analyzed in triple color immunofluorescence. As expected on the basis of the observation that these were the
only cells present in cord blood, CD45RA+CD27+ cells
showed characteristics of unprimed cells since they were CD62L+ and had low CD49d, CD49e, CD29, CD11a/
CD18, and CD58 expression. The majority of cells were
CD28+ and did not express CD45R0 (Table 1 and data
not shown). The CD45RA population had features of antigen-primed cells. CD62L was downregulated, and CD49e,
CD29, and CD58 were upregulated when compared to the unprimed population. The main difference between CD27+
and CD27
cells within the CD45RA
population was the
expression of CD28. Although the majority of CD27+ cells
expressed the antigen, only half of the CD27
cells were
CD28+.
Although CD45R0 was absent from CD45RA+CD27
cells, these T cells had a number of features compatible with
a primed phenotype. CD62L was found to be downregulated, whereas CD49d, CD49e, CD29, CD11a/CD18,
and CD58 were highly expressed. In comparison to the CD45RA
population, expression levels of CD49d and
CD11a/CD18 were even higher on CD45RA+CD27
cells. Remarkably, CD11b and CD57 were present on
>50% of the cells and the majority did not express CD28.
Fas (CD95) was not expressed on CD8+CD45RA+
CD27+ lymphocytes but could be detected on the majority
of CD45RA+CD27 T cells. It should be noted that the
antigen density on the latter population was markedly
lower than on CD8+CD45RA
T cells (Table 1 and Fig. 2
A). Interestingly, Fas ligand mRNA was not present in
CD8+CD45RA+CD27+ T cells but was strongly expressed in CD8+CD45RA+CD27
lymphocytes (Fig. 2 B,
lanes B and C). In contrast, only trace amounts of Fas ligand
mRNA were detected in CD8+CD45RA
CD27+ cells
(Fig. 2 B, lane D).
Finally, it should be noted that a CD8+CD27+ population expressing low levels of CD45RA can be found in
most donors (Fig. 1). Analogous to similar cells within
the CD4+ T cell compartment (34), these cells coexpress
CD45R0 and were found to have an intermediate membrane phenotype between CD45RA+CD27+ and CD45RA
CD27+ T cells (data not shown).
In contrast to naive T cells, which primarily secrete IL-2,
the ability to produce a variety of cytokines is a typical feature of primed T cells (35). Cytokine production capacity
of CD8+ T cell subsets was measured after stimulation for 4 h
with PMA and ionomycin at a single cell level (Fig. 3 and
Table 2). CD8+CD45RA+CD27+ cells paralleled naive
CD4+ cells in that IL-2 was the main cytokine produced
by this subset. In agreement with what would be expected
from primed cells, CD8+CD45RA cells were able to secrete IL-2, IL-4, IFN-
, and TNF-
. IL-4+ cells were exclusively found within this population with both CD27+
and CD27
cells contributing to the total IL-4 secretion.
Also with respect to cytokine expression, the CD45RA+
CD27
subset showed characteristics of antigen-experienced
cells, in that a high percentage of IFN-
and TNF-
producers could be detected. Remarkably, in contrast to
CD45RA
cells, neither IL-2 nor IL-4 production was
found. Since IL-2 production in CD45RA+ cells has been
described to reach its maximum later than in CD45RA
cells (36), we also analyzed 8-h stimulated cells. Indeed, the
frequency of IL-2 producers increased in the CD45RA+
CD27+ subset. However, also at this time point, CD45RA+
CD27
cells did not produce any IL-2. From Fig. 3 it should
be noted that, with respect to the cytokine secretion pattern, cells with a CD27dull expression behave as CD27
T
cells in this assay. Furthermore, expression of surface markers on these cells was also comparable to CD27
cells (data
not shown).
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The results obtained by intracellular cytokine staining
were confirmed by measuring the cytokine content of the
supernatant after 4 d of culture in the presence of PMA and
ionomycin (Table 3). Again, CD45RA+CD27+ T cells
mainly produced IL-2. CD45RA cells secreted all measured cytokines, whereas CD45RA+CD27
cells were only
capable of IFN-
and TNF-
production.
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To test if the distinction in cytokine production profiles
between the two primed CD8+ T cell subpopulations was
reflected in their proliferation capacities, three fractions were
sorted from purified CD8+ T cells, i.e., CD45RA+CD27+,
CD45RA+CD27, and CD45RA
CD27+ cells, and stimulated with a combination of CD2 mAbs supplemented with
various cytokines (Fig. 4). The CD45RA
CD27
population could not be analyzed because of the low number of these cells in most donors (see above). Culture of the sorted cells with the indicated cytokines alone did not result in
proliferation (data not shown). In accordance with a previous
report (18) on stringent activation requirements of unprimed
CD8+ T cells, CD45RA+CD27+ cells were not able to
proliferate in response to CD2 mAbs alone, but addition of
IL-2, -15, -4, and, to a lesser extent, -12 resulted in a vigorous proliferative response, whereas IL-6 and -10 and IFN-
had no effect on proliferation. In contrast, CD2
mAbs alone were sufficient to induce proliferation of
CD45RA
CD27+ cells. IL-2, -15, and -12 further increased proliferation, but IL-4, -6, and -10, and IFN-
only marginally influenced the CD2-induced response.
Proliferation of CD45RA+CD27
cells was only observed
in the presence of either IL-2 or -15. However, the magnitude of the response was lower than that of the other two
populations.
CD45RA+CD27
Our data thus far have shown that CD45RA+
CD27 cells possess a number of phenotypic characteristics
that have been associated with murine (9) and human (11,
15, 16, 37) cytolytic T cells and that these cells secrete
IFN-
and TNF-
, two lymphokines known to play a role
in controlling virus infections (38). Therefore, we addressed
the question of whether these cells behave as CTLs ex vivo.
Purified CD8+ cells were sorted into the distinct subsets and
their cytotoxic capacity was tested in a CD3 mAb-mediated
redirected cytotoxicity assay (Fig. 5). As expected,
unprimed CD45RA+CD27+ cells were not able to lyse the
target cells. CD45RA
CD27+ cells had a low but detectable
cytolytic activity at relatively high effector/target ratios.
Strikingly, however, the CD45RA+CD27
population
had potent cytolytic activity.
Fas-Fas ligand interaction is an important mechanism
used by CTLs for cell-mediated cytotoxicity (39, 40). Next
to this, CTL effectors can kill target cells by releasing a pore-forming protein (perforin) and serine proteases (granzymes),
which are stored in intracellular granules, into the vicinity
of target cell membranes (41, 42). We analyzed freshly isolated CD8+ cells for the presence of perforin and granzyme
B. In accordance with the cytolytic capacities of the different subsets, nearly all cells of the CD45RA+CD27
population contained perforin and granzyme B. Unprimed
CD45RA+CD27+ cells did not contain these enzymes,
whereas low staining could be found within the CD45RA
population (Fig. 6 and Table 4). As already seen in the
analysis of cytokine production, cells with dull CD27 expression showed a similar granzyme B and perforin expression pattern to CD27
cells.
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CTL effectors exert direct ex vivo cytolytic activity, whereas memory cells need restimulation with
antigen to acquire cytotoxic function. Since CD45RA
CD27+ cells, which resemble memory T cells with respect to
membrane phenotype and cytokine secretion ability, showed
no direct ex vivo cytolytic activity, we studied whether cytotoxic effectors could be generated from this population.
To this purpose, PBMCs from an HIV-1 seropositive individual were sorted into CD8+CD45RA+CD27+ and CD8+
CD45RA
CD27+ subsets. CTLs from this individual had
been previously found to recognize an HLA-B57 restricted
epitope of the HIV-1LAI RT aa 244-252 (Klein, M.R.,
unpublished results). Neither CD45RA+CD27+ nor
CD45RA
CD27+ cells showed direct ex vivo cytotoxicity as measured in a redirected kill assay as described above.
To determine the frequency of peptide-specific CTLp within
the distinct subsets, sorted cells were seeded in serial dilutions and stimulated with autologous B-LCL infected with
recombinant vaccinia virus expressing the HIV-1LAI RT gene. On day 15, effector cells were tested for cytotoxicity on
autologous B-LCL pulsed with the HIV-1LAI reverse transcriptase-derived peptide aa 244-252 (Fig. 7). Indeed, cytotoxic effector cells could be generated from the CD45RA
CD27+ subset, which had an enhanced frequency of peptide-specific CTLps (61/106 cells) as compared to the
CD45RA+CD27+ subset (15/106 cells).
CD45RA
Apart from their cytolytic capacities,
CD8+ cells have been shown to exert suppressor activity as
well as helper activity for Ig synthesis (43). Helper capacity
of CD8+ T cell subsets was tested in a CD3 mAb-driven T
cell-dependent B cell differentiation assay (44). When total
CD4+ and CD8+ cells were compared, helper activity was
preferentially found in the CD4+ fraction (Fig. 8 A). However, when CD8+ cells were further separated into the indicated subsets, it was found that the CD45RACD27+
cells were also able to support B cell differentiation, whereas both the CD45RA+CD27+ and the CD45RA+CD27
cells
showed no helper activity.
To test suppressor capacity, sorted CD8+ subsets were added to the autologous coculture of CD4+ and B cells (Fig. 8 B). In accordance with a previous study (43), total CD8+ T cells markedly reduced IgM and IgG synthesis in this system. When the CD8+ cells were further separated into the different subsets, suppressor activity was primarily found in the CD45RA+CD27+ fraction. This effect was not due to enhanced IL-4 consumption of CD8+CD45RA+ CD27+ cells, as addition of exogenous IL-4 did not overcome suppression of the Ig synthesis.
In summary, within the CD8+ compartment, helper and
suppressor activity could be ascribed to CD45RACD27+
and CD45RA+CD27+ T cells, respectively, whereas the
CD45RA+CD27
does not appear to directly influence B
cell differentiation.
In this study, we define discrete subsets (summarized in
Table 5) within the CD8+ T cell population based on expression patterns of CD45RA and CD27. First, similar to
the CD4+ T cell subpopulation, CD45RA+CD27+ T cells
are the only CD8+ lymphocytes present in cord blood, and
the analysis of phenotype and function showed that this
population primarily contains unprimed (naive) cells.
CD8+CD45RACD27+ lymphocytes are T cells that have
several features of antigen-experienced cells. In comparison
to phenotypic data obtained in the murine system, these
CD8+ cells most closely resemble memory-type cells, as
they express intermediate levels of CD11a/CD18 and
CD49d, but have no membrane expression of CD11b (9).
Indeed, this CD8+CD45RA
CD27+ population from an
HIV-1-infected individual contained, compared to CD8+
CD45RA+CD27+ T cells, an enhanced frequency of CTLp
specific for an HIV-1 RT-derived peptide. In line with
this, a study by Merkenschlager and Beverley (17) documented an elevated precursor frequency to recall antigens
within the CD8+CD45RA
CD45R0+ fraction. A second
population that has features of past antigen stimulation has a
CD8+CD45RA+CD27
phenotype. Not only with respect to cytolytic potential, but also regarding surface expression of high levels of CD11b and CD49d and absence
of the lymph node homing receptor CD62L, these cells
fulfill the criteria of CTL effector-type cells that have been
defined in murine models. Further characterization of the CD8+CD45RA+CD27
population allowed us to determine the functional abilities of human CTL-type effector
cells as they occur in vivo. The data show that (a) on a per
cell basis the vast majority of effector cells are able to produce both IFN-
and TNF-
, (b) these cells contain high
amounts of perforin and granzyme B, (c) Fas ligand mRNA
is abundantly expressed in freshly isolated effector cells, and
(d), that these cells exert potent cytotoxic activity without previous in vitro stimulation. Finally, a fourth population
constituted of CD45RA
CD27
T cells can be found in
the CD8+ fraction of all donors. The frequency of these
cells in healthy donors is very low (usually <4%) and therefore not accessible for assays that depend on vast quantities
of purified cells. However, based on membrane phenotype,
cytokine production pattern, and expression of perforin
and granzyme B, it is suggested that this population may
contain both memory- and effector-type cells.
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Based on the finding that the majority of CD8+CD45RA+
CD27 T cells lack CD28 expression, it was not surprising
to find that this subset shares a number of features that have
been previously reported for CD28
lymphocytes (15, 45,
46). Azuma et al. (15) showed that CD8+CD28
T cells
are able to lyse FcR-bearing targets cells in the presence of
CD3 mAb. Moreover, Berthou et al. recently reported that
most of the perforin containing CD8+ lymphocytes lack
the CD28 molecule (47). The current data confirm and extend these observations by showing that not only perforin but also granzyme B and Fas ligand are expressed within
this T cell subset, which is likely to contribute to the lysis
of target cells (39, 40). It is of interest to note that two major costimulatory receptors for T cell growth and cytokine
production, CD28 and CD27, are absent from this effector-type T cell. The absence of CD28 implies that this cell
type is unable to interact productively (in terms of induction of clonal expansion) with professional antigen-presenting cells. This inability is underscored by the observation
that, in marked contrast to both the CD45RA+CD27+ and
CD45RA
CD27+ subsets, CD45RA+CD27
T cells do
not proliferate in response to IL-12, one of the major T cell
stimulatory cytokines secreted by activated antigen-presenting cells (48). Finally, absence of CD27 on these cells also
precludes costimulatory interactions with other, activated
lymphocytes (49). Analysis of the proliferation requirements
showed that CD45RA+CD27
T cells responded poorly
to most stimuli, which is in agreement with functional
studies on the CD8+CD28
T cell subset (15, 46). However, CD45RA+CD27
T cells do respond to IL-2 and to
a lesser extent to IL-15, which suggests that in vivo this
subset might be influenced by Th-derived signals.
In mice, the expression of CD49d nicely separates naive
(low), memory (intermediate), and effector (high expression) T cells, and a study by Christensen et al. has shown
that CD49d plays a critical role in efficient homing of effector T cells during virus infections (13). Also, in humans
CD49d expression correlates well with the distinct CD8+
subsets, CD45RA+CD27 T cells being the ones with the
highest expression. Apart from CD49d, a
1 integrin interacting with vascular cellular adhesion molecule-1 on activated endothelium and also the high expression of the
2
integrins CD11a and CD11b (Table 1) will probably influence the homing properties of these effector-type cells,
which may be different from those of naive or memory
cells. Indeed, CD8+CD45RA+CD27
cells were conspicuously absent from tonsils, whereas CD45RA
CD27+ cells
could readily be demonstrated in this lymphoid tissue (Hamann, D., unpublished data). In line with this observation,
it was reported that CD11b+ cells are present in blood,
liver, and spleen, but absent from tonsil, lymph node, and
thymus (45).
Thus, it would seem that in response to antigen, two
types of primed CD8+ populations may develop. First, an
effector-type population with high levels of granzyme, perforin, and Fas ligand expression that exerts cytolytic activity, which has lost the ability for autocrine proliferation but
has retained the ability to synthesize IFN- and TNF-
,
two cytokines implicated in the neutralization of viruses. Second, a population of memory-type cells, which is not
cytolytic without further activation, produces a wide range
of cytokines and is able to provide helper activity for B cell
differentiation. Kinetic studies in mice showed that effector
T cells can be found relatively early after virus infection (8 d), and decline thereafter. In contrast, memory cells become prominent at a later point (>50 d), and it has been
suggested that effector cells seed the memory pool (9, 50).
However, the relation between these subpopulations in
humans is unclear at this moment. In major contrast to data obtained in the mouse system, effector-type T cell numbers
in humans are relatively stable over time (>18 months,
Rep, M.H.G., unpublished data). In addition, it has been
reported that CD8+CD45RA+CD11abright, CD11b+, and
CD28
T cells increase with age (45, 51, 52) and, in agreement with this (see Table 5 for phenotypic similarities), we
found a similar relationship between age and the percentage of CD8+CD45RA+CD27
T cells (Rep, M.H.G., unpublished data). The reason for the discrepancy in the kinetics of effector-type T cells between mouse and human is
not clear at this moment but it is possible that persistence of
antigen, cross-reactive epitopes (53), and/or stimulatory cytokines plays a role (54). It can be imagined that these
factors will more readily influence the maintenance of a
polyclonal effector T cell population in a natural environment than that of the monoclonal T cell population present
in laboratory animals housed under pathogen-free conditions. It is questionable, based on the in vitro experiments, whether effector T cells that are present in the human circulation have a sizable clonogenic potential. Still, it can be
envisaged that this type of cell contributes to the inhibition
of pathogen spread early in secondary infection through
immediate lysis of infected cells.
In agreement with recent data on CD28 subsets by Monteiro et al. (55), we found shortened telomers in CD8+
CD45RA+CD27 compared to CD8+CD45RA+CD27+
in some donors (Hamann, D., K.C. Wolthers, S.A. Otto,
P.A. Baars, F.M. Miedema, and R.A.W. van Lier, manuscript submitted for publication). This finding shows that
transition to the CD45RA+CD27
subset is accompanied
by cellular division. Cell cycle analysis showed that, analogous to the CD4+ population (23, 56), the CD8+ cells
that are in G2M can only be found in the minute fraction of CD8+CD45RAbrightCD45R0bright cells and not in CD45RA+CD45R0
CD27+ T cells (Baars, P.A., unpublished
data). This finding implicates that CD45RA+CD27
cells
could only develop via a CD45RA+CD45R0+ and a subsequent CD45RA
CD45R0+ stage. However, at this moment we cannot exclude that the transition from CD27+ to
CD27
takes place in compartments, such as solid tissue,
which are not readily available for cell-biological analysis.
Finally, similar to what has been described for CD28
(57)
and CD57+ (16, 37) T cells, we found a limited usage of
V
elements by CD8+CD45RA+CD27
cells (Hamann,
D., K.C. Wolthers, S.A. Otto, P.A. Baars, F.M. Miedema,
and R.A.W. van Lier, manuscript submitted for publication). These findings, combined with the data on telomeric
repeat length, infer that these circulating effector-type cells
in humans are generated through antigen-dependent differentiation and proliferation processes.
Analysis of naive, memory, and CTL effector-type CD8+
T cells seems relevant not only in patients with chronic viral infections such as CMV, hepatitis B, or HIV-1, but also
in patients actively immunized with tumor antigens and after having received an allotransplantation. Patients infected
with CMV have been shown to have an increased percentage of CD57+ cells within the CD8+ population that contains virus-specific CTLs and can be oligoclonal (16). In
HIV-1 infected individuals, an expanded CD28 population within the CD8+ T cell subset has been reported (46).
This population has been found to be primarily CD57+,
exerts potent cytolytic activity, and expresses perforin (46, 58). Interestingly, in contrast to healthy donors where
CD27
and CD28
cells form highly overlapping subsets,
a large population of CD45RA
CD28
cells that still express the CD27 antigen emerges during acute and persistent
viral infections (Roos, M.T.L., manuscript submitted for
publication), which may point to a sequential downregulation of costimulatory molecules during postthymic T cell
differentiation.
Based on the data presented in this paper, we conclude
that unidimensional analysis of CD8+ T cells, using either
CD45RA, CD27, or CD28 mAbs, underestimates the complexity of this subset in humans. Therefore, we propose that combined staining with CD27 and CD45RA mAbs
provides a tool not only for distinguishing unprimed
CD45RA+CD27+ from primed CD45RA cells, but also
for following the dynamics and expansion of a CD45RA+
CD27
cytotoxic effector cell population in CD8+ T cells
in different disease states.
Address correspondence to René A.W. van Lier, Department of Clinical Viro-Immunology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. Phone: 31-20-5123317; FAX: 31-20-5123310.
Received for publication 22 October 1996 and in revised form 15 July 1997.
D. Hamann was supported by the Human Capital and Mobility Programme of the European Community, and P.A. Baars and M.H.G. Rep were supported by the Dutch Society for Support of Research on Multiple Sclerosis.We would like to thank our colleagues Susanne Lens, Kiki Tesselaar, Wiebo van der Graaff, Marijke Roos, Peter Schellekens, and Frank Miedema for stimulating discussions and their help in preparing this manuscript.
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