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*
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada; and
Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, but reduced IL-2Rß, expression. Furthermore, sorted
CD8+ IL-2Rhigh cells secreted less cytokines
than IL-2Rlow cells. These results suggest that the
presence of excessive IL-2 during the activation of naive
CD8+ T cells, while promoting cell cycling initially, may
compromise long-term immunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
TNF, and lymphotoxin) that aid their functions and activate cytotoxic
functions in macrophages (7).
Cytokine production by effector T cells is regulated both positively
and negatively at multiple levels during the development of an immune
response. IL-10 inhibits the cytokine secretion of Th1 cells
(8), whereas IFN- inhibits the proliferation of Th2
cells (9). Similarly, IL-4 inhibits cytokine synthesis of
CD8+ Tc1 cells and abrogates their ability to
proliferate and curtail tumor cell proliferation (10).
Besides cytokines, costimulatory molecules on the surface of APC also
modulate cytokine production by T cells. Stimulation of
CD4+ Th1 or CD8+ Tc1 cells
in the absence of costimulators (e.g., with fixed APC) induces a state
of anergy that is characterized by reduced cytokine synthesis and
reduced proliferation (11, 12). Rapid killing of APC by
the perforin- as well as the Fas-dependent pathway also curtails
cytokine production by CD8+ T cells
(13).
IL-2 is produced by T lymphocytes in response to an antigenic stimulation and is a central regulator of the immune response (14). Although IL-2 has been shown to act as a strong growth factor for T cells, it also renders activated T cells susceptible to activation-induced cell death (15). Effector CD4+ T cells cultured with high doses of IL-2 express reduced amounts of cytokines and have decreased inositol phosphate and diacylglycerol levels and impaired calcium-dependent signaling (16, 17). In the present study we present an interesting paradox between the levels of IL-2 in primary culture of naive T cells (CD8+), and subsequent functions of effectors. Although IL-2 deprivation caused apoptosis and little proliferation initially, the effectors generated under these conditions possessed optimal functions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice, 6–8 wk old, were obtained form Charles River Canada (St. Constant, Canada). C57BL/6-perforin-/-, C3H/HeJ-Fas ligand-/- (FasL-/-),3 and their controls were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in the animal facility of the Institute for Biological Sciences (National Research Council, Ottawa, Canada) in accordance with the guidelines of the Canadian Council on Animal Care.
Cell lines
The mouse B cell line M12.4.1, expressing both class I and class II MHC proteins (H-2d), was obtained from Dr. L. Glimcher (Department of Cancer Biology, Harvard Medical School, Boston, MA). The J774A.1 (mouse macrophage cell line expressing H-2d) cell line was obtained from American Type Culture Collection (Manassas, VA). L1210 cells (H-2d) and L1210 cells transfected with Fas (L1210Fas, H-2d) were provided by Dr. Chris Bleackley (Department of Biochemistry, University of Alberta, Edmonton, Canada). Cell lines were maintained in RPMI 1640 (Life Technologies, Grand Island, NY) plus 8% FBS (HyClone, Logan, UT).
T cell purification
C57BL/6 CD8+ T cells were enriched by removal of B cells, CD4+ T cells, and macrophages on mouse CD8 T cell columns (Cytovax, Edmonton, Canada) according to the manufacturers instructions. Briefly, splenocytes (100 x 106) were incubated with a rat anti-mouse CD4 Ab, loaded onto glass bead columns coated with sheep anti-rat Ig and sheep anti-mouse Ig, and eluted. Cells that passed through the column were 80–90% CD8+ as determined by flow cytometric analysis (EPICS XL, Coulter, Hialeah, FL). CD8+, CD44low (naive) T cells were further purified (>99%) by sorting on a cell sorter (EPICS Elite ESP; Coulter) after staining the cells with PE-labeled rat anti-mouse Ly2 (YTS 169.4) and FITC-labeled rat anti-mouse CD44 (Pgp-1, IM7.8.1) obtained from Cedarlane (Hornby, Canada).
For purification of CD4+ T cells, C57BL/6 splenocytes were incubated with a rat anti-mouse CD8 Ab (YTS 169.4) and processed using Cytovax columns as described above. CD4+, CD44low T cells were further purified ( >99%) by sorting as described above for CD8+ T cells. Cells were stained with PE-labeled rat anti-mouse CD4 (YTS 191.1, Cedarlane Laboratories) and FITC-labeled rat anti-mouse CD44.
Cell cultures
C57BL/6 T cells (H-2b, 1 x 103/well) were cultured with irradiated J774A.1 or M12.4.1 cells (H-2d, 1 x 104/well) in 96-well flat-bottom plates. Cells were cultured in 200 µl of RPMI plus 8% FBS in the presence of IL-12 (1 ng/ml), anti-IL-4 Ab (50 µg/ml), and various concentrations of IL-2 or IL-15. Medium was changed every third day. This stimulation resulted in differentiation and proliferation of effector T cells that remained >99% pure throughout the length of the culture. Between days 7–9 the effectors generated were harvested, washed, counted, and analyzed in various functional assays (in the absence of any cytokines) as described.
Cytokines, Abs, and reagents
Cytokines were obtained from ID Laboratories (London, Canada). Anti-IL-4 Ab was purified on a protein G column (Pharmacia, Uppsala, Sweden) from the supernatants of the 11B11 cell line (18). Con A, PMA, and calcium ionophore (A23187) were obtained from Sigma (St. Louis, MO). Tritiated thymidine (6.7 Ci/mmol) was obtained from ICN Pharmaceuticals (Costa Mesa, CA). Sodium chromate (51Cr; 250–500 mCi/mg) was obtained from Amersham (Oakville, Canada), Propidium iodide was obtained from Calbiochem (La Jolla, CA), and RNase was purchased from Boehringer Mannheim (Laval, Canada). Lympholyte M was obtained from Cedarlane.
Purified anti-mouse CD32/CD16 (FcII/III receptor, 2.4G2 Fc
block), biotin-labeled anti-mouse NK1.1 (PK136), biotin-labeled
anti-mouse CD28 (37.51), FITC-labeled anti-mouse IL-2R
(7D4), and anti-mouse IL-2Rß (TM-ß1) were obtained from
PharMingen (San Diego, CA). FITC-labeled streptavidin and
anti-mouse LFA1 (CD11a, 121/7) were obtained from Cedarlane.
Cytokine assays
For cytokine synthesis, effector T cells (H-2b) were washed, layered on Lympholyte M to remove dead cells, and stimulated (1 x 105/well) with varying numbers of either irradiated allogeneic J774A.1 (H-2d) or M12.4.1 (H-2d) cells or with various amounts of Con A, plate-bound anti-CD3 Abs, or PMA and A23187. Stimulation was conducted in 96-well round-bottom plates in 200 µl of RPMI plus 8% FBS. The cytokines produced were measured in 18-h supernatants as described previously (19, 20).
Thymidine incorporation assays
Allo-specific T cells (H-2b anti-H-2d) were cultured with irradiated J774A.1 macrophage cells (H-2d) in 96-well round-bottom plates in 200 µl of RPMI plus 8% FBS in the presence or the absence of various cytokines. After 24 or 72 h, 1 µCi of tritiated thymidine was added per well. Cells were harvested 18 h after the thymidine pulse, and radioactive incorporation was determined by liquid scintillation counting.
Cytotoxicity assays
T cells were generated as described above and were used as effectors after 7–8 days of culture. M12.4.1, L1210, and L1210Fas target cells (107) were labeled with 51Cr (100 µCi) in 50 µl of RPMI plus 8% FBS medium for 45 min. Targets were washed, various ratios of effectors and targets were cocultured for 4 h in 96-well round-bottom tissue culture plates, the supernatants were collected, and radioactivity was detected by gamma counting. The percent cytotoxicity was calculated using the formula: 100 x [(cpm experimental - cpm spontaneous)/(cpm total - cpm spontaneous)].
Flow cytometric analysis
T cells were incubated on ice (106 cells
in 50 µl of RPMI 1640 plus 1% FBS) with rat anti-mouse CD32/CD16
(FcII/III receptor). After 30 min aliquots were washed and incubated
separately in 50 µl of RPMI plus 1% FBS with the following Abs:
anti-mouse CD4, CD8, LFA1, IL-2R, CD44, NK1.1, and CD28.
Incubation lasted for 30 min on ice. Cells stained with biotinylated
Abs were subsequently incubated with streptavidin-FITC after thorough
washing. Cells were fixed in 1% formaldehyde in PBS and analyzed by
flow cytometry.
For cell cycle analysis, cells were stained with FITC-labeled anti-CD8 Ab for 30 min on ice in 50 µl of RPMI plus 1% FBS. Cells were washed and fixed in 70% ethanol overnight. Cells were stained with the propidium iodide in the presence of RNase A (100 U/ml) and analyzed by flow cytometry. DNA content histograms gated on CD8+ T cells were obtained using EXPO software (Coulter). The percentage of cells in the G2/M phase of the cell cycle was calculated.
The number of apoptotic cells was enumerated using the procedure described by Telford et al. (21). Cells were processed and acquired on the flow cytometer as described above for cell cycle analysis. The apoptotic fraction was measured by gating on CD8+ T cells and by the subdiploid population staining in the region below the large G0/G1 peak.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD8+ CD44low T cells
(1 x 103/well; >99% pure) were stimulated
with irradiated allogeneic J774A.1 cells (1 x
104/well). In this differentiation culture the
generation of effector CD8+ T cells can be
appreciated between days 4–5, depending on the presence of cytokines
in the culture. In the absence of exogenous IL-2 no detectable T cell
proliferation occurred (Fig. 1
a). This may be attributable
to naive CD8+ T cells (as opposed to naive
CD4+ T cells) producing minute amounts of
cytokines (S. Sad, unpublished observations), not sufficient to drive
their differentiation. Addition of increasing amounts of exogenous IL-2
resulted in a corresponding increase in CD8+ T
cell proliferation in a dose-dependent manner. However, IL-15, even at
10 ng/ml, failed to induce significant proliferation.
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b indicates that
at lower cytokine concentrations effector CD8+ T
cells proliferate more strongly in response to IL-15 than to IL-2. At a
cytokine concentration of 1 ng/ml, IL-2 induced 3,171 cpm, whereas
IL-15 induced 24,439 cpm. However, we observed consistently that
CD8+ T cell proliferation in response to IL-15
saturated around a concentration of 1 ng/ml, whereas IL-2 continued to
induce proliferation even at higher cytokine levels. These results
indicate that naive and effector CD8+ T cells
respond differentially to IL-2 and IL-15. IL-2-induced proliferation of CD8+ T cells impairs their subsequent cytokine synthesis ability
As experiments in Fig. 1 clearly suggested that IL-2 plays a
pivotal role in the differentiation of naive CD8+
T cells, we evaluated the optimal amounts of IL-2 required. C57BL/6
CD44low CD8+ T cells were
stimulated with irradiated allogeneic M12.4.1 cells
(H-2d) as described in Fig. 1. On day 6 effector
cells were harvested, washed, and analyzed directly for cell cycle and
the number of apoptotic cells. Fig. 2a clearly indicates that
addition of increasing concentrations of IL-2 enhanced the frequency of
cells going into the G2/M phase of the cell
cycle. On the other hand, Fig. 2b shows that low IL-2
concentrations (0.1 ng/ml) induced maximal apoptosis, with the number
of apoptotic cells declining at IL-2 concentrations of 1 and 10 ng/ml.
These results were further corroborated using the antiapoptotic
cytokine, IL-15. Addition of IL-15 to CD8+ T cell
cultures differentiating in the presence of 0.1 ng/ml IL-2 increased T
cell yield by about 5-fold. However, IL-15 had no effect on T cell
yield when 1 or 10 ng/ml IL-2 was used during the differentiation of
CD8+ T cells (data not shown). Thus, the cell
cycle and apoptosis data taken together indicate that low amounts of
IL-2 promote apoptosis, as opposed to cell cycling, in the primary
culture.
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. After 7 days the numbers of effector cells generated (>99%
pure) were evaluated using a hemocytometer. Fig. 3a shows that the presence of
increasing amounts of IL-2 enhanced the development of effectors. To
evaluate the effects on cytokine synthesis, we washed the effectors,
removed dead cells on Lympholyte M, and restimulated them (1 x
105/well) with Con A (5 µg/ml) for 18 h.
Cytokine synthesis by CD8+ T cells in the
secondary culture inversely correlated with their proliferation in the
primary culture. CD8+ T cells produced maximal
amounts of IL-2 in the secondary culture when they were cultured with
very low levels of IL-2 in the primary culture. On the other hand,
CD8+ effectors generated in the presence of high
IL-2 concentrations in the primary culture produced low cytokine levels
subsequently (Fig. 3b). Addition of IL-15 (10 ng/ml) to
these cultures did not restore their cytokine synthesis defect (data
not shown).
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We then addressed whether naive (CD44low)
CD4+ T cells were also susceptible to the
inhibitory effects of IL-2. C57BL/6 CD44low
CD4+ T cells were stimulated with allogeneic
M12.4.1 cells (H-2d) in the presence of
anti-IL-4, IL-12 and various amounts of IL-2. After 7 days, the
number of effectors generated was counted (Fig. 4a), and they were
restimulated with either Con A (Fig. 4b) or M12.4.1 cells
(Fig. 4c) to determine cytokine synthesis. Interestingly,
IL-2 stimulated the proliferation of effector
CD4+ Th1 cells (Fig. 4a) and also
enhanced their subsequent ability to produce cytokines (Fig. 4, b and c). Thus, IL-2 enhanced the proliferation
of both CD4+ and CD8+
effector cells, but it had contrasting effects on their subsequent
ability to produce cytokines. Although IL-2 enhanced cytokine synthesis
by CD4+ effectors, it inhibited cytokine
synthesis by CD8+ effectors.
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We wanted to establish the extent of inhibition induced by IL-2.
To this end, we addressed the possibility that restimulation of
CD8+ effectors with potent APC/reagents might
restore their cytokine synthesis defect. Allo-specific
(H-2b anti-H-2d)
effector CD8+ T cells were generated in the
presence of various concentrations of IL-2. After their differentiation
into effectors, CD8+ T cells were washed and
restimulated (1 x 105/well) variably as
described in the figure legends. Compared with Con A (Fig. 3b), or activated B cell lymphoma (M12.4.1; data not shown),
restimulation of CD8+ effectors with an activated
allo-macrophage cell line (J774A.1) resulted in quantitatively higher
cytokine levels. Although this may be attributable to the potency of
J774A.1 cells as APCs (10, 22), even under these potent
restimulatory conditions, the pattern of IL-2-induced inhibition of
cytokine synthesis continued (Fig. 5a). Similarly, restimulation
with plate-bound anti-CD3 Abs (Fig. 5b) or with PMA plus
Ca2+ ionophore (A23187; Fig. 5c) did
not restore the IL-2-induced loss of cytokine synthesis. Even the use
of high amounts of Ca2+ ionophore (10 µg/ml)
resulted in only partial restoration of cytokine synthesis (data not
shown).
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The experiments described above indicated that the cytokine
synthesis function of CD8+ T cells is lost due to
exposure of cells to high concentrations of IL-2. We then determined
the effects on the cytotoxic function of CD8+ T
cells. Effector CD8+ T cells
(H-2b anti-H-2d) were
washed, and their cytolytic activity was measured by incubation with
radiolabeled target cells in various proportions for 4 h.
CD8+ T cells from normal mice
(H-2b) showed a strong cytotoxic activity toward
their specific target cells, M12.4.1 (Fig. 6a). The absence of such
cytotoxic activity in perforin-deficient CD8+ T
cells (H-2b) indicates that M12.4.1 target cells
are killed by these CD8+ T cells mainly by the
perforin-dependent mechanism. The presence of increasing amounts of
IL-2 in the primary culture resulted in only a small increase in their
ability to kill by the perforin-dependent pathway. We then addressed
whether target cell killing by the alternate Fas-dependent pathway was
modulated by IL-2. To test the Fas-dependent killing, we used
perforin-deficient CD8+ T cells
(H-2b). Cytotoxicity was tested on targets that
expressed either very low levels of Fas (L1210,
H-2d) or high levels of Fas (L1210Fas,
H-2d). When L1210 cells were used as targets,
Fas-dependent cytotoxicity was minimal as expected. On the other hand,
the expression of Fas by the target cells (L1210Fas) rendered them
susceptible to killing by the CD8+ T cells. The
presence of increasing amounts of IL-2 in the primary culture of naive
CD8+ T cells increased their subsequent ability
to kill targets by the Fas-dependent mechanism (Fig. 6b).
Thus, IL-2 has distinctly opposite effects on the two
CD8+ T cell functions: enhancement of
Fas-dependant cytotoxicity but impairment of cytokine synthesis.
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We have previously demonstrated that killing of APC by
CD8+ T cells results in their reduced cytokine
synthesis (13). Thus, IL-2 induced inhibition of cytokine
synthesis of CD8+ T cells may be attributable to
their increased cytolytic function. To address this question, we
stimulated naive CD8+ T cells (normal,
perforin-deficient, or FasL-deficient) with allo-APC and increasing
concentrations of IL-2 and measured the cytokine synthesis of the
effectors generated. Fig. 7 confirms our
previous observation that perforin-deficient CD8+
T cells produce more IL-2 and IFN-. However, even in the absence of
perforin, IL-2 exposure in primary culture impaired their ability to
produce cytokines in the secondary culture. Furthermore, the amounts of
IL-2 required to induce inhibition of cytokine synthesis were similar
in normal and perforin-deficient CD8+ T cells.
This suggests that IL-2-induced inhibition of cytokine synthesis in
CD8+ T cells may not be related to
perforin-dependent killing. Similar results were obtained with
FasL-deficient CD8+ T cells (Fig. 8). FasL-deficient
CD8+ T cells overall produced more cytokines than
normal control cells. This was particularly evident in FasL-deficient
CD8+ T cells differentiated in high IL-2 amounts,
consistent with the cytotoxicity data presented in Fig. 6b.
However, both normal and FasL-deficient CD8+ T
cells showed similar sensitivity to IL-2-induced inhibition of cytokine
synthesis, indicating that the IL-2-induced increase in Fas-dependent
killing was not the reason for loss of cytokine synthesis.
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CD8+ T cells that were generated with
allo-APC and increasing amounts of IL-2 in the primary culture were
analyzed for the expression of various cell surface markers (Fig. 9). Throughout the culture period, these
cells remained pure without any outgrowth of contaminant populations,
and the cells did not acquire NK1.1 expression. The expression of CD28
was not altered, suggesting that costimulatory pathways may not be
altered as a result of IL-2 exposure. However, IL-2 increased their
expression of LFA1 and CD44 (Fig. 9a). To further rule out
the possibility that the lack of cytokine synthesis could be due to
some other contaminant cells in the culture, we resorted the
CD8+ NK1.1- cells on day 7
and measured their cytokine synthesis after Con A stimulation. After
resorting of the cells, the cytokine production pattern remained the
same. Cells that were precultured with 0.1 ng/ml of IL-2 synthesized
1.24 ng/ml of IL-2 in the secondary culture, whereas cells that were
precultured with 10 ng/ml of IL-2 synthesized <0.05 ng/ml of
IL-2.
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expression in these cells (Fig. 9b). At low IL-2 concentrations (0.1 ng/ml), only 47.4%
CD8+ T cells expressed high levels of IL-2R.
This expression increased substantially to 99.2% at 10 ng/ml IL-2. On
the other hand, CD4+ T cells exhibited
substantial IL-2Rhigh cells (79.1%) even at
low IL-2 concentrations (0.1 ng/ml), resulting in a less dramatic
enhancement with increasing IL-2 (Fig. 9b). An interesting
observation was that IL-2, while enhancing IL-2R expression on
CD8+ cells in a dose-dependent manner,
down-regulated IL-2Rß (Fig. 9c). Thus, IL-2 enhances CD8+ T cell activation as assessed by the expression of surface molecules and cytolytic activity, yet it inhibits IL-2Rß expression and the subsequent ability to produce cytokines.
Sorted IL-2Rlow cells express more cytokines than
IL-2Rhigh cells
We determined whether the purified
IL-2Rlow and
IL-2Rhigh cells differed in their ability to
produce cytokines. Allo-Ag-specific effector CD8+
T cells were derived in the presence of high concentrations (10 ng/ml)
of IL-2. Effectors were sorted for IL-2Rhigh
and IL-2Rlow expression using flow cytometry
and were stimulated with Con A for cytokine synthesis. Fig. 10a shows that on day 7, the
effector CD8+ T cells derived in the presence of
high levels of IL-2 were mainly IL-2Rhigh.
Fig. 10b indicates that even among the cells derived in the
presence of high IL-2 levels, CD8+ T cells that
express low levels of IL-2R -chain produce more IL-2 than cells that
express high levels of IL-2R -chain. This result clearly indicates
that the expression of IL-2R inversely correlates with the
subsequent ability of CD8+ T cells to produce
cytokines.
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Subsequent to the differentiation of naive T cells, effector T
cells have to proliferate extensively to curtail the proliferation of
the pathogens/tumors. Thus, T cells have to continuously produce
cytokines to autonomously aid their proliferation and effector
functions. We addressed whether the lack of cytokine production in
CD8+ T cells as a result of excess IL-2 in
preculture, had any effect on their autonomous proliferative ability.
CD8+ T cells were generated in the presence of
allo-APC and increasing concentrations of IL-2. On day 7 cells were
washed and restimulated with allo-APC in the absence of any exogenous
growth factors. Proliferation was measured after 24 h. Fig. 11a clearly indicates that
the presence of high IL-2 in primary culture induced a proliferative
defect in CD8+ T cells in the secondary culture,
whereas exposure to lesser amounts of IL-2 resulted in stronger
autonomous proliferation. Similar results were obtained at 72 h
after culture (data not shown).
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b).
To determine whether the effectors derived in high levels of IL-2
maintained their proliferation in response to exogenous cytokines, we
measured the proliferation of CD8+ effectors in
response to allo-APC and increasing concentrations of IL-2 or IL-15 in
secondary culture (Fig. 11, c and d).
CD8+ effectors generated in high concentrations
of IL-2 retained their proliferative ability to exogenous cytokines in
vitro, although at a reduced level.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF, and lymphotoxin)
by CD8+ T cells correlates with their cytotoxic
function. For an effective CD8+ T cell response,
in addition to killing infected cells CD8+ T
cells must proliferate sufficiently through the duration of infection
to overpower the pathogen. IL-2 drives the autonomous proliferation of
CD8+ T cells (23, 24). At the same
time, the presence of IL-2 is required to promote T cell
differentiation. We, therefore, analyzed the consequences of
differential IL-2 levels during the priming of naive
CD8+ T cells. Previously, it has been shown that mature effector CD4+ T cell clones lose their ability to produce cytokines upon preculture with high levels of IL-2 (16). This abrogation of cytokine production was associated with decreased inositol phosphate and diacylglycerol production and impaired calcium-dependent signaling (16, 17). Our observations are different from these studies, as we have addressed the effects of IL-2 during the priming of naive CD8+ T cells. Furthermore, our studies highlight the finding that IL-2 differentially affects naive CD4+ and CD8+ T cells; increasing IL-2 inhibits cytokine synthesis by CD8+ cells, whereas it enhances cytokine production by CD4+ cells.
Because cytokine production modulates T cell effector functions, we
addressed the consequences of impaired cytokine synthesis by
CD8+ T cells. We observed that while IL-2 impairs
subsequent cytokine synthesis by CD8+ T cells, it
enhances their cytotoxic activity, particularly by the Fas-dependent
pathway (Fig. 6). Other investigators have also reported that IL-2
enhances Fas-dependent cytotoxicity of T cells (25).
Furthermore, we observed that enhancement of cytotoxic function of
CD8+ T cells with IL-2 correlates with the
increased expression of IL-2R, CD44, and LFA1, indicating the
activated state of these cells (Fig. 9). McKisic et al.
(26) have shown that IL-2 inhibits the cytolytic activity
and cytokine synthesis of IL-2-producing T cell clones. However, we
(20, 27, 28) and others (12, 29) have shown
that exogenous cytokines do not impair the cytotoxic function of
CD8+ T cells. Effector
CD8+ T cells that are treated with IL-4
lose their cytokine synthesis ability, but maintain their short
term cytotoxicity (20). Similarly, anergic cells that fail
to produce IL-2, remain highly cytolytic (12, 29).
Cytokines affect the cytotoxicity of CD8+ T cells
only over the long term, when T cells have to undergo autonomous
proliferation to curtail the proliferation of tumor cells
(10).
Rapid killing of APC by effector CD8+ T cells compromises their stimulation and consequent cytokine synthesis (13). As high levels of IL-2 enhanced the cytotoxicity of CD8+ T cells, we addressed whether the loss of cytokine synthesis was due to their increased cytotoxicity toward APC. However, FasL-deficient and perforin-deficient CD8+ T cells were equally susceptible to IL-2-induced loss of cytokine synthesis.
IL-2, IL-4, and IL-15 all stimulate CD8+ T cell
proliferation. These overlapping activities may be attributed to their
sharing multiple components of the receptors used for binding and
signal transduction. All three cytokines use the same
IL-2Rc chain, but their receptor -chain
usage is specific for each cytokine. Furthermore, IL-2 and IL-15 share
the usage of IL-2R ß-chain. Interestingly, addition of IL-15 to naive
CD8+ cells evoked no T cell proliferation or
differentiation (Fig. 1). More importantly, IL-15 could not overcome
the inhibitory effects of IL-2 (data not shown). This could be partly
due to an IL-2-induced reduction in IL-2Rß expression by
CD8+ T cells (Fig. 9c) and consequent
impairment of IL-15 signal transduction. Furthermore, reduced IL-2Rß
expression in CD8+ effectors may compromise their
long term proliferation and consequent memory development.
One of the differences in the proliferative response of effector
CD8+ T cells to IL-2 vs IL-15, is the observation
that IL-15-induced proliferation saturates around a concentration of 1
ng/ml, whereas IL-2 continues to induce stronger proliferation even
beyond 10 ng/ml. It may be that this unabated proliferation induced by
IL-2 renders the cells nonresponsive. Moreover,
CD8+ T cells that express higher levels of
IL-2R produce lesser amounts of IL-2 on subsequent stimulation than
cells that express lower levels of IL-2R (Fig. 10). However,
CD4+ effectors that are generated with high
amounts of IL-2 have enhanced IL-2R expression but produce elevated
levels of cytokines. Thus, IL-2R expression correlates both
positively (in the case of CD4+ T cells) and
negatively (in the case of CD8+ T cells) with
subsequent T cell functions.
Although high expression of IL-2R may result in increased usage of
IL-2 and, consequently, decreased further production, the effector
CD8+ cells also produced lower amounts of IFN-
(Fig. 5) and TNF (data not shown), suggesting that a different
mechanism may be at play. Furthermore, inhibition of cytokine synthesis
occurred even when effectors were restimulated with agents (Con
A/plate-bound anti-CD3 Abs, in the absence of APC) that induce T
cell activation but not proliferation. Additionally, impairment of
autonomous proliferation of CD8+ effectors (Fig. 11) clearly indicates a functional loss of IL-2 synthesis. To further
confirm that the inhibition of cytokine synthesis was not due to
increased uptake by CD8+ T cells, we irradiated
CD8+ effectors (2500 rad) and restimulated them
with allo-APC for 18 h. Even under these conditions,
CD8+ T cells that were generated in low IL-2 (0.1
ng/ml) produced 1.95 ± 0.32 ng/ml of IL-2, whereas cells
generated in high IL-2 (10 ng/ml) produced <0.05 ng/ml of
IL-2.
Various investigators have also shown that effector CD4+ T cell clones when cultured with high levels of IL-2 undergo activation-induced cell death upon subsequent stimulation with anti-CD3 Abs (15, 30). Interestingly, IL-2-dependent activation-induced cell death occurs only in effector, but not in naive, CD4+ T cells (30). Our results indicate that during the differentiation of naive CD8+ T cells into effectors, the presence of low amounts of IL-2 results in increased numbers of apoptotic cells both during the primary culture as well as in the secondary culture (after restimulation with APC for 24 h). As cytokine production and apoptosis in the secondary culture were measured within the same time frame (18–24 h), the loss of cytokine synthesis by CD8+ T cells appears to be due to defects in signal transduction rather than to the cells undergoing rapid death due to activation. It is, however, conceivable that the failure of effectors to proliferate autonomously due to loss of cytokine production may increase the propensity for apoptosis over the long term.
The lack of ability to produce cytokines and proliferate autonomously
has often been referred to as anergy. Stimulation of T cells with Ag on
fixed APCs results in an anergic state, although the cells maintain
their short term effector function (11, 12, 29). We have
previously shown that IL-4 also induces a similar state in
CD8+ T cells that is characterized by impaired
cytokine synthesis, proliferation, and compromised ability to curtail
the proliferation of tumor cells (10, 20). IL-10 has also
been shown to induce a long term anergy-like state in human
CD4+ T cells (31).
CD8+ T cells proliferate more strongly and
produce more cytokines in the absence of IFN- (S. Sad, unpublished
observations). These results indicate that the induction of an
anergy-like state in T cells may be a phenomenon that occurs with high
levels of various cytokines.
Cytolytic effector CD8+ T cells specific for a
variety of Ags, including viruses, tumors, and exogenous proteins,
persist in the absence of the cognate Ag in vivo and in vitro
(32, 33). The nature of stimulus that drives the
proliferation of memory T cells in the absence of Ag and the role of
IL-2 in the maintenance of memory CD8+ T cells
remain unclear. Although IL-2-deficient mice have normal CTL responses
(34), OVA-specific memory CTLs decline strongly in the
absence of IL-2, (32), suggesting that cell cycling is
important for the maintenance of CD8+ T cell
memory. Our data indicate that IL-2 compromises subsequent cytokine
synthesis in CD8+ T cells, raising questions
about the role of IL-2 in the maintenance of memory cells. It is
possible that the production of IL-2 is precisely regulated in vivo so
that its level is always maintained at low concentrations, sufficient
to drive low level proliferation of cells. Alternatively, IL-2
production by CD8+ T cells may not be important
for maintenance of memory CD8+ T cells, as
CD4+ T cells can produce copious amounts of IL-2
and are less susceptible to IL-2-induced inhibition of cytokine
synthesis. This would mean that CD4+ and
CD8+ T cells have to coexist in vivo to sustain
memory CD8+ T cells. IL-15 has been shown to be
more potent than IL-2 in selectively stimulating the proliferation of
memory CD8+, but not CD4+,
T cells in vivo (35). Our results also demonstrate that
effector, but not naive, CD8+ T cells respond
more strongly to IL-15 than to IL-2 (Fig. 1). Thus, it is possible that
IL-2 might play a more important role during the initiation of an
immune response, whereas at later stages IL-15 might sustain the
circulation of memory CD8+ T cells.
Several observations indicate that CD8+ T cells are often dependent on cytokines produced by CD4+ helper cells (36, 37, 38, 39). CD8+ T cells may be capable of effectively curtailing an acute phase immune response, as effects of IL-2 are less discernable over the short term. However, the chronic course of infection may lead to more CD4+ dependence for sustenance of CD8+ cells.
As opposed to MHC class II, which is selectively expressed on cells, MHC class I is expressed on almost all cells. Because CD8+ T cells are MHC class I restricted, they can be stimulated more readily, and severe CD8+ T cell responses can cause pathology (40, 41). Thus, multiple mechanisms must maintain the regulation of CD8+ T cell responses. As IL-2 is a potent proliferation-promoting cytokine, it is reasonable that its production needs to be precisely regulated. Other mechanisms mediated by IL-4-, perforin-, and Fas-dependent killing also help to regulate CD8+ T cell function (13, 20). The lack of production of cytokines may be a self-regulating mechanism for turning off immune responses. Thus, besides the expression of cytokine patterns, the amounts of individual cytokines in the local microenvironment can have totally different temporal effects.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Subash Sad, Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Room 4105, Ottawa, Ontario, Canada K1A 0R6. E-mail address:
3 Abbreviation used in this paper: FasL, Fas ligand.
Received for publication February 22, 1999. Accepted for publication June 10, 1999.
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References |
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