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Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33136
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- genetic background, this study has separated
the relative outcome of signaling through the TCR and IL-2R. We show
that stimulation through the TCR and CD28 or CD40 ligand directly leads
to T cell activation and several rounds of proliferation in an
IL-2-independent fashion. However, this stimulation is insufficient for
extended T cell growth to multiple cytokines or differentiation into
CTL or IFN-
-secreting effector T cells. IL-2 is required for these
functions in part by regulation of cyclin D3 and granzyme B. Somewhat
less efficiently, IL-4 stimulation of these transgenic T cells
redundantly rescued many of these activities. These data demonstrate a
fundamental requirement for IL-2 and perhaps other common
-chain-dependent cytokines to promote selective gene expression by
Ag-activated T cells for their subsequent growth and differentiation
into effector T lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, , and common -chain (c) subunits (1, 2). Signaling pathways by the IL-2R and IL-15R largely overlap
as both receptors share the and c subunits
(3). Along with the IL-2R and IL-15R, IL-4R, IL-7R, and
IL-9R also use c as a subunit, leading to signaling
redundancy inasmuch as each activates Janus kinase-1 and Janus kinase-3
through their unique subunit and c, respectively
(2, 4, 5). With the exception of the IL-4R, which
activates STAT6, all the other c-dependent cytokines
activate STAT5a, STAT5b, and STAT3. This overlap in signal transduction
results in functional redundancy among these receptors with respect to
stimulation of T cell growth and sensitizing T cells to AICD (6, 7). In most models concerning the activation of mature T lymphocytes, TCR and costimulatory signals move resting T cells to the G1 phase of the cell cycle while cytokine signals, especially IL-2, promote DNA synthesis and subsequent cell division. The primary data supporting this scenario is that IL-2 very efficiently promotes the growth of Ag-activated T cells (8, 9), IL-2 regulates the expression cell cycle proteins (10, 11, 12), and anti-IL-2 and IL-2R Abs block Ag- or mitogen-induced T cell proliferation (13, 14). In the latter case, except under limiting culture conditions, anti-IL-2 blockade does not usually completely block Ag receptor-induced proliferation. This result typically has been suggested to reflect the inefficiency of the Abs, due to the high-affinity interaction of IL-2 with its receptor. Alternatively, such findings might represent IL-2-independent T cell growth, and several recent studies support this notion (15, 16, 17). In any case, the dynamics of T cell activation and the typical experimental systems in use do not allow clear separation of the outcome of TCR and costimulatory T cell activation vs IL-2-dependent effects.
Attempts to discern the role of IL-2 on peripheral T cell function
using T lymphocytes from IL-2- or IL-2R-deficient mice is complicated
by their severe lethal autoimmunity and imbalance in homeostasis that
typically imparts intrinsic T cell functional defects
(18, 19, 20). Recently, we have developed a transgenic (Tg)
mouse on the IL-2R-/- genetic background in
which physiological levels of IL-2R was targeted to the thymus
(16). These animals are long-lived and do not exhibit the
autoimmune syndrome characteristic of IL-2 or IL-2R deficiency, but
their peripheral T cell compartment remains nonresponsive to IL-2.
Therefore, this experimental system offers the opportunity to not only
examine the contribution of IL-2 to peripheral T cell function without
the complication of the severe immunomodulatory pathology, but also
furnishes a genetic approach to separate the outcome of signaling
through the TCR and costimulatory molecules vs the IL-2R. In this
study, we have examined this issue with these IL-2R-deficient T
cells with respect to their growth, death, and differentiation into
effector cells in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Production of Tg mice that expressed wild-type IL-2R under
the control of the proximal lck promoter has been previously
described (16). These mice were backcrossed to
IL-2R+/- mice for five generations, and in
most experiments Tg+
IL-2R-/- or Tg-
IL-2R+/- littermates were used. C57BL/6 mice,
which occasionally were used as controls, and BALB/c mice were obtained
from The Jackson Laboratory (Bar Harbor, ME).
Antibodies
Cy-Chrome-CD8 (53.6.7), Cy-Chrome CD4 (GK1.5), PE-CD4
(GK1.5), PE-IL-2R (TM1), FITC-CD8 (53.5.8),
biotin-anti-rat IgG2a (RG7/1.30), anti-CD28 (37.51), anti-
CD40 ligand (CD40L) (MR1), anti-Bcl-2 (3F11), anti-hamster IgG
(G70-204; G94-56), and PE-streptavidin were obtained from BD PharMingen
(San Diego, CA). FITC-CD4 (GK1.5), biotin-IL-2R (7D4), anti-IL-2
(S4B6) (21), CTLA4Ig (22), and anti-CD3
(145-2C11) were purified and conjugated, as necessary, in our
laboratory. Purified anti-IL-7R (A7R34) (23) was
kindly provided by S. Nishikawa (Kyoto University, Kyoto, Japan).
Anti-Bcl-x (2A1) was kindly provided by L. Boise (University of Miami,
Miami, FL). Rat Ig was obtained from Sigma (St. Louis, MO). Abs to p27
(no. SC-1641), cyclin D2 (no. SC-593), cyclin D3 (no. SC-1641), and
-actin (no. SC-8432) were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). HRP-donkey anti-rabbit Ig and HRP-sheep
anti-mouse Ig were obtained from Pharmacia (Arlington Heights,
IL).
Cell culture
T cells were prepared by negative selection by, first, depletion of B cells on anti-mouse Ig-coated plates, followed by further depletion by incubation at 37°C for 45 min with anti-CD24 (J11D), anti-NK1.1 (PK136) and anti-MHC class II (M5.114) and rabbit C (Accurate Chemicals and Scientific, Westbury, NJ). To enrich for CD4+ T cells, anti-CD8 (HO2.2) was also included in the Ab and C depletion. Viable cells were purified by centrifugation through LymphoLyte M (Accurate Chemicals and Scientific). Accessory cells (AC) were prepared by treatment of normal spleen cells with anti-Thy-1.2 and C at 37°C for 45 min and washed three times with HBSS.
For proliferation assays of primary T cells, spleen cells (2 x 105/well), unfractionated lymph node cells (1 x 105/well) or purified T cells (1 x 105/well) were cultured with complete medium in flat-bottom 96-well plates as previously described (16) with soluble (1 µg/ml) or plate-bound (treated with 3 µg/ml in PBS) anti-CD3, plate-bound anti-CD28 (treated with 3 µg/ml in PBS), AC (1 x 105/well), 10 ng/ml PMA, 50 U/ml IL-2, 500 U/ml IL-4, 25 µg/ml anti-IL-2, 25 µg/ml CTLA4Ig, 25 µg/ml anti-CD40L, or 25 µg/ml rat Ig, as indicated, unless otherwise specified, for 48 h. [3H]Thymidine was added during the last 4 h.
For molecular, anti-CD3-redirected CTL, cytokine-dependent proliferation, cell growth, and cytokine secretions assays, spleen cells were cultured at 2 x 106/well in 24-well flat-bottom culture plates as previously described (16) with anti-CD3, 350 U/ml IL-4, or 50 ng/ml IL-7 for 48 h, as indicated. Allogeneic CTL were prepared exactly as described (16). As required, supernatant fluids were collected, the cells were harvested and washed with PBS, and total RNA (16) or Nonidet P-40 extracts were prepared (24), or the cells were subjected to CTL assay (25). To measure cytokine-dependent proliferation, these activated cells (2 x 104/well) were recultured in 96-well plates with 50 U/ml IL-2, 350 U/ml IL-4, 50 ng/ml IL-7, or no cytokines for 24 h. [3H]Thymidine was added during the last 4 h. In other cases, to measure cytokine-dependent growth or cytokine secretion by effector cells, the anti-CD3-stimulated T blasts were recultured after washing with 100 U/ml IL-2, 350 U/ml IL-4, or 50 ng/ml IL-7 at 1 x 105/ml in 10 ml of medium in a T-25 flask for 4 days. Cell growth was enumerated by counting cells that excluded trypan blue. To measure cytokine production, the IL-4-expanded T cells (1 x 106/well) were recultured with AC (1 x 106/well) and anti-CD3 in 1 ml in 24-well plates. Supernatant fluids were collected 24 h later.
To measure AICD, T cells or CD4 enriched cells (2 x 106/well) were cultured in 24-well flat-bottom plates with anti-CD3 and IL-4 (350 U/ml) for 5 days. Cells were harvested, washed, and recultured (2 x 105/well) with 350 U/ml IL-4 in 96-well flat-bottom culture wells with or without 2 µg/ml Con A for 18 h. Cell death was evaluated by trypan blue dye exclusion.
FACS analysis
FACS analysis was performed as previously described
(26) using a Becton Dickinson (Mountain View, CA) FACScan
and CellQuest software. Typically 10,000 viable cells were analyzed
based on forward- vs side-scatter gating. All FACS staining except
IL-7R was performed by one- or two-step staining, the latter using
biotinylated Abs and PE-streptavidin. IL-7R was detected by
three-step staining by incubations with anti-IL-7R,
biotin-anti-rat-IgG2a, and PE-streptavidin. These cells were also
stained with CyChrome CD4 and FITC-CD8 using Abs that are not
recognized with the anti-rat IgG2a. Spleen cells were labeled with
CFSE as previously described (27). Briefly, spleen cells
(5 x 107/ml) were incubated with 5 µM
CFSE in serum-free medium for 10 min at 37°C, washed 2 times with
culture medium, and cultured as described above for 48 h with
anti-CD3 before FACS analysis.
Cytokine assays
IL-2 was measured in the CTLL bioassay (28) in the
presence of anti-IL-4 (11B11). IFN- and IL-4 were measured by
ELISA using kits from BD PharMingen according to the manufacturer’s
instructions.
Molecular analysis
Northern blot (29) and Western blot
(30) analyses were performed as previously described. In
Western blot analysis, membranes were incubated with the following:
anti-Bcl-2, followed by mouse anti-hamster Ig and HRP-sheep
anti-mouse Ig; anti-p27, anti-cyclin D3, anti-Bcl-x, or
anti--actin, followed by HRP-sheep anti-mouse Ig; or
anti-cyclin D2, followed by HRP-donkey anti-rabbit Ig.
IL-4 binding assay
The indicated cells (10 x 106/tube)
were incubated in duplicate in 1 ml of complete medium
(16) with 50,000 cpm 125I-labeled
IL-4 (70 µCi/µg) for 30 min at 4°C. The cells were washed and the
cell-associated radioactivity was determined by counting in a
scintillation counter. Over 95% of the binding was determined to be
specific as assessed by inhibition by 200 ng/ml unlabeled
IL-4.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wild-type transgenic IL-2R was expressed in the thymus of
IL-2R-/- mice, and these mice will be
referred to as Tg IL-2R-/-. Spleen cells
from Tg IL-2R-/- mice generated substantial
proliferation to soluble anti-CD3 or PMA and IL-4, but essentially
no response to PMA and IL-2 (16). The anti-CD3-induced
response was not inhibited by a mixture of mAbs to IL-2 and
c and was generally 
50% of control response by
IL-2R+/- littermate spleen cells
(16). By contrast, proliferation, cytokine production, and
CTL responses by Tg IL-2R+/- T cells paralleled
that observed for control IL-2R+/- littermate T
cells (Ref. 16 , and data not shown), and for simplicity
these data are not included in this report. A time course study
indicated that both the Tg IL-2R-/- and
IL-2R+/- littermate spleen cells generated similar
proliferative responses to anti-CD3 after 24 h in culture
(Fig. 1
A), but the response by
the Tg IL-2R-/- cells was 50% lower after
48 h in culture. After that time, both responses substantially
decreased, although a somewhat more sustained response was seen for T
cells from the Tg IL-2R-/- mice.
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-/- T cells was a failure of activated T
cells to fully transit the cell cycle and divide, spleen cells from
control and Tg IL-2R-/- mice were labeled with
CSFE and cultured with anti-CD3 for 48 h. As the cells divide,
the fluorescence intensity of the CSFE-labeled cells decreases
proportionally (27). For the recovered cells, a similar
reduction in CSFE staining was noted for T cells from both types of
mice, with a greater decrease in CD8+ cells (Fig. 1
B). The level of decrease in CSFE staining is consistent
with two to three cell divisions after 48 h in culture. Therefore,
although the magnitude of the T cell proliferation to anti-CD3 by
Tg IL-2R-/- cells is reduced at 48 h, the
responsive cells showed near normal capacity to divide during the first
2 days of the response.
The notion that at least some T cell proliferation is independent of
IL-2 in our Tg model is at odds with the observation that spleen cells
from Tg-negative IL-2R-/- mice are extremely
hyproresponsive to activation by anti-CD3 (16). These
poor responses also extend to activation by PMA and ionomycin
(18), suggesting that splenic T cells from
IL-2R-/- mice contain an intrinsic defect
for proliferation. However, the spleens of
IL-2R-/- mice are known to be infiltrated by
a large number of potentially suppressive granulocytes. Therefore, we
tested whether this low response extended to lymph node T cells from
the IL-2R-/- mice (Fig. 1C).
Unlike the spleen, unfractionated lymph node T cells demonstrated
readily measurable proliferative responses to anti-CD3, albeit at
levels still reduced when compared with control T cells. Plate-bound
anti-CD3 was somewhat more effective than soluble anti-CD3 in
inducing proliferation by IL-2R-/- T cells
in unfractionated lymph nodes. The addition of AC to cultures with
soluble anti-CD3 or further cross-linking with plate-bound
anti-CD28 did not significantly enhance the proliferation,
indicating that sufficient costimulatory signals were present in the
lymph node cells. Thus, despite the lymphoadenopathy and high
proportion of activated T cells (18),
IL-2R-/- lymph node T cells proliferated to
anti-CD3, which by necessity was independent of IL-2R-dependent
signaling by IL-2 or IL-15.
IL-2-independent T cell proliferation depends upon TCR and costimulatory signals
To determine which cell surface molecules are required for T cell
proliferation by Tg IL-2R-/- T cells, initially
the proliferation by purified splenic T cells to soluble and
plate-bound anti-CD3 was compared (Fig. 2A). As expected, purified T
cells from both control and Tg IL-2R-/- mice
failed to respond after stimulation with either soluble or plate-bound
anti-CD3, indicating that engagement of only the TCR was
insufficient for T cell proliferation. The addition of AC or
cross-linking with plate-bound anti-CD28 resulted in vigorous
proliferation with the strongest responses in cultures supplemented
with AC. In both cases the responses by the Tg
IL-2R-/- T cells was again about 50% of the
control responses.
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-/- mice, we assessed the capacity of
anti-IL-2, CTLA4Ig, and anti-CD40L to block such responses
(Fig. 2B). Anti-IL-2 inhibited T cell proliferation by
control T cells by 50% while exerting minimal effect on Tg
IL-2R-/- T cells. Therefore, the magnitude of
proliferation by the control T cells in the presence of anti-IL-2
was essentially similar to Tg IL-2R-/- T cells
cultured in the absence of anti-IL-2. When unfractionated spleen
cells were stimulated with soluble anti-CD3, the resulting T cell
proliferation from IL-2R+/- control mice was
partially blocked by anti-IL-2, CTLA4Ig, or anti-CD40L whereas
only CTLA4Ig and anti-CD40L partially blocked the responses by the
Tg IL-2R-/- T cells. However, a mixture of
CTLA4Ig and anti-CD40L substantially (80%) inhibited the
proliferation by both types of T cells. Thus, these data demonstrate
that T cell proliferation by Tg IL-2R-/- T cells
during the first 2 days in culture is dependent upon the TCR and
costimulation through CD28 and CD40/CD40L, but is independent of
IL-2.
IL-2R and IL-4R redundantly promote cytokine responsiveness by
activated T cells
After 48 h in culture, most T cells from Tg
IL-2R-/- mice are activated based on
proliferation, IL-2 secretion, cell division (Figs. 1, 2, and 6), and
expression of CD69 and CD25 (Ref. 16 and Fig. 4). As
expected, these T blasts were unresponsive when restimulated with IL-2
(Fig. 3A and Ref.
16) and, surprisingly, hyporesponsive to IL-4 and IL-7
(Fig. 3A). The poor responses by Tg
IL-2R-/- T blasts to IL-4 or IL-7 was especially
evident during longer-term cultures (Fig. 3B). Thus, in the
absence of IL-2R signaling, not only were these
anti-CD3-activated T cells nonresponsive to IL-2, but they were
also hyporesponsive to IL-4 and IL-7.
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-/- T
blasts after reculture with IL-4 and IL-7 for 24 h (Fig. 3A). It was also possible to grow Tg
IL-2R-/- T cells, albeit less efficiently than
the IL-2R+/- littermate T cells, provided that T
cells were primed with anti-CD3 and exogenous IL-4 and recultured
with IL-4 (Fig. 3B). Collectively, these data demonstrate
that sustained cytokine responsiveness requires that activated T cells
receive cytokine signals that may be redundantly provided by IL-2
or IL-4.
IL-4 sensitizes Tg IL-2R-/- T cells to AICD
Although Tg IL-2R-/- peripheral T cells do
not respond or proliferate to IL-2, it is possible to grow these cells
with IL-4 for at least 5 days (Table I).
When such unfractionated or CD4-enriched T cells were recultured with
Con A or anti-CD3 (not shown) and IL-4, the majority of the cells
died. The levels of AICD were very similar to identically treated
control cells. Thus, IL-4 functioned as a growth factor for Tg
IL-2R-/- T cells and sensitized them to
AICD.
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To begin to define the molecular basis by which the IL-2R and
IL-4R redundantly promote T cell growth, the expression of several
cytokine receptors, cell cycle regulatory proteins, and
anti-apoptotic proteins was determined for T blasts from control
littermate and Tg IL-2R-/- mice. As expected,
IL-2R was selectively not detected for Tg
IL-2R-/- T blasts, and the levels of IL-2R by
the Tg IL-2R-/- cells were markedly diminished
(Fig. 4A), consistent with the
role of IL-2 to up-regulate IL-2R (31). When compared
with T blasts from littermate control mice in four independent
experiments, IL-2R was typically 3- and 16-fold lower on CD4 and CD8
T blasts, respectively. Interestingly, Tg
IL-2R-/- T blasts also showed diminished levels
of IL-7R (Fig. 4A). This finding indicates that IL-2R
signaling is required for maintaining expression of IL-7R and may
partially explain the generally poor proliferative responses by these T
blasts when re-challenged with IL-7 (see Fig. 3). In contrast, Tg
IL-2R-/- T blasts expressed normal levels of
c and IL-4R, the latter as assessed by radiolabeled IL-4
binding (Fig. 4B). Therefore, the relatively poor response
to IL-4 re-stimulation by these T blasts is not due to failed IL-4R
expression.
Unless exogenous IL-4 was added with anti-CD3 at culture
initiation, we have never been successful at inducing proliferation by
the T blasts upon re-stimulation with defined cytokines, unfractionated
cytokine-containing supernatants, or anti-CD3 and AC (data not
shown), and upon reculture for an additional 24 h, these T cells
typically appeared dead (not shown). This failure to survive is not the
result of impaired expression of Bcl-2 or Bcl-x (Fig. 4C).
In fact, the levels of Bcl-x in the Tg IL-2R-/- T
blasts were greater than those seen for littermate control cells.
However, when the expression of several cell cycle regulatory proteins
were examined, Tg IL-2R-/- T blasts selectively
expressed reduced levels of p27 and especially cyclin D3 (Fig. 4C). After normalizing the band intensities to those of
-actin, densitometric analysis of this and two other experiments
revealed that Tg IL-2R-/- cells contained on
average 3.3-fold less cytoplasmic cyclin D3 when compared with the
control T blasts. Thus, IL-2R signaling is required to properly
maintain expression of cyclin D3. The addition of exogenous IL-4 to the
Tg IL-2R-/- T cells did not obviously affect the
expression p27 or cyclin D2, but slightly increased (1.4-fold) the
levels of cyclin D3, but these levels were still lower than the control
T cells. This result suggests that IL-4 up-regulation of cyclin D3 may
also partially account for the capacity of IL-4 to enhance subsequent
IL-4 responsiveness by these activated T cells.
IL-2R and IL-4R redundantly promote generation of T effector
cell function
We previously reported that Tg IL-2R-/- T
cells were greatly impaired in differentiation into CTL (Fig. 5, A and B)
(16). However, addition of IL-4 to these T cells while
they were stimulated with allogeneic spleen cells or anti-CD3
substantially enhanced their levels of alloreactivity (Fig. 5A) or redirected (Fig. 5B) CTL activity. IL-7
was much less efficient in promoting CTL activity by Tg
IL-2R-/- T cells while IL-12- and IL-6-containing
supernatants (not shown) did not mediate such a function. Northern blot
analysis indicated that Tg IL-2R-/- T blasts
lacked granzyme B mRNA and expressed reduced levels of perforin mRNA
(Fig. 5C). Addition of exogenous IL-4 to anti-CD3 Tg
IL-2R-/- T cells induced expression of some
granzyme B mRNA and somewhat enhanced the expression of perforin mRNA,
although the levels of these mRNAs were still lower than seen in the
control T cells. In any case, the ability of IL-4 to enhance CTL
activity by Tg IL-2R-/- T blasts is at least
partially accounted for by the regulation of two genes required for CTL
function.
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-/- T cells was also assessed. After 24
h in culture, anti-CD3 induced generally similar levels of IL-2 by
T cells from Tg IL-2R-/- and
IL-2R+/- littermate mice (Fig. 6A). By contrast, after
48 h, the levels of IL-2 always increased for the Tg
IL-2R-/- T cells while they were always reduced
and often undetectable for the IL-2R+/- littermate
T cells. This reduction in IL-2 is consistent with its consumption by
the activated IL-2R+/- T cells. By contrast, the
increase in IL-2 at 48 h by the Tg IL-2R-/-
T cells indicates that these cells were unable to use the secreted
IL-2.
Unlike IL-2R+/- littermate T cells, those from Tg
IL-2R-/- mice did not secrete IFN- (Fig. 6B). Inclusion of exogenous IL-4 during the culture
diminished the levels of IFN- secreted by the control T cells, as
expected, while it did not significantly affect the amount of IFN-
secreted by the Tg IL-2R-/- T cells. Thus, these
data indicate that signaling through IL-2R is required for IFN-
production in vitro.
After 48 h in culture with anti-CD3, the percentage of
IL-2R+/- and Tg IL-2R-/- T
cells that were CD4 was 31.5 ± 3.6 and 43.5 ± 5.8,
respectively, and the percentage that were CD8 was 42.0 ± 3.6 and
16.3 ± 3.4, respectively, as determined in three experiments; the
inclusion of exogenous IL-4 during this culture did not significantly
alter these percentages (not shown). The lower fraction of activated Tg
IL-2R-/- CD8 is in part due to the lower number
of CD8 T cells in the spleens of these mice that is typically reflected
as a CD4:CD8 ratio of 4:1 rather than the 2:1 ratio of normal mice. In
any case, the failure of anti-CD3 to induce IFN- cannot be
attributed to lack of activation or survival of Tg
IL-2R-/- CD4+ T
cell.
To further explore the capacity of Tg IL-2R-/- T
cells to differentiate into cytokine producing effector cells, the
anti-CD3 plus exogenous IL-4 stimulated T cells were further
cultured for 2–3 days with only exogenous IL-4. These cells were then
restimulated with anti-CD3 and AC, but without exogenous cytokines,
and the resulting supernatant fluids were tested for IFN- and IL-4.
Both the IL-2R+/- and Tg
IL-2R-/- IL-4-expanded T cells produced IFN-,
although on average, the level secreted by the Tg
IL-2RB-/- T cells was 3.6-fold lower (Fig. 6C). Thus, Tg IL-2R-/- T cells do not
express an absolute defect in the production of IFN-. Both the
IL-2R+/- and the Tg IL-2R-/-
T cells produced equivalent levels of IL-4 (Fig. 6D).
Therefore, these culture conditions were not sufficient to completely
polarize cytokine production into a Th1 or Th2 pattern. The higher
amount of IFN- secreted by the IL-2R+/- T cells
may be due to greater numbers of CD8 cells whose IFN- production is
not as readily down-regulated by IL-4 (32). These effector
cells produced modest levels of IL-2 with the highest amount by the Tg
IL-2R-/- T cell (average 3.1 U/ml), which was
about 4-fold higher than detected by the IL-2R+/-
T cells (16). Thus, collectively these data demonstrate
that in the absence of IL-2R function, IL-4 redundantly functions to
promote CTL and IFN- secretion by anti-CD3-activated T
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c-dependent cytokines. The cornerstone for this
conclusion is that highly purified Tg IL-2R-/- T
cells proliferated in response to anti-CD3 and anti-CD28
cross-linking or anti-CD3 in the presence of AC. It is further
supported by the failure of anti-IL-2 or a mixture of anti-IL-2
and c mAbs (16) to inhibit this
proliferation. Furthermore, IL-2 secretion by Tg
IL-2R-/- T cells increased over time. Thus,
unlike control littermate T cells, IL-2 was not consumed at the time
when maximal T cell proliferation occurred. We also showed that lymph
node T cells from IL-2R-/- mice, despite
their severe pathophysiology, proliferated when directly stimulated
through the TCR and CD28 to levels nearly comparable to that usually
detected for Tg IL-2R-/- T cells, but lower than
observed for normal T cells. Similar results were previously noted for
IL-2-/- lymph node T cells (15, 33). The expression of several genes known to be regulated by
IL-2 were also absent or reduced in the anti-CD3-activated Tg
IL-2R-/- T cells. The two most highly
IL-2-dependent targets were IL-2R, especially on
CD8+ T cells, and granzyme B, while the levels of
cyclin D3, IL-7R, and perforin were also consistently reduced.
Lastly, the observations that TCR stimulation led to expansion of lymph
node Tg IL-2R-/- T cells in vivo
(16) and the capacity for Ag to induce substantial clonal
expansion of TCR Tg c-/- T cells
upon adoptive transfer to RAG-/- recipient mice
(34) further demonstrate that TCR activation in vivo leads
to proliferation independent of IL-2 and all other
c-dependent cytokines.
Past studies have shown that Con A or anti-CD3 induced
proliferation by splenic T cells from IL-2-, IL-2R-, and
IL-2R-deficient mice were highly variable, and these responses were
often only 5–10% of control littermate T cells (7, 18, 19, 33). This finding has lead to the conclusion that T cell
proliferation in vitro is largely IL-2 driven, although a portion is
independent of IL-2/IL-2R. It is evident from the anti-IL-2
blocking experiments that 50% of the anti-CD3-induced
proliferation by normal T cells under optimal stimulatory conditions
was dependent upon IL-2. Importantly, the magnitude of the
proliferation of the Tg IL-2R-/- T cells in the
absence or presence of anti-IL-2 or anti-c
(16) was typically 50% of the control responses by normal
littermate T cells. These findings indicate that the proliferation by
the normal T cells is comprised of two components, one independent of
IL-2 and other c-dependent cytokines which is larger
than previously appreciated, and a second IL-2-dependent response.
Therefore, the typical poor polyclonal-indued proliferation by IL-2-,
IL-2R-, and IL-2R-deficient T cells is likely the result of
intrinsic cell defects due to the accompanying autoimmune syndrome. The
substantial blockade of the IL-2-independent T cell proliferation by
the Tg IL-2R-/- T cells by the combination of
anti-CD40L and CTLA4Ig demonstrates that this IL-2-independent
response is largely driven by engagement of the TCR and either CD28 or
CD40L. Recently, human T cells have also been shown to proliferate in
an IL-2-independent fashion that is dependent upon CD28
(17), and proliferation has also been induced in T cells
from c-/- and
IL-2-/- mice after cross-linking CD3 and CD28
(15, 35). The simplest interpretation of these data is
that signaling through the TCR and these costimulatory molecules is
sufficient to directly induce T cell proliferation. In fact, some of
the signal transduction pathways linked to TCR signaling (reviewed in
Ref. 36), e.g., MAP kinase and PI-3-kinase pathways, have
been shown to promote cellular proliferation in numerous other cell
types. Alternatively, we cannot rule out that engagement of TCR, CD40L,
and/or CD28 may have induced another cytokine or cell-cell molecular
interaction that drives this proliferation. So far, we have been
unsuccessful in stimulating proliferation by these
anti-CD3-activated T cells with crude cytokine-containing
supernatants or defined cytokines (data not shown) suggesting that the
anti-CD3-induced response by the Tg IL-2R-/-
T cells in not driven by soluble mediators.
Regardless of what precisely mediated this IL-2R-independent
proliferation, it should be stressed that the growth of such T cells is
limited to two to three cell divisions. Thus, TCR and costimulatory
activation in the absence of IL-2 signal transduction resulted in an
aborted response. Strikingly, these T cells were not competent for
cytokine-dependent expansion mediated by IL-4, even though they
expressed normal levels of this cytokine receptor. Furthermore, in the
absence of exogenous cytokines, Tg IL-2R-/- T
cells did not differentiate into effector T cells, as assessed by their
minimal CTL activity and lack of IFN- secretion. Therefore, these
findings indicate that IL-2 functions in a much broader role than a T
cell growth and survival factor and is responsible for programming
activated T cells for extended cytokine-dependent T cell growth and
differentiation into effector cells.
Consistent with the results herein, many studies have shown an
important role for IL-2 in multiple T cell functions in vitro, yet
paradoxically, the peripheral T cell compartment in these Tg
IL-2R-/- mice, with only minor exceptions, is
outwardly normal (16). Therefore, some of the T cell
activities that require IL-2 in vitro may be provided by redundant
activity in vivo, perhaps by other c-dependent
cytokines. For example, the addition of exogenous IL-4 during the first
2 days with anti-CD3 yielded activated Tg
IL-2R-/- T cells that expanded upon subsequent
culture with IL-4, were sensitive to AICD, and differentiated to CTL as
well as IL-4- and IFN--secreting cells. Thus, IL-4 redundantly
functioned in the absence of IL-2R signaling for these activities in
vitro and is a candidate to provide such an activity in vivo. However,
usually the capacity of IL-4 to rescue these functions by the Tg
IL-2R-/- T cells was still suboptimal when
compared with the responses by littermate control cells. Therefore, it
remains to be determined whether IL-4 serves such a redundant function
in vivo.
The induction of granzyme B by IL-4 at least partially accounts for the
ability of this cytokine to promote CTL differentiation by activated Tg
IL-2R-/- T cells, and identifies granzyme B as a
key IL-2 regulated gene, as suggested by others (37).
Although IL-4 has been shown to function as a T cell growth factor
(38) and sensitizes T cells to AICD (6, 7),
our study showed a requirement for IL-4 to promote these functions by
anti-CD3-activated T cells in the absence of IL-2R signaling.
The lower levels of cyclin D3 and p27 by anti-CD3 stimulated Tg
IL-2R-/- T cells may partially explain the
inability to sustain their growth to c-dependent
cytokines such as IL-4. Other studies have shown that IL-2 induces
cyclin D3 by a STAT5-dependent mechanism (11, 39). The
levels of cyclin D3 in Tg IL-2R-/- T blasts were
somewhat enhanced by IL-4, raising the possibility that proteins that
directly regulate the cell cycle are key targets by which
c-dependent cytokines redundantly function to induce a
cytokine responsive state. We also noted that Bcl-x levels were
actually enhanced in the Tg IL-2R-/- T blasts.
This result is consistent with the requirement of TCR and costimulatory
signals for the expression of Bcl-x (40) and indicates
that IL-2 signaling normally functions to reduce expression of this
anti-apoptotic protein, which will ultimately promote AICD. Thus,
IL-2 signaling during the course of TCR-mediated activation will be
responsible for both the increase and decrease of gene expression in a
highly regulated fashion.
In anti-CD3-induced activation and proliferation by normal T cells,
it is very difficult to separate the outcome of TCR/costimulatory
signals from IL-2R function as the production of IL-2 depends on
costimulation. With respect to cytokine-secreting effector T cells, it
has been generally concluded that IL-2 functions primarily to support
the growth of Ag-activated T cells while IL-12 or IL-4 primarily act to
induce Th1 or Th2 cytokines, respectively. Based on this notion, it was
somewhat unexpected that anti-CD3 failed to induce IFN-
secretion by Tg IL-2R-/- T cells in the absence
of exogenous cytokines as this response was accompanied by substantial
cell proliferation and cell division. Therefore, IL-2 may play a more
direct role beyond just proliferation, at least in vitro, for the
differentiation of effector IFN--secreting T cells. Consistent with
our findings, in some in vitro systems, IL-2 appeared to play a
necessary role to promote both IFN- and IL-4 secretion by developing
T helper cells (41). IL-2 has been reported to induce
IL-12R on T cells (42), which represents one potential
molecular target for IL-2 regulation of IFN-. Upon extended culture,
IL-4 functioned to allow some Tg IL-2R-/- T cells
to secrete both IFN- and IL-4. Thus, redundant pathways operate for
the induction of both CTL activity and IFN- secretion in the absence
of IL-2 function. The molecular basis and cellular target by which IL-4
promotes IFN- secretion remain to be determined.
The study of immune responses to lymphocytic choriomeningitis virus
(LCMV) infection in vivo show some parallels to our evaluation of T
cell responses by Tg IL-2R-/- mice in vitro. In
one study IL-2-/- mice induced protective
immunity to LCMV, although T cells from such mice exhibited reduced CTL
activity (43). Similar analysis of
IL-2-/-/IL-4-/- mice showed an
even greater impairment in LCMV-specific CTL activity
(44). Other investigators also noted impaired T cell
expansion, LCMV-specific CTL induction, and IFN- secretion by
IL-2-/- mice (45, 46). However, in
this case, the IL-2-deficient mice were unable to normally clear LCMV.
The differences in the ability to clear virus in these two studies and
the impaired T cell responses might be partially due to intrinsic T
cell defects due to the related autoimmune syndrome. However, some of
these findings likely represent the biological role for IL-2 in these
immune responses as our results also point to the importance of IL-2
and IL-4 to redundantly support CTL activity as well as a role for IL-2
in promoting IFN- secretion using IL-2R-deficient T cell without
intrinsic functional defects. Thus, further study of T cells from these
Tg IL-2R-/- mice should help clarify the precise
contribution by which IL-2R signaling regulates T cell activation in
vitro and in vivo without complication of immune suppressive autoimmune
disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas Malek, Department of Microbiology and Immunology, University of Miami School of Medicine, P.O. Box 016960 (R138), Miami, FL 33101.
3 Abbreviations used in this paper: AICD, activation-induced cell death; AC, accessory cells; c, common -chain; LCMV, lymphocytic choriomeningitis virus; CD40L, CD40 ligand; Tg, transgenic.
Received for publication September 12, 2000. Accepted for publication November 13, 2000.
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± SE of three mice per group and is normalized to the
proliferation of control IL-2R
