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Department of Pharmacology, University of Bath, Bath, United Kingdom; and Bath Institute for Rheumatic Diseases, Bath, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B and AP-1, but not on NF-AT or
the intact IL-2 promoter. In addition, T cell proliferation was
completely resistant to the actions of the immunosuppressant
cyclosporin A (CsA). Moreover T cell proliferation was unaffected by
the addition of blocking Abs to both IL-2 and the IL-2 receptor,
demonstrating that this form of costimulation by CD28 was independent
of IL-2. We also investigated the effects of stimulating T cell blasts
with CD80 alone and found that there was a limited requirement for IL-2
in this system. We conclude that CD28 costimulation can cause
substantial T cell proliferation in the absence of IL-2, which is
driven by a soluble factor independent of NF-AT
transactivation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD28 is a surface glycoprotein expressed on the majority of T cells that interacts with two known natural ligands (CD80 and CD86) (3). Both ligands are expressed by professional APCs, and both are capable of costimulating responses via CD28. One of the most striking features associated with CD28 costimulation is the ability of this pathway to prevent the induction of a tolerant state known as T cell anergy, an ability closely linked with the induction and secretion of a number of cytokines (3, 4, 5, 6), of which IL-2 appears to be the most important. One interpretation is, therefore, that costimulation of T cell proliferation and prevention of anergy by CD28 is mediated largely by IL-2 production and that IL-2 is responsible for many CD28 effects.
A major feature of signals initiated by CD28 costimulation is the resistance of this pathway to the calcineurin inhibitor, CsA, although the degree of resistance observed has been variable. This may relate to the fact that Abs to CD28 can generate a calcium signal (7, 8) that is dependent on the degree of cross-linking, which then leads to an apparently CsA-sensitive signal. In contrast, it appears that natural ligands do not stimulate calcium elevation. Whether or not CD28 is sensitive to CsA has considerable implications for the interpretation of costimulatory signals, since CsA is known to inhibit nuclear entry of the transcription factor NF-AT that is required for IL-2 gene transcription (9). Thus, if CD28 signals are resistant to CsA, this would suggest that CD28 has either NF-AT-independent effects on IL-2 production, or IL-2-independent costimulatory functions that would therefore dissociate CD28 costimulation from the effects of IL-2-driven proliferation. Attempts to understand the signaling mechanisms of CD28 have identified several pathways, including activation and recruitment of the enzyme phosphoinositide-3-kinase (PI3K)3, as well as a pathway via acidic sphingomyelinase (10, 11, 12). Nonetheless, the involvement of these pathways in costimulation of IL-2 production is still unclear (11, 13, 14).
Downstream of PI3K, the effects of CD28 stimulation include activation
of the ser/thr kinase protein kinase B (PKB) and subsequently p70S6
kinase (15, 16, 17) indicating a possible role for PI3K in
cell cycle progression. In addition, the PI3K pathway also appears to
be involved in CD28 receptor recycling (18), and it is
clear that CD28 costimulation has other functions, such as protection
from cell death and increasing longevity of T cell responses
(19, 20, 21, 22). CD28 costimulatory signals are also thought to
converge with signals from the TCR, resulting in the activation of the
c-Jun N-terminal kinase (JNK), which is required for the generation of
the transcription factor AP-1 (23). This, along with the
influence of transcription factors NF-B and NF-AT, provides the
basis for transcriptional activation of IL-2 gene transcription
(24). Consequently, much of the interpretation of CD28
costimulation has been based on analysis of IL-2 transcription and
production.
To address the role of IL-2 production as a component of CD28
costimulation and to investigate the effects of CsA on CD28 responses
triggered by the natural ligand CD80, we compared two distinct T cell
stimulation protocols, using either PMA and ionomycin or PMA plus CD80
ligand for their ability to stimulate T cells and support IL-2
production. These experiments revealed that T cells proliferated
strongly to both stimuli; however, PMA plus CD80-stimulated
proliferation occurred in a completely CsA-resistant fashion whereas
PMA plus ionomycin was sensitive to CsA. Strikingly, irrespective of
whether CsA was used, PMA- and CD80-stimulated cells displayed a highly
impaired ability to produce IL-2, and, using neutralizing Abs to IL-2
and IL-2 receptor, we observed that proliferation was independent of
IL-2. Consistent with this observation, studies on the IL-2 gene
promoter indicated that PMA plus CD80 could stimulate activity of
NF-B and AP-1 elements, but not NF-AT or the intact IL-2 promoter
activity, while PMA plus ionomycin stimulated all elements. Finally,
using activated T cells stimulated with CD80 alone, we also observed
similar CD28-mediated proliferative responses that also proceeded in a
largely IL-2-independent manner. These data suggest that CD28 is
capable of T cell costimulation independently of IL-2 and NF-AT in a
CsA-resistant manner.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purified resting T cells were isolated from peripheral blood of healthy human donors. PBMC were isolated by density gradient centrifugation at 800 x g for 30 min (Nycomed 1.077 g/ml), and the buoyant layer was isolated. Following two washes with medium, the cells were further purified by adhering to plastic for 1 h at 37°C in complete medium (RPMI 1640 + 10% FCS and antibiotics). Nonadherent cells containing the T cell population were recovered and incubated with mAbs against CD14 (UCHM1), HLA-DR (L243), and CD19 (BU12). Stained cells were magnetically removed using sheep anti-mouse IgG magnetic beads (Dynal, Oslo, Norway). The remaining cells, which comprised the resting T cell population, were used in subsequent experiments. For experiments using T cell blasts, PBMCs were taken following density gradient centrifugation and washing. These were then cultured with superantigen SEA (10 ng/ml) in complete medium. After 4–6 days, blasts were washed and utilized.
Transfectants
Chinese hamster ovary (CHO) cells were transfected with the CD80 cDNA expressed under the control of a CMV promoter, generated as previously described (25). Cells were maintained in glutamine-free DMEM containing 10% FCS, plus penicillin and streptomycin. Before use, cells were trypsinized and washed in PBS and then fixed with 0.025% glutaraldehyde in PBS for 2–3 min. Cells were washed extensively with medium containing 10% FCS, counted, and utilized.
T cell proliferation assays
Proliferation assays were performed in triplicate in 96-well plates in a final volume of 200 µl per well in RPMI 1640 medium containing 10% FCS and antibiotics. T cells (5 x 104) were cultured either alone or stimulated as specified in figure legends or incubated with either CHO or CHO-CD80 cells at a ratio of 3:1 T cell to transfectants. Proliferation was measured by thymidine incorporation after a further 72 h. Where anti-CD28 Abs were used, the Ab 9.3 was added at 1 µg/ml and cross-linked using sheep anti-mouse IgG (5 µg/ml). Assays were incubated at 37°C for 72 h, at which time 50-µl aliquots were removed for IL-2 analysis (DuoSet-ELISA, Genzyme, Boston, MA), and the cells were incubated for a further 18 h with the addition of 1 µCi [3H]thymidine per well. Cells were harvested onto 96-well filter plates using a Packard (Meriden, CT) 96-well harvester, and [3H]thymidine uptake was determined via liquid scintillation counting. Blocking Abs were added at the start of culture in 50-µl volumes to the final concentrations indicated in the figures. Data are plotted as mean values of triplicate wells. SEs were always less than 10% of the mean value.
Carboxy fluorescein diacetate succinimidyl ester labeling
To determine cell division of T cells, resting T cells were labeled with carboxy fluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Leiden, NL). T cells were labeled using 2.5 µM CFSE in PBS for 3 min and then washed twice in complete medium. Cells were then subjected to stimulation as described in the figure legends, using PMA, PMA +CD80, and PMA + ionomycin, and fluorescence was monitored by FACS at various times after stimulation.
Luciferase Assays
Plasmids encoding multimerized NF-AT, NF-B, or AP-1 or the
complete IL-2 promoter were used as previously described
(26). Jurkat cells (5 x 106)
were transiently transfected by electroporation at 320 V, 960 µF in 1
ml of complete medium. After 24 h, Jurkat cells were stimulated
for 8 h as detailed in figure legends or with CHO transfectants at
a 1:3 ratio. Cytoplasmic extracts were performed using luciferase assay
kit (Promega, Madison, WI). Briefly, cells were resuspended in lysis
buffer and incubated at room temperature for 15 min. Two freeze and
thaw cycles in liquid nitrogen aided the cytoplasmic extraction, and
after a brief centrifugation 20 µl of the supernatant was used with
100 µl of luciferase assay reagent. Luminescence was measured
immediately for 10 s with the TD-20/20 Luminometer or using a
Packard Top Count.
FACS analysis
FACS analysis was undertaken using the following Abs at 1 µg/ml: anti-CD2 (OKT11; American Type Culture Collection (ATCC), Manassas, VA), anti-CD25 (HB8784; ATCC), anti-CD69 (55.3.1; Serotec, Oxford, U.K.), and anti-CD28 (YTH913.12; Serotec) The primary Abs were added for 30 min at 4°C, cells were washed in PBS, and the secondary anti-mouse polyvalent FITC (Sigma, St. Louis, MO) was added for 30 min at 4°C. Five thousand events were analyzed by flow cytometry on a Becton Dickinson (Mountain View, CA) FACStarPlus using a 488-nm 100-mW laser.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies have used PMA in synergy with Abs to CD28 to
investigate the role of CD28 as a costimulatory receptor in T cell
activation (7, 27). However, few of these studies have
used the natural ligands of CD28, and confusion has arisen over the
sensitivity of CD28 costimulation to CsA. Since stimulation via the TCR
is inherently CsA sensitive, we utilized the ability of PMA and the
natural ligand CD80 to costimulate proliferative responses to clarify
the effect of CsA on the CD28 pathway. As shown in Fig. 1
, purified T cells proliferated well in
the presence of PMA and CHO-CD80 cells together, although not to either
stimulus alone. Moreover, this type stimulation was resistant to the
immunosuppressive drug CsA, and in a large number of experiments we did
not observe any CsA sensitivity in using this system. In contrast,
stimulation of T cells with PMA and ionomycin was highly sensitive
to CsA. To establish that both forms of stimulation resulted in
expansion of T cells with similar phenotypes, we also performed
flow cytometric analysis of the T cells activated by P + I and PMA +
CD80. These results (Fig. 2) confirmed
that the responding T cells was phenotypically similar in each case and
displayed typical activation markers such as CD69 and CD25. The only
difference observed was that those cells that received CD80 stimulation
expressed a lower level of CD28, which occurs as a result of ligand
engagement and is due to receptor internalization (18, 28). Overall, however, these experiments clearly demonstrated
that costimulation with the natural ligand CD80 resulted in
proliferation of phenotypically normal cells via a CsA-resistant
pathway.
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Given that proliferation of T cells to PMA plus CD80 was
completely CsA resistant, we next tested whether IL-2 production was
similarly unaffected by CsA, as has been suggested previously
(27). We therefore measured the effect of CsA on IL-2
production in response to PMA plus CD80 stimulation. Interestingly, we
observed that resting human T cells stimulated in this manner produced
very limited amounts of IL-2 (Fig. 3),
and this small increase was sensitive to CsA. In contrast, we observed
substantial production of IL-2 after stimulation with PMA and
ionomycin, which was completely inhibited by CsA. These results were
surprising not only because of the ability of T cells to proliferate to
PMA and CD80 despite extremely limited amounts of IL-2 (most notably in
the presence of CsA) but also because previous studies have suggested
that CD28 Abs synergized with PMA to induce IL-2, which was CsA
resistant. Interestingly, when we performed similar experiments using
cross-linked anti-CD28 Abs (Fig. 3), we also observed substantial
IL-2 production, which was not evident using natural ligand. Moreover,
this level of cytokine was sensitive to CsA, indicating that it derived
from a calcineurin-dependent pathway, most likely due to calcium
signals elicited from cross-linking CD28. However, the IL-2 production
stimulated by anti-CD28, while mainly sensitive to CsA, did
reveal some residual CsA-resistant IL-2 production, which may be a
feature of anti-CD28 stimulation. Thus, our results clearly
demonstrated an obvious disparity between the ability of T cell to
proliferate to PMA and CD80 stimulation and their ability to produce
IL-2. This suggested several possibilities; either small amounts of
IL-2 could sustain high levels of proliferation, the T cells consumed
IL-2 more rapidly when stimulated by PMA and CD80, or that T cell
proliferation may not be IL-2 dependent in this system. In addition,
these data highlighted substantial differences between the results
obtained using anti-CD28 Abs and natural ligands.
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B and AP-1 transactivation but not
NF-AT or the intact IL-2 promoter
To further understand the differences in signaling between PMA and
CD80 compared with PMA and ionomycin and their differential effects on
IL-2 production, we next examined the effects of these stimuli on the
transcription factors NFB, AP-1, NF-AT and on the intact IL-2
promoter.
To establish the effect of CD80 on the transactivation of the IL-2
promoter and its components, we used luciferase reporters transiently
transfected into Jurkat cells. As shown in Fig. 4A, PMA alone was unable to
induce substantial NF-B activity without the addition of ionomycin,
and as expected this activation was fully blocked by CsA. In contrast,
CD80 was also able to partially activate NF-B, increasing the
response 
2-fold over PMA alone, but, notably, this enhancement was
resistant to CsA, suggesting that pathways leading from CD28 to NFB
stimulation could be partially responsible for the CsA-resistant
costimulatory effect of CD28. Similar experiments were also performed
using AP-1 reporter constructs. In examining the transactivation of
AP-1 (Fig. 4B), PMA was found to strongly induce AP-1
luciferase activity, which was enhanced by the addition of ionomycin
and blocked by CsA. Significantly, CD80 stimulation also enhanced the
PMA-driven response, and like NFB this was completely resistant to
CsA. Thus, both NFB and AP-1 responses were stimulated by CD28 in a
CsA-resistant manner.
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, C and D, respectively). The NF-AT
transcription factor is known to be dependent on calcium signaling, and
specifically calcineurin, for its translocation to the nucleus, an
event that is blocked by CsA (9). However, a partially
calcineurin-independent mechanism has been suggested to be initiated by
CD28 Abs (29). Using luciferase assays, we observed a
strong NF-AT response to stimulation by PMA and ionomycin that, as
predicted, was completely blocked by CsA. However, in contrast to our
results for NFB and AP-1, we were unable to significantly activate
the NF-AT reporter construct with the PMA and CD80. In addition, using
a reporter construct containing the whole IL-2 promoter (Fig. 4D), similar results were obtained to those with NF-AT. In
this case, however, CD80 was able to slightly enhance IL-2 activation
induced by PMA, although this effect was blocked by CsA, suggesting it
could not account for the CsA-resistant CD28 effect on T cell
proliferation. However, we did observe that, in the presence of P + I,
CD80 stimulation was able to further stimulate the IL-2 promoter,
suggesting that CD28 could only significantly augment activation of the
IL-2 promoter in the context of calcium-dependent signals (data not
shown).
In addition to these experiments, we also directly compared the
abilities of CD80 and anti-CD28 mAb for their abilities to
stimulate NF-AT (E) and the IL-2 promoter (F) constructs. These results
showed similar findings for both constructs in that both PMA/CD80 and
PMA plus CD28 Abs (not cross-linked) were poor stimulators of the
constructs. However, cross-linking of the anti-CD28 Ab using a
sheep anti-mouse Ig consistently gave a 2- to 3-fold stimulation of
both NF-AT and IL-2, suggesting that cross-linking CD28 Abs provides
different signals to the natural ligand. This observation was
consistent with our observations in Fig. 3, suggesting that
cross-linked anti-CD28 Ab and CD80 ligand had different effects on
IL-2 transcription. However, it was notable that the levels of
luciferase activity seen with cross-linked anti-CD28 were at least
10-fold lower than those with PMA and ionomycin in the same assay.
Overall, however these results indicated that both NFB and AP-1
responses were stimulated by CD28 ligation and that the pathways
leading to activation of these transcription factors were CsA
resistant. In contrast, PMA and CD80 did not effectively stimulate
NF-AT or the IL-2 promoter in the absence of a calcium flux, consistent
with our data from normal T cell stimulations, indicating that IL-2
transcription was poorly stimulated in response to PMA and CD80. In
addition, these assays further revealed differences in the ability of
anti-CD28 to stimulate the IL-2 promoter, depending on whether or
not the Ab was cross-linked.
Anti-IL-2 and anti-IL-2R Abs do not inhibit PMA + CD80 proliferation
Taking the above results together, it did not appear that IL-2 was
likely to be responsible for T cell proliferation to PMA and CD80
stimulation. Nonetheless, since it was still formally possible that
PMA- and CD80-stimulated T cells produced small amounts of IL-2
sufficient to drive proliferation, we examined this possibility using
blocking Abs to IL-2 and the IL-2R. As shown in Fig. 5, A and B,
proliferation of the cells stimulated with PMA and CD80 was not
inhibited by blocking Abs to either IL-2 or the IL-2R, demonstrating
that IL-2 was not responsible for the proliferative response to PMA and
CD80. In fact, if anything, these Abs caused slight enhancement of
responses. This lack of inhibition was not due to the inability of the
Abs to block since substantial inhibition was observed in T cell
responses stimulated by either anti-CD3 plus CD80 or with P + I.
However, these Abs only partially inhibited proliferation to PMA and
ionomycin, indicating that, like PMA + CD80, this response was also
only partially IL-2 dependent. Nonetheless, it was clear from these
experiments that IL-2 was not required to sustain proliferation to PMA
and CD80.
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The above situation, where T cells proliferated well despite their
inability to generate significant quantities of IL-2 or activate
components of the IL-2 promoter, was reminiscent of our previous
studies using activated T cells that were capable of responding to CD80
ligation alone (12). Given that PMA stimulation is not a
physiologically relevant stimulus, we wondered whether CD80 stimulation
of T cell blasts might represent a similar situation where IL-2 was not
required. We therefore investigated the effects of CD80 stimulation on
SEA-activated T cell blasts. Accordingly, 5-day-old blasts were washed
and then recultured with either CD80 or CHO control cells, and the
effects of anti IL-2 Abs were tested. As shown in Fig. 6, the CD80-stimulated cells continued to
proliferate whereas the control cells did not. Furthermore, the
addition of blocking anti-IL-2 Abs revealed that this inhibited
only 20% of the response, indicating that IL-2 was also not
essential for T cell proliferation under these circumstances. Thus,
these experiments confirmed the possibility that IL-2-independent T
cell proliferation may also occur under more physiological
circumstances.
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Finally, given that tritiated thymidine incorporation represents
DNA labeling in S phase, but not necessarily actual cell division, we
tested whether PMA- and CD80-stimulated cells divided similarly to
cells from P + I-stimulated cultures. This was done using both cell
counting (data not shown) and assessment of cell division using CFSE
labeling (30). By labeling T cells before cell division
with CFSE, cell division can then be monitored by the decrease in dye
content of the cells as the dye is divided between daughter cells. The
data shown in Fig. 7 indicated that, over
a 7-day period, the CFSE content of the cells decreased over 10-fold in
the PMA + CD80 (Fig. 7B) and the P + I cultures (Fig. 7C). In contrast, the cells stimulated with PMA alone did
not show any decrease in cell staining. In addition, viable cell
numbers increased in the PMA + CD80 and P + I cultures by 6-fold
over the same period. Taken together, these data provided unequivocal
evidence that the T cells were undergoing cell division in response to
PMA + CD80 in a manner that was IL-2 independent.
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Whereas we had established that IL-2 was not responsible for T
cell responses to PMA and CD80, we wished to establish whether
proliferation was dependent on a soluble factor or whether CD28 signals
were driving proliferation directly. To test this, we transferred
supernatants from cultures stimulated with various treatments onto
previously activated day 5 T cell blasts. As shown in Fig. 8, this revealed that treatment with PMA
and CD80 resulted in the secretion of a soluble factor that could cause
proliferation when transferred to T cell blasts. Likewise, the
supernatant from PMA + ionomycin stimulation could also transfer a
proliferative stimulus; however, in keeping with our other
observations, CsA strongly inhibited PMA + ionomycin- but not PMA +
CD80-derived supernatants. Interestingly, these effects were of similar
potency as the addition of IL-2. Thus, this experiment indicated that
cytokines were involved in both forms of proliferation although it
raised the possibility that different cytokines may be involved,
depending on the stimulation conditions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We observed that this form of stimulation was completely CsA resistant, suggesting it did not depend on the phosphatase activity of calcineurin, an enzyme known to be critical for nuclear entry of the transcription factor NF-AT and control of IL-2 gene transcription (31, 32). This lack of effect of CsA on CD28 costimulation is similar to that reported by others (7, 27); however, there are several differences between our observations and those previously. In our experiments we did not observe any sensitivity of CD80-stimulated CD28 to CsA; this contrasts to the observations of Linsley (7), who suggested that a CsA-sensitive pathway may also derive from CD28. We believe these differences may be explained by the use of Abs vs natural ligands. Indeed, our own data using cross-linked anti-CD28 Abs indicate that this type of stimulation does lead to an apparent CsA sensitivity in the CD28 pathway, which while inhibiting IL-2 production does not affect T cell proliferation.
Our findings also contrast somewhat with the findings of June et al. (27), who observed that CD28-costimulated IL-2 mRNA induction was not inhibited by CsA. In our experiments, we observed either limited IL-2 production using ligand or CsA-sensitive IL-2 production from cross-linked Ab. There are several possible explanations for this. One possibility may be that mRNA analysis by Northern blotting does not correlate well with actual IL-2 production, as measured by ELISA. A second alternative is that their autoradiographic exposures were nonlinear, resulting in possible differences not being detected. A third possibility is that there are genuine differences between Ab and ligand stimulation of this pathway; indeed, several differences have been previously reported (33, 34). Overall, however, despite these differences, consistent concepts do emerge from these various studies. Clearly much, if not all, of the CD28 pathway is unaffected by CsA, and in all studies T cell proliferation is not inhibited. In the absence of calcium signaling, as seen using PMA and CD80, we observed that IL-2 induction was significantly impaired. However, IL-2 production generated by Ab cross-linking appears to be sensitive to CsA, in keeping with the known effects of calcium stimulation on NF-AT. Nonetheless, a residual CsA-resistant IL-2 signal was detected using Ab stimulation. Overall, our interpretation of these various data is that the CsA resistance associated with CD28 costimulation is not due to the fact that IL-2 production is unaffected by CsA but that the CD28 pathway responsible for costimulation of T cell proliferation is unaffected and may ultimately be IL-2 independent.
Our findings at the functional level are supported by luciferase assays
of IL-2 gene transcriptional activation. These demonstrated that both
NF-AT and intact IL-2 promoter responses were either not stimulated by
CD80 or stimulated in a manner that was CsA sensitive (Fig. 4, C and D). While there are reports that suggest an
NF-AT response in the presence of CsA (29), these clearly
represented only a minor fraction of NF-AT activation and were observed
using anti-CD28 Abs. Thus, whereas it is possible using Abs to
detect CsA-resistant NF-AT and IL-2 responses, these are not easily
observed using natural ligands. In comparing CD28 Abs and CD80 ligands
directly in luciferase assays in Jurkats, we observed that cross-linked
anti-CD28 stimulated NF-AT and IL-2 luciferase activity to a
greater degree than either soluble anti-CD28 or the CD80 ligand.
This is consistent with our data (Fig. 3), which demonstrated similar
differences between anti-CD28 and CD80 ligand in IL-2 production by
normal T cells. Taken together, these data support the view that
cross-linked CD28 Abs are significantly more potent stimuli for IL-2
production than the natural ligand under some conditions of T cell
stimulation.
In contrast to the NF-AT and IL-2 promoter, we observed CD80
stimulation of NFB and AP-1-driven luciferase reporters in keeping
with other studies implicating CD28 in AP-1 and NFB signaling
(12, 23, 35, 36). In addition we could only observe
substantial effects on IL-2 luciferase by CD80 ligation in the presence
of PI stimulation. Given that CD80 was ineffective with PMA alone, this
suggests that CD28 signals are effective on the whole IL-2 promoter
only in the presence of a calcium-dependent stimulus. These results
would be consistent with a possible effect of CD28-stabilizing mRNA
(37).
Given the apparent discrepancy between proliferation and IL-2
production, we investigated directly the influence of IL-2 on PMA and
CD80 stimulation. Here again, our findings supported the view that IL-2
was not critical to T cell proliferation in this model. Strikingly,
neither anti-IL-2 nor anti-IL2R Abs could inhibit T cell
proliferation, indicating that IL-2 was not involved in T cell
proliferation driven by PMA and CD80. In contrast, these Abs were able
to significantly inhibit proliferation by CD3 and CD80 and P + I.
Despite these surprising findings, our data are similar to those of at
least two previous studies using IL-2 knockout mice. In particular,
studies by Razi-Wolf evaluated the effect of B7 costimulation on
CD4+ T cells in vitro in IL-2-deficient mice
(38). This established that B7 costimulation was still
effective, and furthermore it appeared that this proliferation was
independent of other cytokines, including IL-4 and other common
-chain-utilizing cytokines. Our own preliminary experiments concur
with this finding in that we are unable to detect IL-4 by RT-PCR
analysis. More recently similar findings were reported by Khoruts et
al. (39), which revealed a CD28-dependent IL-2-independent
function triggered by LPS. Thus, despite our use of nonphysiological
stimuli to reveal a discrete CD28 function, it appears that this model
is very similar to functions of CD28 seen in vivo and in vitro on CD28
ligation using T cell blasts.
Given that T cell proliferation in the absence of IL-2 appears to be responsible for inflammatory bowel disease in mice, these studies may provide a useful and manipulable model for understanding the functional behavior of T cells that are able to operate in the absence of IL-2 and in which the negative regulatory influences of IL-2 may be studied.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David M. Sansom, Department of Pharmacology, University of Bath, Bath, BA2 7AY, U.K. E-mail address:
3 Abbreviations used in this paper: PI3K, phosphoinositide-3-kinase; CsA, cyclosporin A; CHO, Chinese hamster ovary; iono, ionomycin; CFSE, carboxy fluorescein diacetate succinimidyl ester; P + I, PMA + ionomycin.
Received for publication October 22, 1998. Accepted for publication June 2, 1999.
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which can be prevented by the immunosuppressant rapamycin. J. Biol. Chem. 269:30077.This article has been cited by other articles:
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  M. Shi, T. H. Lin, K. C. Appell, and L. J. Berg Cell Cycle Progression following Naive T Cell Activation Is Independent of Jak3/Common {gamma}-Chain Cytokine Signals J. Immunol., October 1, 2009; 183(7): 4493 - 4501. [Abstract] [Full Text] [PDF]
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C. Scotta, M. Soligo, C. Camperio, and E. Piccolella FOXP3 Induced by CD28/B7 Interaction Regulates CD25 and Anergic Phenotype in Human CD4+CD25- T Lymphocytes J. Immunol., July 15, 2008; 181(2): 1025 - 1033. [Abstract] [Full Text] [PDF]
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A. Kerstan, N. Armbruster, M. Leverkus, and T. Hunig Cyclosporin A Abolishes CD28-Mediated Resistance to CD95-Induced Apoptosis via Superinduction of Caspase-3 J. Immunol., December 1, 2006; 177(11): 7689 - 7697. [Abstract] [Full Text] [PDF]
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S. Colombetti, V. Basso, D. L. Mueller, and A. Mondino Prolonged TCR/CD28 Engagement Drives IL-2-Independent T Cell Clonal Expansion through Signaling Mediated by the Mammalian Target of Rapamycin. J. Immunol., March 1, 2006; 176(5): 2730 - 2738. [Abstract] [Full Text] [PDF]
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E. Canto, J. L. Rodriguez-Sanchez, and S. Vidal Distinctive response of naive lymphocytes from cord blood to primary activation via TCR J. Leukoc. Biol., December 1, 2003; 74(6): 998 - 1007. [Abstract] [Full Text]
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R. Bommireddy, I. Ormsby, M. Yin, G. P. Boivin, G. F. Babcock, and T. Doetschman TGF{beta}1 Inhibits Ca2+-Calcineurin-Mediated Activation in Thymocytes J. Immunol., April 1, 2003; 170(7): 3645 - 3652. [Abstract] [Full Text] [PDF]
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S. Colombetti, F. Benigni, V. Basso, and A. Mondino Clonal Anergy Is Maintained Independently of T Cell Proliferation J. Immunol., December 1, 2002; 169(11): 6178 - 6186. [Abstract] [Full Text] [PDF]
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 W. W. S. Kum, S. B. Cameron, R. W. Y. Hung, S. Kalyan, and A. W. Chow Temporal Sequence and Kinetics of Proinflammatory and Anti-Inflammatory Cytokine Secretion Induced by Toxic Shock Syndrome Toxin 1 in Human Peripheral Blood Mononuclear Cells Infect. Immun., December 1, 2001; 69(12): 7544 - 7549. [Abstract] [Full Text] [PDF]
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 S. B. Han, S. H. Park, Y. J. Jeon, Y. K. Kim, H. M. Kim, and K. H. Yang Prodigiosin Blocks T Cell Activation by Inhibiting Interleukin-2Ralpha Expression and Delays Progression of Autoimmune Diabetes and Collagen-Induced Arthritis J. Pharmacol. Exp. Ther., November 1, 2001; 299(2): 415 - 425. [Abstract] [Full Text] [PDF]
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E. K. Kapsogeorgou, H. M. Moutsopoulos, and M. N. Manoussakis Functional Expression of a Costimulatory B7.2 (CD86) Protein on Human Salivary Gland Epithelial Cells that Interacts with the CD28 Receptor, but Has Reduced Binding to CTLA4 J. Immunol., March 1, 2001; 166(5): 3107 - 3113. [Abstract] [Full Text] [PDF]
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T. R. Malek, A. Yu, P. Scibelli, M. G. Lichtenheld, and E. K. Codias Broad Programming by IL-2 Receptor Signaling for Extended Growth to Multiple Cytokines and Functional Maturation of Antigen-Activated T Cells J. Immunol., February 1, 2001; 166(3): 1675 - 1683. [Abstract] [Full Text] [PDF]
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