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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 231-240
IMMUNOBIOLOGY
Human Tc1 and Tc2/Tc0 CD8 T-cell clones display distinct cell
surface and functional phenotypes
Milica Vukmanovic-Stejic,
Beejal Vyas,
Patricia Gorak-Stolinska,
Alistair Noble, and
David M. Kemeny
From the Department of Immunology, Guy's, King's, and St. Thomas'
School of Medicine, Rayne Institute, London, United Kingdom.
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Abstract |
It has recently become clear that distinct subsets of CD8 T cells,
analogous to their CD4 counterparts, exist in rodents and humans. To
examine functional differences between human CD8 T-cell subsets, we
generated Tc1, Tc2, and Tc0 T-cell clones from the peripheral blood of
healthy individuals. The majority of CD8 T-cell clones generated
displayed a classic Tc1 phenotype, but 10% to 20% secreted
interleukin (IL)-4 in addition to interferon- (Tc0 phenotype).
Generation of Tc2 clones was dependent on the use of anti-CD3 and
anti-CD28 as the primary stimulus. The cytokine profiles of established
clones remained susceptible to modification by the addition of IL-12
and IL-4. In addition, IL-12 enhanced and IL-4 inhibited the growth of
Tc1 but not Tc2/0 CD8 T-cell clones. Significant functional differences
were observed between the subsets. Tc2/0 clones expressed CD30 and CD40
ligand at a much higher level than Tc1 clones. Both Tc1 and Tc2/0
clones showed comparable cytotoxicity and produced similar levels of
perforin and Fas L. However, Tc2 clones were much more resistant to
activation-induced cell death and less susceptible to
apoptosis by direct Fas ligation. Moreover, Tc1 and Tc2 clones had
opposing effects on the development of CD4 effectors, promoting type 1 and type 2 responses, respectively. These data provide evidence for
profound differences between human CD8 T-cell subsets that may be
important in their functions as cytotoxic or immunoregulatory cells.
(Blood. 2000;95:231-240)
© 2000 by The American Society of Hematology.
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Introduction |
The importance of distinct subsets of CD4 T lymphocytes
in the etiology of a variety of immune-mediated diseases has become clear in the last 10 years. Two distinct populations of mouse T-helper
(Th) clones were first identified by Mosmann et al.1,2 Th1
cells secrete interleukin (IL)-2, interferon (IFN)-, and tumor
necrosis factor (TNF)- and are primarily involved in cell-mediated immune responses, whereas Th2 cells secrete IL-4, IL-5, IL-6, and IL-10
and fulfill an important role in humoral and allergic immune
responses.3 Human CD4 T-cell clones have similar, but not
identically restricted, cytokine profiles.4 Human Th1 and Th2 subsets are usually defined according to IFN-/IL-4 production because the synthesis of IL-2, IL-6, and IL-10 is not stringently restricted to a single subset. Although the expression of type 1 and
type 2 cytokines was initially considered to be mutually exclusive, T
cells expressing both type 1 and type 2 cytokines have been identified
during differentiation5,6 and among terminally
differentiated cells.7
Historically, CD8 T cells have been regarded as a homogeneous
population of cytotoxic cells producing only a limited number of
cytokines. More recently, it has become clear that CD8 T cells have the
potential to produce a much wider array of cytokines, and the existence
of distinct subsets of CD8 T cells that are similar to their CD4
counterparts has been established in the mouse,8-10
rat,11,12 and human.13,14 Analogous to the
Th1/Th2 terminology, these subsets were termed Tc1 and Tc2.
The presence of different cytokines in the T-cell microenvironment
appears to be the major factor that determines the differentiation of
precursor T cells. IL-12, transforming growth factor-, and IFN-
induce differentiation of naive CD4 into Th1 but not Th2 cells, whereas
IL-4 is essential for the differentiation of naive CD4 cells into Th2
cells and inhibits the development of Th1 cells.15-19 IL-6
also has been implicated in Th2 differentiation.20 IFN- and IL-4 also induce the differentiation of naive CD8 cells into type 1 and type 2, respectively, whereas IL-12 promotes the development of Tc1
cells.8,18,21
After differentiation, effector T cells show a stable cytokine pattern
and rarely, if ever, switch to the opposite phenotype or revert to
their precursor state. However, some modifications have been described:
Th0 cells can shift toward Th1 or Th2 in response to
cytokines,22 and human Th2 clones can transiently express
IFN- after IL-12 treatment.23-25 Th1 cells are reported to be irreversible, although mouse Th1 cells can produce IL-4 when
stimulated in the presence of IL-4.24 IL-4 also inhibits the ability of differentiated Tc1 cells to synthesize
IL-226 and down-regulates other functions, including
cytokine synthesis, proliferation, and long-term
cytotoxicity.26,27
In addition to their classic role in the killing of infected cells, CD8
T cells play a role in the regulation of activation and differentiation
of CD4 cells. This regulation could be mediated through secreted
products (cytokines, chemokines) or by cell-cell interaction. CD8 T
cells can alter the balance of Th1/Th2 responses in
vivo28,29 by influencing the development of IL-4- or
IFN--secreting CD4 cells.30,31 In addition, CD8 T cells
appear to play a role in the development of CD4 perforin-mediated
cytotoxicity30 and also have been reported to suppress CD4
proliferative responses32,33 through the inhibition of
costimulatory interactions. CD8 cells are also capable of influencing
other components of the immune response, such as recruitment of
eosinophils into the lungs during respiratory syncytial virus infection
or allergic asthma,31,34,35 activation of
macrophages,36 and regulation of antibody production by B
cells.10,12,37-39
We have generated human CD8 T-cell clones which, according to cytokine
production, can be classified into 3 subsets: Tc1 clones producing
IFN- and a variety of other cytokines but no IL-4, Tc0 clones
producing IL-4 in addition to IFN- and other cytokines, and Tc2
clones producing IL-4 but no IFN-. We investigated the effects of
IL-4 and IL-12 on the proliferation and cytokine production of these
subsets. In addition, the 3 subsets were compared for the
expression of surface markers, cytotoxic capacity, resistance to
activation-induced cell death (AICD), and their ability to direct the
development of CD4 effectors. These data represent the first systematic
analysis of the functional heterogeneity of circulating human CD8 T cells.
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Materials and methods |
Reagents
Lymphoprep was purchased from Nycomed (Birmingham, UK), CD8 and CD4
magnetic beads and Detach-a-bead from Dynal (Wirral, UK), and Hanks
balanced salt solution (HBSS), fetal calf serum (FCS), RPMI 1640, Iscove's Modified Dulbecco's Medium (IMDM),
L-glutamine, and 2-mercaptoethanol from Gibco
BRL (Paisley, UK). Recombinant human IL-2 was purchased from Chiron
(Harefield, UK), IL-4 from Serotec (Oxford, UK), and IL-12 from R&D
systems (Abingdon, UK). Fluorescein isothiocyanate (FITC)- and
phycoerythrin (PE)-labeled antibodies to various cell surface markers
were purchased from Becton Dickinson (Oxford, UK), except for
antibodies to perforin and Fas ligand, which were from Alexis
(Nottingham, UK). All antibody pairs and recombinant cytokines for
enzyme-linked immunosorbent assay (ELISA) were from Cambridge
Biosciences (Cambridge, UK). Anti-CD3 and anti-Fas L (NOK1,2)
monoclonal antibodies (MoAbs) were from Becton Dickinson
(Oxford, UK). Anti-CD28 antibody was from CLB (Amsterdam,
The Netherlands), and anti-Fas (CH11) was from Upstate Biotechnology
(Lake Placid, NY). Antilymphocyte function-associated antigen-1 (LFA-1) MoAb was from Alexis (Nottingham, UK).
Cytotoxicity detection kit lactate dehydrogenase (LDH) was
supplied by Boehringer Mannheim (Lewes, UK), and OKT3 hybridoma was
purchased from European Collection of Animal Cell Cultures (ECACC)
(Salisbury, UK). Staphylococcal enterotoxin B (SEB) was
obtained from Toxin Technologies (Sarasota, FL). All other reagents
were from Sigma-Aldrich Ltd. (Poole, UK).
Purification of CD8 T cells
Peripheral blood (20 to 60 mL) was collected from healthy volunteers
by venipuncture into sodium citrate and diluted 1:1 with HBSS, layered
onto Lymphoprep, and centrifuged at 800g for 20 minutes at
18°C. The interface containing peripheral blood mononuclear cells
(PBMCs) was then collected and washed with HBSS. PBMCs were resuspended
either at 1 × 106/mL in complete media to be used
as feeders or in RPMI 1640/2% FCS at 1 to
5 × 107/mL for the purification of CD8 cells.
CD8 cells were isolated by positive selection using anti-CD8 magnetic
beads, which were added at a 3:1 bead to target cell ratio and mixed by
rotation for 60 minutes at 4°C. Bound CD8 cells were released by
addition of the Detach-a-bead antibody with rotation for 60 minutes at
room temperature. Purified CD8 T cells were then incubated with
anti-CD4 magnetic beads in the same way as described earlier to remove
contaminating CD4 or CD4/CD8 double-positive cells. The purity of the
isolated CD8 T cells was determined by FACScan (Becton Dickinson,
Oxford, UK) and was consistently found to be > 99% pure, as
assessed by staining with PE-labeled anti-CD8 and FITC-labeled
anti-CD4.
Generation of CD8 T-cell clones
Aliquots of purified CD8 cells were prepared at concentrations from
1 × 104/mL to 1 × 101/mL in
complete medium (RPMI 1640, 10% normal human AB serum [HS], 1%
sodium pyruvate, 1% nonessential amino acids, 50 mmol/L
2-mercaptoethanol). Purified CD8 cells were seeded in Terasaki plates
at concentrations of 0.3, 1, 3, 10, and 30 cells/well with
104 irradiated (4000 rad) autologous feeders in a total
volume of 20 µL of complete medium supplemented with
phytohemagglutinin (PHA) at 5 µg/mL and recombinant
human IL-2 at 50 U/mL. On day 5, the cultures were fed with 10 µL of
complete medium and on day 10, the plates were scored for cell growth
and the cloning frequency determined as described by
Taswell.40 Wells deemed to be positive were transferred to
96-well microtiter plates and were restimulated in 200 µL of complete
medium with PHA (5 µg/mL final concentration), IL-2 (50 U/mL final
concentration), and 1 × 105/well irradiated (4000 rad) autologous feeders as before. Every 3 to 4 days, half the medium
was replaced with fresh complete medium supplemented with IL-2 (20 U/mL). Every 14 days, clones were restimulated with PHA and irradiated
feeder cells as before. In some experiments, to determine the effect of
different cytokines on the cloning frequencies and cytokine profiles,
we generated CD8 clones and cultured them in complete medium
supplemented with IL-4 (100 U/mL), IL-2 (50 U/mL) + IL-4 (100 U/mL), or
IL-2 (50 U/mL) + IL-12 (100 U/mL), as described earlier.
Alternatively, purified CD8 cells were first stimulated in bulk
cultures with either plate-bound anti-CD3 and anti-CD28 or anti-CD28
MoAb alone in complete media supplemented with IL-2 (25 U/mL) and IL-4
(50 U/mL). Twenty-four-well plates were coated with phosphate-buffered
saline (PBS) containing 5 µg/mL CD3 and 2 µg/mL CD28 (200 µL/well) overnight at 4°C. After 6 days, CD8 cells were
washed, counted, and plated out at limiting dilution and
stimulated with PHA, IL-2, and autologous feeders, as before.
Flow cytometric analysis
Clones were analyzed for cell surface marker expression using FITC-
and PE-conjugated antibodies to CD3, CD4, CD8, CD28, CD30, and CD40
ligand. Cells were used 14 days after PHA restimulation with and
without phorbol myristate acetate (PMA) and ionomycin treatment (18 hours). Perforin was detected by intracellular staining. Briefly, PMA- and ionomycin-stimulated cells were fixed with 4% formaldehyde and then permeabilized using Becton Dickinson
permeabilization solution before incubation with the labeled antibody.
Fas ligand expression was determined on the surface 6 hours after
stimulation, and also in the presence of Brefeldin A (protein secretion
inhibitor) by intracellular staining, or in the presence of
metalloprotease inhibitor (KB8301) 5 hours after anti-CD3 stimulation.
Data were acquired and analyzed on Becton Dickinson FACSCalibur using
Cellquest software (Becton Dickinson, Oxford, UK).
Cytokine analysis by ELISA
On day 14 of the cycle, clones (1 × 106/well)
were stimulated in 2 mL of complete medium with ionomycin (400 ng/mL)
and PMA (10 ng/mL) for 24 hours. Cell-free supernatants were collected after 24 hours and stored at  20°C. The quantitative
measurement of a range of cytokines was performed using commercially
available ELISA antibody pairs according to the manufacturer's
instructions. Cytokine levels were calculated by reference to standard
curves constructed using recombinant cytokines calibrated against
Quantikine ELISA kit standards (R&D Systems, Abingdon, UK).
Intracellular cytokine staining
Cells were stimulated either with PMA and ionomycin or with
plate-bound anti-CD3 and anti-CD28 MoAbs in the presence of Brefeldin A
at 5 µg/mL. A portion of cells was left unstimulated as a control. After 6 or 18 hours, the cells were washed in PBS/0.2% bovine serum
albumin and fixed in 500 µL of 4% formaldehyde for 20 minutes. After
washing, cells were permeabilized using 250 µL of permeabilization solution (Becton Dickinson) for 10 minutes, incubated with the appropriate antibodies for 30 minutes at 4°C, washed, and fixed with 1% paraformaldehyde.
Cytotoxic assay
Day-14 clones were stimulated using plate-bound anti-CD3 (OKT3
supernatant) overnight. Cells were washed, counted, and adjusted to a
range of concentrations in fresh RPMI with 2% HS. Target cells (OKT3
hybridoma cell line, 2 × 104/well) were cocultured
with effector cells at a range of effector to target ratios (0.3:1 to
50:1) in round-bottom tissue culture plates in 200 µL of RPMI/2% HS.
Supernatants were collected after 4 hours of culture. We used 1%
Triton X to determine maximum lysis, and target cells in medium alone
to determine spontaneous lysis. Spontaneous LDH release by effector
cells also was determined. The levels of LDH were determined using a
Boehringer Mannheim cytotoxicity detection kit according to the
manufacturer's instructions. Percentage lysis was calculated according
to a modified standard formula: (OD [optical density] experimental OD spontaneous targets OD spontaneous effectors)/(OD
maximum OD spontaneous targets) × 100.
Apoptosis induction
Cells (clones at day 14 of the cycle) were purified over Lymphoprep
to remove debris and dead cells. Viable cells were diluted to 1 to
5 × 105 cells/well and stimulated with immobilized
anti-CD3 for 4 to 6 hours. Control cells were left unstimulated. In
some experiments, anti-Fas ligand antibodies (NOK1 and NOK2 at 10 µg/mL) were added to the cultures to determine the involvement of
Fas-Fas ligand interaction in the AICD. In other experiments,
anti-LFA-1 antibody was added (at 1 or 10 µg/mL) to investigate the
importance of adhesion for the induction of AICD in Tc1 clones. After
treatment, the cells were washed in PBS, resuspended in 200 µL of
1× binding buffer (10 mmol/L HEPES/NaOH, pH 7.4, 140 mmol/L NaCl,
2 mmol/L CaCl2), and incubated for 15 minutes in the dark
at room temperature with 2.5 µL annexin V-FITC and 10 µL propidium
iodide (50 µg/mL). Then 300 µL of binding buffer was added to each
tube, and samples were acquired immediately (within 1 hour). Annexin
V-positive, propidium iodide-negative cells were considered
apoptotic. To measure direct anti-Fas induction of apoptosis, we
incubated clones with anti-Fas antibody (CH11 at 1 µg/mL) for 4 hours
at 37°C in 5% CO2 and stained them as described.
Control tubes contained irrelevant IgM antibody.
The effect of IL-4 and IL-12 on proliferation and cytokine
production of Tc1 and Tc0/2 CD8 T-cell clones
We also investigated the effect of different cytokines on
proliferation and cytokine profiles of established CD8 T-cell clones. On day 14 of the cycle, cloned cells were washed and stimulated (1 × 105/well) in 200 µL of complete medium with
PMA (10 ng/mL) and ionomycin (400 ng/mL) in the presence of IL-12 or
IL-4 (0-10 000 U/mL). On day 4, the cells were washed and restimulated
with PMA and ionomycin. After 24 hours, supernatants were collected for
cytokine analysis, and the cells were incubated for another 6 hours in the presence of 3H-thymidine (0.5 µCi/well) and then
harvested. Growth was determined by tritium incorporation, which was
measured using a Canberra Packard Matrix 96 counter (Pangbourne,
Berkshire, UK) and expressed as counts per minute (cpm).
Effects of CD8 clones on CD4 differentiation
CD8 clones were cocultured with autologous CD4 T cells in the
presence of antigen-presenting cells (APC). To ensure
interaction between CD4, CD8, and APC's superantigen, SEB was used as
a stimulus. First, CD8 clones (Tc1 and Tc2) reactive to SEB were
identified. These clones were cocultured with autologous PBMCs depleted
of CD8 cells, at varying ratios in the presence of SEB at 2.5 µg/mL. Two different approaches were used. Short-term cocultures (6 days) were
followed by overnight PMA/ionomycin stimulation in the presence of
Brefeldin A and staining with CD3 APC, CD8 peridinin chloropyll protein
(PerCP), IL-4 PE, and IFN- FITC. By gating on
CD3+CD8 cells, cytokine production of CD4
effector cells could be examined. In the second approach, CD8 clones
and PBMCs depleted of CD8 were cocultured for 14 days at a ratio of
1:1. On day 14, CD4 cells were positively selected using
magnetic beads, as described previously, and the resulting population
was > 99% pure as assessed by CD4/CD8 staining. CD4 cells were
plated at limiting dilution at 0.3 cells/well. The cloning procedure
was followed as described for CD8 clones, and after 2 stimulation
cycles, newly generated CD4 clones were screened for cytokine
production after 18 hours of PMA/ionomycin stimulation.
Data analysis
Cloning frequencies were calculated using weighted mean statistics
as described by Taswell.40 All data are expressed as mean ± SEM. Student's t test and Wilcoxon
signed-rank test were used to assess statistical significance.
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Results |
Generation of distinct subsets of CD8 T-cell clones
Human CD8 clones were generated from highly purified (> 99%)
peripheral blood CD8 T cells using PHA, autologous feeders, and IL-2.
The cloning frequency varied from 0.23 to 0.61 among different experiments and donors. The cytokine profiles were determined by the
concentration of cytokines in the clone culture supernatants 24 hours
after PMA and ionomycin stimulation. Based on IL-4 and IFN-
production, it is possible to group them into 2 subsets: 1 producing
IFN- and no detectable IL-4 (Tc1) and the other producing both
IFN- and IL-4 (Tc0). Of more than 500 clones produced from 5 donors
using this protocol, none failed to secrete IFN-. Furthermore, no
significant difference in the amount of IFN- produced by the 2 subsets was observed17 (17.1 ± 9.4 ng/mL for Tc0 as
compared with 20.5 ± 10 ng/mL for Tc1). The majority (80% to
90%) of the clones produced were Tc1, with approximately 10% to 20%
of clones showing a Tc0 cytokine profile. IL-5 and IL-13 were
predominantly produced by Tc0 clones. Although some Tc1 clones produced
IL-5 (57%) and IL-13 (52%), this was usually at a low level
(mean ± SEM IL-5 production for Tc1 clones: 1302 ± 417
pg/mL compared with 6318.9 ± 1817 pg/mL for Tc0,
P < .02; and 1462 ± 557 pg/mL compared with
4298 ± 769.95 pg/mL for IL-13, P < .005).
Production of other cytokines such as IL-2, granulocyte macrophage
colony-stimulating factor, IL-6, IL-10, and TNF- was not restricted
to a particular subset and was common to most clones. Similar cytokine
profiles were observed in 3 separate cloning experiments from different
donors. Using a modified protocol, a small number of Tc2 clones were
generated from the same donor. Stimulation with anti-CD3 plus anti-CD28
or anti-CD28 alone for 6 days in the presence of IL-2 and IL-4,
followed by a standard limiting dilution procedure, resulted in a small
number of clones (10% to 13%) that were classified as type 2. These
clones either did not produce any detectable IFN-, or IFN-
production was < 5 ng/mL. Cytokine profiles of representative Tc1,
Tc2, and Tc0 clones are shown in Figure 1.
Tc0 clones appear to include cells that produce either IL-4 or IFN-,
or both. This has been described previously in Th0 clones41
and reflects the independent nature of cytokine regulation. Phenotypic
studies were subsequently performed using clones from a single donor.
In our hands, Tc2 clones were very similar to Tc0 clones (in terms of
characteristics and function) and were, in some experiments, grouped
together for further analysis.
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| Fig 1.
Cytokine profiles of representative Tc1, Tc2, and Tc0
clones.
Clones were stimulated with plate-bound anti-CD3 and anti-CD28 MoAbs
for 18 hours in the presence of Brefeldin A. Cells were washed, fixed,
permeabilized, and incubated with anti-IFN- FITC and anti-IL-4 PE
antibodies. Quadrants were set with reference to an unstimulated
control. Stimulated cells stained with isotype control antibodies gave
similar profiles. Percentages of cells positive for the particular
cytokine are indicated.
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IL-12 and IL-4 influence the cytokine profile of CD8 clones
generated
To assess the effects of IL-4 and IL-12 on the cytokine profile of
the clones generated, we conducted parallel cloning experiments with
different cytokine combinations (Table 1).
In the presence of IL-4, both with and without IL-2, the number of
IL-4-secreting clones was increased from 15% to 74% and 70%,
respectively. The proportion of IL-5-producing clones was not
significantly changed (66% in IL-2, 77% in IL-4, 53% in IL-2 + IL-4). IL-4 did not inhibit the generation of IFN--producing clones
and, using this method, none were generated that did not produce
IFN- (Tc2). Mean IFN-, IL-4, and IL-5 production was largely
unaffected. Levels of IL-10 appeared reduced, but this did not reach
statistical significance (P < .09).
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Table 1.
The effect of cytokines (A) and different stimuli (B) on
the cloning frequency and the cytokine profile of human CD8 T-cell
clones
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The presence of IL-12 resulted in a decrease in the proportion of
IL-4-secreting clones from 25% to 12% and, more dramatically, in
IL-5-producing clones from 80% to 22% (Table 1). The percentage of
IL-10-producing clones increased from 19% to 55%, and mean IFN-
production was also significantly increased in IL-12-generated clones
(P < .0001) as compared with clones generated in the
presence of IL-2 alone. The mean IL-4 production was decreased
5-fold (P < .05), and IL-5 production more than 20-fold
(P < .0002).
Tc1 and Tc2 clones differ in their surface marker expression but
show similar levels of cytotoxicity
Cell surface CD8, CD28, CD30, and CD40L expression and intracellular
perforin were determined by flow cytometry 18 hours after stimulation
of clones with PMA and ionomycin (Figure
2). Perforin levels increased from 6 to 24 hours after stimulation, at which point all cells appeared positive. No
difference was observed in the levels of perforin between Tc1 and
Tc2/Tc0 clones at either time. Significant differences were observed in
the levels of CD30 expression: 8 of 11 Tc0 and Tc2 clones showed a high
proportion of CD30-positive cells (40% to 60%), as compared with
< 10% CD30 expression by all the Tc1 clones. Striking differences
also were observed in the levels of CD40 ligand expression. The
majority of Tc2/0 clones tested (8/11) showed significant expression of CD40 ligand (20% to 70% positive cells). Only a low level of
expression was observed (< 10%) on the Tc1 clones. There were no
consistent differences in the levels of CD28 expression.
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| Fig 2.
Tc1 and Tc0 clones differ in their surface marker
expression.
Day-14 clones were stimulated with PMA and ionomycin for 24 hours and
stained using FITC- and PE-labeled antibodies. (A) The surface
phenotype of a representative Tc1 clone (left panel) and Tc2 clone
(right panel). The percentage of cells positive for a particular marker
is indicated. Tc0 clones were found to be very similar to Tc2 clones in
terms of surface markers (not shown). Fas L expression was measured 6 hours after stimulation (A) and 18 hours after stimulation in the
presence of protein secretion inhibitor (B). Perforin was measured by
intracellular staining. Solid lines indicate marker expression; broken
lines indicate isotype control staining.
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Six Tc1 and 6 Tc2/0 clones were further compared for their cytolytic
function. Nonspecific CD3-mediated lysis was measured using the OKT3
hybridoma as target cells at a range of effector to target ratios. All
clones tested showed similar levels of cytotoxicity, and no differences
were observed between Tc1 and Tc2/0 clones (Figure
3).
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| Fig 3.
Tc1 and Tc0 clones show comparable levels of
cytotoxicity.
Six Tc1 and Tc0/2 clones were stimulated with plate-bound anti-CD3 for
18 hours. Cells were washed, counted, and cocultured with OKT3 target
cells at different effector to target cell ratios. After 4 hours,
culture supernatants were collected and LDH levels were measured using
a cytotoxicity detection kit; 1% Triton X was used to determine
maximum lysis, and target cells in medium alone were used to determine
spontaneous lysis. Spontaneous LDH release by effector cells was also
determined. Color development was measured at 490 nm. Percentage lysis
was calculated according to a modified standard formula: (OD
experimental OD spontaneous targets OD spontaneous
effectors)/(OD maximum OD spontaneous targets) × 100.
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Tc1 and Tc2 clones are differentially susceptible to apoptosis
All Tc1 clones tested (> 6) were highly susceptible to AICD after
5 hours of stimulation with anti-CD3 (Figure
4). The percentage of apoptotic cells
induced in this way varied from 14% to 50%, with a mean of
30% ± 3.5%. In contrast, Tc2 clones were much more resistant to
apoptosis. Most Tc2 clones showed complete resistance, whereas others
showed low levels of apoptosis, with a mean of 12% ± 4.3%
(2 representative clones; Tc1 and Tc2 are shown in Figure
4). Anti-CD3-induced AICD was at least partially Fas-Fas L dependent
because a proportion of cells could be rescued by the addition of NOK2
antibody (anti-Fas L MoAb). Interestingly, both groups were
susceptible to induction of apoptosis by direct ligation with anti-Fas
MoAb (CH11), although type 2 clones were less so (mean apoptotic cells
33% ± 3.8% compared with 50% ± 3.6% for Tc1;
P < .02). This indicates that there may be differences in
Fas L expression or function between the subsets. Fas L expression was examined by standard surface staining after 6 hours of
PMA/ionomycin stimulation, under which conditions all clones (Tc1 and
Tc2/0) appeared negative. We assumed that the Fas L was being cleaved and lost from the surface, so we investigated the expression of Fas L after stimulation in the presence of a protein secretion inhibitor (Brefeldin A). Under these conditions, Fas L was readily detectable, but no differences were observed between the subsets (Figure 2B). Furthermore, levels of Fas L expression were the same
after stimulation in the presence of a metalloprotease inhibitor, KB8301, known to prevent the cleavage of Fas L from the surface (data
not shown). This suggests that differences in Fas L function rather
than levels of expression cause differential susceptibility to AICD.
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| Fig 4.
Tc1 and Tc2 clones show differential susceptibility to
apoptosis.
AICD was induced in a number of Tc1 and Tc2 clones by 5 hours of
stimulation with immobilized anti-CD3. After treatment, the cells were
stained with annexin V-FITC and propidium iodide and analyzed as
described in Materials and Methods. The percentage of apoptotic cells
is shown in the lower right quadrants. Induction of apoptosis was
partially blocked by anti-Fas L antibody (NOK2). Apoptosis was also
induced by direct Fas ligation (CH11, anti-Fas MoAb). Two
representative clones are shown.
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The importance of the interaction between LFA-1 and intercellular
adhesion molecule (ICAM)-1 in Th1-mediated B-cell apoptosis has been
demonstrated.42 We investigated whether blocking the homotypic interaction between LFA-1 and ICAM-1 by the addition of
anti-LFA-1 antibody could partially or completely rescue Tc1 clones by
interfering with the induction of AICD. However, we found that blocking
the LFA-1-ICAM-1 interaction had no effect (mean apoptotic cells
40 ± 15 vs 46.3 ± 11 without or with anti-LFA-1, respectively). The anti-LFA-1 antibody was shown to be effective in
blocking a 2-way mixed lymphocyte reaction, indicating that it
effectively blocked cell adhesion at the concentrations used (not shown).
IL-12 increases and IL-4 inhibits proliferation of Tc1 but not Tc0/2
CD8 T-cell clones
The effects of IL-4 and IL-12 on the growth of established Tc1 and
Tc0 CD8 T-cell clones were investigated. Tc1 (n = 8) and Tc0/2
(n = 8) CD8 T-cell clones were stimulated with PMA and ionomycin in
medium containing 0 to 10 000 U/mL of IL-12 or IL-4, as described in
Materials and Methods. IL-12 increased the proliferation of all Tc1
clones (mean increase 117.5% ± 30%) at the highest
concentration of IL-12 (Figure 5).
Conversely, Tc0/2 clones were relatively unaffected by IL-12 (mean
4.0% ± 4.7%). IL-4 had an opposite, although much weaker,
effect. All 8 Tc1 clones tested showed a decrease in proliferation
(mean 38% ± 4.2% at the highest concentration of IL-4
tested), whereas Tc0/2 clones were unaffected, with a mean change in
proliferation of +0.1% ± 5.8% (Figure 5).
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| Fig 5.
The effects of IL-12 and IL-4 on proliferation and
cytokine production of Tc1 and Tc0/2 CD8 T-cell clones.
Cloned cells at 1 × 105/well were incubated with
PMA (10 ng/mL) and ionomycin (400 ng/mL) in the presence of IL-12 or
IL-4 (0 to 10 000 U/mL). All experiments were performed in triplicate.
On day 4, the cells were washed and restimulated with PMA and ionomycin
in the absence of cytokines. At 24 hours, supernatants were collected
and the cells were incubated in the presence of
3H-thymidine (0.5 µCi/well) for 6 hours and then
harvested. Mean cpm values for 8 Tc1 and 8 Tc0/2 clones are shown.
Levels of IFN-, IL-4, IL-5, and IL-10 in the supernatants were
measured by ELISA (as described in Materials and Methods). All results
are expressed as mean ± SEM for 8 clones under control conditions
(filled bars) and at 10 000 U/mL IL-4 or IL-12 (shaded bars).
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IL-12 and IL-4 modulate the cytokine production of Tc1 and
Tc0/2 CD8 T-cell clones
Cytokine production by 8 Tc1 and Tc0/2 clones stimulated with PMA
and ionomycin in medium containing 0 to 10 000 U/mL of IL-12 or IL-4
was examined. IL-12 had a strong modulatory effect on the cytokine
profiles of both Tc1 and Tc2/0 clones. In all of the clones tested,
IL-12 significantly increased IFN- production, in some cases by more
than 4-fold. Interestingly, the most dramatic increases were observed
in some of the Tc0/2 clones, whereas in Tc1 clones, the increase ranged
from 35% to 188% (Figure 5), which could largely be accounted for by
the increase in proliferation of these cells. IL-12 also had a dramatic
effect on IL-10 production. The majority of Tc1 clones (7/8) and half
of the Tc0/2 clones (4/8) produced undetectable amounts of IL-10 in the
absence of IL-12. However, in the presence of IL-12, all but 4 of these
clones showed significant production of IL-10 ranging from 100 to 4000 pg/mL. IL-4 suppressed IFN- production in Tc1 and to a lesser extent
in Tc2/0 clones, although for Tc1 clones, the levels of suppression are
in keeping with the decrease in proliferation that was observed in
these conditions. IL-4 had no effect on IL-10 production by either Tc1
or Tc0/2 clones (Figure 5).
IL-12 significantly decreased IL-5 production by both Tc1 and Tc2/0
clones, whereas IL-4 production was decreased in some, but remained
constant in other, Tc2/0 clones. IL-4 increased IL-5 production by the
majority of Tc1 clones (Figure 5) in the range of 20% to 30% at the
highest IL-4 concentration; however, a few clones showed a more
substantial increase of 2- to 5-fold. Because IL-4 suppresses
proliferation of these clones, these increases may represent much
greater effects on a per-cell basis. The effect on Tc2/0 clones was
variable, with only some clones showing a significant increase in IL-5
production. The addition of IL-4 enhanced subsequent IL-4 production in
most Tc0/2 clones, although only 3 of 8 showed a significant increase
(2- to 4-fold). None of the Tc1 clones could be switched to produce
IL-4.
Tc1 and Tc2 clones exert opposite effects on CD4
differentiation
The effect of Tc1 and Tc2 clones on the differentiation of CD4
effectors was studied in a series of coculture experiments involving
autologous CD8-depleted PBMCs and Tc1 or Tc2 clones stimulated with
SEB. In short-term (6 day) coculture experiments, Tc1 clones strongly
promoted IFN- synthesis and resulted in predominantly Th1 effectors.
Tc2 clones had the opposite effect, enhancing IL-4 production and
down-regulating IFN-, resulting in predominantly type 2 effectors
(Figure 6).
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| Fig 6.
Tc1 and Tc2 clones differentially regulate Th1/Th2
development.
Tc1 ( ) and Tc2 ( ) clones were cocultured with autologous PBMCs
previously depleted of CD8 cells (CD4 + APC) at varying ratios and in
the presence of SEB at 2.5 µg/mL. After 6 days, cultures were
stimulated overnight with PMA/ionomycin in the presence of Brefeldin A
and stained with CD3 APC, CD8 PerCP, IL-4 PE, and IFN- FITC. By
gating on CD3+CD8cells, cytokine
production of CD4 effector cells could be examined. Results are
expressed as percentage of CD4 cells positive for IL-4 or IFN-.
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We also cocultured Tc2 and Tc1 clones with CD8-depleted PBMCs in the
presence of SEB for 14 days, positively selected CD4 cells, and
generated CD4 clones using the PHA/IL-2 protocol. After 2 rounds of
mitogen stimulation, clones were screened for cytokine production.
Significant differences were observed in the levels of secreted
cytokines and in the subset distribution of the generated clones.
Coculture with Tc2 clones resulted in a greater proportion of Th2
clones (from 17% to 43%) and prevented the generation of Th1 clones.
Conversely, coculture with Tc1 cells resulted in an increased
proportion of Th1 (from 13% to 50%) and decreased the proportion of
Th2 clones (from 17% to 6%) (Table 2).
Mean cytokine production of CD4 clones was similarly affected. Clones
generated in the presence of Tc1 cells produced almost 5 times more
IFN- and half the IL-4, whereas clones from Tc2 cocultures produced approximately half the IFN- and more IL-4 and IL-5 than CD4 clones generated from control cultures. Interestingly, coculture with either
type 1 or type 2 CD8 clones decreased the subsequent cloning efficiency
of the CD4 clones (Table 2). No differences were observed in levels of
proliferation or the numbers of CD4 cells recovered in mixed cultures
compared with controls.
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Table 2.
The effect of coculture of CD4 and APC with Tc1 and Tc2
CD8 clones on subsequent cloning frequency, subset distribution, and
average levels of cytokine production of CD4 clones
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Discussion |
The existence of distinct cytokine-producing subsets within the CD8
T-cell population is becoming increasingly accepted. However, little is
known about the immunobiology of these subsets. We have generated a
large number of CD8 clones that can be defined as Tc1 (IFN-, no
IL-4), Tc0 (IFN-, IL-4), or Tc2 (no IFN-, IL-4). The majority of
human CD8 T-cell clones described to date can be classified as Tc1,
although CD8 clones secreting IL-4 have been generated from patients
with lepromatous leprosy,13 cutaneous leishmaniasis,41 HIV,14 and
periodontitis,42 and from the peritoneum of healthy
individuals.43 CD8 T cells that secrete IL-4 and IL-5 are
also present in the lung,44,45 especially in asthmatic
patients.35 The method for generating mitogen-driven CD8
clones from the peripheral blood of healthy donors used in the study
reported here resulted in a majority of clones with a Tc1 cytokine
profile and a minority (10% to 20%) with a Tc0 profile. A small
number of Tc2 clones have been established after a modification of
stimulation conditions, suggesting that the predominance of the type 1 and lack of type 2 phenotype may be, at least in part, an artifact of
the in vitro culture.
The importance of the microenvironment in the development of different
subsets is well documented. It is possible that Tc2 CD8 cells exist in
significant numbers only in certain disease states or are confined to
particular immune compartments. However, our data show that CD8 cells
with the potential to become Tc2 exist, albeit at a low frequency, in
the peripheral blood of normal individuals. As for CD4 cells, CD8 cells
are susceptible to the polarizing effects of IL-4 and IL-12, which
promote type 2/0 and type 1, respectively. However, the majority of CD8
T-cell clones generated from peripheral blood develop from
memory/effector cells, and although polarizing cytokines do influence
the proportion of Tc0/Tc2 clones, they are unable to completely
redirect their development. Medical history and atopic status are
likely to influence the proportion of Tc0/Tc2 clones from each individual.
The effects of cytokines such as IL-4, IFN-, and IL-12 on the
proliferation and cytokine production of established CD8 T-cell populations or clones have not been extensively studied previously. We
observed that IL-12 strongly promoted the proliferation of Tc1 clones,
whereas IL-4 suppressed growth of the clones. The Tc0 and Tc2 clones
were unaffected by either cytokine. We chose PMA and ionomycin
stimulation 14 days after the addition of feeders to ensure that the
response was accessory cell-independent and that only direct effects
of cytokines on CD8 T cells were assessed. Our data clearly demonstrate
that cytokines regulate CD8 responses not only at the level of
differentiation, but also at the level of expansion (proliferation) and
cytokine production of established effector cells. These effects could
serve to maintain and fine-tune polarized CD8 effector responses during
prolonged immune activation.
The data currently available on the specific cell surface phenotypes
associated with different CD8 T-cell subsets are somewhat limited. CD30
has been implicated as a marker for Tc0 and Tc2 CD8 T cells in both
healthy and diseased subjects.46,47 Our findings are in
agreement with these reports because significant expression of CD30 as
well as CD40L was restricted to the Tc2/0 subset. The functional
significance of these costimulatory molecules is, as yet, unclear.
Recent reports have suggested that CD30 may be involved in the
induction of cell death.48-50 The proposed CD30-CD30L pathway appears independent of the Fas-Fas L and TNF pathways and
could play an important role in the deletion of T cells after the resolution of an immune response. Differential expression of CD30
on distinct subsets may serve to preferentially delete or maintain a
specific population, thus influencing the memory T-cell repertoire.
Expression of CD40L coupled with IL-4 production could identify a
subset of cells capable of interacting with and providing help for B
cells,10,51 although this type of interaction may be
compromised by their capacity to kill APC. It is possible that CD40L-expressing Tc2 clones could interact with APC,
inducing IL-12 secretion by macrophages and dendritic
cells,52,53 with subsequent IFN-
production.54 This could serve as a control mechanism for
dampening type 2-mediated immunopathology.
For Th1 and Th2 CD4 T-cell subsets, different cytokine profiles are
closely associated with specific functions; however, such a correlation
has yet to be defined for CD8 T cells. Although mouse Tc1 and Tc2
clones are both reportedly cytotoxic,8,9,55 it appears that
the mechanisms they use for killing, and hence their effectiveness in
eliminating particular targets, may differ. The inability of Tc2 cells
to kill through a Fas-dependent pathway has been reported using
perforin knockout mice.56 Further evidence of reduced
cytolytic activity by Tc214,57 and even noncytolytic CD8
T cells has been described.58 All subsets of our
CD8 clones (Tc1, Tc0, and Tc2) showed efficient anti-CD3-mediated
killing of OKT3 targets and expressed comparable levels of perforin. A clear advantage for Tc2 cytotoxic cells could be their ability to kill
in a type 2 environment where proliferation of Tc1 cells would be
suppressed. This is in agreement with a report by Mosmann27 showing that IL-4 treatment affects proliferation, cytokine secretion, and long-term cytotoxicity of Tc1 cells. In addition, cytotoxic cells
secreting a wide array of cytokines (e.g., Tc2, Tc0) would be capable
of effective killing of infected cells while at the same time providing
additional signals to other cells to help amplify and direct the immune
response. Both Tc1 and Tc2 cells are able to mediate
inflammation,59 which again may be an advantage in
responses to different pathogens as inflammation could be coupled to
different effector functions.
Recent reports indicate that Th1 cells are more susceptible to
activation-induced apoptosis than Th2 cells.60-62 Whereas
some studies found that this was related to differences in the levels of Fas L expression,60,63 others found Fas L levels to be
comparable.61-64 We observed that Tc2 clones are much more
resistant to anti-CD3-mediated AICD. This process appears to be
mediated primarily through the Fas-Fas L interaction and can be partly
abrogated by anti-Fas L antibodies. In our hands, the subsets
expressed comparable levels of Fas ligand, suggesting that differences
in the susceptibility to AICD did not result from the differences in
Fas L expression. This raises the possibility that Fas L on Tc2 cells
is not functional or is only partially functional. Although we have not
investigated Fas-dependent cytotoxicity, this hypothesis is in
agreement with reports56 that Tc2 cells show only marginal
levels of Fas-mediated killing. Alternatively, other proteins that
regulate the Fas-mediated death pathway, such as Fas-associated protein
(FAP), Fas-associated death domain (FADD), and Fas-associated death
domainlike IL-1 converting enzyme (FLICE),61,62
may play a role in protecting Tc2 cells. However, because
anti-Fas L antibody provides only partial rescue in Tc1 cells, other
mechanisms in addition to Fas signaling are likely to be involved in
AICD. Tc2 resistance to AICD was probably not due to a lack of
cell-cell adhesion because blockade of the LFA-1-ICAM-1 interaction
failed to inhibit AICD in Tc1 cells. In certain situations, it may be
desirable to suppress (or terminate) a type 1 inflammatory response or
to promote a humoral immune response. Preferential depletion of Tc1 and
Th1 cells through AICD would be one possible mechanism. It is
interesting to note that the cloning procedure results in a majority of
Tc1 clones despite their increased propensity toward apoptosis. AICD results in death of a proportion of cells while the surviving cells go
on to proliferate.65 This would explain the
relatively low cloning frequency (usually < 50%), indicating that a
proportion of cells die during cloning.
The microenvironment plays a crucial role in directing the T-cell
response toward type 1 or type 2 cytokine secretion. This may be due to
the influence of secreted products (cytokines, chemokines) of resident
cells or APCs or to the nature of direct interactions, including
TcR-ligand affinity, ligand density, and costimulation. We wanted to
investigate the capability of CD8 Tc1 and Tc2 clones to influence the
development of CD4 effectors. Tc1 clones favored the development of CD4
effectors that were Th1-biased, whereas Tc2 clones had the opposite
effect. CD8-dependent suppression of Th2 responses and induction of
IFN- production have been reported in other systems and have mostly
been attributed to the high levels of IFN-.28,29,31,36
However, we have also demonstrated that Tc2 clones not only can promote
Th2 effectors but also can efficiently suppress the development of Th1
cells. The observed effects are consistent with cytokine-mediated
regulation, as Tc2 clones produced IL-4 and IL-10, and Tc1 clones
produced high levels of IFN-, which could act directly or indirectly
on developing CD4 cells. The importance of cell contact, either direct
or through the APC, could not be ruled out and is a matter for further
investigation. Interestingly, both Tc1 and Tc2 clones appeared to have
a suppressive effect on the subsequent generation of CD4 clones,
evident from significantly reduced cloning frequencies. Because
coculture with CD8 clones did not appear to affect the initial
proliferative response or the number of CD4 cells recovered, we
speculate that the CD8 clones, either through secretion of a cytokine
(possibly IL-10) or through inhibition of costimulation, impair the
long-term proliferative capacity of SEB-reactive CD4 cells.
The data described in this article clearly indicate that human CD8 T
cells represent a broad spectrum of cells with a wide array of
potential functions. It seems likely that Tc1 and Tc2 cells will
develop and function under the influence of a particular microenvironment, probably alongside Th1 and Th2 responses,
respectively. In addition to their role in protecting against infection
through the efficient killing of target cells, CD8 T cells themselves have the potential to play an important role in the regulation of the
immune response. They appear capable of influencing the activation or
suppression of other cell types including macrophages,36 eosinophils,31,34,35 and B cells,10,12,37-39,51
and, as we have demonstrated, they can direct the development of CD4 T cells into Th1 and Th2 effectors.
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Footnotes |
Submitted June 8, 1999; accepted August 28, 1999.
This work was supported by grants from the Medical Research
Council, Bayer-Yakuhin, and Glaxo-Wellcome.
Reprints: David M. Kemeny, Department of Immunology,
GKT School of Medicine, Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, United Kingdom; e-mail: david.kemeny{at}kcl.ac.uk.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
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Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL.
Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins.
J Immunol.
1986;136:2348-2357[Abstract].
2.
Cherwinski HM, Schumacher JH, Brown KD, Mosmann TR.
Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies.
J Exp Med.
1987;166:1229-1244[Abstract/Free Full Text].
3.
Mosmann TR, Coffman RL.
TH1 and TH2 ce |
Top
Abstract
Introduction